Posts Tagged ‘Immunotherapy’

Oncolytic Virus Immuno-Therapy: New Approach for a New Class of Immunotherapy Drugs

Curator: Larry H. Bernstein, MD, FCAP


Oncolytic viruses represent a promising novel immunotherapy strategy, which may be optimally combined with existing therapeutic modalities

Oncolytic viruses: a novel form of immunotherapy

Oncolytic viruses are novel anticancer agents, currently under investigation in Phase I–III clinical trials. Until recently, most studies have focused on the direct antitumor properties of these viruses, although there is now an increasing body of evidence that the host immune response may be critical to the efficacy of oncolytic virotherapy. This may be mediated via innate immune effectors, adaptive antiviral immune responses eliminating infected cells or adaptive antitumor immune responses. This report summarizes preclinical and clinical evidence for the importance of immune interactions, which may be finely balanced between viral and tumor elimination. On this basis, oncolytic viruses represent a promising novel immunotherapy strategy, which may be optimally combined with existing therapeutic modalities.
The anticancer activity of viruses has been reported throughout the 20th century. Developments in virology, genetic manipulation and molecular biology have led to a surge of research investigating viruses with oncolytic or antitumor properties over the last 15 years. Several oncolytic viruses are currently in Phase I–III clinical trials [1]. Until recently, despite the multitude of studies investigating direct viral effects upon cancer cells, relatively little attention had been paid to the interaction between oncolytic viruses and the immune system. We discuss the evidence supporting the view that the host immune response is critical to the efficacy of oncolytic virotherapy. The potential of oncolytic viruses to break immunological tumor tolerance, generating antitumor immunity, represents a novel avenue of immunotherapy.
Oncolytic viruses are self-replicating, tumor selective and directly lyze cancer cells [2]. They may be tumor selective in wild-type or attenuated forms or may be engineered to provide tumor selectivity. Naturally occurring oncolytic viruses include the double-stranded RNA reovirus and single-stranded RNA Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV). By contrast, human DNA viruses, including adenoviruses, vaccinia and herpes simplex viruses (HSV) have been genetically modified in a variety of ways to provide tumor selectivity. A diverse range of mechanisms provide tumor specificity, including inactivation of antiviral defences, such as type I IFN responses in many cancer cells, viral deletions permitting replication only in tumor cells that can substitute for viral defects, tumor-selective uptake via upregulated or mutated receptors, and targeting to tumor promoters.

In the majority of clinical trials performed so far, oncolytic viruses have been administered via intratumoral injection. A smaller number of studies have examined regional or intravenous delivery. Clinical experience has demonstrated a favorable toxicity and safety profile and a number of tumor responses, although overall antitumor efficacy has been limited [1]. For example, ONYX-015, a modified adenovirus, has been used in clinical trials with response rates of 0–14% following intratumoral administration [3]. In view of the short history of oncolytic virotherapy, along with recent scientific advances in methods of viral delivery and enhancing antitumor potency, these low levels of single-agent clinical responses provide encouragement for the future.

An increasingly powerful body of evidence supports the ability of the immune system to modify the immunogenicity and behavior of tumors [4]. A host of tumor-associated antigens (TAA) have been characterized [5] and in a single tumor, tumor-infiltrating lymphocytes directed towards multiple TAAs can be identified [6]. Despite these antigenic differences, the antitumor immune response is commonly ineffectual. Tumors can subvert antitumor immunity, generating an immunosuppressive tumor microenvironment by a multitude of mechanisms. These include the induction of Treg cells, secretion of soluble immunosuppressive mediators including nitric oxide, IL-10 and TGF-β and recruitment of myeloid suppressor cells [4]. Matzinger’s ‘danger’ hypothesis proposes that the prime role of the immune system is to respond to cellular or tissue distress as opposed to nonself per se [7]. Several danger signals have been identified, including RNA, DNA, IFN-α, heat-shock proteins, uric acid and hyaluron, providing a mechanistic basis for this hypothesis [8]. On this basis, tumor-associated danger signals are critical to the generation of effective antitumor immunity. In addition to their ability to disrupt immune responses, tumors commonly lack such signals and successful tumor immunotherapy will probably to depend upon their provision. Oncolytic virotherapy represents a potent approach to cancer immunotherapy, combining the enhanced release of TAA via tumor cell death, in the context of danger signals (FIGURE 1).

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Figure 1   Concept of how oncolytic viral infection of tumor cells may lead to the generation of antitumor immune responses

The role of the innate immune response to cancer is double-edged. Chronic inflammatory changes can promote tumor progression via proliferative and proangiogenic signals [9], while by contrast, the infiltration of activated innate inflammatory cells can mediate tumor regression in vivo [10]. Manipulation of the immune environment within a tumor is a potentially critical strategy towards successful tumor immunotherapy [11].

Oncolytic viruses represent prime candidates to enhance the immunogenicity of the tumor microenvironment. As detailed below, oncolytic virotherapy may be immunomodulatory via tumor cell death, production of endogenous danger signals, the release of tumor-derived cytokines and direct effects upon cells of the innate immune system. Evidence from preclinical models suggests that an early influx of immune cells, including macrophages and natural killer (NK) cells, occurs in response to tumor viral therapy [1214]. These changes within the tumor hold the potential to alter the pre-existing immunosuppressive microenvironment, in favor of the generation of therapeutic immune responses. Dendritic cells (DC), the prime antigen-presenting cells and a component of the innate immune response are critical for the subsequent generation of antigen-specific or adaptive immune responses. However, as discussed later, the outcome of the innate response is finely balanced between promotion of tumor clearance and viral clearance limiting efficacy.

Virally induced cell death would be expected to enhance the availability of TAA for uptake by DC. Indeed, viral infection of tumors has been reported to enhance the phagocytosis of tumor-derived material [15,16]. The relationship between the mode of cell death and tumor immunogenicity has, however, been controversial; the immunogenicity of tumors has been reported not to be affected by whether tumor cells are alive, apoptotic or necrotic [17]. Even if the mode of cell death is not an immunogenic determinant, the release of intrinsic cell factors, including heat-shock protein [18], uric acid [19] and bradykinin [20], can be identified as danger signals by DC. Oncolytic viral infection may mediate production of these factors. For example, tumor cell infection by a modified oncolytic adenovirus increases intracellular uric acid levels, activating DC [19].

An array of cytokines provides costimulation for T-cell responses, while by contrast, tumor-derived cytokines, including TGF-β and IL-10, have immunosuppressive properties. In addition, the tumor-derived proinflammatory cytokines VEGF, TNF-α and several chemokines have been linked to promotion of tumor growth [21]. Oncolytic viral infection is likely to alter the balance of cytokines produced and the nature of the subsequent immune response. We have investigated the release of cytokines following infection of melanoma cells with reovirus, a naturally occurring double-stranded RNA virus currently in clinical trials [22]. Reovirus was found to induce secretion of IL-8, RANTES and MIP-1α/β, which play a role in the recruitment of DC, neutrophils and monocytes [23], and of IL-6, which can inhibit the immunosuppressive function of Treg cells [24]. Reovirus additionally reduced tumor secretion of the immunosuppressive cytokine IL-10. The immunogenic property of tumor-conditioned media from reovirus-infected tumor cells (filtered to remove viral particles) was confirmed by their ability to activate DC.

DC & the response to viral infection

The immune system is adept at pathogen recognition and a host of receptors specific for pathogen-associated molecular patterns, including the toll-like receptors (TLR), have been identified [25]. Innate viral recognition can center around viral nucleic acids or viral proteins [25]. DC play a critical role in the early innate immune responses, reciprocally interacting with other innate immune cells, including NK cells [26]. In this context, oncolytic viruses can influence the nature of the innate tumor response. Reovirus-infected DC, for example, enhance NK cytotoxicity towards tumor cells [27].

The effect of viruses upon DC is virus specific: measles and a vaccinia virus strain impair DC phenotype and function [28,29], an oncolytic adenovirus has a neutral effect [30], while reovirus is directly stimulatory to DC [27]. Although the immunomodulatory effects of oncolytic viruses have been investigated to a limited degree, it follows that the immune consequences of therapy with different viruses will vary widely. In addition, the genetic modification of viruses to confer oncolytic specificity may involve interference with virulence genes whose function is to modify the antiviral immune response, including type I interferon response genes [2,31]; alteration of such immunomodulatory genes will alter the consequences of the immune interactions of these modified viruses.

Oncolytic viruses & adaptive antitumor immunity

The innate immune response is thought to provide an important link to the generation of adaptive immune responses. DC are key to this link, taking up TAA, integrating danger signals and presenting antigen in an appropriate costimulatory context to the adaptive arm of the immune system. An adaptive antitumor immune response requires activation of cytotoxic CD8 T cells by DC presenting tumor antigen on MHC class I molecules. The presentation of exogenous antigen in a MHC class I context is termed ‘cross-presentation’. Critically, virally infected cells have been shown to be superior at delivering nonviral antigen for cross-presentation and cross-priming adaptive immune responses in vivo [32]. Intriguingly, recent work has defined a role for TLR-4 receptor ligands (bacterially derived lipopolysaccharide) in enhancing cross-presentation [33]; a similar effect of viral as opposed to bacterial TLR ligands has yet to be explored. Inflammatory stimuli have additionally been shown to enhance antigen processing and the generation of MHC class II complexes, required for CD4+ T-cell help in adaptive immune responses [34,35]; such inflammatory stimuli could be provided by viral tumor infection. Oncolytic virotherapy may therefore enhance immune priming via multiple effects upon DC. There is an emerging body of data from murine and human preclinical research supporting the concept that the efficacy of oncolytic virotherapy is at least partially immune mediated and that antitumor immunity can be generated.

Overall, the antiviral humoral and cellular immune responses may have contrasting consequences. Methods of enhancing viral delivery to tumors or immunomodulation provide an opportunity to alter this balance in favor of therapeutic benefit.

Clinical trials & the immune response

Although preclinical studies have provided support for the concept that the efficacy of oncolytic virotherapy may be dependent upon the host immune response, there are limited data on the immune response following virotherapy from early clinical trials.

Studies of intratumoral administration have provided direct evidence of a cellular immunological response. In a Phase I trial of a second-generation oncolytic HSV expressing GM–CSF injected into subcutaneous metastases from a variety of tumor types, post-treatment biopsies revealed an extensive immune cell infiltrate [54]. Additionally, suggestive of an immune-mediated antitumor effect, was the observation of inflammation in uninjected tumor deposits in four of 30 treated patients. Similarly, in a study of intratumoral administration of a recombinant vaccinia–GM–CSF virus in patients with melanoma deposits, treated lesions were shown to have a dense immune cell infiltrate. The generation of antitumor immunity was implied by the regression of noninjected regional dermal metastases in association with an immune infiltrate in four of seven treated patients [55]. A Phase I study of injection of JX-594, a targeted poxvirus armed with GM–CSF, into primary and metastatic liver tumors has recently been reported with encouraging evidence of activity, with a partial response in three and stable disease in six of ten evaluable patients by Response Evaluation Criteria in Solid Tumors (RECIST) [56]. Consistent with a possible antitumor immune response was the durability of tumor responses. Notably, there was evidence of functional response in noninjected tumors in three of seven evaluable patients by Choi criteria for reduction in Hounsfield units (n = 2) and by reduced 18F-fluorodeoxyglucose (18FDG)-PET signal (n = 1). There was evidence of viral dissemination to noninjected tumor tissue. The responses in injected and noninjected tumor tissue could therefore have been mediated by direct viral oncolysis, antiviral immune responses towards virally infected cells or antitumor immune responses established in the injected lesions.

Oncolytic viruses have been combined with tumor vaccines in an attempt to exploit viral danger signals. Vaccinia virus–melanoma cell lysate vaccines were used in an adjuvant Phase III study of 700 patients following melanoma resection, with no improvement in recurrence or overall survival [57]. A series of clinical studies has been performed by Schirrmacher et al. using a live autologous tumor vaccine infected by NDV irradiated to render tumor cells nonviable [58]. A significant proportion of patients developed antitumor immune responses as assessed by a delayed-type hypersensitivity response to skin prick tests. Phase II studies have been performed in glioblastoma multiforme, melanoma, breast and colorectal cancer with improvements in overall survival by 20–36% at 2–5-year follow-up compared with historical controls. These studies suggest that oncolytic viruses can break immunological tumor tolerance, although Phase III studies are needed to confirm these findings.

Combination therapy may be the optimal context in which to exploit the immunotherapeutic potential of oncolytic viruses. A rationale exists for combination with existing immunotherapy strategies, along with conventional therapy.

Adoptive cellular therapy & viral delivery

The use of cell carriers to chaperone viral particles to the tumor is a promising innovation [51]. Cells of the immune system have proven particularly adept, including cytokine-activated killer cells [52] and T lymphocytes [36]. Adoptive cellular therapy has met with some clinical success, but has been limited by the trafficking to and survival of T cells in the tumor microenvironment [62]. In a mouse model, the combination of oncolytic virus delivery with antigen-specific adoptive T-cell therapy has been shown to improve upon either treatment modality alone [63]. Although yet to be tested in clinical trials, these findings are of significant translational potential.

Immunotherapy combinations

Immunotherapy approaches may be logically combined with virotherapy to enhance antitumor responses.

The host immune response will probably be critical to the efficacy of oncolytic virotherapy, although it is a fine balance between rapid viral elimination and innate and adaptive responses, which may mediate tumor regression. The rational design of combination therapy, modulating the immunological outcome, may hold the key to fulfilling the potential of these novel agents. Clinical trials should be designed to include specific assessment of immune responses to both tumor and viral antigens, and recognize the immunotherapeutic potential of virotherapy in terms of clinical end points and patient selection.

Oncolytic Viruses and Their Application to Cancer Immunotherapy

E. Antonio Chiocca1 and Samuel D. Rabkin2
Cancer Immunol Res April 2014 2; 295

Oncolytic viruses (OV) selectively replicate and kill cancer cells and spread within the tumor, while not harming normal tissue. In addition to this direct oncolytic activity, OVs are also very effective at inducing immune responses to themselves and to the infected tumor cells. OVs encompass a broad diversity of DNA and RNA viruses that are naturally cancer selective or can be genetically engineered. OVs provide a diverse platform for immunotherapy; they act as in situ vaccines and can be armed with immunomodulatory transgenes or combined with other immunotherapies. However, the interactions of OVs with the immune system may affect therapeutic outcomes in opposing fashions: negatively by limiting virus replication and/or spread, or positively by inducing antitumor immune responses. Many aspects of the OV–tumor/host interaction are important in delineating the effectiveness of therapy: (i) innate immune responses and the degree of inflammation induced; (ii) types of virus-induced cell death; (iii) inherent tumor physiology, such as infiltrating and resident immune cells, vascularity/hypoxia, lymphatics, and stromal architecture; and (iv) tumor cell phenotype, including alterations in IFN signaling, oncogenic pathways, cell surface immune markers [MHC, costimulatory, and natural killer (NK) receptors], and the expression of immunosuppressive factors. Recent clinical trials with a variety of OVs, especially those expressing granulocyte macrophage colony-stimulating factor (GM-CSF), have demonstrated efficacy and induction of antitumor immune responses in the absence of significant toxicity. Manipulating the balance between antivirus and antitumor responses, often involving overlapping immune pathways, will be critical to the clinical success of OVs. Cancer Immunol Res; 2(4); 295–300. ©2014 AACR.

Oncolytic virus (OV) therapy is based on selective replication of viruses in cancer cells and their subsequent spread within a tumor without causing damage to normal tissue (1, 2). It represents a unique class of cancer therapeutics with distinct mechanisms of action. The activity of OVs is very much a reflection of the underlying biology of the viruses from which they are derived and the host–virus interactions that have evolved in the battle between pathogenesis and immunity. This provides a diverse set of activities that can be harnessed and manipulated. Typically, OVs fall into two classes: (i) viruses that naturally replicate preferentially in cancer cells and are nonpathogenic in humans often due to elevated sensitivity to innate antiviral signaling or dependence on oncogenic signaling pathways. These include autonomous parvoviruses, myxoma virus (MYXV; poxvirus), Newcastle disease virus (NDV; paramyxovirus), reovirus, and Seneca valley virus (SVV; picornavirus); and (ii) viruses that are genetically manipulated for use as vaccine vectors, including measles virus (MV; paramyxovirus), poliovirus (PV; picornavirus), and vaccinia virus (VV; poxvirus), and/or those genetically engineered with mutations/deletions in genes required for replication in normal but not in cancer cells including adenovirus (Ad), herpes simplex virus (HSV), VV, and vesicular stomatitis virus (VSV; rhabdovirus; refs. 1,3). Genetic engineering has facilitated the rapid expansion of OVs in the past two decades, enabling a broad range of potentially pathogenic viruses to be manipulated for safety and targeting (3). Many of the hallmarks of cancer described by Hanahan and Weinberg (4) provide a permissive environment for OVs; they include sustained proliferation, resisting cell death, evading growth suppressors, genome instability, DNA damage stress, and avoiding immune destruction. In addition, insertion of foreign sequences can endow further selectivity for cancer cells and safety, as well as altering virus tropism through targeting of translation with internal ribosome entry sites (IRES) or microRNAs (PV and VSV), transcription with cell-specific promoter/enhancers (Ad, HSV), or transduction with altered virus receptors (HSV, Ad, MV, and VSV; refs.1, 3). These strategies are also being used to target replication-deficient viral vectors for gene therapy applications in cancer immunotherapy.

OVs have many features that make them advantageous and distinct from current therapeutic modalities: (i) there is a low probability for the generation of resistance (not seen so far), as OVs often target multiple oncogenic pathways and use multiple means for cytotoxicity; (ii) they replicate in a tumor-selective fashion and are relatively nonpathogenic and, in fact, only minimal systemic toxicity has been detected; (iii) virus dose in the tumor increases with time due to in situ virus amplification, as opposed to classical drug pharmacokinetics that decrease with time; and (iv) safety features can be built in, such as drug and immune sensitivity. These features should result in a very high therapeutic index. An important issue for OV therapy is delivery. Although systemic intravenous administration is simpler than intratumoral injection and can target multiple tumors, it has drawbacks, including nonimmune human serum, anti-OV antibodies that preexist for human viruses or can be induced by multiple administrations, lack of extravasation into tumors, and sequestration in the liver (1). Cell carriers [i.e., mesenchymal stromal cells, myeloid-derived suppressor cells (MDSC), neural stem cells, T cells, cytokine-induced killer cells, or irradiated tumor cells] can shield virus from neutralization and facilitate virus delivery to the tumor (5). The effectiveness will vary depending upon the cell phenotype, permissiveness to virus infection, tumor-homing ability, and transfer of infectious virus to tumor cells. To block virus neutralization and extend vascular circulation, viruses can also be coated in nanoparticles (i.e., PEGylation; ref. 1).

OV Immunotherapy

Virus infection and pathogenicity have been major drivers in the evolution of the human immune system, and vaccination against viruses is the quintessential exploitation of adaptive immunity. A major goal of OV-mediated immunotherapy is to activate and redirect functional innate and adaptive immune responses toward the tumor. Interactions between innate and adaptive immune cells and signaling factors (i.e., cytokines and chemokines), often involved in virus infections, play a large role in antitumor immunity or lack thereof, as well as successful immunotherapies (Fig. 1). Virus infection induces an inflammatory response leading to adaptive antivirus immunity. Thus, the immune system was seen initially as a negative factor in OV therapy for limiting virus infection/delivery because of preexisting or therapy-induced immunity, virus replication because of innate antiviral responses, and virus spread because of the infiltration of innate immune cells (6). In addition, most early studies were performed in human xenograft tumor models in immunodeficient mice lacking adaptive immune responses because some viruses were species selective or replicated better in human cells, and because there was availability of a broad diversity of human cancer cell lines. With the use of syngeneic tumor models in immunocompetent mice, it became clear that the consequences of the immune system were complex, but that the induction of antitumor immunity was feasible and efficacious (6). In particular, many OVs act asin situ vaccines, inducing robust, long lasting, and specific adaptive antitumor responses, often CD8+ T cell–mediated (7, 8). Interestingly, adaptive antiviral immunity can enhance antitumor immunity for HSV, but not for VSV (8, 9).

Figure 1.

Figure 1.

Cartoon of OV-mediated effects in tumor. First phase, OV delivered intratumorally or systemically, infects tumor cells (can be blocked by humoral defense systems; antibodies). After infection, OV replicates (can be blocked by innate responses; i.e., IFN-α/β), kills cells often by ICD, and spreads throughout the tumor (can be blocked by innate immune cells, i.e., NK cells and macrophages), eliciting an inflammatory response. When an armed OV is used, the immunomodulatory transgene is expressed (transgene product). Second phase, ICD and inflammation recruit DCs to the tumor, where they take up TAAs and induce an adaptive immune response (T and B cells), which targets the tumor (can be blocked by Tregs and MDSCs). Innate cells such as NK cells also have antitumor activities. Antitumor immune responses can be further enhanced by transgene products. CPA, cyclophosphamide.


The inflammatory cascade and immunogenic cell death (ICD) induced by OV infection of tumors makes OVs particularly powerful inducers of antitumor immunity (8, 10). Among the many different types of cell death, some are immunogenic and characterized by the release of danger-associated molecular patterns (DAMP), such as calreticulin, high-mobility group protein B1 (HMGB1), and ATP, along with tumor-associated antigens (TAA; ref. 10). Multiple forms of ICD have been observed after OV (Ad, VV, HSV, MV, and coxsackievirus) infection of cancer cells, and there is a suggestion that ICD occurs in patients after treatment with oncolytic Ad and temozolomide (11). However, much remains to be learned about the mechanisms of OV-mediated cell death and how it can be exploited to enhance immunogenicity. Inflammation, typically chronic, can also promote tumorigenesis and inhibit T-cell antitumor activity (12). Restraining antiviral immune responses and minimizing pathology, while promoting antitumor immune responses, is a complex and poorly understood balancing act that will dictate OV therapy outcomes. In some cases, where minimal OV replication occurs in mouse tumors (i.e., HSV) or no replication is required (i.e., reovirus; ref. 13), antitumor efficacy is principally due to OV-induced immune responses. Understanding, harnessing, modulating, and/or enhancing OV-mediated immune responses for effective antitumor immunity are major areas in current research that intersect with other immunotherapeutic strategies.

Many viruses express immune evasion genes that enable them to establish infections and spread within their host (14). Mutations in these genes (i.e., HSV Us11, VV E3L, MYXV M156R, Ad VAI, and reovirus σ2/σ3, inhibitors of PKR; HSV ICP0, VV N2, NDV V, and MV V, inhibitors of IRF3; HSV ICP0, MYXV M13L, MV V, PV 3C, and VSV M, inhibitors of NF-κB; VV B8R and MYXV MT-7, inhibitors of IFN-γ; HSV ICP47 and AdE3-19K, inhibitors of MHC class I presentation; MV gp, inhibitor of T cells; and MYXV M128L and MV H, inhibitors of CD46) are likely to enhance the induction of immunity and possibly cross-presentation of TAAs. Such mutations should improve the safety of OVs by making them more visible to the immune system, as well as increasing antitumor immune responses. Conversely, they may diminish virus replication and spread. An additional problem not as easily addressed is OV infection of immune cells, especially dendritic cells (DC), that interferes with their function (15, 16).

Innate Immunity

Although adaptive immunity seems to provide and, in fact, represent even the major mode of anticancer action for OVs, it is also evident that an initial host response against an administered OV could destroy it along with the infected cells before the OV has a chance to replicate and induce cytotoxicity of a magnitude that is sufficient to set up an effective vaccination response (17). Location and site of OV administration is an important determinant of the characteristics of these initial host responses against the OV. For instance, intravenous or intra-arterial administration of OVs, such as recombinant HSV1, leads to its rapid recognition and elimination by the circulating complement and antibodies of the humoral defense system (18, 19). This has also been shown for VV (20), NDV (21), MV (22), and Ad (23, 24). Intratumoral administration can also lead to complement- and antibody-mediated destruction of the OV. In addition, intracellular and microenvironmental antiviral defense responses in infected tumor cells can also greatly limit the magnitude of OV replication (25–31). Finally, innate immune cells can rapidly respond to an administered OV, further limiting its survival and that of OV-infected tumor cells (32–35). In all these models, circumvention of such responses using pharmacologic agents, such as histone deacetylase (HDAC) inhibitors or immunomodulating drugs, or genes that block antiviral defense mechanisms, has led to improved OV replication and tumor cytotoxicity (reviewed in ref. 36). When pharmacologic agents are used, the interference of antiviral responses can be applied in a transient fashion usually right before or at the time of OV administration. This should lead to an initial burst of OV replication leading to tumor cell lysis. As the pharmacologic effects against host innate immunity wane, a large debris field of OVs and tumor antigens could be more promptly recognized by the antiviral host response, leading to a secondary long-term vaccination effect responsible for effective tumor immunity (Fig. 1). However, quantification of responses to OV therapy is a sorely needed area of investigation. For instance, the number of OV-replicative rounds, the tumor cell-OV burst size, the number of OV-replicative tumor foci, and the temporal kinetics of innate response suppression that are needed for an efficient lytic and vaccination effect are still undetermined. In fact, current applications of innate immunity modulation with OV administration remain to be determined in an empirical manner.


Enhancing OV Immunotherapy

Many OVs can accommodate gene insertions and thus can be “armed” with therapeutic transgenes, combining local gene delivery with oncolytic activity (42). Local expression in the tumor obviates toxicity arising from systemic administration of potent immune modulators. GM-CSF, based on its effects in cytokine-transduced cancer cell vaccines (i.e., clinically approved Sipuleucel-T), has been incorporated into a number of OVs [HSV T-Vec, VV JX-594, Ad Ad5/3-D24-GMCSF (43), and CG0070 (44)] that have entered clinical trials (8). GM-CSF–expressing OVs demonstrated only moderate activity in preclinical studies (45, 46), while JX-594 was not compared with a VV lacking GM-CSF (47). Other therapeutic transgenes include interleukin (IL)-2 (NDV, HSV, and parvovirus), IL-12 (Ad and HSV), IL-15 (VSV), IL-18 (HSV), IFN-α/β (Ad, VSV, and VV), soluble CD80 (Ad and HSV), 4-1BB (VV), CD40L (Ad, and no effect with VSV), Flt3L (Ad and HSV), CCL3 (Ad), CCL5 (Ad and VV), and combinations thereof (2). In addition to transgenes that enhance adaptive immune responses, cytokines/chemokines directed at the tumor microenvironment can alter the immune cell balance toward productive therapeutic immunity (Fig. 1). IL-12, a potent antitumor cytokine with antiangiogenic activities, when expressed from oncolytic HSV, reduced neovasculature and tumor regulatory T cells (Treg) and induced T cell–mediated immunity in an immunocompetent cancer stem cell model (48). Expression of a CXCR4 antagonist from oncolytic VV reduced tumor vasculature and accumulation of bone marrow–derived epithelial and myeloid cells and induced antitumor humoral responses (49).

Like many cancer vaccine strategies, OVs expressing TAAs can be used to induce tumor-selective adaptive immune responses. The combination of TAA expression in the tumor and OV-mediated cell killing induces enhanced T-cell migration and activation compared with OV-infected tumor cells expressing the TAA (50). This can be coupled to a prime (replication-deficient Ad or oncolytic Semliki Forest virus expressing a TAA)–boost (oncolytic VSV or VV expressing the same TAA) vaccine strategy, in which the boosted secondary response to the tumor dominates the primary anti-OV response (6, 8). To expand the antigenic repertoire, cDNA libraries from normal tissue (e.g., prostate for prostate tumors) or recurrent tumors have been inserted into VSV, and induced therapeutic immunity (51). Further enhancement was obtained by expressing xenogeneic TAAs (51, 52). The ability of oncolytic VSV expressing TAAs to induce IL-17 in the context of tumor immunity has been exploited to screen tumor cDNA libraries for individual TAAs and optimal TAA combinations, limiting potentially inappropriate responses of whole-cell or cDNA vaccines (53). Developing a similar strategy in a human setting would be a major advance.

A number of immunomodulatory agents have been examined to restrain antiviral immune responses and promote OV replication and spread. Cyclophosphamide can increase OV replication and inhibit tumor growth by suppressing innate immune cell (34) and antibody responses (54), depleting Tregs, and enhancing the antitumor activity of CTLs (Fig. 1; ref.8). A challenge is to identify immunosuppressive strategies that can blunt acute innate cells from blocking virus replication and spread, while permitting sufficient inflammation and cross-priming for robust antitumor immunity. Conversely, it will be of interest to combine OV with chemotherapies that induce ICD (e.g., cyclophosphamide, oxaloplatin, or anthracyclines such as doxorubicin and mitoxantrone), increase tumor cell antigenicity (e.g., gemcitabine, cisplatin, or etoposide) or susceptibility to immune cells (e.g., HDAC inhibitors, paclitaxel, or doxorubicin), or suppress MDSCs (e.g., gemcitabine and paclitaxel) and Tregs (e.g., cyclophosphamide or sunitinib; ref. 55) in immunocompetent preclinical models.

In conclusion, the field of virotherapy is becoming mature in its knowledge of effective anticancer mechanisms in animal tumor models with OVs that are also safe in human clinical trials. It seems that there may soon be a first-in-humans OV approved for use in the United States, which will further stimulate laboratory and clinical endeavors with this therapeutic strategy.


Oncolytic viruses: a new class of immunotherapy drugs.

Oncolytic viruses represent a new class of therapeutic agents that promote anti-tumour responses through a dual mechanism of action that is dependent on selective tumour cell killing and the induction of systemic anti-tumour immunity. The molecular and cellular mechanisms of action are not fully elucidated but are likely to depend on viral replication within transformed cells, induction of primary cell death, interaction with tumour cell antiviral elements and initiation of innate and adaptive anti-tumour immunity. A variety of native and genetically modified viruses have been developed as oncolytic agents, and the approval of the first oncolytic virus by the US Food and Drug Administration (FDA) is anticipated in the near future. This Review provides a comprehensive overview of the basic biology supporting oncolytic viruses as cancer therapeutic agents, describes oncolytic viruses in advanced clinical trials and discusses the unique challenges in the development of oncolytic viruses as a new class of drugs for the treatment of cancer.

Nat Rev Drug Discov. 2015 Sep;14(9):642-62.


Oncolytic Virus-Mediated Immunotherapy: A Combinatorial Approach for Cancer Treatment  

SE Lawler, EA Chiocca    JCO.2015.62.5244


Preclinical Mouse Models for Analysis of the Therapeutic Potential of Engineered Oncolytic Herpes Viruses

MC Speranza, K Kasai, SE Lawler – ILAR Journal, 2016 –
Abstract After more than two decades of research and development, oncolytic herpes
viruses (oHSVs) are moving into the spotlight due to recent encouraging clinical trial data.
oHSV and other oncolytic viruses function through direct oncolytic cancer cell–killing

[HTML] FDA Approves IMLYGIC™(Talimogene Laherparepvec) As First Oncolytic Viral Therapy In The US

J Carroll, D Garde –
THOUSAND OAKS, Calif., Oct. 27, 2015/PRNewswire/–Amgen (AMGN) today announced
that the US Food and Drug Administration (FDA) has approved the Biologics License
Application for IMLYGIC™(talimogene laherparepvec), a genetically modified oncolytic

Other related articles published in this Open Access Online Scientific Journal include the following:

Oncolytic Viruses in Cancer Therapy @ CHI’s PreClinical Congress, June 14, 2016 Westin Boston Waterfront, Boston

Reporter: Aviva Lev-Ari, PhD, RN


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Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator



Microbes Meet Cancer

Understanding cancer’s relationship with the human microbiome could transform immune-modulating therapies.

By Kate Yandell | April 1, 2016


In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types and improved survival to a far greater extent in mice with intact microbiomes.1 The French group, led by INSERM’s Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to develop tumors of the lung.2

The findings incited a flurry of research and speculation about how gut microbes contribute to cancer cell death, even in tumors far from the gastrointestinal tract. The most logical link between the microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or tamp it down, and can modulate immune cells’ vigilance for invaders. Not only does the immune system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.

“We clearly see shifts in the [microbial] community that precede development of tumors,” says microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote cell proliferation, others appear to protect us against cancerous growth. And in some cases, the conditions that spur one cancer may have the opposite effect in another. “It’s become pretty obvious that the commensal microbiota affect inflammation and, through that or through other mechanisms, affect carcinogenesis,” says Trinchieri. “What we really need is to have a much better understanding of which species, which type of bug, is doing what and try to change the balance.”

Gut feeling

In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic gastritis, an inflammation of the stomach lining that often precedes the development of stomach cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the bacterium, now called Helicobacter pylori, was somehow causing the gastritis.3 So committed was Marshall to demonstrating the microbe’s causal relationship to the inflammatory condition that he had his own stomach biopsied to show that it contained no H. pylori, then infected himself with the bacterium and documented his subsequent experience of gastritis.4 Scientists now accept that H. pylori, a common gut microbe that is present in about 50 percent of the world’s population, is responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for stomach cancer.5 Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their work.

H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development, but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal evidence that shifts in the microbiome influence the development of diverse tumor types. “You have a mouse model of carcinogenesis. It works beautifully,” says Trinchieri. “You move to another institution. It works completely differently,” likely because the animals’ microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF

Around the turn of the 21st century, cancer researchers began to systematically experiment with the rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouse’s risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice rapidly developed colon tumors, but that germ-free versions of these mice did not.6 That same year, an MIT-based group led by the late David Schauer demonstrated that infecting mice with the bacterium Citrobacter rodentium spurred colon tumor development.7 And in 2003, MIT’s Susan Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the murine gut microbiome.8

More recent work has documented a similar link between colon cancer and the gut microbiome in humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90 people, some with colon cancer, some with precancerous adenomas, and still others with no disease.9 The researchers found that the feces of people with cancer tended to have an altered composition of bacteria, with an excess of the common mouth microbes Fusobacterium or Porphyromonas. A few months later, Peer Bork of the European Molecular Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of cancer using the relative abundance of 22 bacterial species, including Porphyromonas andFusobacterium.10 They could also use the method to predict colorectal cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers while yielding false positives less than 10 percent of the time. When the two tests were combined, they caught more than 70 percent of cancers.

Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a consequence of tumor development remained unclear. “What comes first, the change in the microbiome or tumor development?” asks Schloss. To investigate this question, he and his colleagues treated mice with microbiome-altering antibiotics before administering a carcinogen and an inflammatory agent, then compared the outcomes in those animals and in mice that had received only the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome, suggesting that resident bacteria were in some way promoting cancer development. And when the researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota from mice already bearing malignancies developed the most tumors of all.11

Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor formation. The researchers determined that the number of tumors corresponded to the unique ways that each antibiotic cocktail modulated the microbiome.12

“We’ve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer,” says Schloss, who hypothesizes that gut bacteria–driven inflammation is to blame for creating an environment that is hospitable to tumor development and growth. Gain or loss of certain components of the resident bacterial community could lead to the release of reactive oxygen species, damaging cells and their genetic material. Inflammation also involves increased release of growth factors and blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)

Recent research has also yielded evidence that the gut microbiota impact the development of cancer in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.

Systemic effects

In the mid-2000s, MIT’s Erdman began infecting a strain of mice predisposed to intestinal tumors withH. hepaticus and observing the subsequent development of colon cancer in some of the animals. To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to develop mammary tumors. “This told us that something really interesting was going on,” Erdman recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more likely to develop mammary tumors than mice not exposed to the bacterium.13The researchers showed that systemic immune activation and inflammation could contribute to mammary tumors in other, less cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer development and progression, thanks to their interactions with the immune system. Some microbes, such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while others, such as some Bacteroides species, help protect against tumors by boosting T-cell infiltration.© EYE OF SCIENCE/SCIENCE SOURCE




At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice coming from different vendors—Taconic Biosciences (formerly Taconic Farms) and the Jackson Laboratory—Gajewski takes advantage of the fact that the animals’ different origins result in different gut microbiomes. “We deliberately stayed away from antibiotics, because we had a desire to model how intersubject heterogeneity [in cancer development] might be impacted by the commensals they happen to be colonized with,” says Gajewski in an email to The Scientist.

Last year, the researchers published the results of a study comparing the progression of melanoma tumors implanted under the mice’s skin, finding that tumors in the Taconic mice grew more aggressively than those in the Jackson mice. When the researchers housed the different types of mice together before their tumors were implanted, however, these differences disappeared. And transferring fecal material from the Jackson mice into the Taconic mice altered the latter’s tumor progression.14

Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mice’s tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria improved their response to the implanted melanoma cells.

“One hypothesis going into the experiments was that we might identify immune-suppressive bacteria, or commensals that shift the immune response towards a character that was unfavorable for tumor control,” says Gajewski.  “But in fact, we found that even a single type of bacteria could boost the antitumor immune response.”


Drug interactions

Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But cancer cells display “self” molecules that can inhibit immune attack. A new type of immunotherapy, dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking either the tumor cells’ surface molecules or the receptors on T?cells that bind to them.


In addition to influencing the development and progression of cancer by regulating inflammation and other immune pathways, resident gut bacteria appear to influence the effectiveness of many cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

  • Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide immunotherapy, work by boosting inflammation. If the microbiome is altered in such a way that inflammation is reduced, these therapeutic agents are less effective.
  • Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer cells are influenced by members of the microbiota that mediate these same cell interactions.
  • Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1 receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells. While treating Taconic mice with PD-L1–targeting antibodies did improve their tumor responses, they did even better when that treatment was combined with fecal transfers from Jackson mice, indicating that the microbiome and the immunotherapy can work together to take down cancer. And when the researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mice’s tumors virtually disappeared.14

Gajewski’s group is now surveying the gut microbiota in humans undergoing therapy with checkpoint inhibitors to better understand which bacterial species are linked to positive outcomes. The researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.

Meanwhile, Zitvogel’s group at INSERM is investigating interactions between the microbiome and another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and germ-free mice had poorer responses to a CTLA-4–targeting antibody compared with mice harboring unaltered microbiomes.15 Particular Bacteroides species were associated with T-cell infiltration of tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals’ responses to the immunotherapy. As an added bonus, treatment with these “immunogenic” Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice transplanted with feces from patients with normal levels of B. fragilis.

“There are bugs that allow the therapy to work, and at the same time, they protect against colitis,” says Trinchieri. “That is very exciting, because not only [can] we do something to improve the therapy, but we can also, at the same time, try to reduce the side effect.”

And these checkpoint inhibitors aren’t the only cancer therapies whose effects are modulated by the microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered microbiomes.1He and his NCI colleague Goldszmid further found that the platinum chemotherapy oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogel’s group has shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its proper function.

Although the mechanisms by which the microbiome influences the effectiveness of such therapies remains incompletely understood, researchers once again speculate that the immune system is the key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?helper 1 cells and a subtype of T?helper 17 cells referred to as “pathogenic,” both of which destroy tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?helper 17 cells, which translocate to the tumor. When the researchers transferred the “pathogenic” T?helper 17 cells into antibiotic-treated mice, the mice’s response to chemotherapy was partly restored.

Microbiome modification

As the link between the microbiome and cancer becomes clearer, researchers are thinking about how they can manipulate a patient’s resident microbial communities to improve their prognosis and treatment outcomes. “Once you figure out exactly what is happening at the molecular level, if there is something promising there, I would be shocked if people don’t then go in and try to modulate the microbiome, either by using pharmaceuticals or using probiotics,” says Michael Burns, a postdoc in the lab of University of Minnesota genomicist Ran Blekhman.

Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however, molding the microbiome is not simple. “It’s a messy, complicated system that we don’t understand,” says Schloss.

So far, studies of the gut microbiome and colon cancer have turned up few consistent differences between cancer patients and healthy controls. And the few bacterial groups that have repeatedly shown up are not present in every cancer patient. “We should move away from saying, ‘This is a causal species of bacteria,’” says Blekhman. “It’s more the function of a community instead of just a single bacterium.”

But the study of the microbiome in cancer is young. If simply adding one type of microbe into a person’s gut is not enough, researchers may learn how to dose people with patient-specific combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by promoting an anti-inflammatory environment in the gut.16

“If it is true that, in humans, we can alter the course of the disease by modulating the composition of the microbiota,” says José Conejo-Garcia of the Wistar Institute in Philadelphia, “that’s going to be very impactful.”

Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she became an associate editor at Cancer Today.


The microbiome doesn’t act in isolation; a patient’s genetic background can also greatly influence response to therapy. Last year, for example, the Wistar Institute’s José Garcia-Conejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5 (TLR5) influences clinical outcomes in cancer patients by changing how the patients’ immune cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).

More than 7 percent of people carry a specific mutation in TLR5 that prevents them from mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their colleagues found that estrogen receptor–positive breast cancer patients who carry the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without the mutation. Among patients with ovarian cancer, on the other hand, those with the TLR5 mutation were more likely to live at least six years after diagnosis than patients who don’t carry the mutation.

Investigating the mutation’s contradictory effects, the researchers found that mice with normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17). But when the researchers knocked out the animals’ microbiomes, these differences in cytokine production disappeared, as did the differences in cancer progression between mutant and wild-type animals.

“The effectiveness of depleting specific populations or modulating the composition of the microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,” says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer prognosis may act via microbiome–immune system interactions. “I think that our paper is just the tip of the iceberg.”


  1. N. Iida et al., “Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment,” Science, 342:967-70, 2013.
  2. S. Viaud et al., “The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide,” Science, 342:971-76, 2013.
  3. J.R. Warren, B. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,”Lancet, 321:1273-75, 1983.
  4. B.J. Marshall et al., “Attempt to fulfil Koch’s postulates for pyloric Campylobacter,” Med J Aust, 142:436-39, 1985.
  5. J. Parsonnet et al., “Helicobacter pylori infection and the risk of gastric carcinoma,” N Engl J Med, 325:1127-31, 1991.
  6. S. Kado et al., “Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor β chain and p53 double-knockout mice,” Cancer Res, 61:2395-98, 2001.
  7. J.V. Newman et al., “Bacterial infection promotes colon tumorigenesis in ApcMin/+ mice,” J Infect Dis, 184:227-30, 2001.
  8. S.E. Erdman et al., “CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice,” Am J Pathol, 162:691-702, 2003.
  9. J.P. Zackular et al., “The human gut microbiome as a screening tool for colorectal cancer,” Cancer Prev Res, 7:1112-21, 2014.
  10. G. Zeller et al., “Potential of fecal microbiota for early-stage detection of colorectal cancer,” Mol Syst Biol, 10:766, 2014.
  11. J.P. Zackular et al., “The gut microbiome modulates colon tumorigenesis,” mBio, 4:e00692-13, 2013.
  12. J.P. Zackular et al., “Manipulation of the gut microbiota reveals role in colon tumorigenesis,”mSphere, doi:10.1128/mSphere.00001-15, 2015.
  13. V.P. Rao et al., “Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice,” Cancer Res, 66:7395, 2006.
  14. A. Sivan et al., “Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy,” Science, 350:1084-89, 2015.
  15. M. Vétizou et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,”Science, 350:1079-84, 2015.



Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, …., Julia Tchou,  Gabriel A. Rabinovich, Jose R. Conejo-Garcia
Cancer cell    12 Jan 2015; Volume 27, Issue 1, p27–40
Figure thumbnail fx1
  • TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by γδ T cells
  • IL-17 drives malignant progression in IL-6-unresponsive tumors
  • TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion
  • A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

see also… Immune Influence

In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment.

By Kate Yandell | April 1, 2016

Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame. Here are some of the hypotheses that have come out of recent research in rodents for how gut bacteria shape immunity and influence cancer.


Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that spur tumor growth and blood vessel formation.

Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead to cancerous growth.


Gut bacteria can also produce factors that lower inflammation and slow tumor growth. Some gut bacteria (e.g., Bifidobacterium)
appear to activate dendritic cells,
which present cancer-cell antigens to T cells that in turn kill the cancer cells.

Read the full story.


Read Full Post »

Pull at Cancer’s Levers

Curator: Larry H. Bernstein, MD, FCAP


Driving Cancer Immunotherapy 

The Stakes in Immuno-Oncology Are Too High for Researchers to Pull at Cancer’s Levers Blindly. Researchers Need a System.

  • Within the past decade or so, a revolutionary idea has emerged in the minds of scientists, physicians, and medical experts. Instead of using man-made chemicals to treat cancer, let us instead unleash the power of our own bodies upon the malignancy.

    This idea is the inspiration behind cancer immunotherapy, which is, according to most experts, a therapeutic approach that involves training the immune system to fight off cancer. In the words of one expert, cancer immunotherapy means “taking the immune system’s inherent properties and turbo charging those to fight cancer.”

    Cancer immunotherapy technologies are being developed to accomplish
    several tasks:

    • Enhance the molecular targeting of cancer cells
    • Report the rate of killing by specific immune agents
    • Direct immune cells toward tumor destruction.

    Since its inception, the field has evolved, and it continues to do so. It began with in vivo investigations of tumor growth and development, and it progressed through laboratory investigations of cellular morphology and survival curves. And now it is adopting pathway analysis to guide therapeutic development and improve patient care.

    To begin to understand cancer immunotherapy, one must understand how the immune system targets tumor cells. One of the prominent adaptive components of the immune system is the T cell, which responds to perceived threats through the massive increase in clonal T cells targeted in some way toward the diseased cell or pathogen.

  • The T-Cell Repertoire

    Adaptive Biotechnologies’ immunoSEQ Assay, a high-throughput research platform for immune system profiling, is designed to generate sequencer-ready libraries using highly optimized primer sets in a multiplex PCR format that targets T- and B-cell receptor genes. This image depicts how the assay’s two-step PCR process can be used to quantify the clonal diversity of immune cells.

    Immunologists call this process VJD rearrangement. It happens during T-lymphocyte development and affects three gene regions, the variable (V), the diversity (D), and the joining (J) regions. This rearrangement of the genetic code allow for the structural diversity in T-cell receptors responsible for antigenic specificity including antigenic targets on tumor cells. In the case of cancer, specificity is complicated because the tumor is actually part of the body itself, one of the reasons cancers naturally evade detection.

    The specificity problem would always hinder attempts to goad the immune system into attacking cancer, scientists realized, unless technologies emerged that could efficiently track the clonal diversity of T cells inside patients. Existing technologies, such as spectratyping, were inadequate.  In 2007, when Dr. Robins and his collaborators began developing the technology, only 10,000 T-cell receptor sequences had been reported in all the literature using older methodologies.

    “The immunology field of the time had no connection with high-throughput sequencing,” notes Dr. Robins, recalling his days as a computational biologist for the Fred Hutchinson Cancer Research Center. “It became clear that instead of using this old technology to look at T-cell receptors, we could just directly sequence them—if we could amplify them correctly.”

    With its first experiment, Dr. Robins’ team ended up with six million T-cell receptor sequences. “Our approach,” Dr. Robins modestly suggests, “kind of changed the scale of what we were able to do.” The team went on to develop advanced multiplex sequencing technology, doing work that essentially started the field of immune sequencing. “Previously,” maintains Dr. Robins, “no one had ever been able to quantitatively do a multiplex PCR.”

    Adaptive Biotechnologies’ product, the ImmunoSEQ® assay, uses several hundred primer pairs to quantify the clonal diversity of T cells. Using this technology, researchers and clinicians can focus on T-cell clones that are expanded specifically in or near a tumor or that are circulating in the blood stream.

    “You obviously can’t get a serial sample of the tumor,” explains Dr. Robins, “but you can get serial samples of blood,” allowing for immune cell repertoire tracking during the progression of a disease. The technology is already being used to assess leukemias in the clinic, directly tracking the leukemia itself based on the massive clonal expansion of a single cancerous B or T cell.

    Eventually, Dr. Robins’ team hopes to monitor serial changes in T cell clones before, during, and after therapeutic intervention. The team has even developed a tumor infiltrating lymphocyte (TIL) assay to examine clones that are attracted to tumors.


Circulating Tumor Cells

“Years ago, they were just interested in what was happening in the tumor,” says Daniel Adams, senior research scientist at Creatv MicroTech. “Now people have realized that the immune system is reacting to the tumor.”

Scientists such as Adams have been tracking tumor cells and tumor-modified stromal cells, as well as components of the non-adaptive immune system, directly within the bloodstream to examine changes that occur over time.

“We can’t go back in to re-biopsy the patient every year, or every time there is a recurrence,” says Adams, “It’s just not feasible.”

That is why Creatv MicroTech, with locations in Maryland and New Jersey, has developed the CellSieve, a mechanical cell filter. The CellSieve, which improves on older technology through better polymers and engineering, isolates circulating tumor cells (CTCs) and stromal cells in order to capture them for further clinical analysis.

Isolation, culture and expansion of cells isolated on CellSieve™. (A) MCF-7 cells spiked into vacutainers, isolated by filtration and cultured on CellSieve for 2-3 weeks. A 3 dimensional cluster attributed to this cell line is seen on the filter. (green=anti-cytokeratin, blue=DAPI) (B) PANC-1 cells spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. PANC-1 is seen growing as a monolayer on the filter. (C) SKBR3 cells are spiked into blood, filtered by CellSieve. The CTCs are identified by presence of anti-cytokeratin and anti-EpCAM, and absence of anti-CD45. After CTCs are counted, cells are subtyped by HER2 FISH. (D) SKBR3 cells are spiked into vacutainers, isolated by filtration and grown on CellSieve for 2-3 weeks. Expanded colonies were directly analyzed as a whole colony and as individual cells, molecularly by HER2/CR17 FISH probes. (E) Circulating stromal cell, e.g. a 70 µm giant cancer associated macrophage can be identified for clinical use, myeloid marker in red. (F) A cell of interest can be identified and restained with immunotherapeutic biomarkers, e.g. PD-L1 (green) and PD-1 (purple). (G) After filtration, cells were identified with histopathological stains (e.g. H&E) for cytological analysis. (H) After H&E, external cell structures were analyzed by SEM. [Creatv MicroTech].


“As a patient goes through therapy, the patient’s resistance builds, and the cancer recurs in different subpopulations,” states Adams. “And after a few years, the original tumor mass is no longer applicable to what is growing in the patient farther down the road.”

Although CTCs are exceedingly rare in the bloodstream, with just one or two in every 5 to 10 mL of blood, and although these cells have a very low viability, the surviving CTCs have a high prognostic value.

“We looked at 30 to 40 breast cancer patients over two years,” reports Adams. “And we showed that if you have a dividing CTC, you have a 90% chance of dying in two years and a 100% chance of dying within two and half years.”

Furthermore, the immune system response can be tracked, says Adams, by examining stromal cells, which can also be collected with the CellSieve filtration device. That is, these cells can be collected serially. Much recent evidence supports the conclusion that stromal cells in the tumor environment co-evolve with the tumor, suggesting that stromal marker changes reflect tumor changes.

“There is this plethora of stromal cells and tumor cells out there in the circulation for you to look at,” declares Adams. “Once the cells are isolated, you can subject them to pathological approaches, biomarker approaches, or molecular approaches—or all of the above.”

A MicroTech Creatv study published in the Royal Society of Chemistry showed the efficacy of following up CTC isolation with techniques such as fluorescence in situ hybridization (FISH), histopathological analysis, and cell culture.

Cancer-Killing Assays

Diverse mechanisms are at play in cancer biology. Our understanding of these mechanisms contributes to a couple of virtuous cycles. It strengthens and is strengthened by diagnostic approaches, such as immune- and tumor-cell monitoring. The same could be said of therapeutic approaches. Cancer biology will inform and be informed by cancer immunotherapies such as adoptive cell transfer. To maintain the virtuous cycle, however, it will be necessary to conduct in vitro testing.

“There is no doubt that immunotherapy is going to play a major role in the treatment of cancer,” says Brandon Lamarche, Ph.D., technical communicator and scientist at ACEA Biosciences. “Regardless of what the route is, what is going to have to happen in terms of the research area is that you need an effective cell-killing assay.”

ACEA Biosciences, a San Diego-based company, has developed a microtiter plate that is coated with gold electrodes across 75% of the well bottoms. When the microtiter plates are placed in the company’s xCELLigence plate reader, the electrodes enable the detection of changes in cell morphology and viability through electrical impedance.

“The instrument provides a weak electric potential to the electrodes on the plate, so you get electrons flowing between these electrodes,” explains Dr. Lamarche. Researchers can then apply reagents or non-adherent immune cell suspensions to adherent cancer cells and examine the effect.

Dr. Lamarche asserts that the xCELLigence system overcomes problems that bedevil competing cell-killing assays. These problems include leaky and radioactive labels, such as chromium 51, and assays that can only provide users with an endpoint for cell killing. “With xCELLigence,” he insists, “you’re getting the full spectrum of what’s happening, and there’s all kinds of subtleties in the cell-killing curves that are very informative in terms of the biology.”

ACEA would like to see the xCELLigence system become the new standard in cell-killing assays from standard research to clinical testing on patient tumors. Dr. Lamarche envisions a day when patient tumor cells are quickly screened with therapeutic scenarios to determine the most efficacious killing option. “xCELLigence technology,” he suggests, “enables you to quickly sample a broad spectrum of conditions with a very simple workflow.”

Bioinformatics of Immuno-Oncology

From monitoring to treatment modalities, the field of cancer immunotherapy is aided by bioinformatics-minded data-mining experts, such as the analysts at Thompson-Reuters who are compiling data archives and applying advanced analytics to find new targets. “Essentially,” says Richard Harrison Ph.D., the company’s chief scientific officer for the life science division, “for every stage within pharmaceutical drug development, we have a database associated with that.”

The analysts at Thompson-Reuters curate and compile databases such as MedaCore and Cortellis, which they provide to their clients to help them with their research and clinical studies. “We can take customer data, and using our tools and our pathway maps, we can help them understand what their data is telling them,” explains Dr. Harrison.

Matt Wampole, Ph.D., a solutions scientist at Thompson-Reuters, spends his days reaching out and working with customers to help them understand and better use the company’s products. “Bench researchers,” he points out, “don’t necessarily know what is upstream of whatever expression change might be leading to a particular change in regulation.” Dr. Wampole indicates that he is part of a “solution team” that aids clients in determining important signaling cascades, regulators, and so on.

“We have a group of individuals who are very ‘skilling’ experts in the field,” Dr. Wampole continues, “including experts in the areas such as biostatistics, data curation, and data analytics. These experts help clients identify models, stratify patients, understand mechanisms, and look into disease mechanisms.”

Dr. Harrison sums up the Thompson-Reuters approach as follows: “We look for master regulators that can serve as both targets and biomarkers.” By examining the gene signatures from both the patient and from curated datasets, in the case of cancer immunotherapy, they hope to segregate patients according to what drugs will work best for them.

  • “We are working with a number of pharmaceutical companies to put our approach into practice for clinical trials,” informs Dr. Harrison. The approach has already been applied in several studies, including one that used data analysis of cell lines to help predict drug response in patients. Another study helped stratify glioblastoma patients.

  • Tumor-Targeted Delivery Platform

    PsiOxus Therapeutics, which is focused on immune therapeutics in oncology, has developed a patented platform for tumor-targeted delivery based on its oncolytic vaccine, Enadenotucirev (EnAd), which can be delivered systemically via intravenous administration.

    According to company officials, EnAd’s anti-cancer scope can be expanded by adding new genes, thereby enabling the creation of a broad range of unique immuno-oncology therapeutics. In a recent study conducted at the University of Oxford, researchers led by Philip G. Jakeman, Ph.D., sought to improve the models for evaluating cancer therapeutics by introducing ex vivo methodologies for research into colorectal cancer.

    The ex vivo approach utilized was able to exploit a major advantage by preserving the three-dimensional architecture of the tumor and its associated compartments, including immune cells. The study, which was presented at the International Summit on Oncolytic Viral Therapeutics in Quebec, showed the tissue slice model can provide a novel means to assessing an oncolytic vaccine in a system that more accurately recapitulates human tumors, provide a more stringent test for oncolytic viruses, such as EnAd, and allow study of the human immune cells within the tumor 3D context.

    By maintaining the components of the tumor immune microenvironment, this new methodology could become useful in analyzing anti-viral responses within tumors, or even in evaluating therapeutics that target immunosuppressive tumor micro-environments, noted the Oxford team.



Deciphering the Cancer Transcriptome

A Rogue’s Gallery of Malignant Outliers May Hide in Transcriptome Profiles That Emphasize Averages


The key link between genomic instability and cancer progression is transcriptome dynamics. The shifts in transcriptome dynamics that contribute to cancer evolution may come down to statistical outliers. [iStock/zmeel]

  • In recent years, scientists have adopted a gene-centric view of cancer, a tendency to see each malignant transformation as the consequence of alterations in a discrete number of genes or pathways. These alterations are, fortunately, absent from healthy cells, but they pervert malignant cells.

    The gene-centric view takes in molecular landscapes illuminated by genomic and transcriptomic technologies. For example, genomes can be cost-effectively sequenced within hours. Such capabilities have made it possible to interrogate associations between genotypes and phenotypes for increasing numbers of conditions, and to collect data from progressively larger patient groups.

    As genomic and transcriptomic technologies rise, they reveal much—but much remains hidden, too. Perhaps these technologies are less like the sun and more like the proverbial streetlight, the one that narrows our searches because we’re inclined to stay in the light, even though what we hope to find may lie in the shadows.

    “Each individual study that looks at the cancer transcriptome is impressive and tells a convincing story, but if we put several high-quality papers together, there are very few genes that overlap,” says Henry H. Heng, Ph.D., professor of molecular medicine, genetics, and pathology at Wayne State University. “This shows that something is wrong.”

  • Distinct Karyotypes

    One of the major observations in Dr. Heng’s lab is that the intra- and intertumor cellular heterogeneity results in nearly every cancer cell having a unique, distinct karyotype, that is, an important but often ignored genotype. “Biological systems need a lot of heterogeneity,” notes Dr. Heng. “People like to think that this is noise, but heterogeneity is a fundamental buffer system for biological function to be achievable. Moreover, it is the key agent for cellular adaptation.”

    To capture the degree of genomic heterogeneity at the genome level and its impact on cancer cell growth, Dr. Heng and colleagues performed serial dilutions to isolate single mouse ovarian surface epithelial cells that had undergone spontaneous transformation. Spectral karyotyping revealed that within a short timeframe each of these unstable cells exhibited a very distinct karyotype. In these unstable cells, cloning at the level of the karyotype was not possible.

    Stable cells exhibited a normal growth distribution, i.e., no subset of stable cells contributed disproportionately to the overall growth of the cell population. In contrast, unstable cell populations showed a non-normal growth distribution, with few cells contributing most to the cell population’s growth. For example, a single unstable colony contributed more than 70% to the cell population’s growth. This finding suggests that although average profiles can be used to describe non-transformed cells, they cannot be taken to represent the biology of malignant cells.

    “Most people who study the transcriptome want to get rid of the noise, but the noise is in fact the strategy that cancer uses to be successful,” explains Dr. Heng. “Each individual cancer cell is very weak but together the entity becomes very robust.”

    In a recent model that Dr. Heng and colleagues proposed, system inheritance visualizes chromosomes not merely as the vehicle for transmitting genetic information, but as the genetic network organizer that shapes the physical interactions between genes in the three-dimensional space. Based on this model, individual genes represent parts of the system. The same genes can be reorganized to form different systems, and chromosomal instability becomes more important than the contribution of individual genes and pathways to cancer biology.

    The vital link between genomic instability and cancer progression is transcriptome dynamics, and the shifts in those dynamics that contribute to cancer evolution may come down to statistical outliers.

    “Transcriptome studies rarely focus on single-cell analyses, which means important outliers are frequently ignored,” declares Dr. Heng. “This preoccupation with uninformative averages explains why we have learned so little despite having examined so many transcriptomes.”

  • Chimeras and Fusion Genes

    “Our focus is on chimeric RNA molecules,” says Laising Yen, Ph.D, assistant professor of pathology at Baylor College of Medicine. “This category of RNAs is very special because their sequences come from different genes.”

    In a study that was designed to capture chimeric RNAs in prostate cancer, Dr. Yen and his colleagues performed high-throughput sequencing of the transcriptomes from human prostate cancer samples. “We found far more chimeric RNAs, in terms of abundance, and a number of species that are not seen in normal tissue,” reports Dr. Yen. This approach identified over 2,300 different chimeric RNA species. Some of these chimeras were present in prostate cancer cell lines, but not in primary human prostate epithelium cells, which points toward their relevance in cancer.

    “Most of these chimeric RNAs do not have a genomic counterpart, which means that they could be produced by trans-splicing,” explains Dr. Yen. During trans-splicing, individual RNAs are generated and trans-spliced together as a single RNA, which provides a mechanism for generating a chimera.

    “The other possibility is that in cancer cells, where gene–gene boundaries are known to become broken, chimeras can be formed by cis-splicing from a very long transcript that encodes several neighboring genes located on the same chromosome,” informs Dr. Yen. Chimeric RNAs formed by either of these two mechanisms can potentially translate into fusion proteins, and these aberrant proteins may have oncogenic consequences.

    Another effort in Dr. Yen’s laboratory focuses on chromosomal aberrations in ovarian cancer. One of the hallmarks of ovarian cancer is the high degree of genomic rearrangement and the increased genomic instability.

    “When we looked at ovarian cancers, we did not find as many chimeric RNAs,” notes Dr. Yen. “But we found many fusion genes.” Gene fusions, similarly to chimeric RNAs, increase the diversity of the cellular proteome, which could be used selectively by cancer cells to increase their rates of proliferation, survival, and migration.

    A recent study in Dr. Yen’s lab identified BCAM-AKT2, a recurrent fusion gene that is specific and unique to high-grade serous ovarian cancer. BCAM-AKT2 is the only fusion gene in this malignancy that was proved to be translated into a fusion kinase in patients, which points toward its functional significance and potential therapeutic value.

    “Recurrent fusion genes, which are repeatedly found in many patients in precisely made forms, indicate that there is a reason that they are present,” concludes Dr. Yen. “This might have important therapeutic implications.”

  • Context-Specific Patterns

    “We contributed to a study of tumor gene expresssion that we are currently revisiting because so much more data has become available,” says Barbara Stranger, Ph.D., assistant professor, Institute for Genomics and Systems Biology, University of Chicago. “The data is being processed in homogenized analytic pipelines, and we can look at many more tumor types across the Cancer Genome Atlas than a few years ago.”

    Previously, Dr. Stranger and colleagues performed expression quantitative trait loci (eQTL) analyses to examine mRNA and miRNA expression in breast, colon, kidney, lung, and prostate cancer samples. This approach identified 149 known cancer risk loci, 42 of which were significantly associated with expression of at least one transcript.

    Causal alleles are being prioritized using a fine-mapping strategy that integrated the eQTL analysis with genome-wide DNAseI hypersensitivity profiles obtained from ENCODE data. These analyses are focusing on capturing differences across tumors and on performing comparisons with normal tissue, and one of the challenges is the lack of normal tissue from the same patients.

    “But still there is a lot of power in these analyses because they are based on large-scale genomic datasets. Also, these tumor datasets can be compared with large-scale normal tissue genomics datasets, such as the NIH’s Genotype-Tissue Expression (GTEx) project,” clarifies Dr. Stranger. “This helps us characterize differences between those tumors and normal tissue in terms of the genetics of gene regulation.”

    An ongoing effort in Dr. Stranger’s laboratory involves elucidating how the effect of genetic polymorphisms is shaped by context. Stimulated cellular states, cell-type differences, cellular senescence, and disease are some of the contexts that are known to impact genetic polymorphisms.

    “We have seen a lot of context specificity,” states Dr. Stranger. “Our observations suggest that a genetic polymorphism can have a specific effect in regulating a particular gene or transcript in one context, and another effect in another context.”

    Another example of cellular context is sex, and an active area of investigation in Dr. Stranger’s lab proposes to dissect the manner in which sex differences shape the regulatory effects of genetic polymorphisms.

    “Thinking about sex-specific differences is not very different from thinking about a different cellular environment,” notes Dr. Stranger.

    The expression of specific transcription factors can be determined by sex; consequently, a polymorphism that interacts with a transcription factor may have functional outcomes that can be seen in only one of the sexes.

    “There are gene-level and gene-splicing differences that we see in normal tissues between males and females, and we want to take the same approach and look at the cancer context to see whether the genetic regulation of gene expression and transcript splicing is different between individuals and whether it has a sex bias,” concludes Dr. Stranger. “Finally, we want to see how that differs in cancer relative to normal tissues.”

    Early Clinical Impact

An increasing number of clinicians are adding the cancer transcriptome to their precision medicine program. They have found that the transcriptome is important in identifying clinically impactful results. [iStock/DeoSum]

“Over the last two years,” says Andrew Kung, M.D., Ph.D., chief of the Division of Pediatric Hematology, Oncology, and Stem Cell Transplantation at Columbia University Medical Center, “we have included the cancer transcriptome as part of our precision medicine program.” Dr. Kung and colleagues developed a clinical genomics test that includes whole-exome sequencing of tumors and normal tissue and RNA-seq of the tumor.

“Our results show that the transcriptome is very important in identifying clinically impactful results,” asserts Dr. Kung. “The technology has really moved from a research tool to real clinical application.” In fact, the test has been approved by New York State for use in cancer patients.

The data from transcriptome profiling has enabled identification of translocations, verification of somatic alterations, and assessment of expression levels of cancer genes.  Dr. Kung and his colleagues are using genomic information for initial diagnosis and prognostic decisions, as well as the investigation of potentially actionable alterations and the monitoring of disease response.

To gain insight into gene-expression changes, transcriptome analysis usually compares two different types of tissues or cells. For example, analyses may attempt to identify differentially expressed genes in cancer cells and normal cells.

“In patients with cancer, we usually do not have access to the normal cell of origin, making it harder to identify the genes that are over- or under-expressed,” explains Dr. Kung. “Fortunately, the vast amounts of existing gene-expression data allow us to identify genes whose expression are most changed relative to models built on the expression data aggregated across large existing datasets.”

These genomic technologies were first used to augment the care of pediatric patients at Columbia. The technologies were so successful that they attracted philanthropic funding, which is being used to expand access to genomic testing to all children with high-risk cancer across New York City.

Other related articles published in this Open Access Online Scientific Journal include the following:

Immunotherapy in Combination, 2016 MassBio Annual Meeting  03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

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New Access to and Global Reach Availability to the New website


UPDATED on 10/12/2016 

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Oncology Drug Development and Personalized Medicine Knowledgebase

  • Comprehensive resource covering all aspects of the global oncology sector
  • Detailed profiles of 5,000 new drugs in development and approved drugs worldwide
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LAKE FOREST, Calif., April 12, 2016 /PRNewswire/ — New Medicine ( has launched New Medicine’s Oncology KnowledgeBASE (, a highly disciplined relational database that provides a comprehensive view of the global oncology sector.   

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Oncology KnowledgeBASE

New Medicine’s Oncology KnowledgeBASE (nm|OK) provides a comprehensive view of the status of the global oncology drug development and personalized medicine sector in terms of:

  • companies developing and marketing therapeutic/in vivo imaging agents and/or in vitro diagnostics
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Future Oncology

Future Oncology

Free_IconFrom June 1995 to August 2008, New Medicine published Future Oncology, a comprehensive analytical newsletter tracking the evolution of global drug development in oncology.  Despite of the incredible amount of effort in this area in the last 20 years, we currently face the same problems that were being tackled then, namely a lack of understanding as to the origins and mechanisms of malignancy.  Despite the incredible global effort in this area and the remarkable scientific breakthroughs in biology and medicine, advanced cancer has remained an incurable disease.  However, although cancer remains undefeated, treatment of this disease has created a huge global market comprised of drugs that, with few exceptions, provide marginal relief at a very high cost.  Because the origins of this disease have remained obscure, there have been numerous approaches popularized at different times as to its treatment.  Future Oncology has tracked these developments over time, from the rise of immunotherapy in the late 1990s to the subsequent discovery of oncogenes and tumor suppressors that shifted the emphasis from the labor intensive immunotherapy and gene transfer approaches to the relative simplicity of the production and delivery of monoclonal antibodies (MAb), oligonucleotides and small molecule drugs.  Although some major advances have led to significant survival gains of patients with hematologic malignancies, they have not produced the same results in the treatment of metastatic solid tumors.

In the meantime, the competitive landscape underwent a major transformation. The archives follow the progress or demise of hundreds of commercial entities globally and hundreds of drugs, among some of the most successful to date as well some noted failures.  The passage of time has produced many surprising winners and a few unexpected losers.  Celgene, an unknown small company in 1995, has become a leading biotech juggernaut.  Rituximab, a relatively low tech transformational therapy for the treatment of hematologic malignancies, developed by the small company Idec and approved in the USA in November 1997, may be considered the most successful anticancer agent to date both for significantly extending survival and for generating billions in sales for its developers and marketers.  Since its first approval in 1995, Rituxan’s total global revenues exceeded $65 billion, including sales in the immunology sector beginning in FY 2013.  Imatinib, launched in 2001, ushered the era of personalized medicine.  Avastin, the first targeted treatment for solid tumors launched by Genentech in 2004, garnered over $60 billion in global revenues to date.

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Curbing Cancer Cell Growth & Metastasis-on-a-Chip’ Models Cancer’s Spread

Curator: Larry H. Bernstein, MD, FCAP


New Approach to Curbing Cancer Cell Growth

Using a new approach, scientists at The Scripps Research Institute (TSRI) and collaborating institutions have discovered a novel drug candidate that could be used to treat certain types of breast cancer, lung cancer and melanoma.

The new study focused on serine, one of the 20 amino acids (protein building blocks) found in nature. Many types of cancer require synthesis of serine to sustain rapid, constant and unregulated growth.

To find a drug candidate that interfered with this pathway, the team screened a large library of compounds from a variety of sources, searching for molecules that inhibited a specific enzyme known as 3-phosphoglycerate dehydrogenase (PHGDH), which is responsible for the first committed step in serine biosynthesis.

“In addition to discovering an inhibitor that targets cancer metabolism, we also now have a tool to help answer interesting questions about serine metabolism,” said Luke L. Lairson, assistant professor of chemistry at TSRI and principal investigator of cell biology at the California Institute for Biomedical Research (CALIBR).

Lairson was senior author of the study, published recently in the Proceedings of the National Academy of Sciences (PNAS), with Lewis Cantley of Weill Cornell Medical College and Costas Lyssiotis of the University of Michigan.

Addicted to Serine

Serine is necessary for nucleotide, protein and lipid biosynthesis in all cells. Cells use two main routes for acquiring serine: through import from the extracellular environment or through conversion of 3-phosphoglycerate (a glycolytic intermediate) by PHGDH.

“Since the late 1950s, it has been known that cancer cells use the process of aerobic glycolysis to generate metabolites needed for proliferative growth,” said Lairson.

This process can lead to an overproduction of serine. The genetic basis for this abundance had remained mysterious until recently, when it was demonstrated that some cancers acquire mutations that increased the expression of PHGDH; reducing PHGDH in these “serine-addicted” cancer cells also inhibited their growth.

The labs of Lewis C. Cantley at Weill Cornell Medical College (in work published in Nature Genetics) and David Sabatini at the Whitehead Institute (in work published in Nature) suggested PHGDH as a potential drug target for cancer types that overexpress the enzyme.

Lairson and colleagues hypothesized that a small molecule drug candidate that inhibited PHGDH could interfere with cancer metabolism and point the way to the development of an effective cancer therapeutic. Importantly, this drug candidate would be inactive against normal cells because they would be able to import enough serine to support ordinary growth.

As Easy as 1-2-800,000

Lairson, in collaboration with colleagues including Cantley, Lyssiotis, Edouard Mullarky of Weill Cornell and Harvard Medical School and Natasha Lucki of CALIBR, screened through a library of 800,000 small molecules using a high-throughput in vitro enzyme assay to detect inhibition of PHGDH. The group identified 408 candidates and further narrowed this list down based on cell-type specific anti-proliferative activity and by eliminating those inhibitors that broadly targeted other dehydrogenases.

With the successful identification of seven candidate inhibitors, the team sought to determine if these molecules could inhibit PHGDH in the complex cellular environment. To do so, the team used a mass spectrometry-based assay (test) to measure newly synthesized serine in a cell in the presence of the drug candidates.

One of the seven small molecules tested, named CBR-5884, was able to specifically inhibit serine synthesis by 30 percent, suggesting that the molecule specifically targeted PHGDH. The group went on to show that CBR-5884 was able to inhibit cell proliferation of breast cancer and melanoma cells lines that overexpress PHGDH.

As expected, CBR-5884 did not inhibit cancer cells that did not overexpress PHGDH, as they can import serine; however, when incubated in media lacking serine, the presence of CBR-5884 decreased growth in these cells.

The group anticipates much optimization work before this drug candidate can become an effective therapeutic. In pursuit of this goal, the researchers plan to take a medicinal chemistry approach to improve potency and metabolic stability.


How Cancer Stem Cells Thrive When Oxygen Is Scarce

(Image: Shutterstock)
image: Shutterstock

Working with human breast cancer cells and mice, scientists at The Johns Hopkins University say new experiments explain how certain cancer stem cells thrive in low oxygen conditions. Proliferation of such cells, which tend to resist chemotherapy and help tumors spread, are considered a major roadblock to successful cancer treatment.

The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.

“There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” said Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center. “That gives us a few more possible targets for drugs that diminish their threat in human cancer.”

A summary of the findings was published online March 21 in the Proceedings of the National Academy of Sciences.

“Aggressive cancers contain regions where the cancer cells are starved for oxygen and die off, yet patients with these tumors generally have the worst outcome. Our new findings tell us that low oxygen conditions actually encourage certain cancer stem cells to multiply through the same mechanism used by embryonic stem cells.”

All stem cells are immature cells known for their ability to multiply indefinitely and give rise to progenitor cells that mature into specific cell types that populate the body’s tissues during embryonic development. They also replenish tissues throughout the life of an organism. But stem cells found in tumors use those same attributes and twist them to maintain and enhance the survival of cancers.

Recent studies showed that low oxygen conditions increase levels of a family of proteins known as HIFs, or hypoxia-inducible factors, that turn on hundreds of genes, including one called NANOG that instructs cells to become stem cells.

Studies of embryonic stem cells revealed that NANOG protein levels can be lowered by a chemical process known as methylation, which involves putting a methyl group chemical tag on a protein’s messenger RNA (mRNA) precursor. Semenza said methylation leads to the destruction of NANOG’s mRNA so that no protein is made, which in turn causes the embryonic stem cells to abandon their stem cell state and mature into different cell types.

Zeroing in on NANOG, the scientists found that low oxygen conditions increased NANOG’s mRNA levels through the action of HIF proteins, which turned on the gene for ALKBH5, which decreased the methylation and subsequent destruction of NANOG’s mRNA. When they prevented the cells from making ALKBH5, NANOG levels and the number of cancer stem cells decreased. When the researchers manipulated the cell’s genetics to increase levels of ALKBH5 without exposing them to low oxygen, they found this also decreased methylation of NANOG mRNA and increased the numbers of breast cancer stem cells.

Finally, using live mice, the scientists injected 1,000 triple-negative breast cancer cells into their mammary fat pads, where the mouse version of breast cancer forms. Unaltered cells created tumors in all seven mice injected with such cells, but when cells missing ALKBH5 were used, they caused tumors in only 43 percent (six out of 14) of mice. “That confirmed for us that ALKBH5 helps preserve cancer stem cells and their tumor-forming abilities,” Semenza said.

How cancer stem cells thrive when oxygen is scarce

The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.

“There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” says Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center.

Chuanzhao Zhang, Debangshu Samanta, Haiquan Lu, John W. Bullen, Huimin Zhang, Ivan Chen, Xiaoshun He, Gregg L. Semenza.
Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.
Proceedings of the National Academy of Sciences, 2016; 201602883     DOI: 10.1073/pnas.1602883113


Pluripotency factors, such as NANOG, play a critical role in the maintenance and specification of cancer stem cells, which are required for primary tumor formation and metastasis. In this study, we report that exposure of breast cancer cells to hypoxia (i.e., reduced O2 availability), which is a critical feature of the tumor microenvironment, induces N6-methyladenosine (m6A) demethylation and stabilization of NANOG mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype. We show that inhibiting the expression of AlkB homolog 5 (ALKBH5), which demethylates m6A, or the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α, which activate ALKBH5 gene transcription in hypoxic breast cancer cells, is an effective strategy to decrease NANOG expression and target BCSCs in vivo.

N6-methyladenosine (m6A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α–dependent expression of AlkB homolog 5 (ALKBH5), an m6A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m6A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.

Specific Proteins Found to Jump Start Spread of Cancer Cells

Metastatic breast cancer cells. [National Cancer Institute]

Scientists at the University of California, San Diego School of Medicine and Moores Cancer Center, with colleagues in Spain and Germany, have discovered how elevated levels of particular proteins in cancer cells trigger hyperactivity in other proteins, fueling the growth and spread of a variety of cancers. Their study (“Prognostic Impact of Modulators of G Proteins in Circulating Tumor Cells from Patients with Metastatic Colorectal Cancer”) is published in Scientific Reports.

Specifically, the international team, led by senior author Pradipta Ghosh, M.D., associate professor at the University of California San Diego School of Medicine, found that increased levels of expression of some members of a protein family called guanine nucleotide exchange factors (GEFs) triggered unsuspected hyperactivation of G proteins and subsequent progression or metastasis of cancer.

The discovery suggests GEFs offer a new and more precise indicator of disease state and prognosis. “We found that elevated expression of each GEF is associated with a shorter, progression-free survival in patients with metastatic colorectal cancer,” said Dr. Ghosh. “The GEFs fared better as prognostic markers than two well-known markers of cancer progression, and the clustering of all GEFs together improved the predictive accuracy of each individual family member.”

In recent years, circulating tumor cells (CTCs), which are shed from primary tumors into the bloodstream and act as seeds for new tumors taking root in other parts of the body, have become a prognostic and predictive biomarker. The presence of CTCs is used to monitor the efficacy of therapies and detect early signs of metastasis.

But counting CTCs in the bloodstream has limited utility, said Dr. Ghosh. “Enumeration alone does not capture the particular characteristics of CTCs that are actually tumorigenic and most likely to cause additional malignancies.”

Numerous efforts are underway to improve the value and precision of CTC analysis. According to Dr. Ghosh the new findings are a step in that direction. First, GEFs activate trimeric G proteins, and second, G protein signaling is involved in CTCs. G proteins are ubiquitous and essential molecular switches involved in transmitting external signals from stimuli into cells’ interiors. They have been a subject of heightened scientific interest for many years.

Dr. Ghosh and colleagues found that elevated expression of nonreceptor GEFs activates Gαi proteins, fueling CTCs and ultimately impacting the disease course and survival of cancer patients.

“Our work shows the prognostic impact of elevated expression of individual and clustered GEFs on survival and the benefit of transcriptome analysis of G protein regulatory proteins in cancer biology,” said Dr. Ghosh. “The next step will be to carry this technology into the clinic where it can be applied directly to deciphering a patient’s state of cancer and how best to treat.”

Metastasis-on-a-Chip’ Models Cancer’s Spread

In the journal Biotechnology Bioengineering, the team reports on its “metastasis-on-a-chip” system believed to be one of the first laboratory models of cancer spreading from one 3D tissue to another.

The current version of the system models a colorectal tumor spreading from the colon to the liver, the most common site of metastasis. Skardal said future versions could include additional organs, such as the lung and bone marrow, which are also potential sites of metastasis. The team also plans to model other types of cancer, such as the deadly brain tumor glioblastoma

To create the system, researchers encapsulated human intestine and colorectal cancer cells inside a biocompatible gel-like material to make a mini-organ. A mini-liver composed of human liver cells was made in the same way. These organoids were placed in a “chip” system made up of a set of micro-channels and chambers etched into the chip’s surface to mimic a simplified version of the body’s circulatory system. The tumor cells were tagged with fluorescent molecules so their activity could be viewed under a microscope.

To test whether the system could model metastasis, the researchers first used highly aggressive cancer cells in the colon organoid. Under the microscope, they saw the tumor grow in the colon organoid until the cells broke free, entered the circulatory system and then invaded the liver tissue, where another tumor formed and grew. When a less aggressive form of colon cancer was used in the system, the tumor did not metastasize, but continued to grow in the colon.

To test the system’s potential for screening drugs, the team introduced Marimastat, a drug used to inhibit metastasis in human patients, into the system and found that it significantly prevented the migration of metastatic cells over a 10-day period. Likewise, the team also tested 5-fluorouracil, a common colorectal cancer drug, which reduced the metabolic activity of the tumor cells.

“We are currently exploring whether other established anti-cancer drugs have the same effects in the system as they do in patients,” said Skardal. “If this link can be validated and expanded, we believe the system can be used to screen drug candidates for patients as a tool in personalized medicine. If we can create the same model systems, only with tumor cells from an actual patient, then we believe we can use this platform to determine the best therapy for any individual patient.”

The scientists are currently working to refine their system. They plan to use 3D printing to create organoids more similar in function to natural organs. And they aim to make the process of metastasis more realistic. When cancer spreads in the human body, the tumor cells must break through blood vessels to enter the blood steam and reach other organs. The scientists plan to add a barrier of endothelial cells, the cells that line blood vessels, to the model.

This concept of modeling the body’s processes on a miniature level is made possible because of advances in micro-tissue engineering and micro-fluidics technologies. It is similar to advances in the electronics industry made possible by miniaturizing electronics on a chip.

Scientists Synthesize Anti-Cancer Agent

A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University
A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University

A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.

The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.

Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.

“Not only does this synthesis render these valuable molecules readily available for biological investigation, but it also allows the previously unknown full structural elucidation of one of them,” Nicolaou said. “The newly developed synthetic technologies will allow us to construct variations for biological evaluation as part of a program to optimize their pharmacological profiles.”

At present, there are no drugs based on trioxacarcins, which damage DNA through a novel mechanism, Nicolaou said.

Trioxacarcins were discovered in the fermentation broth of the bacterial strain Streptomyces bottropensis. They disrupt the replication of cancer cells by binding and chemically modifying their genetic material.

“These molecules are endowed with powerful anti-tumor properties,” Nicolaou said. “They are not as potent as shishijimicin, which we also synthesized recently, but they are more powerful than taxol, the widely used anti-cancer drug. Our objective is to make it more powerful through fine-tuning its structure.”

He said his lab is working with a biotechnology partner to pair these cytotoxic compounds (called payloads) to cancer cell-targeting antibodies through chemical linkers. The process produces so-called antibody-drug conjugates as drugs to treat cancer patients. “It’s one of the latest frontiers in personalized targeting chemotherapies,” said Nicolaou, who earlier this year won the prestigious Wolf Prize in Chemistry.

Fluorescent Nanoparticle Tracks Cancer Treatment’s Effectiveness in Hours

Bevin Fletcher, Associate Editor

Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women's Hospital)

Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women’s Hospital)

Bioengineers at Brigham and Women’s Hospital have developed a new technique to help determine if chemotherapy is working in as few as eight hours after treatment. The new approach, which can also be used for monitoring the effectiveness of immunotherapy, has shown success in pre-clinical models.

The technology utilizes a nanoparticle, carrying anti-cancer drugs, that glows green when cancer cells begin dying. Researchers, using  the “reporter nanoparticles” that responds to a particular enzyme known as caspase, which is activated when cells die, were able to distinguish between a tumor that is drug-sensitive or drug-resistant much faster than conventional detection methods such as PET scans, CT and MRI.  The findings were published online March 28 in the Proceedings of the National Academy of Sciences.

“Using this approach, the cells light up the moment a cancer drug starts working,” co-corresponding author Shiladitya Sengupta, Ph.D., principal investigator in BWH’s Division of Bioengineering, said in a prepared statement.  “We can determine if a cancer therapy is effective within hours of treatment.  Our long-term goal is to find a way to monitor outcomes very early so that we don’t give a chemotherapy drug to patients who are not responding to it.”

Cancer killers send signal of success

Nanoparticles deliver drug, then give real-time feedback when tumor cells die   BY   SARAH SCHWARTZ

New lab-made nanoparticles deliver cancer drugs into tumors, then report their effects in real time by lighting up in response to proteins produced by dying cells. More light (right, green) indicates a tumor is responding to chemotherapy.

Tiny biochemical bundles carry chemotherapy drugs into tumors and light up when surrounding cancer cells start dying. Future iterations of these lab-made particles could allow doctors to monitor the effects of cancer treatment in real time, researchers report the week of March 28 in theProceedings of the National Academy of Sciences.

“This is the first system that allows you to read out whether your drug is working or not,” says study coauthor Shiladitya Sengupta, a bioengineer at Brigham and Women’s Hospital in Boston.

Each roughly 100-nanometer-wide particle consists of a drug and a fluorescent dye linked to a coiled molecular chain. Before the particles enter cells, the dye is tethered to a “quencher” molecule that prevents it from lighting up. When injected into the bloodstream of a mouse with cancer, the nanoparticles accumulate in tumor cells and release the drug, which activates a protein that tears a cancer cell apart. This cell-splitting protein not only kills the tumor cell, but also severs the link between the dye and the quencher, allowing the nanoparticles to glow under infrared light.

Reporter nanoparticle that monitors its anticancer efficacy in real time

Ashish Kulkarnia,b,1,Poornima Raoa,b,Siva Natarajana,b,Aaron Goldman, et al.

The ability to identify responders and nonresponders very early during chemotherapy by direct visualization of the activity of the anticancer treatment and to switch, if necessary, to a regimen that is effective can have a significant effect on the outcome as well as quality of life. Current approaches to quantify response rely on imaging techniques that fail to detect very early responses. In the case of immunotherapy, the early anatomical readout is often discordant with the biological response. This study describes a self-reporting nanomedicine that not only delivers chemotherapy or immunotherapy to the tumor but also reports back on its efficacy in real time, thereby identifying responders and nonresponders early on

The ability to monitor the efficacy of an anticancer treatment in real time can have a critical effect on the outcome. Currently, clinical readouts of efficacy rely on indirect or anatomic measurements, which occur over prolonged time scales postchemotherapy or postimmunotherapy and may not be concordant with the actual effect. Here we describe the biology-inspired engineering of a simple 2-in-1 reporter nanoparticle that not only delivers a cytotoxic or an immunotherapy payload to the tumor but also reports back on the efficacy in real time. The reporter nanoparticles are engineered from a novel two-staged stimuli-responsive polymeric material with an optimal ratio of an enzyme-cleavable drug or immunotherapy (effector elements) and a drug function-activatable reporter element. The spatiotemporally constrained delivery of the effector and the reporter elements in a single nanoparticle produces maximum signal enhancement due to the availability of the reporter element in the same cell as the drug, thereby effectively capturing the temporal apoptosis process. Using chemotherapy-sensitive and chemotherapy-resistant tumors in vivo, we show that the reporter nanoparticles can provide a real-time noninvasive readout of tumor response to chemotherapy. The reporter nanoparticle can also monitor the efficacy of immune checkpoint inhibition in melanoma. The self-reporting capability, for the first time to our knowledge, captures an anticancer nanoparticle in action in vivo.


Cancer Treatment’s New Direction  
Genetic testing helps oncologists target tumors and tailor treatments

Evan Johnson had battled a cold for weeks, endured occasional nosebleeds and felt so fatigued he struggled to finish his workouts at the gym. But it was the unexplained bruises and chest pain that ultimately sent the then 23-year-old senior at the University of North Dakota to the Mayo Clinic. There a genetic test revealed a particularly aggressive form of acute myeloid leukemia. That was two years ago.

The harrowing roller-coaster that followed for Mr. Johnson and his family highlights new directions oncologists are taking with genetic testing to find and attack cancer. Tumors can evolve to resist treatments, and doctors are beginning to turn such setbacks into possible advantages by identifying new targets to attack as the tumors change.

His course involved a failed stem cell transplant, a half-dozen different drug regimens, four relapses and life-threatening side effects related to his treatment.

Nine months in, his leukemia had evolved to develop a surprising new mutation. The change meant the cancer escaped one treatment, but the new anomaly provided doctors with a fresh target, one susceptible to drugs approved for other cancers. Doctors adjusted Mr. Johnson’s treatment accordingly, knocked out the disease and paved the way for a second, more successful stem cell transplant. He has now been free of leukemia for a year.

Now patients with advanced cancer who are treated at major centers can expect to have their tumors sequenced, in hopes of finding a match in a growing medicine chest of drugs that precisely target mutations that drive cancer’s growth. When they work, such matches can have a dramatic effect on tumors. But these “precision medicines” aren’t cures. They are often foiled when tumors evolve, pushing doctors to take the next step to identify new mutations in hopes of attacking them with an effective treatment.

Dr. Kasi and his Mayo colleagues—Naseema Gangat, a hematologist, and Shahrukh Hashmi, a transplant specialist—are among the authors of an account of Mr. Johnson’s case published in January in the journal Leukemia Research Reports.

Before qualifying for a transplant, a patient’s blasts need to be under 5%.

To get under 5%, he started on a standard chemotherapy regimen and almost immediately, things went south. His blast cells plummeted, but “the chemo just wiped out my immune system,”

Then as mysteriously as it began, a serious mycotic throat infection stopped. But Mr. Johnson couldn’t tolerate the chemo, and his blast cells were on the rise. A two-drug combination that included the liver cancer drug Nexavar, which targets the FLT3 mutation, knocked back the blast cells. But the stem cell transplant in May, which came from one of his brothers, failed to take, and he relapsed after 67 days, around late July.

He was put into a clinical trial of an experimental AML drug being developed by Astellas Pharma of Japan. He started to regain weight. In November 2014, doctors spotted the initial signs in blood tests that Mr. Johnson’s cancer was evolving to acquire a new mutation. By late January, he relapsed again , but there was a Philadelphia chromosome mutation,  a well-known genetic alteration associated with chronic myeloid leukemia. It also is a target of the blockbuster cancer drug Gleevec and several other medicines.

Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3) inhibitor therapy with evolving actionable targets

Naseema GangatMark R. LitzowMrinal M. PatnaikShahrukh K. HashmiNaseema Gangat

•   The article reports on a case of AML that underwent clonal evolution.
•   We report on novel acquisition of the Philadelphia t(9;22) translocation in AML.
•   Next generation sequencing maybe helpful in these refractory/relapse cases.
•   Novel FLT3-inhibitor targeted therapies are another option in patients with AML.
•   Personalizing cancer treatment based on evolving targets is a viable option.

For acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215) to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈〉)

The development of kinase inhibitors for the treatment of leukemia has revolutionized the care of these patients. Since the introduction of imatinib for the treatment of chronic myeloid leukemia, multiple other tyrosine kinase inhibitors (TKIs) have become available[1]. Additionally, for acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors [2], [3], [4] and [5]. The article herein reports a unique case of AML that underwent clonal evolution while on a novel FLT3-inhibitor clinical trial.

Our work herein presents clinical and next generation sequencing data at the time of progression to illustrate these important concepts stemming from Darwinian evolution [6]. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process.

Our work focuses on a 23-year-old male who presented with 3 months history of fatigue and easy bruising, a white blood count of 22.0×109/L with 51% circulating blasts, hemoglobin 7.6 g/dL, and a platelet count of 43×109/L. A bone marrow biopsy confirmed a diagnosis of AML. Initial cytogenetic studies identified trisomy 8 in all the twenty metaphases examined. Mutational analysis revealed an internal tandem duplication of the FLT3 gene (FLT3-ITD).

He received standard induction chemotherapy (7+3) with cytarabine (ARA-C; 100 mg/m2for 7 days) and daunorubicin (DNM; 60 mg/m2 for 3 days). His induction chemotherapy was complicated by severe palatine and uvular necrosis of indeterminate etiology (possible mucormycosis).

Bone marrow biopsy at day 28 demonstrated persistent disease with 10% bone marrow blasts (Fig. 1). Due to his complicated clinical course and the presence of a FLT3-ITD, salvage therapy with 5-azacitidine (5-AZA) and sorafenib (SFN) was instituted. Table 1.
The highlighted therapies were employed in this particular case at various time points as shown in Fig. 1.


    • [1]
    • J.E. Cortes, D.W. Kim, J. Pinilla-Ibarz, et al.
    • A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias
    • New Engl. J. Med., 369 (19) (2013), pp. 1783–1796
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    • F. Ravandi, M.L. Alattar, M.R. Grunwald, et al.
    • Phase 2 study of azacytidine plus sorafenib in patients with acute myeloid leukemia and FLT-3 internal tandem duplication mutation
    • Blood, 121 (23) (2013), pp. 4655–4662
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    • N.P. Shah, M. Talpaz, M.W. Deininger, et al.
    • Ponatinib in patients with refractory acute myeloid leukaemia: findings from a phase 1 study
    • Br. J. Haematol., 162 (4) (2013), pp. 548–552
    • [4]
    • Y. Alvarado, H.M. Kantarjian, R. Luthra, et al.
    • Treatment with FLT3 inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated with development of secondary FLT3-tyrosine kinase domain mutations
    • Cancer, 120 (14) (2014), pp. 2142–2149
    • [5]
    • C.C. Smith, C. Zhang, K.C. Lin, et al.
    • Characterizing and overriding the structural mechanism of the Quizartinib-Resistant FLT3 “Gatekeeper” F691L mutation with PLX3397
    • Cancer Discov. (2015)
    • [6]
    • M. Greaves, C.C. Maley
    • Clonal evolution in cancer
    • Nature, 481 (7381) (2012), pp. 306–313




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Myc and Cancer Resistance

Curator: Larry H. Bernstein, MD, FCAP


Myc (c-Myc) is a regulator gene that codes for atranscription factor. The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation.[1]

Myc gene was first discovered in Burkitt lymphoma patients. In Burkitt lymphoma, cancer cells showchromosomal translocations, in which Chromosome 8 is frequently involved. Cloning the break-point of the fusion chromosomes revealed a gene that was similar to myelocytomatosis viral oncogene (v-Myc). Thus, the newfound cellular gene was named c-Myc.


Protein increases signals that protect cancer cells

Researchers have identified a link between the expression of a cancer-related gene and cell-surface molecules that protect tumors from the immune system

Depiction of the Myc protein

The Myc protein, depicted here, is mutated in more than half of all human cancers.   Petarg/Shutterstock


A cancer-associated protein called Myc directly controls the expression of two molecules known to protect tumor cells from the host’s immune system, according to a study by researchers at the Stanford University School of Medicine.

The finding is the first to link two critical steps in the development of a successful tumor: uncontrolled cell growth — when mutated or misregulated, Myc causes an increase in the levels of proteins that promote cell division — and an ability to outwit the immune molecules meant to stop it.

The study was published online March 10 inScience. Dean Felsher, MD, PhD, a professor of oncology and of pathology, is the senior author. The lead author is postdoctoral scholar Stephanie Casey, PhD. The work was conducted in collaboration with researchers at the University of Wurzburg.

“Our findings describe an intimate, causal connection between how oncogenes like Myc cause cancer and how those cancer cells manage to evade the immune system,” Felsher said.

‘Don’t eat me’ and ‘don’t find me’

One of the molecules is the CD47 protein, which researchers in the Stanford laboratory of Irving Weissman, MD, have discovered serves as a “don’t eat me” signal to ward off cancer-gobbling immune cells called macrophages. Weissman is the Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research and the director of Stanford’s Institute for Stem Cell Biology and Regenerative Medicine.

Nearly all human cancers express high levels of CD47 on their surfaces, and an antibody targeting the CD47 protein is currently in phase-1 clinical trials for a variety of human cancers.

The other molecule is a “don’t find me” protein called PD-L1, known to suppress the immune system during cancer and autoimmune diseases but also in normal pregnancy. It’s often overexpressed on human tumor cells. An antibody that binds to PD-L1 has been approved by the U.S. Food and Drug Administration to treat bladder and non-small-cell lung cancer, but it has been shown to be effective in the treatment of many cancers.

Dean Felsher

Programmed death-ligand 1 (PD-L1): an inhibitory immune pathway exploited by cancer

Image of PD-L1 binding to B7.1 and PD-1, deactivating T cell]

In cancer, Myc a usual suspect

Researchers in Felsher’s laboratory have been studying the Myc protein for more than a decade. It is encoded by a type of gene known as an oncogene. Oncogenes normally perform vital cellular functions, but when mutated or expressed incorrectly they become powerful cancer promoters. The Myc oncogene is mutated or misregulated in over half of all human cancers.

In particular, Felsher’s lab studies a phenomenon known as oncogene addiction, in which tumor cells are completely dependent on the expression of the oncogene. Blocking the expression of the Myc gene in these cases causes the complete regression of tumors in animals.

In 2010, Felsher and his colleagues showed that this regression could only occur in animals with an intact immune system, but it wasn’t clear why.

“Since then, I’ve had it in the back of my mind that there must be a relationship between Myc and the immune system,” said Felsher.

Turning off Myc expression

Casey and Felsher decided to see if there was a link between Myc expression and the levels of CD47 and PD-L1 proteins on the surface of cancer cells. To do so, they investigated what would happen if they actively turned off Myc expression in tumor cells from mice or humans. They found that a reduction in Myc caused a similar reduction in the levels of CD47 and PD-L1 proteins on the surface of mouse and human acute lymphoblastic leukemia cells, mouse and human liver cancer cells, human skin cancer cells, and human non-small-cell lung cancer cells. In contrast, levels of other immune regulatory molecules found on the surface of the cells were unaffected.

I’ve had it in the back of my mind that there must be a relationship between Myc and the immune system.

In publicly available gene expression data on tumor samples from hundreds of patients, they found that the levels of Myc expression correlated strongly with expression levels of CD47 and PD-L1 genes in liver, kidney and colorectal tumors.

The researchers then looked directly at the regulatory regions in the CD47 and PD-L1 genes. They found high levels of the Myc protein bound directly to the promoter regions of both CD47 and PD-L1 in mouse leukemia cells, as well as in a human bone cancer cell line. They were also able to verify that this binding increased the expression of the CD47 gene in a human blood cell line.

Possible treatment synergy

Finally, Casey and Felsher engineered mouse leukemia cells to constantly express CD47 or PD-L1 genes regardless of Myc expression status. These cells were better able than control cells to evade the detection of immune cells like macrophages and T cells, and, unlike in previous experiments from Felsher’s laboratory, tumors arising from these cells did not regress when Myc expression was deactivated.

“What we’re learning is that if CD47 and PD-L1 are present on the surfaces of cancer cells, even if you shut down a cancer gene, the animal doesn’t mount an adequate immune response, and the tumors don’t regress,” said Felsher.

The work suggests that a combination of therapies targeting the expression of both Myc and CD47 or PD-L1 could possibly have a synergistic effect by slowing or stopping tumor growth, and also waving a red flag at the immune system, Felsher said.

“There is a growing sense of tremendous excitement in the field of cancer immunotherapy,” said Felsher. “In many cases, it’s working. But it’s not been clear why some cancers are more sensitive than others. Our work highlights a direct link between oncogene expression and immune regulation that could be exploited to help patients.”

The research is an example of Stanford Medicine’s focus on precision health, the goal of which is to anticipate and prevent disease in the healthy and precisely diagnose and treat disease in the ill.

Other Stanford co-authors of the paper are oncology instructor Yulin Li, MD, PhD; postdoctoral scholars Ling Tong, PhD, Arvin Gouw, PhD, and Virginie Baylot, PhD; former research assistant Kelly Fitzgerald; and undergraduate student Rachel Do.

The research was supported by the National Institutes of Health (grants RO1CA089305, CA170378, CA184384, CA105102, P50 CA114747, U56CA112973, U01CA188383, 1F32CA177139 and 5T32AI07290).


The PD-L1 pathway downregulates cytotoxic T-cell activity to maintain immune homeostasis

Under normal conditions, the inhibitory ligands PD-L1 and PD-L2 play an important role in maintaining immune homeostasis.1 PD-L1 and PD-L2 bind to specific receptors on T cells. When bound to their receptors, cytotoxic T-cell activity is downregulated, thereby protecting normal cells from collateral damage.1,2

Image showing PD-L1 binding to B7.1 and PD-1 to deactivate T cells during immune response]


Broadly expressed in multiple tissue types, including hematopoietic, endothelial, and epithelial cells1,4


Receptor expressed on activated T cells and dendritic cells3


Receptor expressed primarily on activated T cells3


Image showing PD-L1 binding to B7.1 and PD-1 to deactivate T cells during immune response]


Restricted expression on immune cells and in some organs, such as the lung and colon1,4,5


Receptor expressed primarily on activated T cells3


Many tumors can exploit the PD-L1 pathway to inhibit the antitumor response

In cancer, the PD-L1/B7.1 and PD-L1/PD-1 pathways can protect tumors from cytotoxic T cells, ultimately inhibiting the antitumor immune response in 2 ways.1-3

  • Deactivating cytotoxic T cells in the tumor microenvironment
  • Preventing priming and activation of new T cells in the lymph nodes and subsequent recruitment to the tumor



Upregulation of PD-L1 can inhibit the last stages of the cancer immunity cycle by deactivating cytotoxic T cells in the tumor microenvironment.1

Activated T cells in the tumor microenvironment release interferon gamma.2

As a result, tumor cells and tumor-infiltrating immune cells overexpress PD-L1.2

PD-L1 binds to T-cell receptors B7.1 and PD-1, deactivating cytotoxic T cells. Once deactivated, T cells remain inhibited in the tumor microenvironment.1,2


PD-L1 overexpression can also inhibit propagation of the cancer immunity cycle by preventing the priming and activation of T cells in the lymph nodes.1-3

PD-L1 expression is upregulated on dendritic cells within the tumor microenvironment.2,3

PD-L1–expressing dendritic cells travel from the tumor site to the lymph node.4

PD-L1 binds to B7.1 and PD-1 receptors on cytotoxic T cells, leading to their deactivation.3


The cancer immunity cycle characterizes the complex interactions between the immune system and cancer

The cancer immunity cycle describes a process of how one’s own immune system can protect the body against cancer. When performing optimally, the cycle is self-sustaining. With subsequent revolutions of the cycle, the breadth and depth of the immune response can be increased.1



  • Oncogenesis leads to the expression of neoantigens that can be captured by dendritic cells
  • Dendritic cells can present antigens to T cells, priming and activating cytotoxic T cells to attack the cancer cells


  • Activated T cells travel to the tumor and infiltrate the tumor microenvironment


  • Activated T cells can recognize and kill target cancer cells
  • Dying cancer cells release additional cancer antigens, propagating the cancer immunity cycle




Image of immunity cycle; explore Genentech cancer immunotherapy research on the cancer immunity cycle



  1. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1-10. PMID: 23890059
  2. Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy—inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res. 2012;18:6580-6587. PMID: 23087408
  3. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008;26:677-704. PMID: 18173375
  4. Motz GT, Coukos G. Deciphering and reversing tumor immune suppression. Immunity. 2013;39:61-73. PMID: 23890064



MYC regulates the antitumor immune response through CD47 and PD-L1

The clinical efficacy of monoclonal antibodies as cancer therapeutics is largely dependent upon their ability to target the tumor and induce a functional antitumor immune response. This two-step process of ADCC utilizes the response of innate immune cells to provide antitumor cytotoxicity triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell. Immunotherapeutics that target NK cells, γδ T cells, macrophages and dendritic cells can, by augmenting the function of the immune response, enhance the antitumor activity of the antibodies. Advantages of such combination strategies include: the application to multiple existing antibodies (even across multiple diseases), the feasibility (from a regulatory perspective) of combining with previously approved agents and the assurance (to physicians and trial participants) that one of the ingredients – the antitumor antibody – has proven efficacy on its own. Here we discuss current strategies, including biologic rationale and clinical results, which enhance ADCC in the following ways: strategies that increase total target–monoclonal antibody–effector binding, strategies that trigger effector cell ‘activating’ signals and strategies that block effector cell ‘inhibitory’ signals.

Keywords: γδ T cells, ADCC, cancer, cytokines, IMiD, immunocytokines, immunomodulators, interleukins, monoclonal antibodies, NK cells, passive immunotherapy

Monoclonal antibodies (mAbs) can target tumor antigens on the surface of cancer cells and have a favorable toxicity profile in comparison with cytotoxic chemotherapy. Expression of tumor antigens is dynamic and inducible through agents such as Toll-like receptor (TLR) agonists, immunomodulatory drugs (IMiDs) and hypomethylating agents [1]. Following binding of the mAb to the tumor antigen, the Fc portion of the mAb interacts with the Fc receptor (FcR) on the surface of effector cells (i.e., NK cells, γδ T cells and macrophages), leading to antitumor cytotoxicity and/or phagocytosis of the tumor cell. FcR interactions can be stimulatory or inhibitory to the killer cell, depending on which FcR is triggered and on which cell. Stimulatory effects are mediated through FcγRI on macrophages, dendritic cells (DCs) and neutrophils, and FcγRIIIa on NK cells, DCs and macrophages. In murine models, the cytotoxicity resulting from FcR activation on a NK cell, γδ T cell and macrophage is responsible for antitumor activity [2]. The role of DCs should be noted: although not considered to be primary ADCC effector cells, they can respond to mAb-bound tumor cells via their own FcR-mediated activation and probably play a significant role in activating effector cells. Preclinical models have shown that, although not the effector cell, DCs are critical to the efficacy of mAb-mediated tumor elimination [3]. Equally, mAb-activated ADCC effector cells can induce DC activation [4] and the importance of this crosstalk is an increasing focus of study [5].

The antitumor effects of mAbs are caused by multiple mechanisms of action, including cell signaling agonism/antagonism, complement activation and ligand sequestration, although ADCC probably plays a predominant role in the efficacy of some mAbs. In a clinical series, a correlation between the affinity of the receptor FcγRIIIa (determined by inherited FcR polymorphisms) and the clinical response to mAb therapy, supporting the significance of the innate immune response [610]. Several strategies could potentially improve the innate response following FcR activation by a mAb (Figure 1):

Quantitatively increasing the density of the bound target, mAb or the effector cells;

Stimulation of the effector cell by targeting the NK cell, γδ T cell and/or macrophage with small molecules, cytokines or agonistic antibodies;

Blocking an inhibitory interaction between the NK cell or macrophage and the tumor cell.


An external file that holds a picture, illustration, etc. Object name is nihms384451f1.jpg

Enhancing ADCC

FcR: Fc receptor; HDACi: Histone deacetylase inhibitor; IMiD: Immunomodulator; KIR: Killer immunoglobulin-like receptor;

The ability of the combination approaches to enhance ADCC is largely determined by the capacity of the mAb to induce ADCC. Since the approval of the first mAb for the treatment of non-Hodgkin’s lymphoma, rituximab (RTX), in 1997, several mAbs have become standard of care for the treatment of both solid tumors and hematologic malignancies, including trastuzumab (TRAST), alemtuzumab, cetuximab, panitumumab and ofatumumab [11]. As noted above, clinical series among lymphoma patients treated with an anti-CD20 mAb (RTX) [6,7], HER2-expressing breast cancer receiving anti-HER2 mAb therapy (TRAST) [8] or colorectal cancer patients treated with an anti-EGFR mAb (cetuximab) [9,10] observed a correlation between clinical benefit and FcγRIIIa genotype, with patients who have higher-affinity polymorphisms demonstrating superior clinical outcomes. By contrast, the anti-EGFR mAb panitumumab does not induce ADCC, owing to a different Fc isotype that does not bind to the FcγRIIIa. Therefore, when considering enhancement of ADCC, such approaches are limited to combinations with mAbs that activate the FcR. Nonetheless, an advantage of this dual therapy strategy is that mAbs yet to be discovered against currently unknown tumor antigens may be combined with the therapeutics discussed herein.

Increasing target–mAb–effector binding

As the central element in the target–mAb–effector cell unit, the mAb seems to be a probable candidate for improvements, either in its antigen-binding or its Fc-binding domains. This approach has been heavily pursued with some degree of success [1215]. Antibody engineering to improve interaction between the target or FcR requires that each new antibody be individually developed and tested as a new entity.

Increasing the antigen target

Tumor cells with a lower density of antigen targets are less responsive to mAbs than higher antigen-expressing diseases [16]. Therefore, it seems logical to try to increase the expression of the target on tumor cells. Antigen expression can be upregulated by cytokines [17], ionizing radiation [18], natural metabolites [19] and hypomethylating agents such as decitabine [20]. In addition, the family of TLR9 agonists known as CpG oligodeoxynucleotides (CpG ODN) can induce CD20 expression on malignant B cells [2123]. Taken together with data showing the activating effect of CpG ODN on effector cells (discussed below), it seems reasonable that the combination of CpG ODN with mAb might have synergistic efficacy. Clinical series, however, have tested CpG ODN administered intravenously or subcutaneously and have observed little efficacy in Phase I and II studies [2426] in low-grade lymphoma. One possible limitation of these studies has been their application to diseases (primarily follicular and mantle cell lymphoma) known to already have high expression of the relevant antigen (CD20). It is plausible that increasing antigen expression on low antigen-expressing diseases such as chronic lymphocytic leukemia could have a greater increase in relative efficacy. To this end, monotherapy studies have recently been undertaken [27,301] and should lead to combination trials.


Effector cells: γδ T cells

The role of NK cells and macrophages in mediating ADCC has been well established; however, only recently have γδ T cells been found to play a role as ADCC effectors. Typically, this population is considered as a minor subset (<5% of circulating T cells), although they may infiltrate tumors of epithelial origin preferentially and constitute a large portion of the tumor-infiltrating lymphocytes in cancers such as breast carcinoma. The combination of HLA-unrestricted cytotoxicity against multiple tumor cell lines of various histologies, secretion of cytolytic granules and proinflammatory cytokines such as TNF-α, IL-17 and IFN-γ make γδ T cells potentially potent antitumor effectors [32,33].


TLR agonists    

In addition to its aforementioned induction of CD20, CpG ODN also indirectly augments innate immune function. TLRs are specialized to recognize pathogen-associated molecular patterns; they stimulate plasmacytoid DCs and B cells [53], and one of many plasmacytoid DC responses to stimulation by CpG ODNs is activation of local NK cells, thus improving spontaneous cytotoxicity and ADCC [54]. CpG ODN effects on NK cells appeared to be indirect and IFN-γ production by T cells (possibly in response to plasmacytoid DC activation) has been hypothesized as the intermediary of NK cell activation.


Immunomodulatory drugs

IMiDs have shown clinical activity in multiple hematologic malignancies despite their primary mechanism of action being unclear. Among their biologic effects (particularly lenalidomide) there are demonstrable and pleiotropic effects on immune cells and signaling molecules. These include enhancement of in vitro NK cell- and monocyte-mediated ADCC on RTX-coated [68] as well as TRAST- and cetuximab-coated tumor cells [69]. In vivo studies in a human lymphoma severe combined immune deficiency mouse model demonstrated significant increases in NK cell recruitment to tumors mediated via microenvironment cytokine changes and augmented RTX-associated ADCC [70]. Studies suggest that IMiD activation of NK cells occurs indirectly; partly via IL-2 induction by T cells [71]. Clinically, a recent study noted significant increases in peripheral blood NK cells, NK cell cytotoxicity and serum IL-2, IL-15 and GM-CSF [72], the potential ADCC-promoting effects of which are discussed below.



PD-1 is a negative regulatory member of the CD28 superfamily expressed on the surface of activated T cells, B cells, NK cells and macrophages, similar to but more broadly regulatory than CTLA-4. Its two known ligands, PD-L1 and PD-L2, are both expressed on a variety of tumor cell lines. The PD-1–PD-L1 axis modulates the NK cell versus multiple myeloma effect, as seen by its blockade enhancing NK cell function against autologous primary myeloma cells, seemingly through effects on NK cell trafficking, immune complex formation with myeloma cells and cytotoxicity specifically toward PD-L1(+) tumor cells [179]. Two anti-PD-1 mAbs (BMS-936558 and CT-011) are currently in clinical trials, the latter in a combination study with RTX for patients with low-grade follicular lymphoma [314].

ConclusionThe recent approval of an anti-CTLA4 mAb has demonstrated that modulating the immune response can improve patient survival [180,181]. As the immune response is a major determinant of mAb efficacy, the opportunity now exists to combine mAb therapy with IMiDs to enhance their antitumor efficacy. Remarkable advances in the basic science of cellular immunology have increased our understanding of the effector mechanisms of mAb antitumor efficacy. Whereas the earliest iterations of such combinations, for example IL-2 and GM-CSF, may have augmented both effector and suppressive cells, newer approaches such as IL-15 and TLR agonists may more efficiently activate effector cells while minimizing the influence of suppressive cells. Despite these encouraging rationale and preliminary data, clinical evidence is still required to demonstrate whether combination therapies will increase the antitumor effects of mAb.

Still, this approach is unique in combining a tumor-targeting therapy, the mAb, with an immune-enhancing therapy. If successful, these therapies may be combined with multiple mAbs in routine practice, as well as novel mAbs yet to be developed. Various approaches including augmenting antigen expression, stimulating the innate response and blocking inhibitory signals are being explored to determine the optimal synergy with mAb therapies. Therapies targeting NK cells, γδ T cells, macrophages and DCs may ultimately be used in combination to further augment ADCC. Encouraging preclinical studies have led to a number of promising therapeutics, and the results of proof-of-concept clinical trials are eagerly awaited.

PD-L1, other targeted therapies await more standardized IHC

February 2016—Immunohistochemistry is heading down a path toward more standardization, and that’s essential as it plays an increasing role in rapidly expanding immunotherapy, says David L. Rimm, MD, PhD, professor of pathology and of medicine (oncology) and director of translational pathology at Yale University School of Medicine. As a co-presenter of a webinar produced by CAP TODAY in collaboration with Horizon Diagnostics, titled “Immunohistochemistry Through the Lens of Companion Diagnostics” (, he analyzes the core challenges of IHC’s adaptation to the needs of precision medicine: binary versus continuous IHC, measuring as opposed to counting or viewing by the pathologist, automation, and assay performance versus protein measurement.

“Immunohistochemistry is 99 percent binary already,” Dr. Rimm points out. “There are only a few assays in our labs—ER, PR, HER2, Ki-67, and maybe a few more—where we really are looking at a continuous curve or a level of expression.”

Two criteria in the 2010 ASCO/CAP guidelines on ER and PR testing in breast cancer patients are key, he says: 1) the percentage of cells staining and 2) any immunoreactivity. “The first is hard to estimate, but the guidelines recommend the use of greater than or equal to one percent of cells that are immunoreactive. That means they could have a tiny bit of signal or they could have a huge amount of signal and they would be considered immunoreactive, which thereby makes this a binary test.”

Having the test be binary can be a problem for companion diagnostic purposes because any immunoreactivity is dependent on the laboratory threshold and counterstain. For example, if two of the same spots, serial sections on a tissue microarray, were shown side by side, one with and one without the hematoxylin counterstain, “you might see the counterstain make this positive test into a negative by eye, which is a potential problem with IHC when you have a binary stain.” (Fig. 1).


Dr. Rimm describes a small study done with three different CLIA-certified labs, each using a different FDA-approved antibody and measuring about 500 breast cancer cases on a tissue microarray. The study showed there can be fairly significant discordance between labs—between 18 and 30 percent discordance—in terms of the cases that were positive. “In fact, if we look at outcome, 18 percent of the cases were called positive in Lab Two but were negative in Lab Three. Lab Three showed outcomes similar to the double positives whereas Lab Two had false-negatives.” This is an important problem that occurs when we try to binarize our immunohistochemistry, he says.

Counting is more variable in a real-world setting due to the variability of the threshold for considering a case positive. “You can easily calculate that if your threshold was five percent, then you’d have 70 percent positive cells. And you would easily call this positive. But if you added more hematoxylin because that’s how your pathologist liked it, then perhaps you’d only have 30 percent positive. So this is the risk of using thresholds.” (Fig. 2).


Although this is done in all of immunohistochemistry today, Dr. Rimm thinks it is an important consideration as IHC transitions to more standardized form. “An H score—intensity times area, which has been attempted many times, can’t be done by human beings. Pathologists try but have failed.”

“We can’t do those intensities by eye. We have to measure them with a machine. But we get a very different piece of information content when we measure intensity, as opposed to measuring the percentage of cells above a threshold. In sum, more information is present in a measurement than in counting.”

Pathologists read slides for a living, so it’s uncomfortable to think about giving that up in order to use a machine to measure the slides. “But I think if we want to serve our clients and our patients, we really owe them the accuracy of the 21st century as opposed to the methods of the 20th century.” (Fig. 3).

A shows comparison of a quantitative fluorescence score on the x axis versus an H-score on the y axis. Note the noncontinuous nature of human estimation of intensity times area (H-score). B) The survival curve in a population of lung cancer cases using the H-score. C) The survival curve in the same population using the quantitative score. (Source: David Rimm, MD, PhD)

A shows comparison of a quantitative fluorescence score on the x axis versus an H-score on the y axis. Note the noncontinuous nature of human estimation of intensity times area (H-score). B) The survival curve in a population of lung cancer cases using the H-score. C) The survival curve in the same population using the quantitative score. (Source: David Rimm, MD, PhD)

Among the currently available quantitative measuring devices are the Visiopharm, VIAS (Ventana), Aperio (Leica), InForm (Perkin-Elmer), and Definiens platforms. “We use the platform invented in my lab, called Aqua [Automated Quantitative Analysis], but this is now owned by Genoptix/Novartis. Genoptix intends to provide commercial tests using Aqua internally,” Dr. Rimm says, “as well as enable platform and commercial testing through partnership with additional reference lab providers.

“There are many quantification platforms,” he adds, “and I believe that any of them, used properly, can be effective in measurement.”

(Of the 265 participants in the CAP PM2 Survey, 2015 B mailing, who reported using an imaging system for quantification, 4.6 percent use VIAS, 4.1 percent use ACIS, 0.8 use Applied Imaging, and 10 percent use “other” imaging systems. Of the 1,359 Survey participants who responded to the question about use of an imaging system to analyze hormone receptor slides, 1,094, or 80.5 percent, reported not using any imaging system for quantification.)

Says Dr. Rimm: “The first platform we used to try to quantitate some DAB stain slides was actually the Aperio Nuclear Image Analysis algorithm. But the problem with DAB is that you can’t see through it. And so inherently it’s physically flawed as a method for accurate measurement.” He compares DAB to looking at stacks of pennies from above, where their height and quantity can’t be surmised, as opposed to from the side, where their numbers can be accurately estimated. “This is why I don’t use, in general, DAB-type technologies or any chromogen.”

Fluorescence doesn’t have this problem, and that is the reason Dr. Rimm began using fluorescence as a quantitative method. “We try to be entirely quantitative without any feature extraction. So we define epithelial tumors using a mask of cytokeratin. We define a mask by bleeding and dilating, filling some holes, and then ultimately measure the intensity of each cell, or of each target we’re looking for. In this case, in a molecularly defined compartment.”

Compartments can be defined by any type of molecular interactions. “We defined DAPI-positive pixels as nuclei, and we measure the intensity of the estrogen receptor within the compartment. And that gives us an intensity over an area or the equivalent of a concentration.” Many other fluorescent tools can be used in this same manner, but he cautions against use of fluorescent tools that group and count. “That’s a second approach that can be used, but the result gives you a count instead of a measurement.”

When comparing a pathologist’s reading versus a quantitative immunofluorescence score, he notes, pathologists actually don’t generate a continuous score. Instead, pathologists tend to use groups. “We tend to use a 100 or a 200 or an even number. We never say, ‘Well, it’s 37 percent positive.’ We say, ‘It’s 40 percent positive,’ because we know we can’t reproducibly tell 37 from 38 from 40 percent positive.”

The result of that is a noncontinuous scoring result, which doesn’t give the information content of quantitative measurement. A comparison between the two methods shows that at times, where quantitative measurement shows a significant difference in outcome, nonquantitative measure or an H-score difference may not show a difference in outcome. (Fig. 3 illustrates this concept.)

“Pathologists tend to group things, and we also tend to overestimate. It’s not that pathologists are bad readers. It’s just the tendency of the human eye because of our ability to distinguish different intensities and the subtle difference between intensities. But even if you compare two quantitative methods, you can see that the method where light absorbance occurs—that is the percent positive nuclei by Aperio, which is a chromogen-based method—tends to saturate. This is, in fact, amplified dramatically when you look at something with a wide dynamic range like HER2.” (Fig. 4).


In one study, researchers found less than one percent discordance—essentially no discordance—between two antibodies (Dekker TJ, et al. Breast Cancer Res. 2012;14[3]:R93). But looking at these results graphed quantitatively, you would see a very different result, Dr. Rimm says. “You can see a whole group of cases down below where there’s very low extracellular domain and very high cytoplasmic domain. In fact, some of these cases have essentially no extracellular domain, but high levels of cytoplasmic domain, and other cases have roughly equal levels of each” (Carvajal-Hausdorf DE, et al. J Natl Cancer Inst.2015;107[8]:pii:djv136).

Recent studies by Dr. Rimm’s group have shown this to have clinical implications. He looked at patients treated with trastuzumab in the absence of chemotherapy, in an unusual study called the HeCOG (Hellenic Cooperative Oncology Group) trial.

“We found that patients who had high levels of both extracellular and intracellular domain have much more benefit than patients who are missing the extracellular domain and thereby missing the trastuzumab binding site.” Follow-up studies are being done to validate this finding in larger cohorts.

Preanalytical variables, Dr. Rimm emphasizes, can have significant effects on IHC results, and more than 175 of them have been identified. “These are basically all the things we can’t control, which is the ultimate argument for standardization.”

In a surprising study by Flory Nkoy, et al., he says, it was shown that breast cancer specimens were more likely to be ER negative if the patient’s surgery was on a Friday because there was a higher ER-negative rate on Friday than on Monday. “So how could that be? Well, it was clearly the fact that the tissue was sitting over the weekend. And when it sat over the weekend, the ER positivity rate was going down” (Arch Pathol Lab Med. 2010;134:606–612).

Another study showed that after one hour, four hours, and eight hours of storage at room temperature, you lose significant amounts of staining, Dr. Rimm says. “And perhaps the best nonquantitative study or H-score-based study of this phenomenon was done by Isil Yildiz-Aktas, et al., where a significant decrease in the estrogen receptor score was found after only three hours in delay to fixation” (Mod Pathol. 2012;25:1098–1105).

How long the slide is left to sit after it is cut is another preanalytical variable to be concerned with. “In the clinical lab, that’s not often a problem since we cut them, then stain them right away. But in a research setting, a fresh-cut slide can look very different from a slide that’s two days old, six days old, or 30 days old, where a 2+ spot on a breast cancer patient becomes negative after 30 days sitting on a lab bench. So those are both key variables to be mindful of.”

One solution for those preanalytic variables is trying to prevent delayed time to fixation. “And probably time to fixation is one of the main preanalytic variables, although it’s only one of the many hundreds of variables. The method we use to try to get around this problem is to use core biopsies or allow rapid and complete fixation, and then other things can be done.”

Finally, he warns, don’t cut your tissue until right before you stain it. “If you’re asked to send a tissue out to a collaborator or someone who is going to use it for research purposes later, we recommend coring and re-embedding the core, or sending the whole block. Unstained sections, when not properly stored in a vacuum, will ultimately be damaged by hydration or oxidation, both of which lead to loss of antigenicity.”

The crux of the matter is assay performance versus protein measurement, Dr. Rimm says. “In the last six to nine months, we really are faced with this problem in spades, as PD-L1 has become a very important companion diagnostic.”

There are now four PD-L1 drugs with complementary or companion diagnostic tests (Fig. 5). One of the FDA-approved drugs, nivolumab (Opdivo, Bristol-Myers Squibb), for example, uses a clone called 28-8, which is provided by Dako in an assay, a complementary diagnostic assay, and with the following suggested scoring system: one percent, five percent, or 10 percent. In contrast, pembrolizumab (Keytruda, Merck) is also now FDA-approved but requires a companion diagnostic test that uses a different antibody, although the same Dako Link 48 platform. This diagnostic has a different scoring system of less than one percent, one to 49 percent, and 50 percent and over.

Two other companies, Roche/Genentech and AstraZeneca, also have drugs in trials that may or may not have companion diagnostic testing, though both have already identified a partner and a unique antibody (neither of those listed above) and companion diagnostic testing scores used in their clinical trials.

“So what’s a pathologist to do?” Dr. Rimm says. “Well, there are a few problems with this. First of all, what we really should be doing is measuring PD-L1. That’s the target and that’s what should ultimately predict response. But instead what we’re stuck with, through the intricacies of the way our field has grown and our legacy, is closed-system assays. While these probably do measure PD-L1, we do not know how these compare to each other.” Two parallel large multi-institutional studies are addressing this issue now, he says.

There are solutions for managing these closed-system assays to be sure the assay is working in your lab and that you can get the right answer, Dr. Rimm says. His laboratory uses a closed-system assay for PD-L1, relying not on the defined system but rather on a test system it has developed in doing a study with different investigators.

Sample runs by these different investigators show the potentially high variability, he says. “In a scan of results, no one would deny which spots are the positive spots and which are the negative.” But the difference in staining prevents accurate measurement of these things and shows the variability inherent even in a closed-box system.

A comparison of two closed-box systems, the SP1 run on the Discovery Ultra on Ventana, and the SP1, same antibody, run on the Dako closed-box system, also shows that, in fact, there’s not 100 percent agreement using same-day, same-FDA-cleared antibody staining and different autostainers. So automation may not solve the problem, Dr. Rimm notes (Fig. 6).


“When running these in a quantitative fashion and measuring them quantitatively, there are actually differences in the way these closed-box systems run. And so you, as the pathologist, have to be the one who makes sure your assays are correct, your thresholds are correct, and your measurements are accurate.”

The way to do that, he believes, is to use standardization or index arrays. An index array of HER2 that his laboratory developed has 3+ amplified, 2+ amplified, not amplified, and so on from 80 cases in the lab’s archive, shown stained with immunofluorescence and quantitative and DAB stain. “It was only with this standardization array, run every time we ran our stainer, that we were able to draw the conclusions in the previous study about extracellular versus cytoplasmic domain.”

Companies have realized the importance of this, and specifically companies like NantOmics (formerly OncoPlexDx) have realized they can exactly quantitate the amount of tissue on a slide using a specialized mass spectrometry method, he says. “They can actually give you amol/µg of total protein.”

He and colleagues are working with NantOmics now to try to convert from amols to protein to average quantitative fluorescent scores to help build these standards and make standard arrays more accurate. “This is still a work in progress, but I believe this is ultimately the kind of accuracy that can standardize all of our labs. We have shown that the quantitative fluorescence system is truly linear and quantitative for EGFR measurements when using mass spectrometry as a gold standard.” They are preparing to submit a manuscript with this data.

In the interim, Dr. Rimm’s laboratory has begun working also with Horizon Diagnostics, employing Horizon’s experimental 15-spot positive-control array. “When you use this array and quantitate it with quantitative fluorescence, you get a very interesting profile. If a cut point is set at one point, you would see three clearly positive cells or spots and 12 clearly negative spots with two different antibodies. But is that the threshold?”

“In fact, using a little higher score and a very quantitative test, you might find that the threshold may, in fact, be a little bit lower than that.” It turns out that only three of these 12 spots are true negatives. The others at least have some level of RNA, and some have a lot. “So how do we handle these? And are these behaving the same way with multiple antibodies?” Parallel results, finding nearly the same threshold case, have been found using SP142 from Ventana, E1L3N from Cell Signaling, and SP263 from Ventana.

Studies to address those issues are still in the early stage, he says. He cautions that there is variance in these assays, and more work is being done to reproduce the data. “But I think the important point is that, using these kinds of arrays, you can definitively determine whether your lab has the same cut point as every other lab. And were we to quantitate this with mass spectrometry, we would know exactly the break point for use in the future.”

Dr. Rimm’s laboratory has also built its own PD-L1 index tissue microarray with a number of its own tumor slides ranging from very low to very high expressors, a series of cell lines, and including some placenta-positive controls on normal tumor. He has found that generating an index array has advantages, and he encourages other laboratories to prepare their own index arrays to increase the accuracy and reproducibility of their laboratory-developed tests. “You can produce these in your own lab so that you can be sure you can standardize your tests run in your clinical lab from day to day and week to week as part of an LDT.”

“If we think about it, there really are no clinical antibodies today that are truly quantitative,” Dr. Rimm says. “And when there are, new protocols will be required, but I believe those protocols are now in existence. We just await the clinical trials that require truly quantitative protein measurement or in situ proteomics.”

In that process of moving toward in situ proteomics, suggests web-inar co-presenter Clive Taylor, MD, DPhil, professor of pathology in the Keck School of Medicine at the University of Southern California, FDA approval, per se, will not solve any of the problems discussed in the webinar. (See the January 2016 issue for the full report of Dr. Taylor’s presentation.) “I think what the FDA approval will do is demand that we find solutions to these problems ourselves. The FDA’s attitude is, to a large degree, dependent on the claim. So if we just use immunohistochemistry as a simple stain, then the FDA classes that as sort of class I, level 1. And we can do that [IHC stain] without having to get preapproval by the FDA.

“On the other hand, if we take something like the well-established HercepTest, where based on the result of that test alone, it’s decided whether or not the patient gets treatment, treatment that’s very expensive and treatment that has benefits and…side effects. That claim is, in fact, a very high-level claim. And for that, the FDA is demanding high-level data, which I think is entirely appropriate,” Dr. Taylor says.

Most of these upcoming companion diagnostics, if not all, he says, will be regarded by the FDA as class III, high level or high complexity. They will require a premarket approval study in conjunction with a clinical trial. And the FDA will demand high standards of control and performance, eventually. “There are not many labs that can produce those high standards as in-house or lab-developed tests today. And even the companies currently in trials are not producing the improved performance level for these tests that we are talking about today, as being required for high-quality quantitative and reproducible companion diagnostics. Eventually, I am convinced we will have to do that. It’s just that it will take time to get there.”

The FDA can only approve what is brought to it, Dr. Rimm points out. And so a true, fully quantitative IHC-based assay has presumably never been submitted, or at least never been approved by the FDA. “What we’re seeing instead are the assays that the FDA has approved, which are well defined and rigorously submitted. However, the result is a closed system that we use, which may or may not accurately measure PD-L1 on the slide, depending upon preanalytic variables and individual laboratories’ methods.”

“So questions keep popping up. And I can only say that we, as pathologists, have the final responsibility to our patients. And while it may not be recommended and it may change in the future, right now lab-derived tests or LDTs may be more accurate than FDA-approved platforms.”

“If you think about it, in molecular diagnostics where I’m familiar with EFGR and BRAF and KRAS tests, in that testing setting, less than 25 percent of the labs that do that test actually use the FDA-approved test,” Dr. Rimm says. “The remainder of the labs do their own LDTs, including our labs here at Yale.”

It wouldn’t surprise him if the same thing happens for PD-L1. “I’m aware of at least two labs—and we probably will be the third—that devise our own LDT for PD-L1 testing using the standards I’ve discussed, using array-type controls to be sure that our levels are correct, and then using a scoring system that we derived.”

“We aren’t really in a position to know at the time that we receive a piece of lung cancer tissue whether the oncologist is going to use pembrolizumab, which requires a companion diagnostic, or nivolumab, or the other drugs, which may or may not require a companion diagnostic. So in that sense, we’re almost bound to use an LDT,” Dr. Rimm says, since his lab can’t actually run four different potentially incongruent, though FDA-approved, tests for PD-L1.

Until a truly quantitative approach is developed and submitted to the FDA and approved, Dr. Taylor believes we won’t see things changing. “The algorithms that currently are approved have been approved on the basis that they can produce a similar result to a consensus group of pathologists. So they’re only as good as the pathologists.”

“As Dr. Rimm has discussed, I actually believe we can get a much better result than the pathologists can get with their naked eye. We have to get away from comparing it to what we currently can do and start to try to construct a proper test, just like we did in the clinical lab 30 years ago when we automated the clinical lab,” Dr. Taylor says. “We need to automate anatomic pathology, including the sample preparation, the assay process, and the reading, all three together in a closed system. And we’re nibbling away at the edges of it. We’ll get there, but it’ll take some time.”

Dr. Rimm is skeptical that the diagnostics field has learned any lessons from HercepTest and the companion diagnostics world of almost 20 years ago. “The submissions to the FDA for PD-L1 look very similar to what was submitted in 1998 for the HercepTest, the companion diagnostic test for trastuzumab [Herceptin]. And that’s disappointing. I think that is 20-year-old technology and we can do better. But even if we want to use the 20- or 40-year-old DAB-based technology, we should still be standardizing it and having a mechanism for standardization and having defined thresholds.”

As future FDA submissions come in, Dr. Rimm hopes that “even if they’re not quantitated, they can be standardized as to where the thresholds occur, so that we can be sure we deliver the best possible care to patients. And in the interim, I think we, as pathologists, will have to do that standardization with an LDT to be sure we’re giving our best results.”

Dr. Taylor warns that there is only a limited number of labs in the country and in the world that will be able to produce these LDTs, because of the complexity. “The FDA has already said in a position paper that it believes it may have to regulate LDTs to some extent. And what that will mean is that in the validation process, your own LDT will start to approach what is required for an FDA-approved test. And most labs are in no position to be able to do that.”

“So I think we’re going to come to a blending here, all forced by companion diagnostics. This is in situ proteomics,” Dr. Taylor says. “It’s a new test, essentially. It’s not straightforward immunohistochemistry, but a new test. And I think the fluorescence approach that Dr. Rimm has used has a lot of advantages in relating signal to target in terms of figure out what the best test is and stop comparing it to the pathologists. We should compare it to the best assay we can produce.”

With respect to the PD-L1 problem, Dr. Rimm notes, “I would point out that there is a so-called ‘Blueprint’ for comparison of the different antibodies and the different FDA assays, or potentially FDA-submitted tests anyway, to see how equivalent they are.” Similarly, he adds, the National Comprehensive Cancer Network recently issued a press release describing a multi-institutional study to assess the FDA-approved assay but also including an LDT (the Cell Signaling antibody E1L3N using the Leica Bond staining platform).

He points to a newly published study by his group (McLaughlin J, et al. JAMA Oncol. 2016;2[1]:46–54), finding that objective determination of PD-L1 protein levels in non-small cell lung cancer reveals heterogeneity within tumors and prominent interassay variability or discordance. The authors concluded that future studies measuring PD-L1 quantitatively in patients treated with anti-PD-1 and anti PD-L1 therapies may better address the prognostic or predictive value of these biomarkers. With future rigorous studies, including tissues with known responses to anti-PD-1 and anti-PD-L1 therapies, researchers could determine the optimal assay, PD-L1 antibody, and the best cut point for PD-L1 positivity.

Other work that will probably come out in mid-2016 from Dr. Rimm’s group has shown that expression of PD-L1 is largely bimodal, he says. “That is, there’s a group of patients that express a lot, and then there’s another group of patients that expresses a little or none.”

So time will tell how PD-L1 will be scored. “But if you look at the data from the Merck study and their cut point of greater than 50 percent, or even the cut point from the AstraZeneca studies of greater than 25 percent, you’re really dichotomizing the population into patients who are truly PD-LI positive from patients who are negative or almost negative.”

“Of course, we don’t want to miss patients in that negative to almost-negative group who will respond,” Dr. Rimm says. “On the other hand, we probably will have fairly good specificity and sensitivity with the assay defined by Merck and Dako with 22C3 as was recently published” (Robert C, et al. N Engl J Med. 2015;372[26]:2521–2532).

Many difficulties lie ahead, as researchers try to weigh the merits of different drugs with different approved tests on different platforms, involving different antibodies, Dr. Taylor says. “Does the lab try to set up four different PD-L1s, and if we only have one platform and not another, what do we do about that?” He thinks the tests may often be sent out to larger reference labs or academic centers as a result.

Dr. Rimm confirms that his own lab’s LDT—although literally thousands of PD-L1 tests have been conducted using it—is not yet up and running in the Yale CLIA laboratory, and in the meantime the IHC slides are being sent out to a commercial vendor.

Eventually, Dr. Taylor believes, the pressure of these dilemmas will lead the diagnostics field to develop an immunoassay on tissue sections. “We’ve never been forced to do that before, but once we are, that will produce a huge change in diagnostic capability and research capability.”

Anti–PD-1/PD-L1 therapy of human cancer: past, present, and future

Lieping Chen and

The cDNA of programmed cell death 1 (PD-1) was isolated in 1992 from a murine T cell hybridoma and a hematopoietic progenitor cell line undergoing apoptosis (1). Genetic ablation studies showed that deficiencies in PD-1 resulted in different autoimmune phenotypes in various mouse strains (2, 3). PD-1–deficient allogeneic T cells with transgenic T cell receptors exhibited augmented responses to alloantigens, indicating that the PD-1 on T cells plays a negative regulatory role in response to antigen (2).

Several studies contributed to the discovery of the molecules that interact with PD-1. In 1999, the B7 homolog 1 (B7-H1, also called programmed death ligand-1 [PD-L1]) was identified independently from PD-1 using molecular cloning and human expressed-sequence tag database searches based on its homology with B7 family molecules, and it was shown that PD-L1 acts as an inhibitor of human T cell responses in vitro (4). These two independent lines of study merged one year later when Freeman, Wood, and Honjo’s laboratories showed that PD-L1 is a binding and functional partner of PD-1 (5). Next, it was determined that PD-L1–deficient mice (Pdl1 KO mice) were prone to autoimmune diseases, although this strain of mice did not spontaneously develop such diseases (6). It became clear later that the PD-L1/PD-1 interaction plays a dominant role in the suppression of T cell responses in vivo, especially in the tumor microenvironment (7, 8).

In addition to PD-L1, another PD-1 ligand called B7-DC (also known as PD-L2) was also identified by the laboratories of Pardoll (9) and Freeman (10). This PD-1 ligand was found to be selectively expressed on DCs and delivered its suppressive signal by binding PD-1. Mutagenesis studies of PD-L1 and PD-L2 molecules guided by molecular modeling revealed that both PD-L1 and PD-L2 could interact with other molecules in addition to PD-1 and suggested that these interactions had distinct functions (11). The functional predictions from these mutagenesis studies were later confirmed when PD-L1 was found to interact with CD80 on activated T cells to mediate an inhibitory signal (12, 13). This finding came as a surprise because CD80 had been previously identified as a functional ligand for CD28 and cytotoxic T lymphocyte antigen-4 (CTLA-4) (14, 15). PD-L2 was also found to interact with repulsive guidance molecule family member b (RGMb), a molecule that is highly enriched in lung macrophages and may be required for induction of respiratory tolerance (16). With at least five interacting molecules in the PD-1/PD-L1 pathway (referred to as the PD pathway) (Figure 1), further studies will be required to understand the relative contributions of these molecules during activation or suppression of T cells.

The PD pathway. The PD pathway has at least 5 interacting molecules. PD-...

The PD pathway.

The PD pathway has at least 5 interacting molecules. PD-L1 and PD-L2, with different expression patterns, were identified as ligands of PD-1, and the interaction of PD-L1 or PD-L2 with PD-1 may induce T cell suppression. PD-L1 was found to interact with B7-1 (CD80) on activated T cells and inhibit T cell activity. PD-L2 has a second receptor, RGMb; initially, this interaction activates T cells, but it subsequently induces respiratory tolerance. PD-L1 on tumor cells can also act as a receptor, and the signal delivered from PD-1 on T cells can protect tumor cells from cytotoxic lysis.

The discovery of the PD pathway did not automatically justify its application to cancer therapy, especially after the initial PD-1–deficient mouse studies, which suggested that PD-1 deficiency increases the incidence of autoimmune diseases (2, 3). In our initial work to characterize PD-L1 and its function, PDL1 mRNA was found to be broadly expressed in various tissues (17). However, normal human tissues seldom express PD-L1 protein on their cell surface, with the exception of tonsil (17), placenta (18), and a small fraction of macrophage-like cells in lung and liver (17), suggesting that, under normal physiological conditions, PDL1 mRNA is under tight posttranscriptional regulation. In sharp contrast, PD-L1 protein is abundantly expressed on the cell surface in various human cancers, as indicated by immunohistochemistry in frozen human tumor sections. Additionally, the pattern of PD-L1 expression was found to be focal rather than diffuse in most human cancers (17). In fact, the majority of in vitro–cultured tumor lines of both human and mouse origin are PD-L1–negative on the cell surface, despite overwhelming PD-L1 signal in specimens that are freshly isolated from patients with cancer (17, 19). This discrepancy was explained by the finding that IFN-γ upregulates PD-L1 on the cell surface of normal tissues and in various tumor lines (7, 17, 19). It was widely thought that IFN-γ typically promotes, rather than suppresses, T cell responses by stimulating antigen processing and presentation machinery (20, 21); therefore, the role of IFN-γ in downregulating immune responses in the tumor microenvironment via induction of PD-L1 was not well accepted until more recently. This finding is vital to our current understanding of the unique immunology that takes place in the tumor microenvironment and provided an important clue that led to the “adaptive resistance” hypothesis (see below) that explains this pathway’s mechanism of action to evade tumor immunity.

Due to the lack of cell surface expression of PD-L1 on most cultured tumor lines, it is necessary to reexpress PD-L1 on the surface using transfection to recapitulate the effects of cell surface PD-L1 in human cancers and to create models to study how tumor-associated PD-L1 interacts with immune cells. We now know that cancer cells and other cells in the tumor microenvironment can upregulate the expression of PD-L1 after encountering T cells, mostly via IFN-γ, which may make the transfection-mediated expression of PD-L1 unnecessary in some tumor models. Nevertheless, our results demonstrated that PD-L1+ human tumor cells could eliminate activated effector T cells (Teffs) via apoptosis in coculture systems, and this effect could be blocked by inclusion of an anti-human PD-L1 mAb (clone 2H1). Next, we generated a hamster mAb (clone 10B5) against mouse PD-L1 to block its interaction with T cells and test its role in tumor immunity in vitro and in vivo. We demonstrated that progressive growth of PD-L1+ murine P815 tumors in syngeneic mice could be suppressed using anti–PD-L1 mAb (17). Altogether, these studies represented the initial attempt at using mAb to block the PD pathway as an approach for cancer therapy. These proof-of-concept studies (17) were confirmed by several subsequent studies. A study from Nagahiro Minato’s laboratory showed that the J558L mouse myeloma line constitutively expressed high levels of cell surface PD-L1 and the growth of these cells in syngeneic BALB/c mice could be partially suppressed by administering anti–PD-L1 mAb (22). Our laboratory showed that regression of progressively growing squamous cell carcinomas in syngeneic mice could also be suppressed using a combination of adoptively transferred tumor-draining lymphocytes and anti–PD-L1 mAb (23). Furthermore, the Zou laboratory demonstrated that ovarian cancer–infiltrating human T cells could be activated in vitro using DCs, which showed enhanced activity in the presence of anti–PD-L1 mAb; upon transfer, these cells could eliminate established human ovarian cancers in immune-deficient mice (24). These early studies established the concept that the PD pathway could be used by tumors to escape immune attack in the tumor microenvironment. More importantly, these studies built a solid foundation for the development of anti-PD therapy for the treatment of human cancers.  …..

Anti-PD therapy has taken center stage in immunotherapies for human cancer, especially for solid tumors. This therapy is distinct from the prior immune therapeutic agents, which primarily boost systemic immune responses or generate de novo immunity against cancer; instead, anti-PD therapy modulates immune responses at the tumor site, targets tumor-induced immune defects, and repairs ongoing immune responses. While the clinical success of anti-PD therapy for the treatment of a variety of human cancers has validated this approach, we are still learning from this pathway and the associated immune responses, which will aid in the discovery and design of new clinically applicable approaches in cancer immunotherapy.


PD-1 Pathway Inhibitors: Changing the Landscape of Cancer Immunotherapy

Dawn E. Dolan, PharmD, and Shilpa Gupta, MD

Background: Immunotherapeutic approaches to treating cancer have been evaluated during the last few decades with limited success. An understanding of the checkpoint signaling pathway involving the programmed death 1 (PD-1) receptor and its ligands (PD-L1/2) has clarified the role of these approaches in tumor-induced immune suppression and has been a critical advancement in immunotherapeutic drug development. Methods: A comprehensive literature review was performed to identify the available data on checkpoint inhibitors, with a focus on anti–PD-1 and anti–PD-L1 agents being tested in oncology. The search included Medline, PubMed, the registry, and abstracts from the American Society of Clinical Oncology meetings through April 2014. The effectiveness and safety of the available anti–PD-1 and anti–PD-L1 drugs are reviewed. Results: Tumors that express PD-L1 can often be aggressive and carry a poor prognosis. The anti–PD-1 and anti–PD-L1 agents have a good safety profile and have resulted in durable responses in a variety of cancers, including melanoma, kidney cancer, and lung cancer, even after stopping treatment. The scope of these agents is being evaluated in various other solid tumors and hematological malignancies, alone or in combination with other therapies, including other checkpoint inhibitors and targeted therapies, as well as cytotoxic chemotherapy. Conclusions: The PD-1/PD-L1 pathway in cancer is implicated in tumors escaping immune destruction and is a promising therapeutic target. The development of anti–PD-1 and anti–PD-L1 agents marks a new era in the treatment of cancer with immunotherapies. Early clinical experience has shown encouraging activity of these agents in a variety of tumors, and further results are eagerly awaited from completed and ongoing studies.


Role of PD-1/PD-L1 Pathway PD-1 is an immunoinhibitory receptor that belongs to the CD28 family and is expressed on T cells, B cells, monocytes, natural killer cells, and many tumor-infiltrating lymphocytes (TILs)10; it has 2 ligands that have been described (PD-L1 [B7H1] and PD-L2 [B7-DC]).11 Although PD-L1 is expressed on resting T cells, B cells, dendritic cells, macrophages, vascular endothelial cells, and pancreatic islet cells, PD-L2 expression is seen on macrophages and dendritic cells alone.10 Certain tumors have a higher expression of PD-L1.12 PD-L1 and L2 inhibit T-cell proliferation, cytokine production, and cell adhesion.13 PD-L2 controls immune T-cell activation in lymphoid organs, whereas PD-L1 appears to dampen T-cell function in peripheral tissues.14 PD-1 induction on activated T cells occurs in response to PD-L1 or L2 engagement and limits effector T-cell activity in peripheral organs and tissues during inflammation, thus preventing autoimmunity. This is a crucial step to protect against tissue damage when the immune system is activated in response to infection.15-17 Blocking this pathway in cancer can augment the antitumor immune response.18 Like the CTLA-4, the PD-1 pathway down-modulates Tcell responses by regulating overlapping signaling proteins that are part of the immune checkpoint pathway; however, they function slightly differently.14,16 Although the CTLA-4 focuses on regulating the activation of T cells, PD-1 regulates effector T-cell activity in peripheral tissues in response to infection or tumor progression.16 High levels of CTLA-4 and PD-1 are expressed on regulatory T cells and these regulatory T cells and have been shown to have immune inhibitory activity; thus, they are important for maintaining self-tolerance.16 The role of the PD-1 pathway in the interaction of tumor cells with the host immune response and the PD-L1 tumor cell expression may provide the basis for enhancing immune response through a blockade of this pathway.16 Drugs targeting the PD-1 pathway may provide antitumor immunity, especially in PD-L1 positive tumors. Various cancers, such as melanoma, hepatocellular carcinoma, glioblastoma, lung, kidney, breast, ovarian, pancreatic, and esophageal cancers, as well as hematological malignancies, have positive PD-L1 expression, and this expression has been correlated with poor prognosis.8,19 Melanoma and kidney cancer are prototypes of immunogenic tumors that have historically been known to respond to immunotherapeutic approaches with interferon alfa and interleukin 2. The CTLA-4 antibody ipilimumab is approved by the US Food and Drug Administration for use in melanoma. Clinical activity of drugs blocking the PD-1/PD-L1 pathway has been demonstrated in melanoma and kidney cancer.20-24 In patients with kidney cancer, tumor, TIL-associated PD-L1 expression, or both were associated with a 4.5-fold increased risk of mortality and lower cancer-specific survival rate, even after adjusting for stage, grade, and performance status.18,19,25,26 A correlation between PD-L1 expression and tumor growth has been described in patients with melanoma, providing the rationale for using drugs that block the PD-1/PD-L1 pathway.19,27 Historically, immunotherapy has been ineffective in cases of non–small-cell lung cancer (NSCLC), which has been thought to be a type of nonimmunogenic cancer; nevertheless, lung cancer can evade the immune system through various complex mechanisms.28 In patients with advanced lung cancer, the peripheral and tumor lymphocyte counts are decreased, while levels of regulatory T cells (CD4+), which help suppress tumor immune surveillance, have been found at higher levels.29-32 Immune checkpoint pathways involving the CTLA-4 or the PD-1/PD-L1 are involved in regulating T-cell responses, providing the rationale for blocking this pathway in NSCLC with antibodies against CTLA-4 and the PD-1/PD-L1 pathway.32 Triple negative breast cancer (TNBC) is an aggressive subset of breast cancer with limited treatment options. PD-L1 expression has been reported in patients with TNBC. When PD-L1 expression was evaluated in TILs, it correlated with higher grade and larger-sized tumors.33 Tumor PD-L1 expression also correlates with the infiltration of T-regulatory cells in TNBC, findings that suggest the role of PD-L1–expressing tumors and the PD-1/PD-L1–expressing TILs in regulating immune response in TNBC.34


Preclinical evidence exists for the complementary roles of CTLA-4 and PD-1 in regulating adaptive immunity, and this provides rationale for combining drugs targeting these pathways.44-46 Paradoxically and originally believed to be immunosuppressive, new data allow us to recognize that cytotoxic agents can antagonize immunosuppression in the tumor microenvironment, thus promoting immunity based on the concept that tumor cells die in multiple ways and that some forms of apoptosis may lead to an enhanced immune response.8,15 For example, nivolumab was combined with ipilimumab in a phase 1 trial of patients with advanced melanoma.46 The combination had a manageable safety profile and produced clinical activity in the majority of patients, with rapid and deep tumor regression seen in a large proportion of patients. Based on the results of this study, a phase 3 study is being undertaken to evaluate whether this combination is better than nivolumab alone in melanoma (NCT01844505). Several other early-phase studies are underway to explore combinations of various anti–PD-1/PD-L1 drugs with other therapies across a variety of tumor types (see Tables 1 and 2), possibly paving the way for future combination studies.


Development of PD-1/PD-L1 Pathway in Tumor Immune Microenvironment and Treatment for Non-Small Cell Lung Cancer

Jiabei He, Ying Hu, Mingming Hu & Baolan Li

Lung cancer is currently the leading cause of cancer-related death in worldwide, non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. Surgery, platinum-based chemotherapy, molecular targeted agents and radiotherapy are the main treatment of NSCLC. With the strategies of treatment constantly improving, the prognosis of NSCLC patients is not as good as before, new sort of treatments are needed to be exploited. Programmed death 1 (PD-1) and its ligand PD-L1 play a key role in tumor immune escape and the formation of tumor microenvironment, closely related with tumor generation and development. Blockading the PD-1/PD-L1 pathway could reverse the tumor microenvironment and enhance the endogenous antitumor immune responses. Utilizing the PD-1 and/or PD-L1 inhibitors has shown benefits in clinical trials of NSCLC. In this review, we discuss the basic principle of PD-1/PD-L1 pathway and its role in the tumorigenesis and development of NSCLC. The clinical development of PD-1/PD-L1 pathway inhibitors and the main problems in the present studies and the research direction in the future will also be discussed.

Lung cancer is currently the leading cause of cancer-related death in the worldwide. In China, the incidence and mortality of lung cancer is 5.357/10000, 4.557/10000 respectively, with nearly 600,000 new cases every year1. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, the early symptoms of patients with NSCLC are not very obvious, especially the peripheral lung cancer. Though the development of clinic diagnostic techniques, the majority of patients with NSCLC have been at advanced stage already as they are diagnosed. Surgery is the standard treatment in the early stages of NSCLC, for the advanced NSCLC, the first-line therapy is platinum-based chemotherapy. In recent years, patients with specific mutations may effectively be treated with molecular targeted agents initially. The prognosis of NSCLC patients is still not optimistic even though the projects of chemotherapy as well as radiotherapy are continuously ameliorating and the launch of new molecular targeted agents is never suspended, the five-year survival rate of NSCLC patients is barely more than 15%2, the new treatment is needed to be opened up.

During the last few decades, significant efforts of the interaction between immune system and immunotherapy to NSCLC have been acquired. Recent data have indicated that the lack of immunologic control is recognized as a hallmark of cancer currently. Programmed death-1 (PD-1) and its ligand PD-L1 play a key role in tumor immune escape and the formation of tumor microenvironment, closely related with tumor generation and development. Blockading the PD-1/PD-L1 pathway could reverse the tumor microenvironment and enhance the endogenous antitumor immune responses.

In this review, we will discuss the PD-1/PD-L1 pathway from the following aspects: the basic principle of PD-1/PD-L1 pathway and its role in the tumorigenesis and development of NSCLC, the clinical development of several anti-PD-1 and anti-PD-L1 drugs, including efficacy, toxicity, and application as single agent, or in combination with other therapies, the main problems in the present studies and the research direction in the future.


Cancer as a chronic, polygene and often inflammation-provoking disease, the mechanism of its emergence and progression is very complicated. There are many factors which impacted the development of the disease, such as: environmental factors, living habits, genetic mutations, dysfunction of the immune system and so on. At present, increasing evidence has revealed that the development and progression of tumor are accompanied by the formation of special tumor immune microenvironment. Tumor cells can escape the immune surveillance and disrupt immune checkpoint of host in several methods, therefore, to avoid the elimination from the host immune system. Human cancers contain a number of genetic and epigenetic changes, which can produce neoantigens that are potentially recognizable by the immune system3, thus trigger the body’s T cells immune response. The T cells of immune system recognize cancer cells as abnormal primarily, generate a population of cytotoxic T lymphocytes (CTLs) that can traffic to and infiltrate cancers wherever they reside, and specifically bind to and then kill cancer cells. Effective protective immunity against cancer depends on the coordination of CTLs4. Under normal physiological conditions, there is a balance status in the immune checkpoint molecule which makes the immune response of T cells keep a proper intensity and scope in order to minimize the damage to the surrounding normal tissue and avoid autoimmune reaction. However, numerous pathways are utilized by cancers to up-regulate the negative signals through cell surface molecules, thus inhibit T-cell activation or induce apoptosis and promote the progression and metastasis of cancers5. Increasing experiments and clinical trails show that immunotherapeutic approaches utilizing antagonistic antibodies to block checkpoint pathways, can release cancer inhibition and facilitate antitumor activity, so as to achieve the purpose of treating cancer.

The present research of immune checkpoint molecules are mainly focus on cytotoxic T lymphocyte-associated antigen 4 (CLTA-4), Programmed death-1 (PD-1) and its ligands PD-L1 (B7H1) and PD-L2 (B7-DC). CTLA-4 regulates T cell activity in the early stage predominantly, and PD-1 mainly limits the activity of T-cell in the tumor microenvironment at later stage of tumor growth6. Utilizing the immune checkpoint blockers to block the interactions between PD-1 and its ligands has shown benefits in clinical trials, including the NSCLC patients. PD-1 and its ligands have been rapidly established as the currently most important breakthrough targets in the development of effective immunotherapy.

PD-1/PD-L1 pathway and its expression, regulation

PD-1 is a type 1 trans-membrane protein that encoded by the PDCD1 gene7. It is a member of the extended CD28/CTLA-4 immunoglobulin family and one of the most important inhibitory co-receptors expressed by T cells. The structure of the PD-1 includes an extracellular IgV domain, a hydrophobic trans-membrane region and an intracellular domain. The intracellular tail includes separate potential phosphorylation sites that are located in the immune receptor tyrosine-based inhibitory motif (ITIM) and in the immunoreceptor tyrosine-based switch motif (ITSM). Mutagenetic researches indicated that the activated ITSM is essential for the PD-1 inhibitory effect on T cells8. PD-1 is expressed on T cells, B cells, monocytes, natural killer cells, dendritic cells and many tumor-infiltrating lymphocytes (TILs)9. In addition, the research of Francisoet et al. showed that PD-1 was also expressed on regulatory T cells (Treg) and able to facilitate the proliferation of Treg and restrain immune response10.

PD-1 has two ligands: PD-L1 (also named B7-H1; CD274) and PD-L2 (B7-DC; CD273), that are both coinhibitory. PD-L1 is expressed on resting T cells, B cells, dendritic cells, macrophage, vascular endothelial cells and pancreatic islet cells. PD-L2 expression is seen on macrophages and dendritic cells alone and is far less prevalent than PD-L1 across tumor types. It shows much more restricted expression because of its more restricted tissue distribution. Differences in expression patterns suggest distinct functions in immune regulation across distinct cell types. The restricted expression of PD-L2, largely to antigen-presenting cells, is consistent with a role in regulating T-cell priming or polarization, whereas broad distribution of PD-L1 suggests a more general role in protecting peripheral tissues from excessive inflammation.

PD-L1 is expressed in various types of cancers, especially in NSCLC11,12, melanoma, renal cell carcinoma, gastric cancer, hepatocellular as well as cutaneous and various leukemias, multiple myeloma and so on13,14,15. It is present in the cytoplasm and plasma membrane of cancer cells, but not all cancers or all cells within a cancer express PD-L116,17. The expression of PD-L1 is induced by multiple proinflammatory molecules, including types I and II IFN-γ, TNF-α, LPS, GM-CSF and VEGF, as well as the cytokines IL-10 and IL-4, with IFN-γ being the most potent inducer18,19. IFN-γ and TNF-α are produced by activated type 1 T cells, and GM-CSF and VEGF are produced by a variety of cancer stromal cells, the tumor microenvironment upregulates PD-L1 expression, thereby, promotes immune suppression. This latter effect is called “adaptive immune resistance”, because the tumor protects itself by inducing PD-L1 in response to IFN-γ produced by activated T cells17. PD-L1 is regulated by oncogenes, also known as the inherent immune resistance. PD-L1 expression is suppressed by the tumor suppressor gene: PTEN (phosphatase and tension homolog deleted on chromosome ten) gene. Cancer cells frequently contain mutated PTEN, which can activate the S6K1 gene, thus results in PD-L1 mRNA to polysomes increase greatly20, hence increases the translation of PD-L1 mRNA and plasma membrane expression of PD-L1. Parsa et al.’s research also demonstrated that neuroglioma with PTEN gene deletion regulate PD-L1 expression at the translational level by activating the PI3K/AKT downstream mTOR-S6K1signal pathway and, hence increase the PD-L1 expression21. Micro-RNAs also translationally regulate PD-L1 expression. MiRNA-513 is complementary to the 3′ untranslated region of PD-L1 and prevents PD-L1 mRNA translation22. In addition, a later literature reported that in the model of melanoma, the up-regulation of PD-L1 is closely related to the CD8 T cell, independent of regulation by oncogenes13. Noteworthily, the PD-L1 can bind to T cell expressed CD80, and at this point CD80 is a receptor instead of ligand to transmit negative regulated signals23.


PD-1/PD-L1 mediate immune suppression by multiple mechanisms

Like the CTLA-4, the PD-1/PD-L1 pathway down-modulates T-cell response by regulating overlapping signal proteins in the immune checkpoint pathway. However, their functions are slightly different24. The CTLA-4 focuses on regulating the activation of T cells, while PD-1 regulates effector T-cell activity in peripheral tissues in response to infection or tumor progression25. Tregs that high-level expression of PD-1 have been shown to have immune inhibitory activity, thus, they are important for maintaining self-tolerance. In normal human bodies, this is a crucial step to protect against tissue damage when the immune system is activated in response to infection26. However, in response to immune attack, cancer cells overexpress PD-L1 and PD-L2. They bind to PD-1 receptor on T cells, inhibiting the activation of T-cells, thus suppressing T-cell attack and inducing tumor immune escape. Thus tumor cells effectively form a suitable tumor microenvironment and continue to proliferate27. PD-1/PD-L1 pathway regulates immune suppression by multiple mechanisms, specific performance of the following: Induce apoptosis of activated T cells: PD-1 reduces T cell survival by impacting apoptotic genes. During T cell activation, CD28 ligation sustains T cell survival by driving expression of the antiapoptotic gene Bcl-xL. PD-1 prevents Bcl-xL expression by inhibiting PI3K activation, which is essential for upregulation of Bcl-xL. Early studies demonstrated that PD-L1+ murine and human tumor cells induce apoptosis of activated T cells and that antibody blocking of PD-L1 can decrease the apoptosis of T cells and facilitate antitumor immunity28,16. Facilitate T cell anergy and exhaustion: A research shown that the occurrence of tumor is associated with chronic infection29. According to the study of chronic infection, PD-1 overexpressed on the function exhausted T cells, blocking the PD-1/PD-L1 pathway can restore the proliferation, secretion and cytotoxicity30. In addition, later research demonstrated that the exhaustion of TILs in the tumor microenvironment is closely related to the PD-L1 expression of tumor cells, myeloid cells derived from tumor31. Enhance the function of regulatory T cells: PD-L1 can promote the generation of induced Tregs by down-regulating the mTOR, AKT, S6 and the phosphorylation of ERK2 and increasing PTEN, thus restrain the activity of effector T-cell32. Blocking the PD-1/PD-L1 pathway can increase the function of effector CD8 T-cell and inhibt the function of Tregs, bone marrow derived inhibition cells, thus enhance the anti-tumor response. Inhibit the proliferation of T cells: PD-1 ligation also prevents phosphorylation of PKC-theta, which is essential for IL-2 production33, and arrests T cells in the G1 phase, blocking proliferation. PD-1 mediates this effect by activating Smad3, a factor that arrests cycling34. Restrain impaired T cell activation and IL-2 production: PD-1/PD-L1 blocks the downstream signaling events triggered by Ag/MHC engagement of the TCR and co-stimulation through CD28, resulting in impaired T cell activation and IL-2 production. Signaling through the TCR requires phosphorylation of the tyrosine kinase ZAP70. PD-1 engagement reduces the phosphorylation of ZAP70 and, hence, inhibits downstream signaling events. In addition, signaling through PD-1 also prevents the conversion of functional CD8+ T effector memory cells into CD8+ central memory cells35 and, thus, reduces long-term immune memory that might protect against future metastatic disease. PD-L1 also promotes tumor progression by reversing signaling through CD80 into T cells. CD80-PD-L1 interactions restrain self-reactive T cells in an autoimmune setting36, therefore, their inhibition may facilitate antitumor immunity.

Researches on the mechanism of PD-1/PD-L1 pathway mediating immune escape are still ongoing, especially the mechanism of PD-L2 is still unclear. These researches provide the theoretical basis and research direction for the further immunotherapy targets research.


Anti-PD-1 antibodies


Nivolumab (BMS-936558, Brand name: Opdivo) is a human monoclonal IgG4 antibody that essentially lacks detectable antibody-dependent cellular cytotoxicity (ADCC). Inhibition by monoclonal antibody of PD-1 on CD8+ TILs within lung cancers can restore cytokine secretion and T-cell proliferation48. Results of a larger phase I study in 296 patients (236 patients evaluated) reported that the objective response (complete or partial responses) of patients with NSCLC was 18%. A total of 65% of responders had durable responses lasting for more than 1 year. Stable disease lasting 24 weeks was seen in patients with NSCLC. PD-L1expression was tested in 42 patients: 9 of 25(36%) patients whose PD-L1 expression positive were objectively response to PD-1 blockade treatment, while the remaining 17 nonresponsive patients were negative45.

In another early phase I trial of nivolumab49, an objective response was observed in 22 patients (17%; 95% CI, 11%–25%) in a dose-expansion cohort of 129 previously treated patients with advanced NSCLC. Six additional patients who had an unconventional immune-related response were not included. Moreover, the median duration of response was exceptional for 17 months. Although the median PFS in the cohort was 2.3 months and the median overall survival was 9.9 months, it seemed clear that those who responded had sustained benefit. Specifically, the 2-year overall survival rate was 24%, and many remained in remission after completing 96 weeks of continuous therapy.

Single-agent trials of nivolumab are planning or ongoing on NSCLC (NCT01721759, NCT02066636). In addition, there are clinical randomized trials which focus on the comparison of nivolumab and plain-based combination chemotherapy (NCT02041533, NCT01673867). In March 4, 2015, nivolumab was approved by the US Food and Drug Administration for treatment of patients with metastatic NSCLC (squamous cell carcinoma), when progression of their diseases during or after chemotherapy with platinum-based drugs.


Pembrolizumab (MK-3475) is a highly selective, humanized monoclonal antibody with activity against PD-1 that contains a mutation at C228P designed to prevent Fc-mediated ADCC. It is now in the clinical research phases for patients with advanced solid tumors. Its safety and efficacy were evaluated in a phase I clinical trial of KEYNOTE-001. The best response according of 38 cases of patients which initially accepted pembrolizumab 10 mg/kg q3wwas 21% (based on RECIST1.1 evaluation) and the median PFS of responder still has not reached until 62 weeks. The researchers also found that the antitumor activity of pembrolizumab was associated with the PD-L1expression44,50. The critical values of the expression of PD-L1 will be validated in 300 cases of patients which will soon been rolled into the study.

Clinical trial of pembrolizumab monotherapy is ongoing for patients with NSCLC (NCT01840579). Randomized trials comparing pembrolizumab to combination chemotherapy (NCT02142738) or single-agent docetaxel (NCT01905657) are ongoing in PD-L1 positive patients with NSCLC.

Pidilizumab (CT-011)

Pidilizumab is a humanized IgG-1K recombinant anti-PD-1 monoclonal antibody that has demonstrated antitumor activity in mouse cancer models. In a first-in-human phase I dose-escalation study in patients with only advanced hematologic cancers, there is no clinical trials of NSCLC presently51.


Anti-PD-L1 antibodies

Another therapeutic method based on the PD-1/PD-L1 pathway is by specific binding between antibody and PD-L1, thus preventing its activity. It was speculated that utilizing PD-L1 as therapeutic target maybe accompanied by less toxicity in part by modulating the immune response selectively in the tumor microenvironment. However, since PD-L2 expressed by tumor cells or some other tumor-associated molecules may play a role in mediating PD-1-expressing lymphocytes, it is conceivable that the magnitude of the anti-tumor immune response could also be blunted.


BMS-936559/MDX1105 is a fully humanized, high affinity, IgG4 monoclonal antibody that react specifically with PD-L1, thus inhibiting the binding of PD-L1 and PD-1, CD80 (which binds not only PD-L1 but also CTLA-4 and CD28). Initial results from a multicenter and dose-escalation phase I trial of 207 patients(including 75 cases of patients with NSCLC) showed durable tumor regression (objective response rate of 6%–17%) and prolonged stabilization of disease (12%–41% at 24weeks) in patients with advanced cancers, including NSCLC, melanoma and kidney cancer. In patients with NSCLC, there were five objective responses (in 4 patients with the nonsquamous subtype and 1 with the squamous subtype) at doses of 3 mg/kg and 10 mg/kg, with response rates of 8% and 16%, respectively. Six additional patients with NSCLC had stable disease lasting at least 24 weeks52.


MPDL3280A is a human IgG1 antibody that targets PD-L1. Its Fc component has been engineered to not activate antibody-dependent cell cytotoxicity. In a recently reported phase I study, a 21% response rate was noted in patients with metastatic melanoma, RCC or NSCLC53, including several patients who demonstrated shrinkage of tumor within a few days of initiating treatment.

Fifty-two patients were enrolled in an expansion cohort of the phase I trial of MPDL3280A, 62% of them were heavily pretreated NSCLC (≥3 lines of systemic therapy) and the ORR was 22%54. Analysis of biomarker data from archival tumor samples demonstrated a correlation between PD-L1 status and response and lack of progressive disease55.


MEDI4736 is a human IgG1 antibody that binds specifically to PD-L1, thus preventing PD-L1 binding to PD-1 and CD80. Interim results from a phase I trial reported no colitis or pneumonitis of any grade, with several durable remissions, including NSCLC patients56. An ongoing phase I dose-escalation study (NCT01693562) of MEDI-4736 in 26 patients, 4 partial responses (3 in patients with NSCLC and 1 with melanoma) were observed and 5 additional patients exhibited lesser degrees of tumor shrinkage. The disease control rate at 12 weeks was 46%57. Expansion cohorts was opened in Sep 2013, 10 mg/kg q2w dose. 151 patients was enrolled so far in the expansion cohorts, tumor shrinkage was reported as early as the first assessment at 6 weeks and among the 13 patients with NSCLC, responses were sustained at 10 or more to 14.9 or more months58. In the NSCLC expansion cohort, the response rate was 16% in 58 evaluable patients and the disease control rate at 12 weeks was 35% with responses seen in all histologic subtypes as well as in a smaller proportion of PD-L1- tumors.

On the basis of the favorable toxicity profile and promising activity in a heavily pretreated NSCLC population, a global Phase III placebo controlled trial using the 10 mg/kg biweekly dose has been launched in Stage III patients who have not progressed following chemo-radiation (NCT02125461). The primary outcome measures are overall survival and progression-free survival.


AMP-224 was a B7-DC-Fc fusion protein which can block the PD-1 receptor competitively59. Some NSCLC patients were included in a first-in-man phase I trial of this fusion protein drug. A dose-dependent reduction in PD-1-high TILs was observed at 4 hours and 2 weeks after drug administration60.


A variety of approaches for combining PD-1/PD-L1 pathway inhibitors with other therapeutic methods have been explored over the past few years in an effort to offer more feasible therapeutic options for clinic to improve treatment outcomes. Approaches have included combinations with other immune checkpoint inhibitors, immunostimulatory cytokines (e.g. IFN-y) cytotoxic chemotherapy, platinum-based chemotherapy, radiotherapy, anti-angiogenic inhibitors, tumor vaccine and small-molecule molecularly targeted therapies many with promising results61,62. Studies indicated that PD-1/PD-L1 pathway inhibitors were most effective when combined with treatments that activating the immune system63.

Preclinical evidence exists for the complementary roles of CTLA-4 and PD-1 in regulating adaptive immunity, and this provides rationale for combining drugs targeting these pathways. In a Phase I study in 46 chemotherapy-naive patients with NSCLC, four cohorts of patients received ipilimumab (3 mg/kg) plus nivolumab for four cycles followed by nivolumab 3 mg/kg intravenously every 2 weeks. The ORR was 22% and did not correlate with PD-L1 status64.

In another Phase I study, 56 patients with advanced NSCLC were assigned based on histology to four cohorts to receive nivolumab (5–10 mg/kg) intravenously every 3 weeks plus one of four concurrent standard “platinum doublet” chemotherapy regimens. No dose de-escalation was required for dose-limiting toxicity. The ORR was 33–50% across arms and the 1-year OS rates were promising at 59–87%65.


The research of cancer immunotherapy provides a new wide space for cancer treatment (including NSCLC), and compared with other therapeutic method, immunotherapy has its unique advantages, such as: relative safety, effectivity, less and low grade side effect and so on. Especially with the discovery and continued in-depth study of PD-1/PD-L1 pathway in the immune regulation mechanism, many significative conclusions were reported. Data from many clinical trails suggest that some patients with NSCLC have been benefited from the drugs of anti-PD-1 and anti-PD-L1 already. However, summarized what have been discussed above, only a small fraction of patients benefit from PD-1 or PD-L1 inhibitors treatment. But with the continuous studies on biomarker and combined treatment in PD-1/PD-L1 pathway, new research progress will be acquired as well. We will make significant progress on treatment and in control of NSCLC.


Prospects for Targeting PD-1 and PD-L1 in Various Tumor Types     

Table 1: Selected Anti–PD-1 and Anti–PD-L1 Antibodies
Table 2: Selected Adverse Events
Table 3: Selected Clinical Trials for Metastatic Melanoma
Table: 4 Selected Trials for Metastatic Renal Cell Carcinoma
Table 5: Selected Trials for Non–Small-Cell Lung Cancer (NSCLC )
Table 6: Selected Trials for Other Tumor Types

Immune checkpoints, such as programmed death ligand 1 (PD-L1) or its receptor, programmed death 1 (PD-1), appear to be Achilles’ heels for multiple tumor types. PD-L1 not only provides immune escape for tumor cells but also turns on the apoptosis switch on activated T cells. Therapies that block this interaction have demonstrated promising clinical activity in several tumor types. In this review, we will discuss the current status of several anti–PD-1 and anti–PD-L1 antibodies in clinical development and their direction for the future.

Several PD-1 and PD-L1 antibodies are in clinical development (Table 1). Overall, they are very well tolerated; most did not reach dose-limiting toxicity in their phase I studies. As listed in Table 2, no clinically significant difference in adverse event profiles has been seen between anti–PD-1 and anti–PD-L1 antibodies. Slightly higher rates of infusion reactions (11%) were observed with BMS-936559 (anti–PD-L1) than with BMS-96558 (nivolumab). In an early stage of a nivolumab phase I study, there was concern about fatal pneumonitis.[7] It has been hypothesized that PD-1 interaction with PD-L2 expressed on the normal parenchymal cells of lung and kidney provides unique negative signaling that prevents autoimmunity.[8] Thus, anti–PD-1 antibody blockage of such an interaction may remove this inhibition, allowing autoimmune pneumonitis or nephritis. Anti–PD-L1 antibody, however, would theoretically leave PD-1–PD-L2 interaction intact, preventing the autoimmunity caused by PD-L2 blockade. With implementation of an algorithm to detect early signs of pneumonitis and other immune-related adverse events, many of these side effects have become manageable. However, it does require discerning clinical attention to detect potentially fatal side effects. In terms of antitumor activity, both anti–PD-1 and anti–PD-L1 antibodies have shown responses in overlapping multiple tumor types. Although limited to a fraction of patients, most responses, when observed, were rapid and durable.

– See more at:


Immune Checkpoint Blockade in Cancer Therapy

Michael A. PostowMargaret K. Callahan and Jedd D. Wolchok

Immunologic checkpoint blockade with antibodies that target cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and the programmed cell death protein 1 pathway (PD-1/PD-L1) have demonstrated promise in a variety of malignancies. Ipilimumab (CTLA-4) and pembrolizumab (PD-1) are approved by the US Food and Drug Administration for the treatment of advanced melanoma, and additional regulatory approvals are expected across the oncologic spectrum for a variety of other agents that target these pathways. Treatment with both CTLA-4 and PD-1/PD-L1 blockade is associated with a unique pattern of adverse events called immune-related adverse events, and occasionally, unusual kinetics of tumor response are seen. Combination approaches involving CTLA-4 and PD-1/PD-L1 blockade are being investigated to determine whether they enhance the efficacy of either approach alone. Principles learned during the development of CTLA-4 and PD-1/PD-L1 approaches will likely be used as new immunologic checkpoint blocking antibodies begin clinical investigation.

CTLA-4 was the first immune checkpoint receptor to be clinically targeted (Fig 1) Normally, after T-cell activation, CTLA-4 is upregulated on the plasma membrane where it functions to downregulate T-cell function through a variety of mechanisms, including preventing costimulation by outcompeting CD28 for its ligand, B7, and also by inducing T-cell cycle arrest.15 Through these mechanisms and others, CTLA-4 has an essential role in maintaining normal immunologic homeostasis, as evidenced by the fact that mice deficient in CTLA-4 die from fatal lymphoproliferation.6,7 Recognizing the role of CTLA-4 as a negative regulator of immunity, investigators led studies demonstrating that antibody blockade of CTLA-4 could result in antitumor immunity in preclinical models.8,9

Fig 1.


Article SOURCE:

Fig 1.

The cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) immunologic checkpoint. T-cell activation requires antigen presentation in the context of a major histocompatibility complex (MHC) molecule in addition to the costimulatory signal achieved when B7 on an antigen-presenting cell (dendritic cell shown) interacts with CD28 on a T cell. Early after activation, to maintain immunologic homeostasis, CTLA-4 is translocated to the plasma membrane where it downregulates the function of T cells.

On the basis of this preclinical rationale, two antibodies targeting CTLA-4, ipilimumab (Bristol-Myers Squibb, Princeton, NJ) and tremelimumab (formerly Pfizer, currently MedImmune/AstraZeneca, Wilmington, DE), entered clinical development. Early reports of both agents showed durable clinical responses in some patients.1012Unfortunately, despite a proportion of patients experiencing a durable response, tremelimumab did not statistically significantly improve overall survival, which led to a negative phase III study comparing tremelimumab to dacarbazine/temozolomide in patients with advanced melanoma.13 It is possible that the lack of an overall survival benefit was a result of the crossover of patients treated with chemotherapy to an expanded access ipilimumab program or a result of the dosing or scheduling considerations of tremelimumab.

Ipilimumab, however, was successful in improving overall survival in two phase III studies involving patients with advanced melanoma.14,15 Although the median overall survival was only improved by several months in each of these studies, landmark survival after treatment initiation favored ipilimumab; in the first phase III study, 18% of patients were alive after 2 years compared with 5% of patients who received the control treatment of gp100 vaccination.14 More recently reported pooled data from clinical trials of ipilimumab confirm that approximately 20% of patients will have long-term survival of at least 3 years after ipilimumab therapy, with the longest reported survival reaching 10 years.1618

For patients with other malignancies, CTLA-4 antibody therapy has also shown some benefits. Ipilimumab, in combination with carboplatin and paclitaxel in a phased treatment schedule, showed improved progression-free survival compared with carboplatin and paclitaxel alone for patients with non–small-cell lung cancer.19Several patients with pancreatic cancer had declines in CA 19-9 when ipilimumab was given with GVAX (Aduro, Berkeley, CA),20and ipilimumab has also resulted in responses in patients with prostate cancer.21 Unfortunately, a phase III study in patients with castrate-resistant prostate cancer who experienced progression on docetaxel chemotherapy demonstrated that after radiotherapy, ipilimumab did not improve overall survival compared with placebo.22 Although this study is felt to have been a negative study, ipilimumab may have conferred a benefit to patients with favorable prognostic features, such as the absence of visceral metastases, but this requires further study. Another CTLA-4–blocking antibody, tremelimumab, has shown responses in patients with mesothelioma, and ongoing trials are under way.23

CTLA-4 blockade has also been administered together with other immunologic agents, such as the indoleamine 2,3-dioxygenase inhibitor INCB024360,106 the oncolytic virus talimogene laherparepvec,107 and granulocyte-macrophage colony-stimulating factor,108 with encouraging early results. We expect subsequent studies involving engineered T-cell–based therapies and checkpoint blockade.

Other promising data involve CTLA-4 combinations with PD-1 blockade. A phase I study of ipilimumab and nivolumab in patients with melanoma resulted in a high durable response rate and impressive overall survival compared with historical data.109,110Although the most recently reported grade 3 or 4 toxicity rate in patients with melanoma was 64%, which is higher than either ipilimumab or nivolumab individually,111 the vast majority of these irAEs were asymptomatic laboratory abnormalities of unclear clinical consequence. For example, elevations in amylase or lipase were reported in 21% of patients, none of whom met clinical criteria for a diagnosis of pancreatitis. The rate of grade 3 or 4 diarrhea was 7%, which is approximately similar to the rate of grade 3 or 4 diarrhea with ipilimumab monotherapy at 3 mg/kg. Whether ipilimumab and nivolumab improve overall survival compared with either nivolumab or ipilimumab alone remains the subject of an ongoing phase III randomized trial, and investigations of the combination of ipilimumab and nivolumab (and tremelimumab and MEDI4736) are ongoing in many other cancers.

Immunotherapy with checkpoint-blocking antibodies targeting CTLA-4 and PD-1/PD-L1 has improved the outlook for patients with a variety of malignancies. Despite the promise of this approach, many questions remain, such as the optimal management of irAEs and how best to evaluate combination approaches to determine whether they will increase the efficacy of CTLA-4 or PD-1/PD-L1 blockade alone. Themes from the experience with CTLA-4 and PD-1/PD-L1 will likely be relevant for investigations of novel immunologic checkpoints in the future.

This is a very important article, Dr. Larry.

It fits so beautiful with our work on Molecules in Development Table.

Thank you


This image depicts the process of metastasis in a mouse tumor, where tumor cells (green) have helped to reorganize the collagen into aligned fibers (blue) that provide the structural support for motility. This helps the tumor cells to enter blood vessels (red), ultimately leading to the formation of metastases in other organs.

This image depicts the process of metastasis in a mouse tumor, where tumor cells (green) have helped to reorganize the collagen into aligned fibers (blue) that provide the structural support for motility. This helps the tumor cells to enter blood vessels (red), ultimately leading to the formation of metastases in other organs.  Image: Madeleine Oudin and Jeff Wyckoff

Paving the way for metastasis

Cancer cells remodel their environment to make it easier to reach nearby blood vessels.

Anne Trafton | MIT News Office     March 15, 2016


A new study from MIT reveals how cancer cells take some of their first steps away from their original tumor sites. This spread, known as metastasis, is responsible for 90 percent of cancer deaths.

Studying mice, the researchers found that cancer cells with a particular version of the Mena protein, called MenaINV (invasive), are able to remodel their environment to make it easier for them to migrate into blood vessels and spread through the body. They also showed that high levels of this protein are correlated with metastasis and earlier deaths among breast cancer patients.

Finding a way to block this protein could help to prevent metastasis, says Frank Gertler, an MIT professor of biology and a member of the Koch Institute for Integrative Cancer Research.

“That’s something that I think would be very promising, because we know that when we genetically remove MenaINV, the tumors become nonmetastatic,” says Gertler, who is the senior author of a paper describing the findings in the journal Cancer Discovery.

Madeleine Oudin, a postdoc at the Koch Institute, is the paper’s lead author.

On the move

For cancer cells to metastasize, they must first become mobile and then crawl through the surrounding tissue to reach a blood vessel. In the new study, the MIT team found that cancer cells follow the trail of fibronectin, a protein that is part of the “extracellular matrix” that provides support for surrounding cells. Fibronectin is found in particularly high concentrations around the edges of tumors and near blood vessels.

“Cancer cells within a tumor environment are constantly faced with differences in fibronectin concentrations, and they need to be able to move from low to high concentrations to reach the blood vessels,” Oudin says.

MenaINV, an alternative form of the normal Mena protein, is key to this process. MenaINV includes a segment not found in the normal version, and this makes it bind more strongly to a receptor known as alpha-5 integrin, which is found on the surfaces of tumor cells and nearby supporting cells, and recognizes fibronectin.

When MenaINV attaches to this receptor, it promotes the binding of fibronectin to the same receptors. Fibronectin is normally a tangled protein, but when it binds to cell surfaces, it gets stretched out into long bundles. This stimulates the organization of collagen, another extracellular matrix protein, into stiff fibrils that radiate from the edges of the tumor.

This pattern, which is typically seen in tumors that are more aggressive, essentially paves the way for tumor cells to move toward blood vessels.

“If you have curly, coiled collagen, that’s associated with a good outcome, but if it gets realigned into these really straight long fibers, that provides highways for these cells to migrate on,” Oudin says.

In studies of mice, cells with the invasive form of Mena were better able to recognize and crawl toward higher concentrations of fibronectin, moving along the collagen pathways, while cells without MenaINV did not move toward the higher concentrations.

Predicting metastasis

The researchers also looked at data from breast cancer patients and found that high levels of MenaINV and fibronectin are associated with metastasis and earlier death. However, there was no link between the normal version of Mena and earlier death.

Gertler’s lab had previously developed antibodies that can detect the normal and invasive forms of Mena, which are now being developed for testing patient biopsy samples. Such tests could help doctors to determine whether a patient’s tumor is likely to spread or not, and possibly to guide the patient’s treatment. In addition, scientists may be able to develop drugs that inhibit MenaINV, which could be useful for treating cancer or preventing it from metastasizing.

The researchers now hope to study how MenaINV may contribute to other types of cancers. Preliminary studies suggest that it plays a similar role in lung and colon cancers as that seen in breast cancer. They are also investigating how the choice between the two forms of the Mena protein is regulated, and how other proteins found in the extracellular matrix might contribute to cancer cell migration.

Facilitating Tumor Cell Migration

Researchers identify a modified form of a migration-regulating protein in cancer cells that remodels the tumor microenvironment to promote metastasis.
By Catherine Offord | March 16, 2016

Emerging evidence suggests that metastasis—the spread of cancer from one organ or tissue to another—is aided by a significant remodeling of the cancer cells’ surroundings. Now, researchers at MIT have made progress toward understanding the mechanisms involved in this process by highlighting the role of a protein that reorganizes the tumor’s extracellular matrix to facilitate cellular migration into blood vessels. The findings were published yesterday (March 15) in Cancer Discovery.

Using a mouse model, the team showed that a cancer-cell-expressed protein called MenaINV—a mutated, “invasive” form of the cell-migration-modulator Mena—binds more strongly than its normal equivalent to a receptor on tumor and nearby support cells. The binding rearranges fibronectin in the tumor microenvironment, which in turn triggers the reorganization of collagen in the extracellular matrix into linear fibers radiating from the tumor.

This collagen restructuring is key in facilitating the migration of tumor cells to the blood vessels, from where they can disseminate throughout the body.

Tumor cell-driven extracellular matrix remodeling enables haptotaxis during metastatic progression

Madeleine J. Oudin1Oliver Jonas1Tatsiana Kosciuk1Liliane C. Broye1Bruna C. Guido1Jeff Wyckoff1, …., James E. Bear2 and Frank B. Gertler1,*
Cancer Discov CD-15-1183  Jan 25, 2016

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration.


Researchers Find Link Between Death of Tumor-support Cells and Cancer Metastasis       Fri, 02/19/2016

The images show tumors that have metastasized to the lungs (image b) and bones (image d) in mice that had CAFs eliminated after 10 days. (Credit: Biju Parekkadan, Massachusetts General Hospital)

The images show tumors that have metastasized to the lungs (image b) and bones (image d) in mice that had CAFs eliminated after 10 days. (Credit: Biju Parekkadan, Massachusetts General Hospital)

Researchers have discovered that eliminating cells thought to aid tumor growth did not slow or halt the growth of cancer tumors. In fact, when the cancer-associated fibroblasts (CAFs), were eliminated after 10 days, the risk of metastasis of the primary tumor to the lungs and bones of mice increased dramatically. Scientists used bioengineered CAFs equipped with genes that caused those cells to self-destruct at defined moments in tumor progression. The study, published in Scientific Reports on Feb. 19, was conducted by researchers funded by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) at Massachusetts General Hospital (MGH). NIBIB is part of the National Institutes of Health.

What causes cancer to grow and metastasize is not well understood by scientists. CAFs are thought to be fibroblast cells native to the body that cancer cells hijacks and use to sustain their growth. However, because fibroblasts are found throughout the human body, it can be difficult to follow and study cancer effects on these cells.

“This work underscores two important things in solving the puzzle that is cancer,” said Rosemarie Hunziker, Ph.D., program director for Tissue Engineering at NIBIB. “First, we are dealing with a complex disease with so many dimensions that we are really only just beginning to describe it.  Second, this approach shows the power of cell engineering — manipulating a key cell in the cancer environment has led to a significant new understanding of how cancer grows and how it might be controlled in the future.”

Biju Parekkadan, Ph.D., assistant professor of surgery and bioengineering at MGH, and his team designed an experiment with the goal of better understanding the cellular environment in which tumors exist (called tumor microenvironment or TME), and the role of CAFs in tumor growth. In an effort to understand whether targeting CAFs could limit the growth of breast cancer tumors implanted in mice, they bioengineered CAFs with a genetic “kill switch.” The cells were designed to die when exposed to a compound that was not toxic to the surrounding cells.

Parekkadan and his team chose two different stages of tumor growth in which the CAFs were killed off after the tumor was implanted. When the CAFs were eliminated on the third or fourth day, they found no major difference in tumor growth or risk of metastasis compared with the tumors where the CAFs remained. However, there was an increase in tumor-associated macrophages — cells that have been associated with metastasis — in this early stage.

When the team waited to eliminate the CAFs until the 10th or 11th day, they discovered that in addition to the increase in macrophages, the cancer was more likely to spread to the lungs and bones of the mice. The unexpected results from this experiment could spur more research into the role of CAFs in cancer growth and metastasis.

More research may reveal whether or not there is a scientific basis for targeting CAFs for destruction — and if so, the awareness that timing matters when it comes to the response of the tumor. While neither treatment affected the growth of the initial tumor, it is important to understand that most cancer deaths result from metastases to vital organs rather than from the direct effects of the primary tumor.



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Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator



GEN News Highlights   Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The researchers discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells. “We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” Professor Patrizia Agostinis explains. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.” [©KU Leuven Laboratory of Cell Death Research & Therapy, Dr. Abhishek D. Garg]


Scientists from KU Leuven in Belgium say they have shown that next-generation cell-based immunotherapy may offer new hope in the fight against brain cancer.

Cell-based immunotherapy involves the injection of a therapeutic anticancer vaccine that stimulates the patient’s immune system to attack the tumor. Thus far, the results of this type of immunotherapy have been mildly promising. However, Abhishek D. Garg and Professor Patrizia Agostinis from the KU Leuven department of cellular and molecular medicine believe they have found a novel way to produce more effective cell-based anticancer vaccines.

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The investigators discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells.

“We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” explains Prof. Agostinis. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.”

Combined with chemotherapy, this novel cell-based immunotherapy drastically increased the survival rates of mice afflicted with brain tumors. Almost 50% of the mice were completely cured. None of the mice treated with chemotherapy alone became long-term survivors.

“The major goal of any anticancer treatment is to kill all cancer cells and prevent any remaining malignant cells from growing or spreading again,” says Professor Agostinis. “This goal, however, is rarely achieved with current chemotherapies, and many patients relapse. That’s why the co-stimulation of the immune system is so important for cancer treatments. Scientists have to look for ways to kill cancer cells in a manner that stimulates the immune system. With an eye on clinical studies, our findings offer a feasible way to improve the production of vaccines against brain tumors.”

The team published its study (“Dendritic Cell Vaccines Based on Immunogenic Cell Death Elicit Danger Signals and T Cell–Driven Rejection of High-Grade Glioma”) in Science Translational Medicine.


Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell–driven rejection of high-grade glioma


SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma


Cortical GABAergic excitation contributes to epileptic activities around human glioma


Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.


Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.


Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies


Magnetic Resonance Metabolic Imaging of Glioma


Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

Cancer immunotherapy has gained considerable momentum over the past 5 years, owing predominantly to the discovery of immune checkpoint inhibitors. These inhibitors are designed to release the brakes of the immune system that under physiological conditions prevent auto-immunity by negatively regulating cytotoxic T lymphocyte (CTL) function. Following the FDA approval of the anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibody (mAb) ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2, 3).

However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.

Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (68). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6, 1012).

There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL- and Th1-driven antitumor immunity.

One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (1419). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14, 1719). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20, 21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed “eat me” signal calreticulin (ecto-CRT), the “find me” signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21, 22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).

Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables (Tables11 and and2).2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.

A list of prominent enhancers of immunogenicity and ICD inducers applied in DC vaccine setups and their associations with DAMPs and DC biology.
A list of preclinical tumor models and clinical studies for evaluation of the in vivo potency of DC vaccines loaded with immunogenically killed tumor cells.
The Impact of DC Biology on the Efficacy of DC Vaccines

Over the past years, different DC vaccine parameters have been shown to impact the clinical effectiveness of DC vaccinations. In the next section, we will elaborate on some promising adaptations of the DC preparation protocol.

Given the labor-intensive ex vivo culturing protocol of monocyte-derived DCs and inspired by the results of the Provenge study, several groups are currently exploiting the use of blood-isolated naturally circulating DCs (7678). In this context, De Vries et al. evaluated the use of antigen-loaded purified plasmacytoid DCs for intranodal injection in melanoma patients (79). This strategy was feasible and induced only very mild side effects. In addition, the overall survival of vaccinated patients was greatly enhanced as compared to historical control patients. However, it still remains to be determined whether this strategy is more efficacious than monocyte-derived DC vaccine approaches (78). By contrast, experiments in the preclinical GL261 high-grade glioma model recently showed that vaccination with tumor antigen-loaded myeloid DCs resulted in more robust Th1 responses and a stronger survival benefit as compared to mice vaccinated with their plasmacytoid counterparts (80).

In view of their strong potential to stimulate cytotoxic T cell responses, several groups are currently exploring the use of Langerhans cell-like DCs as sources for DC vaccines (8183). These so-called IL-15 DCs can be derived from CD14+monocytes by culturing them with IL-15 (instead of the standard IL-4). Recently, it has been shown that in comparison to IL-4 DCs, these cells have an increased capacity to stimulate antitumor natural killer (NK) cell cytotoxicity in a contact- and IL-15-dependent manner (84). NK cells are increasingly being recognized as crucial contributors to antitumor immunity, especially in DC vaccination setups (85, 86). Three clinical trials are currently evaluating these Langerhans cell-type DCs in melanoma patients (NCT00700167, NCT 01456104, and NCT01189383).

Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio- and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87, 88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89, 90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in high-grade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202).

With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93, 94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional (95). Furthermore, the frequency of vaccination can also influence the vaccine’s immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58, 96).

Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97, 98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97, 98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99, 100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumor-antigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100, 103). Binding of CD40 on DCs to CD40L on CD4+ helper T cells licenses DCs and enables them to prime CD8+ effector T cells.

A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide “off-the-shelf” availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immune-escape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

Toward a More Immunogenic Tumor Cell Cargo

The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze–thaw cycles to induce primary necrosis of cancer cells (8, 93). Freeze–thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.

Immunogenic Treatment Modalities

Tables Tables11 and and22 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells (Table (Table1).1). Table Table22 lists the preclinical and clinical studies that investigated their in vivo potential. Figure Figure11 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

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A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1- and cytotoxic T lymphocyte (CTL)-driven tumor rejection.

Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidation-associate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28, 29) (Table (Table1).1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30, 31).

One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as high-grade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze–thaw cycles (to induce necrosis of cancer cells) or a combination of freeze–thaw cycles and subsequent high-dose γ-irradiation (8, 18) (Table (Table2).2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze–thaw lysate (in comparison to freeze–thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) (Table (Table2).2). ….

Heat Shock Treatment

Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41–43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) (Table (Table1).1). …

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Figure 2

A schematic representation of immunogenic cell death (ICD)-based DC vaccines. ICD causes cancer cells to emit a spatiotemporally defined pattern of danger signals. Upon loading of these ICD-undergoing cancer cells onto DCs, they induce extensive phagocytosis and antigen uptake. Loaded DCs show enhanced phenotypic and functional maturation and immunization with these ICD-based DC vaccines instigates Th1-, Th17-, and cytotoxic T lymphocyte (CTL)-driven antitumor immunity in vivo.
Inducers of Immunogenic Cell Death

Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20, 21, 113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20, 21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. …

Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables (Tables11 and and2).2). Figure Figure22 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.


Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. …


The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. …

High-hydrostatic pressure

High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHP-treated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42).  …

Oncolytic Viruses

Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).

Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) (Table (Table1)1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). …

Photodynamic therapy

Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). …

Combinatorial Regimens

In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). ….

Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. ….


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