Advertisements
Feeds:
Posts
Comments

Posts Tagged ‘Glioma’


Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

GEN News Highlights   Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer

http://www.genengnews.com/gen-news-highlights/advanced-immunotherapeutic-method-shows-promise-against-brain-cancer/81252433/

 

http://www.genengnews.com/Media/images/GENHighlight/Mar3_2016_LeuvenLab_CellDeathMouseBrain6232214015.jpg

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.

An external file that holds a picture, illustration, etc.
Object name is fimmu-06-00663-g001.jpg

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712296/bin/fimmu-06-00663-g001.jpg

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). …

An external file that holds a picture, illustration, etc.
Object name is fimmu-06-00663-g002.jpg

http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=4712296_fimmu-06-00663-g002.jpg

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.

Radiotherapy

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. …

Shikonin

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. ….

 

Advertisements

Read Full Post »


Neuron directed nanoparticle transporters

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Nanoparticle transport system for genes thwarts brain cancer in rats

     Sep 15, 2015    https://www.linkedin.com/pulse/nanoparticle-transport-system-genes-thwarts-brain-hossein-panahi

 

(Nanowerk News) Researchers have designed a nanoparticle transport system for gene delivery that destroys deadly brain gliomas in a rat model, significantly extending the lives of the treated animals. The nanoparticles are filled with genes for an enzyme that converts a prodrug called ganciclovir into a potent destroyer of the glioma cells.Glioma is one of the most lethal human cancers, with a five year survival rate of just 12%, and no reliable treatment. Advances in the understanding of the molecular processes that cause these tumors has resulted in therapies aimed at delivering specific genes into tumors – genes that make proteins to kill or suppress the growth of the tumor. Currently this approach relies heavily on using viruses to deliver the anti-tumor genes into the target cancer cells. Unfortunately, viral delivery poses significant safety risks including toxicity, activation of the patient’s immune system against the virus, and the possibility of the virus itself encouraging tumors to develop.

“Efforts to treat glioma with traditional drug and radiation therapies have not been very successful,” says Jessica Tucker, Ph.D., NIBIB Director for the Program in Gene and Drug Delivery Systems and Devices. “The ability to successfully deliver genes using these biodegradable nanoparticles, rather than potentially harmful viruses, is a significant step that reinvigorates the potential for gene therapy to treat deadly gliomas as well as other cancers.”
Nanoparticle transport system for genes thwarts brain cancer in rats

https://media.licdn.com/mpr/mpr/shrinknp_800_800/AAEAAQAAAAAAAAJ5AAAAJDNkNGY4NDg2LTc3OWEtNGY3Zi1hMThjLTliZGQ1ZGNkYTZhOQ.png

 

Scheme for nanoparticle gene therapy. A.Nanoparticles carrying DNA encoding HSVtk are injected into rat gliomas B. Convection-enhanced delivery (CED) pressure during injection distributes nanoparticles (yellow dots) throughout the tumor (pink) C. The nanoparticles make thymidine kinase, which is spread throughout the tumor (yellow) D. Ganciclovir, delivered into the rat’s blood system, reacts with the thymidine kinase to kill and shrink the tumor.
Jordan Green, Ph.D., of the Johns Hopkins University School of Medicine Biomedical Engineering Department and a senior author of the work, and his international team describe their findings in the February 24 issue of ACS Nano(“Polymeric Nanoparticles for Nonviral Gene Therapy Extend Brain Tumor Survival in Vivo”). The collaborators include colleagues from the Johns Hopkins University School of Medicine Departments of Neurosurgery, Oncology, Ophthalmology, and Pathology, as well as Tang Du Hospital in China, University of the Negevin, Israel, and the Instituto Neurologico C. Besta in Italy.
Biodegradable nanoparticles have recently shown promise as a method to deliver genes into cells. Their use for delivery avoids many of the problems associated with viral gene delivery. To demonstrate virus free delivery, the first goal of the group was to develop a nanoparticle that could efficiently carry DNA encoding a gene known as HSVtk into cells. The HSVtk gene produces an enzyme that turns the compound ganciclovir–which by itself has no effect on cancer cells — into a compound that is toxic to actively dividing brain cancer cells.
A number of polymer structures were tested for their ability to deliver DNA into two rat glioma cell lines. Among the many polymers tried, the one known as PBAE 447 was found to be the most efficient in delivering the HSVtk gene into the cultured rat glioma cells. Furthermore, when combined with ganciclovir, the HSVtk-encoding nanoparticles were 100% effective in killing both of the glioma cell lines grown in the laboratory.
Next, the gene therapy system was tested in live rats with brain gliomas. Because it is important that the nanoparticles spread throughout the entire tumor, they were infused into the rat gliomas using convection-enhanced delivery (CED). The method involves injection into the tumor and the application of a pressure gradient, which efficiently disperses the nanoparticles throughout the tumors.
To test the tumor-killing ability of the system, the tumor-bearing rats were given systemic administration of ganciclovir for two days, then CED was used to infuse the HSVtk-encoding nanoparticles into the rat gliomas, and systemic ganciclovir treatment continued for eight more days. The treatment resulted in shrinkage of the tumors and a significant increase in survival when compared with control glioma-bearing animals that did not receive the combination treatment.
“The results provide the first demonstration of a successful non-viral nanomedicine method for HSVtk/ganciclovir treatment of brain cancer,” stated Green. “Next steps will include enhancing the efficiency of this nanoparticle delivery system and evaluating the technology in additional brain cancer animal models.”
In the future, the investigators envision that doctors would administer this therapy during the surgery commonly used to treat glioma in humans. They are also interested in testing the ability to deliver other cancer-killing genes and whether the nanoparticles could be successfully administered systemically — which could broaden the use of the therapy for a wide range of solid tumors and systemic cancers.
Source: National Institute of Biomedical Imaging and Bioengineering

Nanoparticle transport system for genes thwarts brain cancer in rats

 

Read Full Post »


Accumulation of 2-hydroxyglutarate is not a biomarker for malignant progression of IDH-mutated low grade gliomas 

Larry H Bernstein, MD, FCAP, Curator

LPBI

Accumulation of 2-hydroxyglutarate is not a biomarker for malignant progression in IDH-mutated low-grade gliomas

Neuro Oncol. 2013 Jun; 15(6): 682–690.
Online 2013 Feb 14. doi: 10.1093/neuonc/not006

Objectives

To determine whether accumulation of 2-hydroxyglutarate in IDH-mutated low-grade gliomas (LGG; WHO grade II) correlates with their malignant transformation and to evaluate changes in metabolite levels during malignant progression.

Methods

Samples from 54 patients were screened for IDH mutations: 17 patients with LGG without malignant transformation, 18 patients with both LGG and their consecutive secondary glioblastomas (sGBM; n = 36), 2 additional patients with sGBM, 10 patients with primary glioblastomas (pGBM), and 7 patients without gliomas. The cellular tricarboxylic acid cycle metabolites, citrate, isocitrate, 2-hydroxyglutarate, α-ketoglutarate, fumarate, and succinate were profiled by liquid chromatography–tandem mass spectrometry. Ratios of 2-hydroxyglutarate/isocitrate were used to evaluate differences in 2-hydroxyglutarate accumulation in tumors from LGG and sGBM groups, compared with pGBM and nonglioma groups.

Results

IDH1 mutations were detected in 27 (77.1%) of 37 patients with LGG. In addition, in patients with LGG with malignant progression (n = 18), 17 patients were IDH1 mutated with a stable mutation status during their malignant progression. None of the patients with pGBM or nonglioma tumors had an IDH mutation. Increased 2-hydroxyglutarate/isocitrate ratios were seen in patients with IDH1-mutated LGG and sGBM, in comparison with those with IDH1-nonmutated LGG, pGBM, and nonglioma groups. However, no differences in intratumoral 2-hydroxyglutarate/isocitrate ratios were found between patients with LGG with and without malignant transformation. Furthermore, in patients with paired samples of LGG and their consecutive sGBM, the 2-hydroxyglutarate/isocitrate ratios did not differ between both tumor stages.

Conclusion

Although intratumoral 2-hydroxyglutarate accumulation provides a marker for the presence of IDHmutations, the metabolite is not a useful biomarker for identifying malignant transformation or evaluating malignant progression.

Keywords: α-ketoglutarate, IDH1 mutations, liquid chromatography–tandem mass spectrometry, low-grade gliomas, secondary glioblastomas, 2-hydroxyglutarate

Low-grade gliomas (LGG) occur in the cerebral hemispheres and represent 10%–15% of all astrocytic brain tumors.1 Despite long-term survival in many patients, 50%–75% of patients with LGG eventually die of either progression of a low-grade tumor or transformation to a malignant glioma.2 The time to progression can vary from a few months to several years,35 and the median survival among patients with LGG ranges from 5 to 10 years.6,7 Among several risk factors, only age, histology, tumor location, and Karnofsky performance index have generally been accepted as prognostic factors for patients with LGG.8,9 As a prognostic molecular marker, only 1p19q codeletion was identified as such in pure oligodendrogliomas. However, this association was not seen in either astrocytomas or oligoastrocytomas.10

Somatic mutations in human cytosolic isocitrate dehydrogenases 1 (IDH1) were first described in 2008 in ∼12% of glioblastomas11 and later in acute myeloid leukemia, in which the reported mutations were missense and specific for a single R132 residue.11,12 Some gliomas lacking cytosolic IDH1 mutations were later observed to have mutations in IDH2, the mitochondrial homolog of IDH1.12IDH mutations are the most commonly mutated genes in many types of gliomas, with incidences of up to 75% in grade II and grade III gliomas.13,14 Further frequent mutations in patients with LGG were recently identified, including inactivating alterations in alpha thalassemia/mental retardation syndrome X-linked (ATRX), inactivating mutations in 2 suppressor genes, homolog of Drosophila capicua (CIC) and far-upstream binding protein 1 (FUBP1), in about 70% of grade II gliomas and 57% of sGBM.1517 The association between ATRX mutations with IDHmutations and the association between CIC/FUBP1 mutations and IDH mutations and 1p/19q loss are especially common among the grade II-III gliomas and remarkably homogeneous in terms of genetic alterations and clinical characteristics.16

It was thought that IDH mutations might be a prognostic factor in LGG, predicting a prolonged survival from the beginning of the disease.1823 However, this assumption, as shown in our and other earlier studies, had to be corrected because survival among patients who have LGG with IDH mutations is only improved after transformation to secondary high-grade gliomas.18,19,24 Furthermore, it had already been demonstrated that an IDH mutation is not a biomarker for further malignant transformation in LGG.18 IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and reduce NADP to NADPH.25 The mutations inactivate the standard enzymatic activity of IDH112 and confer novel activity on IDH1 for conversion of α-KG and NADPH to 2-hydroxyglutarate (2HG) and NADP+, supporting the evidence thatIDH1 and 2 are proto-oncogenes. This gain of function causes an accumulation of 2HG in glioma and acute myeloid leukemia samples.26,27 The 2HG levels in cancers with IDH mutations are found to be consistently elevated by 10–100-fold, compared with levels in samples lacking mutations of IDH1 or IDH2.26,28Nevertheless, how exactly the production or accumulation of 2HG by mutant IDH might drive cancer development is not well understood.

In the present study, we postulate that intratumoral 2HG could be a useful biomarker that predicts the malignant transformation of WHO grade II LGG. We therefore screened for IDH mutations in patients with LGG and measured the accumulation of 2HG in 2 populations of patients, patients with LGG with and without malignant transformation, with use of liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, we compared the concentrations of 2HG in LGG and their consecutive secondary glioblastomas (sGBM) to evaluate changes in metabolite levels during the malignant progression.

Go to:Methods and Materials

….

According to aforementioned criteria, a total of 72 tumor samples from 54 patients were analyzed (Table 1). The samples were from 17 patients with LGG without malignant transformation, 18 patients with both LGG and consecutive sGBM (n = 36), 2 additional patients with sGBM, 10 patients with pGBM, and 7 patients with nonglioma tumors. The nonglioma samples comprised 3 meningiomas, 2 metastases of breast cancers, 1 cavernoma, and 1 reactive gliosis from a patient with epilepsy.

Table 1.

Patient characteristics

DNA Isolation and IDH Mutation Detection

Tumor tissue samples were taken intraoperatively and were snap frozen at −80°C. To ensure a tumor cell content of at least 80% for nucleic acid extraction, control slides stained with hematoxylin and eosin were examined by the local neuropathologist. IDH1 and IDH2 mutations were assessed using direct DNA sequencing, as reported previously.18

Progression and Survival

Progression-free survival (PFS) was defined as the time from first diagnosis of an LGG to tumor progression or end of follow-up. Time to malignant transformation was defined as the time from the day of first surgery for an LGG to the day of surgery for malignant progression to a secondary high-grade glioma. Overall survival (OS) was the time from the day of first surgery to death or end of follow-up. All patient data were updated on June 15, 2012.

LC-MS/MS Analysis of Tricarboxylic Acid Cycle (TCA) Metabolites

Instrumentation included an AB Sciex QTRAP 5500 triple quadruple mass spectrometer coupled to a high-performance liquid chromatography (HPLC) system from Shimadzu containing a binary pump system, an autosampler, and a column oven. Targeted analyses of citrate, isocitrate, α-ketoglutarate (α-KG), succinate, fumarate (Sigma-Aldrich), and 2-hydroxyglutarate (2HG; SiChem GmbH) were performed in multiple reaction monitoring (MRM) scan mode with use of negative electrospray ionization (-ESI). Expected mass/charge ratios (m/z), assumed as [M-H+], were m/z 190.9, m/z 191.0, m/z 145.0, m/z 116.9, m/z 114.8, and m/z 147.0 for citrate, isocitrate, α-KG, succinate, fumarate, and 2HG, respectively. For quantification, ratios of analytes and respective stable isotope-labeled internal standards (IS) (Table 2) were used. For quantification of isocitrate and 2HG, stable isotope-labeled succinate was used as IS because of unavailability of labeled analogs. MRM transitions are summarized in Table 2.

Table 2.

MRM transitions and respective fragmentation parameters

Results  

Patient Characteristics

According to the malignant progression of the LGG, patients were divided into two groups: group 1 patients with LGG (LGG1) without malignant transformation and group 2 patients with LGG (LGG2) with histologically confirmed malignant progression.

Among 35 patients with LGG who were included in this study, 19 (54%) were women, and 16 (46%) were men. Furthermore, histologically, 6 patients had oligoastrocytomas, and the remaining 29 patients had diffuse astrocytomas.

The median age of all patients with LGG was 37.4 years at the time of the first diagnosis. Patients in LGG2 had a median age of 37.1 years, which did not differ significantly from that of patients in LGG1, who had a median age of 41.4 years.

The median time to malignant transformation among patients in the LGG2 group was 3.35 years (range, 2.5–5.4 years). The median OS among all patients with LGG was 13.1 years (11.4 years in LGG1 and 13.1 years in LGG2; P = .97).

IDH1 Mutation and Outcome

An IDH1 mutation was detected in 27 of 35 patients with LGG (77.1%), in 10 of 17 patients in LGG1 (59%), and in 17 of 18 patients in LGG2 (95%). In all cases, IDH1 mutations were found on R132. IDH2mutations were not detected in any of the patients. The IDH1 mutation status was stable during progression from LGG to sGBM in all patients in LGG2. None of the patients with pGBM or nonglioma had an IDHmutation. Patients with LGG with an IDH1 mutation had a median PFS of 3.3 years, which was comparable to that among patients with wild-type LGG (2.8 years; P > .05). Furthermore, the OS among patients with LGG with an IDH1 mutation was not statistically different at 13.0 years compared with that among patients with LGG without an IDH1 mutation, who had an OS of 9.3 years (P = .66).

LC-MS/MS Profiling of TCA Metabolites

TCA metabolites, citrate, isocitrate, α-ketoglutarate, succinate, fumarate, and 2-hydroxyglutarate were measured in glioma samples with and without an IDH1 mutation, in samples identified as primary GBM, and in nonglioma brain tumor specimens (Fig. 1). No differences in citrate, isocitrate, α-KG, succinate, and fumarate concentrations were found when comparing all of the latter groups. Concentrations of 2HG, a side product in IDH1-mutated gliomas, were 20–34-fold higher in IDH1-mutated gliomas (0.64–0.81 µmol/g), compared with non–IDH1-mutated LGG1 (P ≤ .001). No differences were observed between IDH1-mutated gliomas and IDH1-nonmutated LGG2 and sGBM, caused by strongly elevated 2HG levels in either 1 or 2 samples in these groups, respectively. Furthermore, in IDH1-mutated gliomas, 2HG concentrations were a mean of 20 times higher than in pGBM and nongliomas (P ≤ .001) (Fig. 1). No differences were observed between the single groups of IDH1-mutated gliomas LGG1, LGG2, and sGBM in relation to 2HG concentration.

Fig. 1.

Dot-box and whisker plots show concentration ranges for TCA metabolites measured in IDH1-nonmutated (IDH1wt) and IDH1-mutated (IDH1mut) LGG and sGBM and in pGBM and nonglioma tumor specimens; boxes span the 25th and 75th percentiles with median, whiskers

To detect possible differences among the IDH1-mutated LGG1, LGG2, and sGBM, the α-KG/isocitrate and 2HG/isocitrate ratios were used in additional tests. Therefore, the direct precursor-product relation would correct for all differences possibly expected during pre-analytical processing. To prove this, analyte ratios ofIDH1-mutated and nonmutated gliomas were compared. IDH1-mutated gliomas showed a 2HG/isocitrate ratio that was 13 times higher (P ≤ .001) (Fig. 2A), which corresponds to a lower accumulation of 2HG inIDH1-nonmutated gliomas. α-KG/isocitrate ratios were determined to be approximately 10-fold higher inIDH1-mutated gliomas than in IDH1-nonmutated gliomas (P = .005) (Fig. 2B), which also implies lower accumulation of α-KG in IDH1-nonmutated gliomas.

Fig. 2.

2-Hydroxyglutarate to isocitrate ratios (A) and α-ketoglutarate to isocitrate ratios (B) for IDH1-nonmutated (IDH1wt) and IDH1-mutated (IDH1mut) gliomas (LGG and sGBM); boxes span the 25th and 75th percentiles with median, and whiskers represent

2HG/isocitrate and α-KG/isocitrate ratios, respectively, were calculated in all 8 specimen groups (Fig. 3). In addition to the differences in 2HG/isocitrate ratios of IDH1-mutated and nonmutated gliomas (Fig. 2A), the ratios in IDH1-mutated gliomas were 4–9 times higher, compared with those in pGBM (P ≤ .001), and 3–6 times higher, compared with those in non-glioma tumor specimens, which was not statistically significant (Fig. 3A). In detail, ratios of 2HG and isocitrate were established to be 13, 9.4, and 22 times higher in IDH1-mutated LGG1, LGG2, and their consecutive sGBM, respectively, than in IDH1-nonmutated LGG1 (Fig. 3A). No significant differences were observed between IDH1-mutated gliomas and IDH1-nonmutated LGG2 and sGBM. The comparison of 2HG/isocitrate ratios between IDH1-nonmutated gliomas and IDH1-mutated LGG2 and sGBM showed no statistically significant differences. However, a trend toward higher ratios inIDH1-mutated LGG1/2 was seen. Furthermore, no differences could be determined by comparing 2HG/isocitrate ratios measured in the groups of IDH1-mutated LGG1 and LGG2. Although 2HG/isocitrate ratios in IDH1-mutated secondary glioblastomas are 1.7 and 2.3 times higher than in the LGG1 and LGG2 groups, respectively, no statistically significant differences were observed.

Fig. 3.

2-Hydroxyglutarate to isocitrate (A, C) and α-ketoglutarate to isocitrate (B, D) ratios for groups of IDH1-nonmutated (IDH1wt) and mutated gliomas (IDH1mut), pGBM, and nongliomas; dot-box and whisker plots (A, B) span the 25th and 75th percentiles

The absence of a straight trend to higher 2HG/isocitrate ratios during malignant progression is shown by paired analysis of IDH1-mutated LGG2 and their consecutive sGBM (Fig. 3C). Similar findings were observed using the α-KG/isocitrate ratios. Although significant differences were found, with ratios approximately 10 times higher in IDH1-mutated glioblastomas than in IDH1-nonmutated glioblastomas (Fig. 2B), it was not possible to differentiate among the 3 IDH1-mutated glioblastoma groups LGG1, LGG2, and their consecutive sGBM with use of this analyte ratio (Fig. 3B and D).

Go to:Discussion     

On the basis of a comprehensive analysis of cellular TCA metabolites from several cohorts of patients with glioma and nonglioma, our study provides evidence that the level of 2HG accumulation is not suitable as an early biomarker for distinguishing patients with LGG in relation to their course of malignancy. To our knowledge, this is the first report of a paired analysis of 2HG levels in LGG and their consecutive sGBM showing stable 2HG accumulation during malignant progression. This fact assumes that malignant transformation of IDH-mutated LGG appears to be independent of their intracellular 2HG accumulation. Considering these results, we could not stratify patients with LGG into subgroups with distinct survival.

To date, little is known about biomarkers that may predict malignant transformation and, consequently, predict survival in patients with LGG. The investigation of biomarkers in this patient group is relevant because treatment interventions can be tailored to prolong survival, minimize treatment-related adverse effects, and, accordingly, maximize quality of life. In many previous studies, IDH mutations, as the most commonly detected mutations in LGG, were observed to be a significant prognostic factor in patients with glioma, often relating to improved survival among patients with LGG.1823 However, only patients withIDH-mutated LGG with malignant progression have a prolonged OS.18,19,24 In an earlier conducted study, the analysis of 2 groups of patients with LGG with and without malignant transformation failed to provide a significant influence from the IDH mutation, neither on the PFS nor on the OS.18 In agreement with these data, in a recent study, we showed again that PFS and OS among patients with LGG with and without anIDH mutation did not differ significantly, despite the malignant transformation in LGG2. This result is not unexpected because the same patient population was analyzed in both studies.

Accumulation of 2HG in IDH1-mutated gliomas was first described by Dang et al.26 2HG accumulation is an important marker of IDH1/2-mutated gliomas and other neoplasms.26,29,30 Furthermore, it was assumed that 2HG accumulation might be a potential systemic biomarker of gliomas, but it was not detectable in serum samples from patients with glioma.31 Nevertheless, it has become possible in the meantime to detect 2HG production using magnetic resonance spectroscopy in a noninvasive manner to identify patients withIDH1 mutant brain tumors.32

Because 2HG accumulation provides one of the few potential read-outs for mutant IDH enzymatic activity, we suspected that different intratumoral levels of 2HG accumulation in patients with LGG (especially in those with an IDH mutation) may affect their clinical course in relation to malignant transformation. Therefore, intratumoral concentrations of 2HG and other TCA metabolites were quantified by LC-MS/MS, showing concentrations in IDH1-mutated glioma samples that were comparable to the levels described by Dang et al.26 As previously shown,22 no differences in metabolite levels were observed, with the exception of 2HG with increased accumulation in IDH-mutated gliomas, compared with IDH-nonmutated specimens. However, the fold-difference in 2HG levels between IDH1 mutant and IDH1 wild-type LGG in our study was smaller than in some other studies.26,30 As a reason for this issue, we identified 3 samples of IDH1 wild-type LGG and sGBM with strongly elevated 2HG levels. The fact that some IDH1 wild-type tumors might accumulate 2HG was previously described by Wise et al,33 who reported that the increased IDH2-dependent carboxylation of glutamine-derived α-KG in hypoxia is associated with a concomitant increased synthesis of 2HG in cells with wild-type IDH1 and IDH2. Thus, they concluded that, in further support of the increased mitochondrial reductive glutamine metabolism that they observed in hypoxia, the incubation in hypoxia can lead to elevated 2HG levels in cells lacking IDH1/2 mutations.

The ratio of 2HG to isocitrate and the ratio of α-KG to isocitrate were provided. The use of these ratios can function as an internal control because isocitrate is the direct precursor of α-KG and 2HG. However, intracellular accumulation of both metabolites (2HG and of α-KG) did not differ between both IDH-mutated LGG groups with and without malignant progression. In the same way, detection of 2HG concentrations and the 2HG/isocitrate ratios in patients with LGG and their consecutive sGBM were comparable to values during malignant progression. Correspondingly, all other assessed TCA metabolites remained stable during the malignant progression of the LGG to sGBM. Therefore, 2HG accumulation appears, at least for now, merely to represent a highly correlative and stable marker for an emerging class of somatic mutations in theIDH enzymes from the early stage of glioma development and after malignant transformation to high-grade gliomas.

An expected lower level of α-KG in IDH-mutated LGG and sGBM, in comparison with IDH-nonmutated LGG, pGBM, and nonglioma tumors, was not detected in our study. This is in concordance with a similar finding by Dang et al,26 who showed unaffected α-KG levels in whole-tumor cell lysates. However, the latter finding was in contrast to reported results by Zhao et al,34 who showed that forced expression of mutant IDHin cultured cells led to a dose-dependent decrease in α-ketoglutarate levels. A possible explanation for this finding is the mono-allelic heterozygous mutations of the IDH1 gene leading to expression of both wild-type and mutant IDH1 in a single cell and, therefore, to production of 2HG and α-KG at the same time in every mutated cell.11,12 Another possible reason is the reported evidence that the biochemical effects of mutantIDH1 on α-KG-dependent enzymes are not principally attributable to depletion of α-KG but are a competitive antagonism with α-KG.3537 Thus, Xu et al postulated that IDH1 mutations alone do not reduce cellular level of α-KG sufficiently to have a significant tumorigenic consequence, but nonetheless these mutations sensitize α-KG–dependent dioxygenases to the inhibitory effect by the large amounts of intratumoral accumulated 2HG.35

Whether 2HG acts as a mutagen or plays a distinct role in gliomagenesis remains to be determined. Dang et al predicted that patients with LGG may benefit from the therapeutic inhibition of 2HG production, resulting in the slowing or halting of conversion of LGG into a lethal secondary glioblastoma, thus changing the course of the disease.26 However, our data confirm similar values of 2HG accumulation in the different LGG groups (with and without malignant progression) and present comparable ranges of 2HG in the low-grade and high-grade tumor stages. In addition, our study used the ratio of 2HG to isocitrate, which might provide a more sensitive screening tool for IDH-mutated LGG than an increase in the absolute concentration of 2HG.

Finally, more work is needed to provide valuable clues about the precise role that 2HG might play in the initiation and progression of LGG. Moreover, the value of 2HG as a useful biomarker for diagnosis or monitoring of the treatment response of LGG has not yet been realized.

Read Full Post »


Mitochondrial Isocitrate Dehydrogenase and Variants

Writer and Curator: Larry H. Bernstein, MD, FCAP 

2.1.4      Mitochondrial Isocitrate Dehydrogenase (IDH) and variants

2.1.4.1 Accumulation of 2-hydroxyglutarate is not a biomarker for malignant progression of IDH-mutated low grade gliomas

Juratli TA, Peitzsch M, Geiger K, Schackert G, Eisenhofer G, Krex D.
Neuro Oncol. 2013 Jun;15(6):682-90
http://dx.doi.org:/10.1093/neuonc/not006

Low-grade gliomas (LGG) occur in the cerebral hemispheres and represent 10%–15% of all astrocytic brain tumors.1 Despite long-term survival in many patients, 50%–75% of patients with LGG eventually die of either progression of a low-grade tumor or transformation to a malignant glioma.2 The time to progression can vary from a few months to several years,35 and the median survival among patients with LGG ranges from 5 to 10 years.6,7 Among several risk factors, only age, histology, tumor location, and Karnofsky performance index have generally been accepted as prognostic factors for patients with LGG.8,9 As a prognostic molecular marker, only 1p19q codeletion was identified as such in pure oligodendrogliomas. However, this association was not seen in either astrocytomas or oligoastrocytomas.10

Somatic mutations in human cytosolic isocitrate dehydrogenases 1 (IDH1) were first described in 2008 in ∼12% of glioblastomas11 and later in acute myeloid leukemia, in which the reported mutations were missense and specific for a single R132 residue.11,12 Some gliomas lacking cytosolic IDH1 mutations were later observed to have mutations in IDH2, the mitochondrial homolog of IDH1.12 IDH mutations are the most commonly mutated genes in many types of gliomas, with incidences of up to 75% in grade II and grade III gliomas.13,14 Further frequent mutations in patients with LGG were recently identified, including inactivating alterations in alpha thalassemia/mental retardation syndrome X-linked (ATRX), inactivating mutations in 2 suppressor genes, homolog of Drosophila capicua (CIC) and far-upstream binding protein 1 (FUBP1), in about 70% of grade II gliomas and 57% of sGBM.1517 The association between ATRX mutations with IDHmutations and the association between CIC/FUBP1 mutations and IDH mutations and 1p/19q loss are especially common among the grade II-III gliomas and remarkably homogeneous in terms of genetic alterations and clinical characteristics.16

It was thought that IDH mutations might be a prognostic factor in LGG, predicting a prolonged survival from the beginning of the disease.1823 However, this assumption, as shown in our and other earlier studies, had to be corrected because survival among patients who have LGG with IDH mutations is only improved after transformation to secondary high-grade gliomas.18,19,24 Furthermore, it had already been demonstrated that an IDH mutation is not a biomarker for further malignant transformation in LGG.18 IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and reduce NADP to NADPH.25 The mutations inactivate the standard enzymatic activity of IDH112 and confer novel activity on IDH1 for conversion of α-KG and NADPH to 2-hydroxyglutarate (2HG) and NADP+, supporting the evidence thatIDH1 and 2 are proto-oncogenes. This gain of function causes an accumulation of 2HG in glioma and acute myeloid leukemia samples.26,27 The 2HG levels in cancers with IDH mutations are found to be consistently elevated by 10–100-fold, compared with levels in samples lacking mutations of IDH1 or IDH2.26,28Nevertheless, how exactly the production or accumulation of 2HG by mutant IDH might drive cancer development is not well understood.

In the present study, we postulate that intratumoral 2HG could be a useful biomarker that predicts the malignant transformation of WHO grade II LGG. We therefore screened for IDH mutations in patients with LGG and measured the accumulation of 2HG in 2 populations of patients, patients with LGG with and without malignant transformation, with use of liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, we compared the concentrations of 2HG in LGG and their consecutive secondary glioblastomas (sGBM) to evaluate changes in metabolite levels during the malignant progression.

Objectives: To determine whether accumulation of 2-hydroxyglutarate in IDH-mutated low-grade gliomas (LGG; WHO grade II) correlates with their malignant transformation and to evaluate changes in metabolite levels during malignant progression. Methods: Samples from 54 patients were screened for IDH mutations: 17 patients with LGG without malignant transformation, 18 patients with both LGG and their consecutive secondary glioblastomas (sGBM; n = 36), 2 additional patients with sGBM, 10 patients with primary glioblastomas (pGBM), and 7 patients without gliomas. The cellular tricarboxylic acid cycle metabolites, citrate, isocitrate, 2-hydroxyglutarate, α-ketoglutarate, fumarate, and succinate were profiled by liquid chromatography-tandem mass spectrometry. Ratios of 2-hydroxyglutarate/isocitrate were used to evaluate differences in 2-hydroxyglutarate accumulation in tumors from LGG and sGBM groups, compared with pGBM and nonglioma groups. Results: IDH1 mutations were detected in 27 (77.1%) of 37 patients with LGG. In addition, in patients with LGG with malignant progression (n = 18), 17 patients were IDH1 mutated with a stable mutation status during their malignant progression. None of the patients with pGBM or nonglioma tumors had an IDH mutation. Increased 2-hydroxyglutarate/isocitrate ratios were seen in patients with IDH1-mutated LGG and sGBM, in comparison with those with IDH1-nonmutated LGG, pGBM, and nonglioma groups. However, no differences in intratumoral 2-hydroxyglutarate/isocitrate ratios were found between patients with LGG with and without malignant transformation. Furthermore, in patients with paired samples of LGG and their consecutive sGBM, the 2-hydroxyglutarate/isocitrate ratios did not differ between both tumor stages. Conclusion: Although intratumoral 2-hydroxyglutarate accumulation provides a marker for the presence of IDH mutations, the metabolite is not a useful biomarker for identifying malignant transformation or evaluating malignant progression.

LC-MS/MS Analysis of Tricarboxylic Acid Cycle (TCA) Metabolites

Instrumentation included an AB Sciex QTRAP 5500 triple quadruple mass spectrometer coupled to a high-performance liquid chromatography (HPLC) system from Shimadzu containing a binary pump system, an autosampler, and a column oven. Targeted analyses of citrate, isocitrate, α-ketoglutarate (α-KG), succinate, fumarate (Sigma-Aldrich), and 2-hydroxyglutarate (2HG; SiChem GmbH) were performed in multiple reaction monitoring (MRM) scan mode with use of negative electrospray ionization (-ESI). Expected mass/charge ratios (m/z), assumed as [M-H+], were m/z 190.9, m/z 191.0, m/z 145.0, m/z 116.9, m/z 114.8, and m/z 147.0 for citrate, isocitrate, α-KG, succinate, fumarate, and 2HG, respectively. For quantification, ratios of analytes and respective stable isotope-labeled internal standards (IS) (Table 2) were used. For quantification of isocitrate and 2HG, stable isotope-labeled succinate was used as IS because of unavailability of labeled analogs. MRM transitions are summarized in Table 2.

IDH1 Mutation and Outcome

An IDH1 mutation was detected in 27 of 35 patients with LGG (77.1%), in 10 of 17 patients in LGG1 (59%), and in 17 of 18 patients in LGG2 (95%). In all cases, IDH1 mutations were found on R132. IDH2mutations were not detected in any of the patients. The IDH1 mutation status was stable during progression from LGG to sGBM in all patients in LGG2. None of the patients with pGBM or nonglioma had an IDH mutation. Patients with LGG with an IDH1 mutation had a median PFS of 3.3 years, which was comparable to that among patients with wild-type LGG (2.8 years; P > .05). Furthermore, the OS among patients with LGG with an IDH1 mutation was not statistically different at 13.0 years compared with that among patients with LGG without an IDH1 mutation, who had an OS of 9.3 years (P = .66).

LC-MS/MS Profiling of TCA Metabolites

TCA metabolites, citrate, isocitrate, α-ketoglutarate, succinate, fumarate, and 2-hydroxyglutarate were measured in glioma samples with and without an IDH1 mutation, in samples identified as primary GBM, and in nonglioma brain tumor specimens (Fig. 1). No differences in citrate, isocitrate, α-KG, succinate, and fumarate concentrations were found when comparing all of the latter groups. Concentrations of 2HG, a side product in IDH1-mutated gliomas, were 20–34-fold higher in IDH1-mutated gliomas (0.64–0.81 µmol/g), compared with non–IDH1-mutated LGG1 (P ≤ .001). No differences were observed between IDH1-mutated gliomas and IDH1-nonmutated LGG2 and sGBM, caused by strongly elevated 2HG levels in either 1 or 2 samples in these groups, respectively. Furthermore, in IDH1-mutated gliomas, 2HG concentrations were a mean of 20 times higher than in pGBM and nongliomas (P ≤ .001) (Fig. 1). No differences were observed between the single groups of IDH1-mutated gliomas LGG1, LGG2, and sGBM in relation to 2HG concentration.

Fig. 1.  Dot-box and whisker plots show concentration ranges for TCA metabolites measured in IDH1-nonmutated (IDH1wt) and IDH1-mutated (IDH1mut) LGG and sGBM and in pGBM and nonglioma tumor specimens

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661092/bin/not00601.gif

To detect possible differences among the IDH1-mutated LGG1, LGG2, and sGBM, the α-KG/isocitrate and 2HG/isocitrate ratios were used in additional tests. Therefore, the direct precursor-product relation would correct for all differences possibly expected during pre-analytical processing. To prove this, analyte ratios ofIDH1-mutated and nonmutated gliomas were compared. IDH1-mutated gliomas showed a 2HG/isocitrate ratio that was 13 times higher (P ≤ .001) (Fig. 2A), which corresponds to a lower accumulation of 2HG inIDH1-nonmutated gliomas. α-KG/isocitrate ratios were determined to be approximately 10-fold higher inIDH1-mutated gliomas than in IDH1-nonmutated gliomas (P = .005) (Fig. 2B), which also implies lower accumulation of α-KG in IDH1-nonmutated gliomas.

2-hydroxyglutarate-to-isocitrate-ratios

2-hydroxyglutarate-to-isocitrate-ratios

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661092/bin/not00602.jpg

Fig. 2.  2-Hydroxyglutarate to isocitrate ratios (A) and α-ketoglutarate to isocitrate ratios (B) for IDH1-nonmutated (IDH1wt) and IDH1-mutated (IDH1mut) gliomas (LGG and sGBM); boxes span the 25th and 75th percentiles with median, and whiskers represent the 10th and 90th percentiles with points as outliers. Abbreviations: LGG, low-grade gliomas; sGBM, secondary glioblastomas.

2HG/isocitrate and α-KG/isocitrate ratios, respectively, were calculated in all 8 specimen groups (Fig. 3). In addition to the differences in 2HG/isocitrate ratios of IDH1-mutated and nonmutated gliomas (Fig. 2A), the ratios in IDH1-mutated gliomas were 4–9 times higher, compared with those in pGBM (P ≤ .001), and 3–6 times higher, compared with those in non-glioma tumor specimens, which was not statistically significant (Fig. 3A). In detail, ratios of 2HG and isocitrate were established to be 13, 9.4, and 22 times higher in IDH1-mutated LGG1, LGG2, and their consecutive sGBM, respectively, than in IDH1-nonmutated LGG1 (Fig. 3A). No significant differences were observed between IDH1-mutated gliomas and IDH1-nonmutated LGG2 and sGBM. The comparison of 2HG/isocitrate ratios between IDH1-nonmutated gliomas and IDH1-mutated LGG2 and sGBM showed no statistically significant differences. However, a trend toward higher ratios inIDH1-mutated LGG1/2 was seen. Furthermore, no differences could be determined by comparing 2HG/isocitrate ratios measured in the groups of IDH1-mutated LGG1 and LGG2. Although 2HG/isocitrate ratios in IDH1-mutated secondary glioblastomas are 1.7 and 2.3 times higher than in the LGG1 and LGG2 groups, respectively, no statistically significant differences were observed.   Fig. 3.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661092/bin/not00603.gif

The absence of a straight trend to higher 2HG/isocitrate ratios during malignant progression is shown by paired analysis of IDH1-mutated LGG2 and their consecutive sGBM (Fig. 3C). Similar findings were observed using the α-KG/isocitrate ratios. Although significant differences were found, with ratios approximately 10 times higher in IDH1-mutated glioblastomas than in IDH1-nonmutated glioblastomas (Fig. 2B), it was not possible to differentiate among the 3 IDH1-mutated glioblastoma groups LGG1, LGG2, and their consecutive sGBM with use of this analyte ratio (Fig. 3B and D).

On the basis of a comprehensive analysis of cellular TCA metabolites from several cohorts of patients with glioma and nonglioma, our study provides evidence that the level of 2HG accumulation is not suitable as an early biomarker for distinguishing patients with LGG in relation to their course of malignancy. To our knowledge, this is the first report of a paired analysis of 2HG levels in LGG and their consecutive sGBM showing stable 2HG accumulation during malignant progression. This fact assumes that malignant transformation of IDH-mutated LGG appears to be independent of their intracellular 2HG accumulation. Considering these results, we could not stratify patients with LGG into subgroups with distinct survival.

2.1.4.2 An Inhibitor of Mutant IDH1 Delays Growth and Promotes Differentiation of Glioma Cells

Rohle D1, Popovici-Muller J, Palaskas N, Turcan S, Grommes C, et al.
Science. 2013 May 3; 340(6132):626-30
http://dx.doi.org:/10.1126/science.1236062

The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant–but not IDH1-wild-type–glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.

Somatic mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified in multiple human cancers, including glioma (12), sarcoma (34), acute myeloid leukemia (56), and others. All mutations map to arginine residues in the catalytic pockets of IDH1 (R132) or IDH2 (R140 and R172) and confer on the enzymes a new activity: catalysis of alpha-ketoglutarate (2-OG) to the (R)-enantiomer of 2-hydroxyglutarate (R-2HG) (78). R-2HG is structurally similar to 2-OG and, due to its accumulation to millimolar concentrations in IDH1-mutant tumors, competitively inhibits 2-OG–dependent dioxygenases (9).

The mechanism by which mutant IDH1 contributes to the pathogenesis of human glioma remains incompletely understood. Mutations in IDH1 are found in 50 to 80% of human low-grade (WHO grade II) glioma, a disease that progresses to fatal WHO grade III (anaplastic glioma) and WHO grade IV (glioblastoma) tumors over the course of 3 to 15 years. IDH1 mutations appear to precede the occurrence of other mutations (10) and are associated with a distinctive gene-expression profile (“proneural” signature), DNA hypermethylation [CpG island methylator phenotype (CIMP)], and certain clinicopathological features (1113). When ectopically expressed in immortalized human astrocytes, R132H-IDH1 promotes the growth of these cells in soft agar (14) and induces epigenetic alterations found in IDH1-mutant human gliomas (15,16). However, no tumor formation was observed when R132H-IDH1 was expressed from the endogenousIDH1 locus in several cell types of the murine central nervous system (17).

To explore the role of mutant IDH1 in tumor maintenance, we used a compound that was identified in a high-throughput screen for compounds that inhibit the IDH1-R132H mutant homodimer (fig. S1 and supplementary materials) (18). This compound, subsequently referred to as AGI-5198 (Fig. 1A), potently inhibited mutant IDH1 [R132H-IDH1; half-maximal inhibitory concentration (IC50), 0.07 µM) but not wild-type IDH1 (IC50 > 100 µM) or any of the examined IDH2 isoforms (IC50 > 100 µM) (Fig. 1B). We observed no induction of nonspecific cell death at the highest examined concentration of AGI-5198 (20 µM).

Fig. 1 An R132H-IDH1 inhibitor blocks R-2HG production and soft-agar growth of IDH1-mutant glioma cells

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985613/bin/nihms504357f1.jpg

an-r132h-idh1-inhibitor-blocks-r-2hg-production-and-soft-agar-growth-of-idh1-mutant-glioma-cells

an-r132h-idh1-inhibitor-blocks-r-2hg-production-and-soft-agar-growth-of-idh1-mutant-glioma-cells

(A) Chemical structure of AGI-5198. (B) IC50 of AGI-5198 against different isoforms of IDH1 and IDH2, measured in vitro. (C) Sanger sequencing chromatogram (top) and comparative genomic hybridization profile array (bottom) of TS603 glioma cells. (D) AGI-5198 inhibits R-2HG production in R132H-IDH1 mutant TS603 glioma cells. Cells were treated for 2 days with AGI-5198, and R-2HG was measured in cell pellets. R-2HG concentrations are indicated above each bar (in mM). Error bars, mean ± SEM of triplicates. (E and F) AGI-5198 impairs soft-agar colony formation of (E) IDH1-mutant TS603 glioma cells [*P < 0.05, one-way analysis of variance (ANOVA)] but not (F) IDH1–wild-type glioma cell lines (TS676 and TS516). Error bars, mean ± SEM of triplicates.

We next explored the activity of AGI-5198 in TS603 glioma cells with an endogenous heterozygous R132H-IDH1 mutation, the most common IDH mutation in glioma (2). TS603 cells were derived from a patient with anaplastic oligodendroglioma (WHO grade III) and harbor another pathognomomic lesion for this glioma subtype, namely co-deletion of the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q) (19) (Fig. 1C). Measurements of R-2HG concentrations in pellets of TS603 glioma cells demonstrated dose-dependent inhibition of the mutant IDH1 enzyme by AGI-5198 (Fig. 1D). When added to TS603 glioma cells growing in soft agar, AGI-5198 inhibited colony formation by 40 to 60% (Fig. 1E). AGI-5198 did not impair colony formation of two patient-derived glioma lines that express only the wild-type IDH1allele (TS676 and TS516) (Fig. 1F), further supporting the selectivity of AGI-5198.

After exploratory pharmacokinetic studies in mice (fig. S2), we examined the effects of orally administered AGI-5198 on the growth of human glioma xenografts. When given daily to mice with established R132H-IDH1 glioma xenografts, AGI-5198 [450 mg per kg of weight (mg/kg) per os] caused 50 to 60% growth inhibition (Fig. 2A). Treatment was tolerated well with no signs of toxicity during 3 weeks of daily treatment (fig. S3). Tumors from AGI-5198– treated mice showed reduced staining with an antibody against the Ki-67 protein, a marker used for quantification of tumor cell proliferation in human brain tumors. In contrast, staining with an antibody against cleaved caspase-3 showed no differences between tumors from vehicle and AGI-5198–treated mice (fig. S4), suggesting that the growth-inhibitory effects of AGI-5198 were primarily due to impaired tumor cell proliferation rather than induction of apoptotic cell death. AGI-5198 did not affect the growth of IDH1 wild-type glioma xenografts (Fig. 2B).

Fig. 2 AGI-5198 impairs growth of IDH1-mutant glioma xenografts in mice

http://www.ncbi.nlm.nih.gov/corecgi/tileshop/tileshop.fcgi?p=PMC3&id=735048&s=43&r=3&c=2

AGI-5198 impairs growth of IDH1-mutant glioma xenografts in mice

AGI-5198 impairs growth of IDH1-mutant glioma xenografts in mice

Given the likely prominent role of R-2HG in the pathogenesis of IDH-mutant human cancers, we investigated whether intratumoral depletion of this metabolite would have similar growth inhibitory effects onR132H-IDH1-mutant glioma cells as AGI-5198. We engineered TS603 sublines in which IDH1–short hairpin RNA (shRNA) targeting sequences were expressed from a doxycycline-inducible cassette. Doxycycline had no effect on IDH1 protein levels in cells expressing the vector control but depleted IDH1 protein levels by 60 to 80% in cells infected with IDH1-shRNA targeting sequences (Fig. 2C). We next injected these cells into the flanks of mice with severe combined immunodeficiency and, after establishment of subcutaneous tumors, randomized the mice to receive either regular chow or doxycycline-containing chow. As predicted from our experiments with AGI-5198, doxycycline impaired the growth of TS603 glioma cells expressing inducible IDH1-shRNAs in soft agar (fig. S5) and in vivo (Fig. 2D) but had no effect on the growth of tumors expressing the vector control (fig. S6). Immunohistochemistry (IHC) with a mutant-specific R132H-IDH1 antibody confirmed depletion of the mutant IDH1 protein in IDH1-shRNA tumors treated with doxycycline. This was associated with an 80 to 90% reduction in intratumoral R-2HG levels, similar to the levels observed in TS603 glioma xenografts treated with AGI-5198 (fig. S7). Knockdown of the IDH1 protein in R132C-IDH1-mutant HT1080 sarcoma cells similarly impaired the growth of these cells in vitro and in vivo (fig. S8).

Fig. 3 AGI-5198 promotes astroglial differentiation in R132H-IDH1  mutant cells
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985613/bin/nihms504357f3.jpg

The gene-expression data suggested that treatment of IDH1-mutant glioma xenografts with AGI-5198 promotes a gene-expression program akin to gliogenic (i.e., astrocytic and oligodendrocytic) differentiation. To examine this question further, we treated TS603 glioma cells ex vivo with AGI-5198 and performed immunofluorescence for glial fibrillary acidic protein (GFAP) and nestin (NES) as markers for astrocytes and undifferentiated neuroprogenitor cells, respectively. .. We investigated whether blockade of mutant IDH1 could restore this ability, and this was indeed the case (Fig. 3D). These results indicate that mIDH1 plays an active role in restricting cellular differentiation potential, and this defect is acutely reversible by blockade of the mutant enzyme.

In the developing central nervous system, gliogenic differentiation is regulated through changes in DNA and histone methylation (24). Mutant IDH1 can affect both epigenetic processes through R-2HG mediated suppression of TET (ten-eleven translocation) methyl cytosine hydroxylases and Jumonji-C domain histone demethylases (JHDMs). We therefore sought to define the epigenetic changes that were associated with the acute growth-inhibitory effects of AGI-5198 in vivo. .. Treatment of mice with AGI-5198 resulted in dose-dependent reduction of intratumoral R-2HG with partial R-2HG reduction at the 150 mg/kg dose (0.85 ± 0.22 mM) and near-complete reduction at the 450 mg/kg dose (0.13 ± 0.03 mM) (Fig. 4A).

Fig. 4 Dose-dependent inhibition of histone methylation in IDH1-mutant gliomas after short term treatment with AGI-5198

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985613/bin/nihms504357f4.gif

We next examined whether acute pharmacological blockade of the mutant IDH1 enzyme reversed the CIMP, which is strongly associated with IDH1-mutant human gliomas (12). ..  On a genome-wide scale, we observed no statistically significant change in the distribution of β values between AGI-5198– and vehicle-treated tumors (Fig. 4B) (supplementary materials).
We next examined the kinetics of histone demethylation after inhibition of the mutant IDH1 enzyme. The histone demethylases JMJD2A and JMJD2C, which remove bi- and trimethyl marks from H3K9, are significantly more sensitive to inhibition by the R-2HG oncometabolite than other 2-OG–dependent oxygenases (891425). Restoring their enzymatic activity in IDH1-mutant cancer cells would thus be expected to require near-complete inhibition of R-2HG production. Consistent with this prediction, tumors from the 450 mg/kg AGI-5198 cohort showed a marked decrease in H3K9me3 staining, but there was no decrease in H3K9me3 staining in tumors from the 150 mg/kg AGI-5198 cohort (Fig. 4C) (fig. S11). Of note, AGI-5198 did not decrease H3K9 trimethylation in IDH1–wild-type glioma xenografts (fig. S12A) or in normal astrocytes (fig. S12B), demonstrating that the effect of AGI-5198 on histone methylation was not only dose-dependent but also IDH1-mutant selective.

Because the inability to erase repressive H3K9 methylation can be sufficient to impair cellular differentiation of nontransformed cells (16), we examined the TS603 xenograft tumors for changes in the RNA expression of astrocytic (GFAP, AQP4, and ATP1A2) and oligodendrocytic (CNP and NG2) differentiation markers by real-time polymerase chain reaction (RT-PCR). Compared with vehicletreated tumors, we observed an increase in the expression of astroglial differentiation genes only in tumors treated with 450 mg/kg AGI-5198 (Fig. 4D).

In summary, we describe a tool compound (AGI-5198) that impairs the growth of R132H-IDH1-mutant, but not IDH1 wild-type, glioma cells. This data demonstrates an important role of mutant IDH1 in tumor maintenance, in addition to its ability to promote transformation in certain cellular contexts (1426). Effector pathways of mutant IDH remain incompletely understood and may differ between tumor types, reflecting clinical differences between these disorders. Although much attention has been directed toward TET-family methyl cytosine hydroxylases and Jumonji-C domain histone demethylases, the family of 2-OG–dependent dioxygenases includes more than 50 members with diverse functions in collagen maturation, hypoxic sensing, lipid biosynthesis/metabolism, and regulation of gene expression (27).

2.1.4.3 Detection of oncogenic IDH1 mutations using MRS

OC Andronesi, O Rapalino, E Gerstner, A Chi, TT Batchelor, et al.
J Clin Invest. 2013;123(9):3659–3663
http://dx.doi.org:/10.1172/JCI67229

The investigation of metabolic pathways disturbed in isocitrate dehydrogenase (IDH) mutant tumors revealed that the hallmark metabolic alteration is the production of D-2-hydroxyglutarate (D-2HG). The biological impact of D-2HG strongly suggests that high levels of this metabolite may play a central role in propagating downstream the effects of mutant IDH, leading to malignant transformation of cells. Hence, D-2HG may be an ideal biomarker for both diagnosing and monitoring treatment response targeting IDH mutations. Magnetic resonance spectroscopy (MRS) is well suited to the task of noninvasive D-2HG detection, and there has been much interest in developing such methods. Here, we review recent efforts to translate methodology using MRS to reliably measure in vivo D-2HG into clinical research.

Recurrent heterozygous somatic mutations of the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes were recently found by genome-wide sequencing to be highly frequent (50%–80%) in human grade II–IV gliomas (12). IDH mutations are also often observed in several other cancers, including acute myeloid leukemia (3), central/periosteal chondrosarcoma and enchondroma (4), and intrahepatic cholangiocarcinoma (5). The identification of frequent IDH mutations in multiple cancers suggests that this pathway is involved in oncogenesis. Indeed, increasing evidence demonstrates that IDH mutations alter downstream epigenetic and genetic cellular signal transduction pathways in tumors (67). In gliomas, IDH1 mutations appear to define a distinct clinical subset of tumors, as these patients have a 2- to 4-fold longer median survival compared with patients with wild-type IDH1 gliomas (8). IDH1 mutations are especially common in secondary glioblastoma (GBM) arising from lower-grade gliomas, arguing that these mutations are early driver events in this disease (9). Despite aggressive therapy with surgery, radiation, and cytotoxic chemotherapy, average survival of patients with GBM is less than 2 years, and less than 10% of patients survive 5 years or more (10).

The discovery of cancer-related IDH1 mutations has raised hopes that this pathway can be targeted for therapeutic benefit (1112). Methods that can rapidly and noninvasively identify patients for clinical trials and determine the pharmacodynamic effect of candidate agents in patients enrolled in trials are particularly important to guide and accelerate the translation of these treatments from bench to bedside. Magnetic resonance spectroscopy (MRS) can play an important role in clinical and translational research because IDH mutated tumor cells have such a distinct molecular phenotype (13,14).

The family of IDH enzymes includes three isoforms: IDH1, which localizes in peroxisomes and cytoplasm, and IDH2 and IDH3, which localize in mitochondria as part of the tricarboxylic acid cycle (11). All three wild-type enzymes catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG), using the cofactor NADP+ (IDH1 and IDH2) or NAD+(IDH3) as the electron acceptor. To date, only mutations of IDH1 and IDH2 have been identified in human cancers (11), and only one allele is mutated. In gliomas, about 90% of IDH mutations involve a substitution in IDH1 in which arginine 132 (R132) from the catalytic site is replaced by a histidine (IDH1 R132H), known as the canonical IDH1 mutation (8). A number of noncanonical mutations such as IDH1 R132C, IDH1 R132S, IDH1 R132L, and IDH1 R132G are less frequently present. Arginine R172 in IDH2 is the corresponding residue to R132 in IDH1, and the most common mutation is IDH2 R172K. In addition to IDH2 R172K, IDH2 R140Q has also been observed in acute myeloid leukemia. Although most IDH1 mutations occur at R132, a small number of mutations producing D-2-hydroxyglutarate (D-2HG) occur at R100, G97, and Y139 (15). However, only a single residue is mutated in either IDH1 or IDH2 in a given tumor.

IDH mutations result in a very high accumulation of the oncometabolite D-2HG in the range of 5- to 35-mM levels, which is 2–3 orders of magnitude higher than D-2HG levels in tumors with wild-type IDH or in healthy tissue (13). All IDH1 G97, R100, R132, and Y139 and IDH2 R140 and R172 mutations confer a neomorphic activity to the IDH1/2 enzymes, switching their activity toward the reduction of αKG to D-2HG, using NADPH as a cofactor (15). The gain of function conferred by these mutations is possible because in each tumor cell a copy of the wild-type allele exists to supply the αKG substrate and NADPH cofactor for the mutated allele.

A cause and effect relationship between IDH mutation and tumorigenesis is probable, and D-2HG appears to play a pivotal role as the relay agent. Evidence is mounting that high levels of D-2HG alter the biology of tumor cells toward malignancy by influencing the activity of enzymes critical for regulating the metabolic (14) and epigenetic state of cells (671618). D-2HG may act as an oncometabolite via competitive inhibition of αKG-dependent dioxygenases (16). This includes inhibition of histone demethylases and 5-methlycytosine hydroxylases (e.g., TET2), leading to genome-wide alterations in histone and DNA hypermethylation as well as inhibition of hydroxylases, resulting in upregulation of HIF-1 (19). The effects of D-2HG have been shown to be reversible in leukemic transformation (18), which gives further evidence that treatments that lower D-2HG could be a valid therapeutic approach for IDH-mutant tumors. In addition to increased D-2HG, widespread metabolic disturbances of the cellular metabolome have been measured in cells with IDH mutations, including changes in amino acid concentration (increased levels of glycine, serine, threonine, among others, and decreased levels of aspartate and glutamate), N-acetylated amino acids (N-acetylaspartate, N-acetylserine, N-acetylthreonine), glutathione derivatives, choline metabolites, and TCA cycle intermediates (fumarate, malate) (14). These metabolic changes might be exploited for therapy. For example, IDH mutations cause a depletion of NADPH, which lowers the reductive capabilities of tumor cells (20) and perhaps makes them more susceptible to treatments that create free radicals (e.g., radiation) (21).

In vivo MRS of D-2HG in IDH mutant tumors

D-2HG may be an optimal biomarker for tumors with IDH mutations, as it ideally fulfills several important requirements: (a) there is virtually no normal D-2HG background — in cells without IDH mutations, D-2HG is produced as an error product of normal metabolism and is only present at trace levels; (b) 99% of tumors with IDH mutations have increased levels of D-2HG by several orders of magnitude; (c) the only other known cause of elevated 2HG is hydroxyglutaric aciduria (in this case, high L-2HG caused by a mutation in 2HG dehydrogenase), which is a rare inborn error of metabolism that presents with a different clinical phenotype and marked developmental anomalies in early childhood. Hence, tumors displaying increased levels of D-2HG are unlikely to represent false-positive cases for IDH mutations. Furthermore, this raises the possibility that D-2HG levels could also be used to quantify and predict the efficacy of drugs targeting mutant IDH1 for antitumor therapy (1115). In fact, it is hard to find a similar example of another tumor biomarker metabolite that is so well supported by the underlying biology.

The high levels of D-2HG observed in IDH1-mutant gliomas are amenable to detection by in vivo MRS. Given that the detection threshold of in vivo MRS is around 1 mM (1 μmol/g, wet tissue), D-2HG should be measurable only in situations in which it accumulates due to IDH1 mutations. Conversely, D-2HG is not expected to be detectable in tumors in which IDH1 is not mutated or in healthy tissues. In addition, ex vivo MRS measurements of intact biopsies (22) or extracts reach higher sensitivity 0.1–0.01 mM (0.1–0.01 μmol/g) and can be used as a cheaper and faster alternative to mass spectrometry.

Recently, reliable detection of D-2HG using in vivo 1H MRS was demonstrated in glioma patients (2930). Andronesi et al. reported the unambiguous detection of D-2HG in mutant IDH1 glioma in vivo using 2D correlation spectroscopy (COSY) and J-difference spectroscopy (29). In 2D COSY the overlapping signals are resolved along a second orthogonal chemical shift dimension (3132), and in the case of D-2HG, the cross-peaks resulting from the scalar coupling of Hα-Hβ protons show up in a region that is free of the contribution of other metabolites in both healthy and wild-type tumors. While 2D COSY retains all the metabolites in the spectrum, J-difference spectroscopy (2533) takes the opposite approach instead by focusing on the metabolite of interest, such as D-2HG, and selectively applying a narrow-band radiofrequency pulse to selectively refocus the Hα-Hβ scalar coupling evolution, then removing the contribution of overlapping metabolites. In this case a 1D difference spectrum with the Hα signal of D-2HG is detected at 4.02 ppm. Both methods have strengths and weaknesses: 2D COSY has the highest resolving power to disentangle overlapping metabolites, but has less sensitivity and quantification is more complex; J-difference spectroscopy has increased sensitivity, and quantification is straightforward, but it is susceptible to subtraction errors.

In Table 1, a comparison is made among the published methods for D-2HG detection. Results selected from the literature are shown in Figure 1. Besides the approaches discussed thus far, other methods are available in the in vivo MRS armamentarium that could be perhaps explored for reliable detection of 2D-HG, such as multiple-quantum filtering sequences (3435) and a variety of 2D spectroscopic methods (3639).

Table 1 Summary of in vivo 1H MRS methods used in the literature for detection of D-2HG in patients with mutant IDH glioma

http://dm5migu4zj3pb.cloudfront.net/manuscripts/67000/67229/small/JCI67229.t1.gif

Figure 1 In vivo D-2HG measurements: (A) J-difference spectroscopy with MEGA-LASER sequence in a patient with GBM with mutant IDH1. Adapted with permission from Science Translational Medicine (29). (B) Spectral editing with PRESS sequence of TE 97 ms (TE1: 32 ms, TE2: 65 ms) in a patient with mutant IDH1 oligodendroglioma. Adapted with permission from Nature Medicine (30). (C) Spectra acquired with PRESS sequence of TE 30 ms in a patient with mutant IDH1 anaplastic astrocytoma. Adapted with permission from Journal of Neuro-Oncology (24). Cho, choline; Cre, creatine; Gln, glutamine; Glu, glutamate; Lac, lactate; MM, macromolecules; NAA, N-acetyl- aspartate.

http://dm5migu4zj3pb.cloudfront.net/manuscripts/67000/67229/small/JCI67229.f1.gif

Ex vivo MRS of D-2HG in tumors with IDH mutations

The panoply of methods and ability of ex vivo MRS (50) to detect D-2HG in patient samples is far superior to in vivo MRS because the above list of limitations and artifacts is not of concern.

Metabolic profiling of intact tumor biopsies as small as 1 mg can be performed with high-resolution magic angle spinning (HRMAS) (5153). HRMAS preserves the integrity of the samples that can be further analyzed with immunohistochemistry, genomics, or other metabolic profiling tools such as mass spectrometry. Detection of D-2HG in mutant IDH1 glioma was confirmed by ex vivo HRMAS experiments (295455). In addition to D-2HG, ex vivo HRMAS studies can detect quantitative and qualitative changes for a large number of metabolites in IDH mutated tumors (5455).

The example of IDH1 mutations is a perfect illustration of the rapid pace of progress brought to the medical sciences by the power and advances of modern technology: genome-wide sequencing, metabolomics, and imaging.

In vivo MRS has the unique ability to noninvasively probe IDH mutations by measuring the endogenously produced oncometabolite D-2HG. As an imaging-based technique, it has the benefit of posing minimal risk to the patients, can be performed repeatedly as many times as necessary, and can probe tumor heterogeneity without disturbing the internal milieu. To date, in vivo MRS is the only imaging method that is specific to IDH mutations — existing PET or SPECT radiotracers are not specific (5657), IDH-targeted agents for in vivo molecular imaging do not yet exist, and the prohibitive cost of radiotracers will likely limit their clinical development.
2.1.4.4 Hypoxia promotes IDH-dependent carboxylation of α-KG to citrate to support cell growth and viability

DR Wise, PS Ward, JES Shay, JR Cross, Joshua J Grube, et al.
PNAS | Dec 6, 2011; 108(49):19611–19616
http://www.pnas.org/cgi/doi/10.1073/pnas.1117773108

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitantincreased synthesisof2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells areunable toproliferate.The reductive carboxylation ofglutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

Citrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O2), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (4, 5). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD+, which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.

This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (6, 7). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabolism based on his observation that hypoxic yeast cells preferred to convert glucose into lactic acid rather than burning it in an oxidative fashion. The molecular basis forthis shift in mammalian cells has been linked to the activity of the transcription factor HIF1 (8–10). Stabilization of the labile HIF1α subunit occurs in hypoxia. It can also occur in normoxia through several mechanisms including loss of the von Hippel-Lindau tumor suppressor (VHL), a common occurrence in renal carcinoma(11). Although hypoxia and/or HIF1α stabilization is a common feature of multiple cancers, to date the source of citrate in the setting of hypoxia or HIF activation has not been determined. Here, we study the sources of hypoxic citrate synthesis in a glioblastoma cell line that proliferates in profound hypoxia (0.5% O2). Glucose uptake and conversion to lactic acid increased in hypoxia. However, glucose conversion into citrate dramatically declined. Glutamine consumption remained constant in hypoxia, and hypoxic cells were addicted to the use of glutamine in hypoxia as a source of α-ketoglutarate. Glutamine provided the major carbon source for citrate synthesis during hypoxia. However, the TCA cycle-dependent conversion of glutamine into citric acid was significantly suppressed. In contrast, there was a relative increase in glutamine-dependent citrate production in hypoxia that resulted from carboxylation of α-ketoglutarate. This reductive synthesis required the presence of mitochondrial isocitrate dehydrogenase 2 (IDH2). In confirmation of the reverse flux through IDH2, the increased reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia was associated with increased synthesis of 2HG. Finally, constitutive HIF1α-expressing cells also demonstrated significant reductive carboxylation-dependent synthesis of citrate in normoxia and a relative defect in the oxidative conversion of glutamine into citrate. Collectively, the data demonstrate that mitochondrial glutaminemetabolismcanbereroutedthroughIDH2-dependent citrate synthesis in support of hypoxic cell growth.

Some Cancer Cells Can Proliferate at 0.5% O2 Despite a Sharp Decline in Glucose-Dependent Citrate Synthesis. At 21% O2, cancer cells have been shown to synthesize citrate by condensing glucose-derived acetyl-CoA with glutamine-derived oxaloacetate through the activity of the canonical TCA cycle enzyme citrate synthase (4). In contrast, less is known regarding the synthesis of citrate by cells that can continue proliferating in hypoxia. The glioblastoma cellline SF188 is able to proliferate at 0.5% O2 (Fig.1A),a level of hypoxia that is sufficient to stabilize HIF1α (Fig. 1B) and predicted to limit respiration (12, 13). Consistent with previous observations in hypoxic cells, we found that SF188 cells demonstrated increased lactate production when incubated in hypoxia
(Fig. 1C), and the ratio of lactate produced to glucose consumed increased demonstrating an increase in the rate of anaerobic glycolysis. When glucose-derived carbon in the form of pyruvate is converted to lactate, it is diverted away from subsequent metabolism that can contribute to citrate production. However, we observed that SF188 cells incubated in hypoxia maintain their intracellular citrate to ∼75% of the level maintained under normoxia (Fig. 1D). This prompted an investigation of how proliferating cells maintain citrate production under hypoxia. Increased glucose uptake and glycolytic metabolism are critical elements of the metabolic response to hypoxia. To evaluate the contributions made by glucose to the citrate pool under normoxia or hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 10 mM [U-13C] glucose. Following a 4-h labeling period, cellular metabolites were extracted and analyzed for isotopic enrichment.

Fig. 1. SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis. (A) SF188 cells were plated in complete medium equilibrated with 21% O2 (Normoxia) or 0.5% O2 (Hypoxia), total viable cells were counted 24 h and 48 h later (Day 1 and Day 2), and population doublings were calculated. Data are the mean ± SEM of four independent experiments. (B) Western blot demonstrates stabilized HIF1α protein in cells cultured in hypoxia compared with normoxia. (C) Cells were grown in normoxia or hypoxia for 24 h, after which culture medium was collected. Medium glucose and lactate levels were measured and compared with the levels in fresh medium. (D) Cells were cultured for 24 h as in C. Intracellular metabolism was then quenched with 80% MeOH prechilled to −80 °C that was spiked with a 13C-labeled citrate as an internal standard. Metabolites were then extracted, and intracellular citrate levels were analyzed with GC-MS and normalized to cell number. Data for C and D are the mean ± SEM of three independent experiments. (E) Model depicting the pathway for cit+2 production from [U-13C] glucose. Glucose uniformly 13Clabeled will generate pyruvate+3. Pyruvate+3 can be oxidatively decarboxylated by PDH to produce acetyl-CoA+2, which can condense with unlabeled oxaloacetate to produce cit+2. (F) Cells were cultured for 24 h as in C and D, followed by an additional 4 h of culture in glucose-deficient medium supplemented with 10 mM [U-13C]glucose. Intracellular metabolites were then extracted, and 13C-enrichment in cellular citrate was analyzed by GCMS and normalized to the total citrate pool size. Data are the mean ± SD of three independent cultures from a representative of two independent experiments. *P < 0.05, ***P < 0.001

Fig. 2. Glutamine carbon is required for hypoxic cell viability and contributes to increased citrate production through reductive carboxylation relative to oxidative metabolism in hypoxia. (A) SF188 cells were cultured for 24 h in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2 (Hypoxia). Culture medium was then removed from cells and analyzed for glutamine levels which were compared with the glutamine levels in fresh medium. Data are the mean ± SEM of three independent experiments. (B) The requirement for glutamine to maintain hypoxic cell viability can be satisfied by α-ketoglutarate. Cells were cultured in complete medium equilibrated with 0.5% O2 for 24 h, followed by an additional 48 h at 0.5% O2 in either complete medium (+Gln), glutamine-deficient medium (−Gln), or glutamine-deficient medium supplemented with 7 mM dimethyl α-ketoglutarate (−Gln +αKG). All medium was preconditioned in 0.5% O2. Cell viability was determined by trypan blue dye exclusion. Data are the mean and range from two independent experiments. (C) Model depicting the pathways for cit+4 and cit+5 production from [U-13C]glutamine (glutamine+5). Glutamine+5 is catabolized to α-ketoglutarate+5, which can then contribute to citrate production by two divergent pathways. Oxidative metabolism produces oxaloacetate+4, which can condense with unlabeled acetyl-CoA to produce cit+4. Alternatively, reductive carboxylation produces isocitrate+5, which can isomerize to cit+5. (D) Glutamine contributes to citrate production through increased reductive carboxylation relative to oxidative metabolism in hypoxic proliferating cancer cells. Cells were cultured for 24 h as in A, followed by 4 h of culture in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in cellular citrate was quantitated with GC-MS. Data are the mean ± SD of three independent cultures from a representative of three independent experiments. **P < 0.01.

Fig. 3. Cancer cells maintain production of other metabolites in addition to citrate through reductive carboxylation in hypoxia. (A) SF188 cells were cultured in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2 (Hypoxia) for 24 h. Intracellular metabolism was then quenched with 80% MeOH prechilled to −80 °C that was spiked with a 13C-labeled citrate as an internal standard. Metabolites were extracted, and intracellular aspartate (asp), malate (mal), and fumarate (fum) levels were analyzed with GC-MS. Data are the mean± SEM of three independent experiments. (B) Model for the generation of aspartate, malate, and fumarate isotopomers from [U-13C] glutamine (glutamine+5). Glutamine+5 is catabolized to α-ketoglutarate+5. Oxidative metabolism of α-ketoglutarate+5 produces fumarate+4, malate+4, and oxaloacetate (OAA)+4 (OAA+ 4 is in equilibrium with aspartate+4 via transamination). Alternatively, α-ketoglutarate+5 can be reductively carboxylated to generate isocitrate+5 and citrate+5. Cleavage of citrate+5 in the cytosol by ATP-citrate lyase (ACL) will produce oxaloacetate+3 (in equilibrium with aspartate+3). Oxaloacetate+3 can be metabolized to malate+3 and fumarate+3. (C) SF188 cells were cultured for 24 h as in A, and then cultured for an additional 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C] glutamine. 13C enrichment in cellular aspartate, malate, and fumarate was determined by GC-MS and normalized to the relevant metabolite total pool size. Data shown are the mean ± SD of three independent cultures from a representative of three independent experiments. **P < 0.01, ***P < 0.001.

Glutamine Carbon Metabolism Is Required for Viability in Hypoxia. In addition to glucose, we have previously reported that glutamine can contribute to citrate production during cell growth under normoxic conditions (4). Surprisingly, under hypoxic conditions, we observed that SF188 cells retained their high rate of glutamine consumption (Fig. 2A). Moreover, hypoxic cells cultured in glutamine-deficient medium displayed a significant loss of viability (Fig. 2B). In normoxia, the requirement for glutamine to maintain viability of SF188 cells can be satisfied by α-ketoglutarate, the downstream metabolite of glutamine that is devoid of nitrogenous groups (14). α-ketoglutarate cannot fulfill glutamine’s roles as a nitrogen source for nonessential amino acid synthesis or as an amide donor for nucleotide or hexosamine synthesis, but can be metabolized through the oxidative TCA cycle to regenerate oxaloacetate, and subsequently condense with glucose-derived acetyl-CoA to produce citrate. To test whether the restoration of carbon from glutamine metabolism in the form of α-ketoglutarate could rescue the viability defect of glutamine-starved SF188 cells even under hypoxia, SF188 cells incubated in hypoxia were cultured in glutamine-deficient medium supplemented with a cell-penetrant form of α-ketoglutarate (dimethyl α-ketoglutarate). The addition of dimethyl α-ketoglutarate rescued the defect in cell viability observed upon glutamine withdrawal (Fig. 2B). These data demonstrate that, even under hypoxic conditions, when the ability of glutamine to replenish oxaloacetate through oxidative TCA cycle metabolism is diminished, SF188 cells retain their requirement for glutamine as the carbon backbone for α-ketoglutarate. This result raised the possibility that glutamine could be the carbon source for citrate production through an alternative, nonoxidative, pathway in hypoxia.

Cells Proliferating in Hypoxia Preferentially Produce Citrate Through Reductive Carboxylation Rather than Oxidative Metabolism. To distinguish the pathways by which glutamine carbon contributes to citrate production in normoxia and hypoxia, SF188 cells were incubated in normoxia or hypoxia and cultured in medium containing 4 mM [U-13C] glutamine. After 4 h of labeling, intracellular metabolites were extracted and analyzed by GC-MS. In normoxia,the cit+4 pool constituted the majority of the enriched citrate in the cell. Cit+4 arises from the oxidative metabolism of glutamine-derived α-ketoglutarate+5 to oxaloacetate+4 and its subsequent condensation with unenriched, glucose-derived acetyl-CoA (Fig.2C and D). Cit+5 constituted a significantly smaller pool than cit+4 in normoxia. Conversely, in hypoxia, cit+5 constituted the majority of the enriched citrate in the cell. Cit+5 arises from the reductive carboxylation of glutamine-derived α-ketoglutarate+5 to isocitrate+5, followed by the isomerization of isocitrate+5 to cit+5 by aconitase. The contribution of cit+4 to the total citrate pool was significantly lower in hypoxia than normoxia, and the accumulation of other enriched citrate species in hypoxia remained low. These data support the role of glutamine as a carbon source for citrate production in normoxia and hypoxia.

Cells Proliferating in Hypoxia Maintain Levels of Additional Metabolites Through Reductive Carboxylation. Previous work has documented that, in normoxic conditions, SF188 cells use glutamine as the primary anaplerotic substrate, maintaining the pool sizes of TCA cycle intermediates through oxidative metabolism (4). Surprisingly, we found that, when incubated in hypoxia, SF188 cells largely maintained their levels of aspartate (in equilibrium with oxaloacetate), malate, and fumarate (Fig. 3A). To distinguish how glutamine carbon contributes to these metabolites in normoxia and hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 4 mM [U-13C] glutamine. After a 4-h labeling period, metabolites were extracted and the intracellular pools of aspartate, malate, and fumarate were analyzed by GC-MS. In normoxia, the majority of the enriched intracellular asparatate, malate, and fumarate were the +4 species, which arise through oxidative metabolism of glutamine-derived α-ketoglutarate (Fig. 3 B and C). The +3 species, which can be derived from the citrate generated by the reductive carboxylation of glutamine derived α-ketoglutarate, constituted a significantly lower percentage of the total aspartate, malate, and fumarate pools. By contrast, in hypoxia, the +3 species constituted a larger percentage of the total aspartate, malate, and fumarate pools than they did in normoxia. These data demonstrate that, in addition to citrate, hypoxic cells preferentially synthesize oxaloacetate, malate, and fumarate through the pathway of reductive carboxylation rather than the oxidative TCA cycle.

IDH2 Is Critical in Hypoxia for Reductive Metabolism of Glutamine and for Cell Proliferation.We hypothesized that the relative increase in reductive carboxylation we observed in hypoxia could arise from the suppression of α-ketoglutarate oxidation through the TCA cycle. Consistent with this, we found that α-ketoglutarate levels increased in SF188 cells following 24 h in hypoxia (Fig. 4A). Surprisingly, we also found that levels of the closely related metabolite 2-hydroxyglutarate (2HG) increased in hypoxia, concomitant with the increase in α-ketoglutarate under these conditions. 2HG can arise from the noncarboxylating reduction of α-ketoglutarate (Fig. 4B). Recent work has found that specific cancer-associated mutations in the active sites of either IDH1 or IDH2 lead to a 10- to 100-fold enhancement in this activity facilitating 2HG production (15–17), but SF188 cells lack IDH1/2 mutations. However, 2HG levels are also substantially elevated in the inborn error of metabolism 2HG aciduria, and the majority of patients with this disease lack IDH1/2 mutations. As 2HG has been demonstrated to arise in these patients from mitochondrial α-ketoglutarate (18), we hypothesized that both the increased reductive carboxylation of glutamine-derived α-ketoglutarate to citrate and the increased 2HG accumulation we observed in hypoxia could arise from increased reductive metabolism by wild-type IDH2 in the mitochondria.

Fig. 4. Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2. (A) α-ketoglutarate and 2HG increase in hypoxia. SF188 cells were cultured in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2 (Hypoxia) for 24 h. Intracellular metabolites were then extracted, cell extracts spiked with a 13C-labeled citrate as an internal standard, and intracellular α-ketoglutarate and 2HG levels were analyzed with GC-MS. Data shown are the mean ± SEM of three independent experiments. (B) Model for reductive metabolism from glutamine-derived α-ketoglutarate. Glutamine+5 is catabolized to α-ketoglutarate+5. Carboxylation of α-ketoglutarate+5 followed by reduction of the carboxylated intermediate (reductive carboxylation) will produce isocitrate+5, which can then isomerize to cit+5. In contrast, reductive activity on α-ketoglutarate+5 that is uncoupled from carboxylation will produce 2HG+5. (C) IDH2 is required for reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia. SF188 cells transfected with a siRNA against IDH2 (siIDH2) or nontargeting negative control (siCTRL) were cultured for 2 d in complete medium equilibrated with 0.5% O2.(Upper) Cells were then cultured at 0.5% O2 for an additional 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in intracellular citrate and 2HG was determined and normalized to the relevant metabolite total pool size. (Lower) Cells transfected and cultured in parallel at 0.5% O2 were counted by hemocytometer (excluding nonviable cells with trypan blue staining) or harvested for protein to assess IDH2 expression by Western blot. Data shown for GC-MS and cell counts are the mean ± SD of three independent cultures from a representative experiment. **P < 0.01, ***P < 0.001.

Reprogramming of Metabolism by HIF1 in the Absence of Hypoxia Is Sufficient to Induce Increased Citrate Synthesis by Reductive Carboxylation Relative to Oxidative Metabolism. The relative increase in the reductive metabolism of glutamine-derived α-ketoglutarate at 0.5% O2 may be explained by the decreased ability to carry out oxidative NAD+-dependent reactions as respiration is inhibited (12, 13). However, a shift to preferential reductive glutamine metabolism could also result from the active reprogramming of cellular metabolism by HIF1 (8–10), which inhibits the generation of mitochondrial acetyl-CoA necessary for the synthesis of citrate by oxidative glucose and glutamine metabolism (Fig. 5A). To better understand the role of HIF1 in reductive glutamine metabolism, we used VHL-deficient RCC4 cells, which display constitutive expression of HIF1α under normoxia (Fig. 5B).

Fig. 5. Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate. (A) Model depicting how HIF1 signaling’s inhibition of pyruvate dehydrogenase (PDH) activity and promotion of lactate dehydrogenase-A (LDH-A) activity can block the generation of mitochondrial acetyl-CoA from glucose-derived pyruvate, thereby favoring citrate synthesis from reductive carboxylation of glutamine-derived α-ketoglutarate. (B) Western blot demonstrating HIF1α protein in RCC4 VHL−/− cells in normoxia with a nontargeting shRNA (shCTRL), and the decrease in HIF1α protein in RCC4 VHL−/− cells stably expressing HIF1α shRNA (shHIF1α). (C) HIF1-induced reprogramming of glutamine metabolism. Cells from B at 21% O2 were cultured for 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. Intracellular metabolites were then extracted, and 13C enrichment in cellular citrate was determined by GC-MS. Data shown are the mean ± SD of three independent cultures from a representative of three independent experiments. ***P < 0.001.

Compared with glucose metabolism, much less is known regarding how glutamine metabolism is altered under hypoxia. It has also remained unclear how hypoxic cells can maintain the citrate production necessary for macromolecular biosynthesis. In this report, we demonstrate that in contrast to cells at 21% O2, where citrate is predominantly synthesized through oxidative metabolism of both glucose and glutamine, reductive carboxylation of glutamine carbon becomes the major pathway of citrate synthesis in cells that can effectively proliferate at 0.5% O2. Moreover, we show that in these hypoxic cells, reductive carboxylation of glutamine-derived α-ketoglutarate is dependent on mitochondrial IDH2. Although others have previously suggested the existence of reductive carboxylation in cancer cells (19, 20), these studies failed to demonstrate the intracellular localization or specific IDH isoform responsible for the reductive carboxylation flux. Recently, we identified IDH2 as an isoform that contributes to reductive carboxylation in cancer cells incubated at 21% O2 (16), but remaining unclear were the physiological importance and regulation of this pathway relative to oxidative metabolism, as well as the conditions where this reductive pathway might be advantageous for proliferating cells. Here we report that IDH2-mediated reductive carboxylation of glutamine-derived α-ketoglutarate to citrate is an important feature of cells proliferating in hypoxia. Moreover, the reliance on reductive glutamine metabolism can be recapitulated in normoxia by constitutive HIF1 activation in cells with loss of VHL. The mitochondrial NADPH/NADP+ ratio required to fuel the reductive reaction through IDH2 can arise from the increased NADH/NAD+ ratio existing in the mitochondria under hypoxic conditions (21, 22), with the transfer of electrons from NADH to NADP+ to generate NADPH occurring through the activity of the mitochondrial transhydrogenase (23).

In further support of the increased mitochondrial reductive glutamine metabolism that we observe in hypoxia, we report here that incubation in hypoxia can lead to elevated 2HG levels in cells lacking IDH1/2 mutations. 2HG production from glutamine-derived α-ketoglutarate significantly decreased with knockdown of IDH2, supporting the conclusion that 2HG is produced in hypoxia by enhanced reverse flux of α-ketoglutarate through IDH2in a truncated, noncarboxylating reductive reaction. However,other mechanisms may also contribute to 2HG elevation in hypoxia. These include diminished oxidative activity and/or enhanced reductive activity of the 2HG dehydrogenase, a mitochondrial enzyme that normally functions to oxidize 2HG back to α-ketoglutarate (25). The level of 2HG elevation we observe in hypoxic cells is associated with a concomitant increase in α-ketoglutarate, and is modest relative to that observed in cancers with IDH1/2 gain-of-function mutations. Nonetheless, 2HG elevation resulting from hypoxia in cells with wild-type IDH1/2 may hold promise as a cellular or serum biomarker for tissues undergoing chronic hypoxia and/or excessive glutamine metabolism.

2.1.4.5 IDH mutation impairs histone demethylation and results in a block to cell differentiation.

C Lu, PS Ward, GS Kapoor, D Rohle, S Turcan, et al.
Nature 483, 474–478 (22 Mar 2012)
http://dx.doi.org:/10.1038/nature10860

Recurrent mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been identified in gliomas, acute myeloid leukaemias (AML) and chondrosarcomas, and share a novel enzymatic property of producing 2-hydroxyglutarate (2HG) from α-ketoglutarate1, 2, 3, 4, 5, 6. Here we report that 2HG-producing IDH mutants can prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. In tumour samples from glioma patients, IDH mutations were associated with a distinct gene expression profile enriched for genes expressed in neural progenitor cells, and this was associated with increased histone methylation. To test whether the ability of IDH mutants to promote histone methylation contributes to a block in cell differentiation in non-transformed cells, we tested the effect of neomorphic IDH mutants on adipocyte differentiation in vitro. Introduction of either mutant IDH or cell-permeable 2HG was associated with repression of the inducible expression of lineage-specific differentiation genes and a block to differentiation. This correlated with a significant increase in repressive histone methylation marks without observable changes in promoter DNA methylation. Gliomas were found to have elevated levels of similar histone repressive marks. Stable transfection of a 2HG-producing mutant IDH into immortalized astrocytes resulted in progressive accumulation of histone methylation. Of the marks examined, increased H3K9 methylation reproducibly preceded a rise in DNA methylation as cells were passaged in culture. Furthermore, we found that the 2HG-inhibitable H3K9 demethylase KDM4C was induced during adipocyte differentiation, and that RNA-interference suppression of KDM4C was sufficient to block differentiation. Together these data demonstrate that 2HG can inhibit histone demethylation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.

Figure 1: IDH mutations are associated with dysregulation of glial differentiation and global histone methylation.

http://www.nature.com/nature/journal/v483/n7390/carousel/nature10860-f1.2.jpg

Figure 2: Differentiation arrest induced by mutant IDH or 2HG is associated with increased global and promoter-specific H3K9 and H3K27 methylation.

http://www.nature.com/nature/journal/v483/n7390/carousel/nature10860-f2.2.jpg

Figure 3: IDH mutation induces histone methylation increase in CNS-derived cells and can alter cell lineage gene expression.

http://www.nature.com/nature/journal/v483/n7390/carousel/nature10860-f3.2.jpg
2.1.4.6 Isocitrate dehydrogenase mutations in leukemia

McKenney AS, Levine RL.
J Clin Invest. 2013 Sep; 123(9):3672-7
http://dx.doi.org:/1172/JCI67266

Recent genome-wide discovery studies have identified a spectrum of mutations in different malignancies and have led to the elucidation of novel pathways that contribute to oncogenic transformation. The discovery of mutations in the genes encoding isocitrate dehydrogenase (IDH) has uncovered a critical role for altered metabolism in oncogenesis, and the neomorphic, oncogenic function of IDH mutations affects several epigenetic and gene regulatory pathways. Here we discuss the relevance of IDH mutations to leukemia pathogenesis, therapy, and outcome and how mutations in IDH1 and IDH2 affect the leukemia epigenome, hematopoietic differentiation, and clinical outcome.

Mutations in isocitrate dehydrogenase (IDH) have been identified in a spectrum of human malignancies. Mutations in IDH1 were first identified in an exome resequencing analysis of patients with colorectal cancer (1). Shortly thereafter, recurrent IDH1 and IDH2 mutations were found in patients with glioma, most commonly in patients who present with lower-grade gliomas (2). IDH1 mutations were subsequently discovered in patients with acute myeloid leukemia (AML) through whole genome sequencing (3), which was followed by the identification of somatic IDH2 mutations in patients with AML (46). Further studies revealed that IDH mutations induce a neomorphic function to produce the oncometabolite 2-hydroxyglutarate (2HG) (78), which can inhibit many cellular processes (910). In particular, the ability of 2HG to alter the epigenetic landscape makes IDH a prototypical target for prognostic studies and drug targeting in leukemias.

IDH proteins catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG, also known as 2-oxoglutarate). IDH3 primarily functions as the allosterically regulated, rate-limiting enzymatic step in the TCA cycle, while the other two isoforms, which are mutated in cancer, utilize this catalytic process in additional contexts including metabolism and glucose sensing (IDH1) and regulation of oxidative respiration (IDH2) (1112). Loss-of-function mutations in other TCA cycle components have previously been identified in other types of cancer, specifically in mutations in fumarate hydratase (FH) and succinate dehydrogenase (SDH). As such, many hypothesized that IDH1/2 mutations would result in loss of metabolic activity, and indeed, enzymatic studies confirmed that the mutant protein’s ability to perform its native function is markedly attenuated, as measured by reduced production of αKG or NADPH (1314).

However, the genetic data relating to these mutations were more consistent with gain-of-function mutation: all of the observed alterations are somatic, heterozygous mutations that occur at highly conserved positions, which appear to be functionally equivalent between different isoforms. This discrepancy was resolved when metabolic profiling showed that the IDH1 mutant protein catalyzes a neomorphic reaction that converts αKG to 2HG. 2HG can be detected at high levels in gliomas harboring these mutations (4), and the accumulation of 2HG was further found to be common to oncogenic IDH mutations (8). This finding indicated that 2HG may serve as a potential functional biomarker of IDH mutation, and later, metabolomics analysis of 2HG content in patient samples led to the identification of IDH2 mutations in leukemias (6). IDH mutant proteins have been proposed to form a heterodimer with the remaining wild-type IDH isoform (7814), which is consistent with genetic data showing retention of the wild-type allele in IDH-mutant cancers.

The discovery of the neomorphic function of IDH opened the doors for true investigation into the implications of these mutations and the resultant intracellular accumulation of 2HG. 2HG is thought to competitively inhibit the activity of a broad spectrum of αKG-dependent enzymes with known and postulated roles in oncogenic transformation. Some targets, such as the prolyl 4-hydroxylases, have unclear implications in leukemia pathogenesis. However, the recent demonstration that alterations in epigenetic factors occur in the majority of acute leukemias led to investigations of the effects of 2HG on the jumonji C domain histone-modifying enzymes and the newly characterized tet methylcytosine dioxygenase (TET) family of methylcytosine hydroxylases. Importantly, expression of IDH or exposure to chemically modified, cell-permeable 2HG affects hematopoietic differentiation, likely due to changes in epigenetic regulation that induce reversible alterations in differentiation states (15).

TET1 was initially discovered as a binding partner of mixed-lineage leukemia (MLL) in patients with MLL-translocated AML (1617). However, the function of the TET gene family and its role in leukemogenesis remained unknown until TET1 was shown to catalyze αKG-dependent addition of a hydroxyl group to methylated cytosines (18), which precedes DNA demethylation and results in altered epigenetic control (10,1824). TET enzymes have further been shown to catalyze conversion of 5-methylcytosine (5mC) to 5-formylcytosine (5fC) or 5-carboxylcytosine (5cC) (2526). These data suggest that loss of TET2 enzymatic function can lead to aberrant cytosine methylation and epigenetic silencing in malignant settings. TET2mutations were initially found in array-comparative genomic hybridization and genome-wide SNP arrays, which identified microdeletions containing this gene in a patient with myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS) (27). This discovery was followed by the identification of somatic missense, nonsense, and frameshift TET2 mutations in patients with MDS, MPN, AML, and other myeloid malignancies (2730). Most TET2 alleles result in nonsense/frameshift mutations, which result in loss of TET2 catalytic function (31), consistent with a tumor suppressor function in myeloid malignancies.

When 2HG was hypothesized to affect specific enzymatic processes in oncogenesis, AML patients were observed to harbor IDH1/2 and TET mutations in a mutually exclusive manner (9). Of note, exploration into the functional relationship between these mutant IDH proteins and the function of TET2 ultimately suggested a role for 2HG in inhibiting TET enzymatic function. IDH- or TET2-mutant patient samples are characterized by increased global hypermethylation of DNA and transcriptional silencing of genes with hypermethylated promoters. Expression of these IDH-mutant alleles in experimental models was further observed to result in increased methylation, reduced hydroxymethylation, and impaired TET2 function (9). Finally, in biochemical assays, 2HG was shown to directly inhibit TET2 as well as other αKG-dependent enzymes (10). These data demonstrate that a key feature of IDH1/2 mutations in hematopoietic cells is to impair TET2 function and disrupt DNA methylation (​Figure1).

Figure 1 Normal IDH functions to convert isocitrate to αKG in the Krebs cycle.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754251/bin/JCI67266.f1.gif

mutations have been observed with IDH1_2 mutations leukemias

mutations have been observed with IDH1_2 mutations leukemias

Many mutations have been observed in conjunction with IDH1/2 mutations in different types of leukemia.

In de novo adult AML, these mutations should be observed in the context of other prognostic indicators such as CEBPA, NPM1, and DNMT3A mutation. In AML that progresses from MPN, IDH1/2 mutations can be examined separately from the mutations responsible for MPN (such as JAK2 or MPL mutations) using paired pre- and post-transformation samples. Evidence supports a role for IDH1/2 hotspot mutations in leukemic transformation.

http://www.ncbi.nlm.nih.gov/pmc/articles/instance/3754251/bin/JCI67266.f2.gif

Conditional loss of Tet2 expression in mice results in a chronic myelomonocytic leukemia (CMML) phenotype and in increased hematopoietic self-renewal in vivo (32). Of note, in vitro systems have shown that TET2 silencing and expression of IDH1/2 mutant alleles leads to impaired hematopoietic differentiation and expansion of stem/progenitor cells (9). More recently, IDH1 (R132H) conditional knockin mice with hematopoietic-specific recombination were analyzed and found to have myeloid expansion, although they did not develop overt AML. This suggests that IDH mutations by themselves cannot promote overt transformation, and that additional genetic, epigenetic, and/or microenvironmental factors are needed to cooperate with mutant IDH alleles to promote hematologic malignancies. The hematopoietic defects included increased numbers of hematopoietic stem cells and myeloid progenitor cells, and a DNA methylation signature that was similar to observed patterns in primary AML patients with IDH1 mutations (33). While many models of IDH-mutant leukemia have shown potential, future models that incorporate the complexity seen in human patients are needed, as discussed below. More recently, the effects of IDH1/2 mutations on hematopoietic cell lines were replicated using exogenously applied 2HG, which was rendered permeable to the cell membrane by esterification. The Kaelin group used this system to dissect the role of 2HG in the αKG-dependent pathways that may be affected in IDH mutation, and to show that the effects are reversible (34). Tools such as these will help advance our understanding of the biology of IDH mutations and, by extension, the potential therapies that may affect mutant IDH and the downstream pathways. Indeed, given the recent description of mutant-selective IDH1/2 inhibitors (3437), the development of genetically accurate models of IDH mutant–mediated leukemogenesis will be critical to evaluate the effects of targeted therapies in mice with AML and subsequently in the clinical context.

2.1.4.7 The Common Feature of Leukemia-Associated IDH1 and IDH2 Mutations – a Neomorphic Enzyme Activity Converting α-Ketoglutarate to 2-Hydroxyglutarate

PS Ward, J Patel, DR Wise, O Abdel-Wahab, BD Bennett, HA Coller, et al.
Cancer Cell 2010 Mar 16; 17(3):225–234
http://dx.doi.org/10.1016/j.ccr.2010.01.020

Highlights

  • All IDH mutations reported in cancer share a common neomorphic enzymatic activity
  • Both wild-type IDH1 and IDH2 are required for cell proliferation
  • IDH2 R140Q mutations occur in 9% of AML cases
  • Overall, IDH2 mutations appear more common than IDH1 mutations in AML

 

Summary

The somatic mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) observed in gliomas can lead to the production of 2-hydroxyglutarate (2HG). Here, we report that tumor 2HG is elevated in a high percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Surprisingly, less than half of cases with elevated 2HG possessed IDH1 mutations. The remaining cases with elevated 2HG had mutations in IDH2, the mitochondrial homolog of IDH1. These data demonstrate that a shared feature of all cancer-associated IDH mutations is production of the oncometabolite 2HG. Furthermore, AML patients with IDH mutations display a significantly reduced number of other well characterized AML-associated mutations and/or associated chromosomal abnormalities, potentially implicating IDH mutation in a distinct mechanism of AML pathogenesis.

Significance

Most cancer-associated enzyme mutations result in either catalytic inactivation or constitutive activation. Here we report that the common feature of IDH1 and IDH2 mutations observed in AML and glioma is the acquisition of an enzymatic activity not shared by either wild-type enzyme. The product of this neomorphic enzyme activity can be readily detected in tumor samples, and we show that tumor metabolite analysis can identify patients with tumor-associated IDH mutations. Using this method, we discovered a 2HG-producing IDH2 mutation, IDH2 R140Q, that was present in 9% of serial AML samples. Overall, IDH1 and IDH2 mutations were observed in over 23% of AML patients.

Mutations in human cytosolic isocitrate dehydrogenase I (IDH1) occur somatically in > 70% of grade II-III gliomas and secondary glioblastomas, and in 8.5% of acute myeloid leukemias (AML) (Mardis et al., 2009 and Yan et al., 2009). Mutations have also been reported in cancers of the colon and prostate (Kang et al., 2009 and Sjoblom et al., 2006). To date, all reported IDH1 mutations result in an amino acid substitution at a single arginine residue in the enzyme’s active site, R132. A subset of intermediate grade gliomas lacking mutations in IDH1 has been found to harbor mutations in IDH2, the mitochondrial homolog of IDH1. The IDH2 mutations that have been identified in gliomas occur at the analogous residue to IDH1 R132, IDH2 R172. Both IDH1 R132 and IDH2 R172 mutants lack the wild-type enzyme’s ability to convert isocitrate to α-ketoglutarate (Yan et al., 2009). To date, all reported IDH1 or IDH2 mutations are heterozygous, with the cancer cells retaining one wild-type copy of the relevant IDH1 or IDH2 allele. No patient has been reported with both an IDH1 and IDH2 mutation. These data argue against the IDH mutations resulting in a simple loss of function.

Normally both cytosolic IDH1 and mitochondrial IDH2 exist as homodimers within their respective cellular compartments, and the mutant proteins retain the ability to bind to their respective wild-type partner. Therefore, it has been proposed that mutant IDH1 can act as a dominant negative against wild-type IDH1 function, resulting in a decrease in cytosolic α-ketoglutarate levels and leading to an indirect activation of the HIF-1α pathway (Zhao et al., 2009). However, recent work has provided an alternative explanation. The R132H IDH1 mutation observed in gliomas was found to display a gain of function for the NADPH-dependent reduction of α-ketoglutarate to R(–)-2-hydroxyglutarate (2HG) ( Dang et al., 2009). This in vitro activity was confirmed when 2HG was found to be elevated in IDH1-mutated gliomas. Whether this neomorphic activity is a common feature shared by IDH2 mutations was not determined.

IDH1 R132 mutations identical to those reported to produce 2HG in gliomas were recently reported in AML (Mardis et al., 2009). These IDH1 R132 mutations were observed in 8.5% of AML patients studied, and a significantly higher percentage of mutation was observed in the subset of patients whose tumors lacked cytogenetic abnormalities. IDH2 R172 mutations were not observed in this study. However, during efforts to confirm and extend these findings, we found an IDH2 R172K mutation in an AML sample obtained from a 77-year-old woman. This finding confirmed that both IDH1 and IDH2 mutations can occur in AML and prompted us to more comprehensively investigate the role of IDH2 in AML.

The present study was undertaken to see if IDH2 mutations might share the same neomorphic activity as recently reported for glioma-associated IDH1 R132 mutations. We also determined whether tumor-associated 2HG elevation could prospectively identify AML patients with mutations in IDH. To investigate the lack of reduction to homozygosity for either IDH1 or IDH2 mutations in tumor samples, the ability of wild-type IDH1 and/or IDH2 to contribute to cell proliferation was examined.

IDH2 Is Mutated in AML

A recent study employing a whole-genome sequencing strategy in an AML patient resulted in the identification of somatic IDH1 mutations in AML (Mardis et al., 2009). Based on the report that IDH2 mutations were also observed in the other major tumor type in which IDH1 mutations were implicated (Yan et al., 2009), we sequenced the IDH2 gene in a set of de-identified AML DNA samples. Several cases with IDH2 R172 mutations were identified. In the initial case, the IDH2 mutation found, R172K, was the same mutation reported in glioma samples. It has been recently reported that cancer-associated IDH1 R132 mutants display a loss-of-function for the use of isocitrate as substrate, with a concomitant gain-of-function for the reduction of α-ketoglutarate to 2HG (Dang et al., 2009). This prompted us to determine if the recurrent R172K mutation in IDH2 observed in both gliomas and leukemias might also display the same neomorphic activity. In IDH1, the role of R132 in determining IDH1 enzymatic activity is consistent with the stabilizing charge interaction of its guanidinium moiety with the β-carboxyl group of isocitrate (Figure 1A). This β-carboxyl is critical for IDH’s ability to catalyze the interconversion of isocitrate and α-ketoglutarate, with the overall reaction occurring in two steps through a β-carboxyl-containing intermediate (Ehrlich and Colman, 1976). Proceeding in the oxidative direction, this β-carboxyl remains on the substrate throughout the IDH reaction until the final decarboxylating step which produces α-ketoglutarate.

IDH1 R132 and IDH2 R172 Are Analogous Residues

IDH1 R132 and IDH2 R172 Are Analogous Residues

http://ars.els-cdn.com/content/image/1-s2.0-S153561081000036X-gr1.jpg

Figure 1. IDH1 R132 and IDH2 R172 Are Analogous Residues that Both Interact with the β-Carboxyl of Isocitrate

(A) Active site of crystallized human IDH1 with isocitrate.

(B) Active site of human IDH2 with isocitrate, modeled based on the highly homologous and crystallized pig IDH2 structure. For (A) and (B), carbon 6 of isocitrate containing the β-carboxyl is highlighted in cyan, with remaining isocitrate carbons shown in yellow. Carbon atoms of amino acids (green), amines (blue), and oxygens (red) are also shown. Hydrogen atoms are omitted from the figure for clarity. Dashed lines depict interactions < 3.1 Å, corresponding to hydrogen and ionic bonds. Residues coming from the other monomer of the IDH dimer are denoted with a prime (′) symbol.

To understand how R172 mutations in IDH2 might relate to the R132 mutations in IDH1 characterized for gliomas, we modeled human IDH2 based on the pig IDH2 structure containing bound isocitrate (Ceccarelli et al., 2002). Human and pig IDH2 protein share over 97% identity and all active site residues are identical. The active site of human IDH2 was structurally aligned with human IDH1 (Figure 1). Similar to IDH1, in the active site of IDH2 the isocitrate substrate is stabilized by multiple charge interactions throughout the binding pocket. Moreover, like R132 in IDH1, the analogous R172 in IDH2 is predicted to interact strongly with the β-carboxyl of isocitrate. This raised the possibility that cancer-associated IDH2 mutations at R172 might affect enzymatic interconversion of isocitrate and α-ketoglutarate similarly to IDH1 mutations at R132.

Mutation of IDH2 R172K Enhances α-Ketoglutarate-Dependent NADPH Consumption

To test whether cancer-associated IDH2 R172K mutations shared the gain of function in α-ketoglutarate reduction observed for IDH1 R132 mutations (Dang et al., 2009), we overexpressed wild-type or R172K mutant IDH2 in cells with endogenous wild-type IDH2 expression, and then assessed isocitrate-dependent NADPH production and α-ketoglutarate-dependent NADPH consumption in cell lysates. As reported previously (Yan et al., 2009), extracts from cells expressing the R172K mutant IDH2 did not display isocitrate-dependent NADPH production above the levels observed in extracts from vector-transfected cells. In contrast, extracts from cells expressing a comparable amount of wild-type IDH2 markedly increased isocitrate-dependent NADPH production (Figure 2A). However, when these same extracts were tested for NADPH consumption in the presence of α-ketoglutarate, R172K mutant IDH2 expression was found to correlate with a significant enhancement to α-ketoglutarate-dependent NADPH consumption. Vector-transfected cell lysates did not demonstrate this activity (Figure 2B). Although not nearly to the same degree as with the mutant enzyme, wild-type IDH2 overexpression also reproducibly enhanced α-ketoglutarate-dependent NADPH consumption under these conditions.

Expression of R172K Mutant IDH2 Results in Enhanced α-Ketoglutarate-Dependent Consumption of NADPH

Expression of R172K Mutant IDH2 Results in Enhanced α-Ketoglutarate-Dependent Consumption of NADPH

http://ars.els-cdn.com/content/image/1-s2.0-S153561081000036X-gr2.jpg

Figure 2. Expression of R172K Mutant IDH2 Results in Enhanced α-Ketoglutarate-Dependent Consumption of NADPH

(A) 293T cells transfected with wild-type or R172K mutant IDH2, or empty vector, were lysed and subsequently assayed for their ability to generate NADPH from NADP+ in the presence of 0.1 mM isocitrate.

(B) The same cell lysates described in (A) were assayed for their consumption of NADPH in the presence of 0.5 mM α-ketoglutarate. Data for (A) and (B) are each representative of three independent experiments. Data are presented as the mean and standard error of the mean (SEM) from three independent measurements at the indicated time points.

(C) Expression of wild-type and R172K mutant IDH2 was confirmed by western blotting of the lysates assayed in (A) and (B). Reprobing of the same blot with IDH1 antibody as a control is also shown.

Mutation of IDH2 R172K Results in Elevated 2HG Levels

R172K mutant IDH2 lacks the guanidinium moiety in residue 172 that normally stabilizes β-carboxyl addition in the interconversion of α-ketoglutarate and isocitrate. Yet R172K mutant IDH2 exhibited enhanced α-ketoglutarate-dependent NADPH consumption in cell lysates (Figure 2B). A similar enhancement of α-ketoglutarate-dependent NADPH consumption has been reported for R132 mutations in IDH1, resulting in conversion of α-ketoglutarate to 2HG (Dang et al., 2009). To determine whether cells expressing IDH2 R172K shared this property, we expressed IDH2 wild-type or IDH2 R172K in cells. The accumulation of organic acids, including 2HG, both within cells and in culture medium of the transfectants was then assessed by gas-chromatography mass spectrometry (GC-MS) after MTBSTFA derivatization of the organic acid pool. We observed a metabolite peak eluting at 32.5 min on GC-MS that was of minimal intensity in the culture medium of IDH2-wild-type-expressing cells, but that in the medium of IDH2-R172K-expressing cells had a markedly higher intensity approximating that of the glutamate signal (Figures 3A and 3B). Mass spectra of this metabolite peak fit that predicted for MTBSTFA-derivatized 2HG, and the peak’s identity as 2HG was additionally confirmed by matching its mass spectra with that obtained by derivatization of commercial 2HG standards (Figure 3C). Similar results were obtained when the intracellular organic acid pool was analyzed. IDH2 R172K expressing cells were found to have an approximately 100-fold increase in the intracellular levels of 2HG compared with the levels detected in vector-transfected and IDH2-wild-type-overexpressing cells (Figure 3D). Consistent with previous work, IDH1-R132H-expressing cells analyzed in the same experiment had comparable accumulation of 2HG in both cells and in culture medium. 2HG accumulation was not observed in cells overexpressing IDH1 wild-type (data not shown).

Figure 3. Expression of R172K Mutant IDH2 Elevates 2HG Levels within Cells and in Culture Medium

(A and B) 293T cells transfected with IDH2 wild-type (A) or IDH2 R172K (B) were provided fresh culture medium the day after transfection. Twenty-four hours later, the medium was collected, from which organic acids were extracted, purified, and derivatized with MTBSTFA. Shown are representative gas chromatographs for the derivatized organic acids eluting between 30 to 34 min, including aspartate (Asp) and glutamate (Glu). The arrows indicate the expected elution time of 32.5 min for MTBSTFA-derivatized 2HG, based on similar derivatization of a commercial R(-)-2HG standard. Metabolite abundance refers to GC-MS signal intensity.

(C) Mass spectrum of the metabolite peak eluting at 32.5 min in (B), confirming its identity as MTBSTFA-derivatized 2HG. The structure of this derivative is shown in the inset, with the tert-butyl dimethylsilyl groups added during derivatization highlighted in green. m/e indicates the mass (in atomic mass units) to charge ratio for fragments generated by electron impact ionization.

(D) Cells were transfected as in (A) and (B), and after 48 hr intracellular metabolites were extracted, purified, MTBSTFA-derivatized, and analyzed by GC-MS. Shown is the quantitation of 2HG signal intensity relative to glutamate for a representative experiment. See also Figure S1.

http://ars.els-cdn.com/content/image/1-s2.0-S153561081000036X-gr3.jpg

Mutant IDH2 Produces the (R) Enantiomer of 2HG

Cancer-associated mutants of IDH1 produce the (R) enantiomer of 2HG ( Dang et al., 2009). To determine the chirality of the 2HG produced by mutant IDH2 and to compare it with that produced by R132H mutant IDH1, we used a two-step derivatization method to distinguish the stereoisomers of 2HG by GC-MS: an esterification step with R-(−)-2-butanolic HCl, followed by acetylation of the 2-hydroxyl with acetic anhydride ( Kamerling et al., 1981). Test of this method on commercial S(+)-2HG and R(−)-2HG standards demonstrated clear separation of the (S) and (R) enantiomers, and mass spectra of the metabolite peaks confirmed their identity as the O-acetylated di-(−)-2-butyl esters of 2HG (see Figures S1A and S1B available online). By this method, we confirmed the chirality of the 2HG found in cells expressing either R132H mutant IDH1 or R172K mutant IDH2 corresponded exclusively to the (R) enantiomer ( Figures S1C and S1D).

Leukemic Cells Bearing Heterozygous R172K IDH2 Mutations Accumulate 2HG

IDH2 Is Critical for Proliferating Cells and Contributes to the Conversion of α-Ketoglutarate into Citrate in the Mitochondria

A peculiar feature of the IDH-mutated cancers described to date is their lack of reduction to homozygosity. All tumors with IDH mutations retain one IDH wild-type allele. To address this issue we examined whether wild-type IDH1 and/or IDH2 might play a role in either cell survival or proliferation. Consistent with this possibility, we found that siRNA knockdown of either IDH1 or IDH2 can significantly reduce the proliferative capacity of a cancer cell line expressing both wild-type IDH1 and IDH2 ( Figure 4A).

Both IDH1 and IDH2 Are Critical for Cell Proliferation

Both IDH1 and IDH2 Are Critical for Cell Proliferation

http://ars.els-cdn.com/content/image/1-s2.0-S153561081000036X-gr4.jpg

Figure 4. Both IDH1 and IDH2 Are Critical for Cell Proliferation

(A) SF188 cells were treated with either of two unique siRNA oligonucleotides against IDH1 (siIDH1-A and siIDH1-B), either of two unique siRNA oligonucleotides against IDH2 (siIDH2-A and siIDH2-B), or control siRNA (siCTRL), and total viable cells were counted 5 days later. Data are the mean ± SEM of four independent experiments. In each case, both pairs of siIDH nucleotides gave comparable results. A representative western blot from one of the experiments, probed with antibody specific for either IDH1 or IDH2 as indicated, is shown on the right-hand side.

(B) Model depicting the pathways for citrate +4 (blue) and citrate +5 (red) formation in proliferating cells from [13C-U]-L-glutamine (glutamine +5).

(C) Cells were treated with two unique siRNA oligonucleotides against IDH2 or control siRNA, labeled with [13C-U]-L-glutamine, and then assessed for isotopic enrichment in citrate by LC-MS. Citrate +5 and Citrate +4 refer to citrate with five or four 13C-enriched atoms, respectively. Reduced expression of IDH2 from the two unique oligonucleotides was confirmed by western blot. Blotting with actin antibody is shown as a loading control.

(D) Cells were treated with two unique siRNA oligonucleotides against IDH3 (siIDH3-A and siIDH3-B) or control siRNA, and then labeled and assessed for isotopic citrate enrichment by GC-MS. Shown are representative data from three independent experiments. Reduced expression of IDH3 from the two unique oligonucleotides was confirmed by western blot. In (C) and (D), data are presented as mean and standard deviation of three replicates per experimental group.

The genetic analysis of these tumor samples revealed two neomorphic IDH mutations that produce 2HG. Among the IDH1 mutations, tumors with IDH1 R132C or IDH1 R132G accumulated 2HG. This result is not unexpected, as a number of mutations of R132 to other residues have also been shown to accumulate 2HG in glioma samples (Dang et al., 2009).

The other neomorphic allele was unexpected. All five of the IDH2 mutations producing 2HG in this sample set contained the same mutation, R140Q. As shown in Figure 1, both R140 in IDH2 and R100 in IDH1 are predicted to interact with the β-carboxyl of isocitrate. Additional modeling revealed that despite the reduced ability to bind isocitrate, the R140Q mutant IDH2 is predicted to maintain its ability to bind and orient α-ketoglutarate in the active site (Figure 6). This potentially explains the ability of cells with this neomorph to accumulate 2HG in vivo. As shown in Figure 5, samples containing IDH2 R140Q mutations were found to have accumulated 2HG to levels 10-fold to 100-fold greater than the highest levels detected in IDH wild-type samples.

Figure 5. Primary Human AML Samples with IDH1 or IDH2 Mutations Display Marked Elevations of 2HG

http://ars.els-cdn.com/content/image/1-s2.0-S153561081000036X-gr5.jpg

Structural Modeling of R140Q Mutant IDH2

Structural Modeling of R140Q Mutant IDH2

Figure 6.  Structural Modeling of R140Q Mutant IDH2

(A) Active site of human wild-type IDH2 with isocitrate replaced by α-ketoglutarate (α-KG). R140 is well positioned to interact with the β-carboxyl group that is added as a branch off carbon 3 when α-ketoglutarate is reductively carboxylated to isocitrate.

(B) Active site of R140Q mutant IDH2 complexed with α-ketoglutarate, demonstrating the loss of proximity to the substrate in the R140Q mutant. This eliminates the charge interaction from residue 140 that stabilizes the addition of the β-carboxyl required to convert α-ketoglutarate to isocitrate.

IDH2 Mutations Are More Common Than IDH1 Mutations in AML

  • Neomorphic Enzymatic Activity to Produce 2HG Is the Shared Feature of IDH1 and IDH2 Mutations
  • 2HG as a Screening and Diagnostic Marker
  • Maintaining At Least One IDH1 and IDH2 Wild-Type Allele May Be Essential for Transformed Cells
  • 2HG as an Oncometabolite

Read Full Post »


Imaging of Non-tumorous and Tumorous Human Brain Tissues

Reporter and Curator: Dror Nir, PhD

The point of interest in the article I feature below is that it represents a potential building block in a future system that will use full-field optical coherence tomography during brain surgery to improve the accuracy of cancer lesions resection. The article is featuring promising results for differentiating tumor from normal brain tissue in large samples (order of 1–3 cm2) by offering images with spatial resolution comparable to histological analysis, sufficient to distinguish microstructures of the human brain parenchyma.  Easy to say, and hard to make…:) –> Intraoperative apparatus to guide the surgeon in real time during resection of brain tumors.

 

Imaging of non-tumorous and tumorous human brain tissues with full-field optical coherence tomography 

Open Access Article

Osnath Assayaga1Kate Grievea1Bertrand DevauxbcFabrice HarmsaJohan Palludbc,Fabrice ChretienbcClaude BoccaraaPascale Varletbc;  a Inserm U979 “Wave Physics For Medicine” ESPCI -ParisTech – Institut Langevin, 1 rue Jussieu, 75005, b France, Centre Hospitalier Sainte-Anne, 1 rue Cabanis 75014 Paris, France

c University Paris Descartes, France.

Abstract

A prospective study was performed on neurosurgical samples from 18 patients to evaluate the use of full-field optical coherence tomography (FF-OCT) in brain tumor diagnosis.

FF-OCT captures en face slices of tissue samples at 1 μm resolution in 3D to a penetration depth of around 200 μm. A 1 cm2 specimen is scanned at a single depth and processed in about 5 min. This rapid imaging process is non-invasive and requires neither contrast agent injection nor tissue preparation, which makes it particularly well suited to medical imaging applications.

Temporal chronic epileptic parenchyma and brain tumors such as meningiomas, low-grade and high-grade gliomas, and choroid plexus papilloma were imaged. A subpopulation of neurons, myelin fibers and CNS vasculature were clearly identified. Cortex could be discriminated from white matter, but individual glial cells such as astrocytes (normal or reactive) or oligodendrocytes were not observable.

This study reports for the first time on the feasibility of using FF-OCT in a real-time manner as a label-free non-invasive imaging technique in an intraoperative neurosurgical clinical setting to assess tumorous glial and epileptic margins.

Abbreviations

  • FF-OCT, full field optical coherence tomography;
  • OCT, optical coherence tomography

Keywords

Optical imaging; Digital pathology; Brain imaging; Brain tumor; Glioma

1. Introduction

1.1. Primary CNS tumors

Primary central nervous system (CNS) tumors represent a heterogeneous group of tumors with benign, malignant and slow-growing evolution. In France, 5000 new cases of primary CNS tumors are detected annually (Rigau et al., 2011). Despite considerable progress in diagnosis and treatment, the survival rate following a malignant brain tumor remains low and 3000 deaths are reported annually from CNS tumors in France (INCa, 2011). Overall survival from brain tumors depends on the complete resection of the tumor mass, as identified through postoperative imaging, associated with updated adjuvant radiation therapy and chemotherapy regimen for malignant tumors (Soffietti et al., 2010). Therefore, there is a need to evaluate the completeness of the tumor resection at the end of the surgical procedure, as well as to identify the different components of the tumor interoperatively, i.e. tumor tissue, necrosis, infiltrated parenchyma (Kelly et al., 1987). In particular, the persistence of non-visible tumorous tissue or isolated tumor cells infiltrating brain parenchyma may lead to additional resection.

For low-grade tumors located close to eloquent brain areas, a maximally safe resection that spares functional tissue warrants the current use of intraoperative techniques that guide a more complete tumor resection. During awake surgery, speech or fine motor skills are monitored, while cortical and subcortical stimulations are performed to identify functional areas (Sanai et al., 2008). Intraoperative MRI provides images of the surgical site as well as tomographic images of the whole brain that are sufficient for an approximate evaluation of the abnormal excised tissue, but offers low resolution (typically 1 to 1.5 mm) and produces artifacts at the air-tissue boundary of the surgical site.

Histological and immunohistochemical analyses of neurosurgical samples remain the current gold standard method used to analyze tumorous tissue due to advantages of sub-cellular level resolution and high contrast. However, these methods require lengthy (12 to 72 h), complex multiple steps, and use of carcinogenic chemical products that would not be technically possible intra-operatively. In addition, the number of histological slides that can be reviewed and analyzed by a pathologist is limited, and it defines the number and size of sampled locations on the tumor, or the surrounding tissue.

To obtain histology-like information in a short time period, intraoperative cytological smear tests are performed. However tissue architecture information is thereby lost and the analysis is carried out on only a limited area of the sample (1 mm × 1 mm).

Intraoperative optical imaging techniques are recently developed high resolution imaging modalities that may help the surgeon to identify the persistence of tumor tissue at the resection boundaries. Using a conventional operating microscope with Xenon lamp illumination gives an overall view of the surgical site, but performance is limited by the poor discriminative capacity of the white light illumination at the surgical site interface. Better discrimination between normal and tumorous tissues has been obtained using fluorescence properties of tumor cells labeled with preoperatively administered 5-ALA. Tumor tissue shows a strong ALA-induced PPIX fluorescence at 635 nm and 704 nm when the operative field is illuminated with a 440 nm-filtered lamp. More complete resections of high-grade gliomas have been demonstrated using 5-ALA fluorescence guidance (Stummer et al., 2000), however brain parenchyma infiltrated by isolated tumor cells is not fluorescent, reducing the interest of this technique when resecting low-grade gliomas.

Refinement of this induced fluorescence technique has been achieved using a confocal microscope and intraoperative injection of sodium fluorescein. A 488 nm laser illuminates the operative field and tissue contact analysis is performed using a handheld surgical probe (field of view less than 0.5 × 0.5 mm) which scans the fluorescence of the surgical interface at the 505–585 nm band. Fluorescent isolated tumor cells are clearly identified at depths from 0 to 500 μm from the resection border (Sanai et al., 2011), demonstrating the potential of this technique in low-grade glioma resection.

Reviewing the state-of-the-art, a need is identified for a quick and reliable method of providing the neurosurgeon with architectural and cellular information without the need for injection or oral intake of exogenous markers in order to guide the neurosurgeon and optimize surgical resections.

1.2. Full-field optical coherence tomography

Introduced in the early 1990s (Huang et al., 1991), optical coherence tomography (OCT) uses interference to precisely locate light deep inside tissue. The photons coming from the small volume of interest are distinguished from light scattered by the other parts of the sample by the use of an interferometer and a light source with short coherence length. Only the portion of light with the same path length as the reference arm of the interferometer, to within the coherence length of the source (typically a few μm), will produce interference. A two-dimensional B-scan image is captured by scanning. Recently, the technique has been improved, mainly in terms of speed and sensitivity, through spectral encoding (De Boer et al., 2003Leitgeb et al., 2003 and Wojtkowski et al., 2002).

A recent OCT technique called full-field optical coherence tomography (FF-OCT) enables both a large field of view and high resolution over the full field of observation (Dubois et al., 2002 and Dubois et al., 2004). This allows navigation across the wide field image to follow the morphology at different scales and different positions. FF-OCT uses a simple halogen or light-emitting diode (LED) light source for full field illumination, rather than lasers and point-by-point scanning components required for conventional OCT. The illumination level is low enough to maintain the sample integrity: the power incident on the sample is less than 1 mW/mm2 using deep red and near infrared light. FF-OCT provides the highest OCT 3D resolution of 1.5 × 1.5 × 1 μm3 (X × Y × Z) on unprepared label-free tissue samples down to depths of approximately 200 μm–300 μm (tissue-dependent) over a wide field of view that allows digital zooming down to the cellular level. Interestingly, it produces en face images in the native field view (rather than the cross-sectional images of conventional OCT), which mimic the histology process, thereby facilitating the reading of images by pathologists. Moreover, as for conventional OCT, it does not require tissue slicing or modification of any kind (i.e. no tissue fixation, coloration, freezing or paraffin embedding). FF-OCT image acquisition and processing time is less than 5 min for a typical 1 cm2 sample (Assayag et al., in press) and the imaging performance has been shown to be equivalent in fresh or fixed tissue (Assayag et al., in press and Dalimier and Salomon, 2012). In addition, FF-OCT intrinsically provides digital images suitable for telemedicine.

Numerous studies have been published over the past two decades demonstrating the suitability of OCT for in vivo or ex vivo diagnosis. OCT imaging has been previously applied in a variety of tissues such as the eye (Grieve et al., 2004 and Swanson et al., 1993), upper aerodigestive tract (Betz et al., 2008Chen et al., 2007 and Ozawa et al., 2009), gastrointestinal tract (Tearney et al., 1998), and breast tissue and lymph nodes (Adie and Boppart, 2009Boppart et al., 2004Hsiung et al., 2007Luo et al., 2005Nguyen et al., 2009Zhou et al., 2010 and Zysk and Boppart, 2006).

In the CNS, published studies that evaluate OCT (Bizheva et al., 2005Böhringer et al., 2006Böhringer et al., 2009Boppart, 2003 and Boppart et al., 1998) using time-domain (TD) or spectral domain (SD) OCT systems had insufficient resolution (10 to 15 μm axial) for visualization of fine morphological details. A study of 9 patients with gliomas carried out using a TD-OCT system led to classification of the samples as malignant versus benign (Böhringer et al., 2009). However, the differentiation of tissues was achieved by considering the relative attenuation of the signal returning from the tumorous zones in relation to that returning from healthy zones. The classification was not possible by real recognition of CNS microscopic structures. Another study showed images of brain microstructures obtained with an OCT system equipped with an ultra-fast laser that offered axial and lateral resolution of 1.3 μm and 3 μm respectively (Bizheva et al., 2005). In this way, it was possible to differentiate malignant from healthy tissue by the presence of blood vessels, microcalcifications and cysts in the tumorous tissue. However the images obtained were small (2 mm × 1 mm), captured on fixed tissue only and required use of an expensive large laser thereby limiting the possibility for clinical implementation.

Other studies have focused on animal brain. In rat brain in vivo, it has been shown that optical coherence microscopy (OCM) can reveal neuronal cell bodies and myelin fibers (Srinivasan et al., 2012), while FF-OCT can also reveal myelin fibers (Ben Arous et al., 2011), and movement of red blood cells in vessels (Binding et al., 2011).

En face images captured with confocal reflectance microscopy can closely resemble FF-OCT images. For example, a prototype system used by Wirth et al. (2012) achieves lateral and axial resolution of 0.9 μm and 3 μm respectively. However small field size prevents viewing of wide-field architecture and slow acquisition speed prohibits the implementation of mosaicking. In addition, the poorer axial resolution and lower penetration depth of confocal imaging in comparison to FF-OCT limit the ability to reconstruct cross-sections from the confocal image stack.

This study is the first to analyze non-tumorous and tumorous human brain tissue samples using FF-OCT.

2. Materials and methods

2.1. Instrument

The experimental arrangement of FF-OCT (Fig. 1A) is based on a configuration that is referred to as a Linnik interferometer (Dubois et al., 2002). A halogen lamp is used as a spatially incoherent source to illuminate the full field of an immersion microscope objective at a central wavelength of 700 nm, with spectral width of 125 nm. The signal is extracted from the background of incoherent backscattered light using a phase-shifting method implemented in custom-designed software. This study was performed on a commercial FF-OCT system (LightCT, LLTech, France).

 

Fig 1

Capturing “en face” images allows easy comparison with histological sections. The resolution, pixel number and sampling requirements result in a native field of view that is limited to about 1 mm2. The sample is moved on a high precision mechanical platform and a number of fields are stitched together (Beck et al., 2000) to display a significant field of view. The FF-OCT microscope is housed in a compact setup (Fig. 1B) that is about the size of a standard optical microscope (310 × 310 × 800 mm L × W × H).

2.2. Imaging protocol

All images presented in this study were captured on fresh brain tissue samples from patients operated on at the Neurosurgery Department of Sainte-Anne Hospital, Paris. Informed and written consent was obtained in all cases following the standard procedure at Sainte-Anne Hospital from patients who were undergoing surgical intervention. Fresh samples were collected from the operating theater immediately after resection and sent to the pathology department. A pathologist dissected each sample to obtain a 1–2 cm2 piece and made a macroscopic observation to orientate the specimen in order to decide which side to image. The sample was immersed in physiological serum, placed in a cassette, numbered, and brought to the FF-OCT imaging facility in a nearby laboratory (15 min distant) where the FF-OCT images were captured. The sample was placed in a custom holder with a coverslip on top (Fig. 1C, D). The sample was raised on a piston to rest gently against the coverslip in order to flatten the surface and so optimize the image capture. The sample is automatically scanned under a 10 × 0.3 numerical aperture (NA) immersion microscope objective. The immersion medium is a silicone oil of refractive index close to that of water, chosen to optimize index matching and slow evaporation. The entire area of each sample was imaged at a depth of 20 μm beneath the sample surface. This depth has been reported to be optimal for comparison of FF-OCT images to histology images in a previous study on breast tissue (Assayag et al., in press). There are several reasons for the choice of imaging depth: firstly, histology was also performed at approximately 20 μm from the edge of the block, i.e. the depth at which typically the whole tissue surface begins to be revealed. Secondly, FF-OCT signal is attenuated with depth due to multiple scattering in the tissue, and resolution is degraded with depth due to aberrations. The best FF-OCT images are therefore captured close to the surface, and the best matching is achieved by attempting to image at a similar depth as the slice in the paraffin block. It was also possible to capture image stacks down to several hundred μm in depth (where penetration depth is dependent on tissue type), for the purpose of reconstructing a 3D volume and imaging layers of neurons and myelin fibers. An example of such a stack in the cerebellum is shown as a video (Video 2) in supplementary material. Once FF-OCT imaging was done, each sample was immediately fixed in formaldehyde and returned to the pathology department where it underwent standard processing in order to compare the FF-OCT images to histology slides.

2.3. Matching FF-OCT to histology

The intention in all cases was to match as closely as possible to histology. FF-OCT images were captured 20 μm below the surface. Histology slices were captured 20 μm from the edge of the block. However the angle of the inclusion is hard to control and so some difference in the angle of the plane always exists when attempting matching. Various other factors that can cause differences stem from the histology process — fixing, dehydrating, paraffin inclusion etc. all alter the tissue and so precise correspondence can be challenging. Such difficulties are common in attempting to match histology to other imaging modalities (e.g. FF-OCT Assayag et al., in press; OCT Bizheva et al., 2005; confocal microscopy Wirth et al., 2012).

An additional parameter in the matching process is the slice thickness. Histology slides were 4 μm in thickness while FF-OCT optical slices have a 1 μm thickness. The finer slice of the FF-OCT image meant that lower cell densities were perceived on the FF-OCT images (in those cases where individual cells were seen, e.g. neurons in the cortex). This difference in slice thickness affects the accuracy of the FF-OCT to histology match. In order to improve matching, it would have been possible to capture four FF-OCT slices in 1 μm steps and sum the images to mimic the histology thickness. However, this would effectively degrade the resolution, which was deemed undesirable in evaluating the capacities of the FF-OCT method.

3. Results

18 samples from 18 adult patients (4 males, 14 females) of age range 19–81 years have been included in the study: 1 mesial temporal lobe epilepsy and 1 cerebellum adjacent to a pulmonary adenocarcinoma metastasis (serving as the non-tumor brain samples), 7 diffuse supratentorial gliomas (4 WHO grade II, 3 WHO grade III), 5 meningiomas, 1 hemangiopericytoma, and 1 choroid plexus papilloma. Patient characteristics are detailed in Table 1.

 

Table 1

3.1. FF-OCT imaging identifies myelinated axon fibers, neuronal cell bodies and vasculature in the human epileptic brain and cerebellum

The cortex and the white matter are clearly distinguished from one another (Fig. 2). Indeed, a subpopulation of neuronal cell bodies (Fig. 2B, C) as well as myelinated axon bundles leading to the white matter could be recognized (Fig. 2D, E). Neuronal cell bodies appear as dark triangles (Fig. 2C) in relation to the bright surrounding myelinated environment. The FF-OCT signal is produced by backscattered photons from tissues of differing refractive indices. The number of photons backscattered from the nuclei in neurons appears to be too few to produce a signal that allows their differentiation from the cytoplasm, and therefore the whole of the cell body (nucleus plus cytoplasm) appears dark.

Fig 2

 

Myelinated axons are numerous, well discernible as small fascicles and appear as bright white lines (Fig. 2E). As the cortex does not contain many myelinated axons, it appears dark gray. Brain vasculature is visible (Fig. 2F and G), and small vessels are distinguished by a thin collagen membrane that appears light gray. Video 1 in supplementary material shows a movie composed of a series of en face 1 μm thick optical slices captured over 100 μm into the depth of the cortex tissue. The myelin fibers and neuronal cell bodies are seen in successive layers.

The different regions of the human hippocampal formation are easily recognizable (Fig. 3). Indeed, CA1 field and its stratum radiatum, CA4 field, the hippocampal fissure, the dentate gyrus, and the alveus are easily distinguishable. Other structures become visible by zooming in digitally on the FF-OCT image. The large pyramidal neurons of the CA4 field (Fig. 3B) and the granule cells that constitute the stratum granulosum of the dentate gyrus are visible, as black triangles and as small round dots, respectively (Fig. 3D).

 

Fig 3

In the normal cerebellum, the lamellar or foliar pattern of alternating cortex and central white matter is easily observed (Fig. 4A). By digital zooming, Purkinje and granular neurons also appear as black triangles or dots, respectively (Fig. 4C), and myelinated axons are visible as bright white lines (Fig. 4E). Video 2 in supplementary material shows a fly-through movie in the reconstructed axial slice orientation of a cortex region in cerebellum. The Purkinje and granular neurons are visible down to depths of 200 μm in the tissue.

 

Fig 4

3.2. FF-OCT images distinguish meningiomas from hemangiopericytoma in meningeal tumors

The classic morphological features of a meningioma are visible on the FF-OCT image: large lobules of tumorous cells appear in light gray (Fig. 5A), demarcated by collagen-rich bundles (Fig. 5B) which are highly scattering and appear a brilliant white in the FF-OCT images. The classic concentric tumorous cell clusters (whorls) are very clearly distinguished on the FF-OCT image (Fig. 5D). In addition the presence of numerous cell whorls with central calcifications (psammoma bodies) is revealed (Fig. 5F). Collagen balls appear bright white on the FF-OCT image (Fig. 5H). As the collagen balls progressively calcify, they are consumed by the black of the calcified area, generating a target-like image (Fig. 5H). Calcifications appear black in FF-OCT as they are crystalline and so allow no penetration of photons to their interior.

Fig 5

Mesenchymal non-meningothelial tumors such as hemangiopericytomas represent a classic differential diagnosis of meningiomas. In FF-OCT, the hemangiopericytoma is more monotonous in appearance than the meningiomas, with a highly vascular branching component with staghorn-type vessels (Fig. 6A, C).

Fig 6

3.3. FF-OCT images identify choroid plexus papilloma

The choroid plexus papilloma appears as an irregular coalescence of multiple papillas composed of elongated fibrovascular axes covered by a single layer of choroid glial cells (Fig. 7). By zooming in on an edematous papilla, the axis appears as a black structure covered by a regular light gray line (Fig. 7B). If the papilla central axis is hemorrhagic, the fine regular single layer is not distinguishable (Fig. 7C). Additional digital zooming in on the image reveals cellular level information, and some nuclei of plexus choroid cells can be recognized. However, cellular atypia and mitosis are not visible. These represent key diagnosis criteria used to differentiate choroid plexus papilloma (grade I) from atypical plexus papilloma (grade II).

Fig 7

3.4. FF-OCT images detect the brain tissue architecture modifications generated by diffusely infiltrative gliomas

Contrary to the choroid plexus papillomas which have a very distinctive architecture in histology (cauliflower-like aspect), very easily recognized in the FF-OCT images (Fig. 7A to G), diffusely infiltrating glioma does not present a specific tumor architecture (Fig. 8) as they diffusely permeate the normal brain architecture. Hence, the tumorous glial cells are largely dispersed through a nearly normal brain parenchyma (Fig. 8E). The presence of infiltrating tumorous glial cells attested by high magnification histological observation (irregular atypical cell nuclei compared to normal oligodendrocytes) is not detectable with the current generation of FF-OCT devices, as FF-OCT cannot reliably distinguish the individual cell nuclei due to lack of contrast (as opposed to lack of resolution). In our experience, diffuse low-grade gliomas (less than 20% of tumor cell density) are mistaken for normal brain tissue on FF-OCT images. However, in high-grade gliomas (Fig. 8G–K), the infiltration of the tumor has occurred to such an extent that the normal parenchyma architecture is lost. This architectural change is easily observed in FF-OCT and is successfully identified as high-grade glioma, even though the individual glial cell nuclei are not distinguished.

Fig 8

4. Discussion

We present here the first large size images (i.e. on the order of 1–3 cm2) acquired using an OCT system that offer spatial resolution comparable to histological analysis, sufficient to distinguish microstructures of the human brain parenchyma.

Firstly, the FF-OCT technique and the images presented here combine several practical advantages. The imaging system is compact, it can be placed in the operating room, the tissue sample does not require preparation and image acquisition is rapid. This technique thus appears promising as an intraoperative tool to help neurosurgeons and pathologists.

Secondly, resolution is sufficient (on the order of 1 μm axial and lateral) to distinguish brain tissue microstructures. Indeed, it was possible to distinguish neuron cell bodies in the cortex and axon bundles going towards white matter. Individual myelin fibers of 1 μm in diameter are visible on the FF-OCT images. Thus FF-OCT may serve as a real-time anatomical locator.

Histological architectural characteristics of meningothelial, fibrous, transitional and psammomatous meningiomas were easily recognizable on the FF-OCT images (lobules and whorl formation, collagenous-septae, calcified psammoma bodies, thick vessels). Psammomatous and transitional meningiomas presented distinct architectural characteristics in FF-OCT images in comparison to those observed in hemangiopericytoma. Thus, FF-OCT may serve as an intraoperative tool, in addition to extemporaneous examination, to refine differential diagnosis between pathological entities with different prognoses and surgical managements.

Diffuse glioma was essentially recognized by the loss of normal parenchyma architecture. However, glioma could be detected on FF-OCT images only if the glial cell density is greater than around 20% (i.e. the point at which the effect on the architecture becomes noticeable). The FF-OCT technique is therefore not currently suitable for the evaluation of low tumorous infiltration or tumorous margins. Evaluation at the individual tumor cell level is only possible by IDH1R132 immunostaining in IDH1 mutated gliomas in adults (Preusser et al., 2011). One of the current limitations of the FF-OCT technique for use in diagnosis is the difficulty in estimating the nuclear/cytoplasmic boundaries and the size and form of nuclei as well as the nuclear-cytoplasmic ratio of cells. This prevents precise classification into tumor subtypes and grades.

To increase the accuracy of diagnosis of tumors where cell density measurement is necessary for grading, perspectives for the technique include development of a multimodal system (Harms et al., 2012) to allow simultaneous co-localized acquisition of FF-OCT and fluorescence images. The fluorescence channel images in this multimodal system show cell nuclei, which increase the possibility of diagnosis and tumor grading direct from optical images. However, the use of contrast agents for the fluorescence channel means that the multimodal imaging technique is no longer non-invasive, and this may be undesirable if the tissue is to progress to histology following optical imaging. This is a similar concern in confocal microscopy where use of dyes is necessary for fluorescence detection (Wirth et al., 2012).

In its current form therefore, FF-OCT is not intended to serve as a diagnostic tool, but should rather be considered as an additional intraoperative aid in order to determine in a short time whether or not there is suspicious tissue present in a sample. It does not aim to replace histological analyses but rather to complement them, by offering a tool at the intermediary stage of intraoperative tissue selection. In a few minutes, an image is produced that allows the surgeon or the pathologist to assess the content of the tissue sample. The selected tissue, once imaged with FF-OCT, may then proceed to conventional histology processing in order to obtain the full diagnosis (Assayag et al., in press and Dalimier and Salomon, 2012).

Development of FF-OCT to allow in vivo imaging is underway, and first steps include increasing camera acquisition speed. First results of in vivo rat brain imaging have been achieved with an FF-OCT prototype setup, and show real-time visualization of myelin fibers (Ben Arous et al., 2011) and movement of red blood cells in vessels (Binding et al., 2011). To respond more precisely to surgical needs, it would be preferable to integrate the FF-OCT system into a surgical probe. Work in this direction is currently underway and preliminary images of skin and breast tissue have been captured with a rigid probe FF-OCT prototype (Latrive and Boccara, 2011).

In conclusion, we have demonstrated the capacity of FF-OCT for imaging of human brain samples. This technique has potential as an intraoperative tool for determining tissue architecture and content in a few minutes. The 1 μm3 resolution and wide-field down to cellular-level views offered by the technique allowed identification of features of non-tumorous and tumorous tissues such as myelin fibers, neurons, microcalcifications, tumor cells, microcysts, and blood vessels. Correspondence with histological slides was good, indicating suitability of the technique for use in clinical practice for tissue selection for biobanking for example. Future work to extend the technique to in vivo imaging by rigid probe endoscopy is underway.

The following are the supplementary data related to this article.

Video 1.  Shows a movie composed of a series of en face 1 μm thick optical slices captured over 100 μm into the depth of the cortex tissue. The myelin fibers and neuronal cell bodies are seen in successive layers. Field size is 800 μm × 800 μm.

Video 2.  Shows a fly-through movie in the reconstructed cross-sectional orientation showing 1 μm steps through a 3D stack down to 200 μm depth in cerebellum cortical tissue. Purkinje and granular neurons are visible as dark spaces. Field size is 800 μm × 200 μm.

Acknowledgments

The authors wish to thank LLTech SAS for use of the LightCT Scanner.

References

 

Adie and Boppart, 2009

Adie, Boppart

Optical Coherence Tomography for Cancer Detection

SpringerLink (2009), pp. 209–250

Assayag et al., in press

Assayag et al.

Large field, high resolution full field optical coherence tomography: a pre-clinical study of human breast tissue and cancer assessment

Technology in Cancer Research & Treatment TCRT Express, 1 (1) (2013), p. e600254http://dx.doi.org/10.7785/tcrtexpress.2013.600254

Beck et al., 2000

Beck et al.

Computer-assisted visualizations of neural networks: expanding the field of view using seamless confocal montaging

Journal of Neuroscience Methods, 98 (2) (2000), pp. 155–163

Ben Arous et al., 2011

Ben Arous et al.

Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy

Journal of Biomedical Optics, 16 (11) (2011), p. 116012

Full Text via CrossRef

Betz et al., 2008

C.S. Betz et al.

A set of optical techniques for improving the diagnosis of early upper aerodigestive tract cancer

Medical Laser Application, 23 (2008), pp. 175–185

Binding et al., 2011

Binding et al.

Brain refractive index measured in vivo with high-NA defocus-corrected full-field OCT and consequences for two-photon microscopy

Optics Express, 19 (6) (2011), pp. 4833–4847

Bizheva et al., 2005

Bizheva et al.

Imaging ex vivo healthy and pathological human brain tissue with ultra-high-resolution optical coherence tomography

Journal of Biomedical Optics, 10 (2005), p. 011006 http://dx.doi.org/10.1117/1.1851513

Böhringer et al., 2006

Böhringer et al.

Time domain and spectral domain optical coherence tomography in the analysis of brain tumor tissue

Lasers in Surgery and Medicine, 38 (2006), pp. 588–597 http://dx.doi.org/10.1002/lsm.20353

Böhringer et al., 2009

Böhringer et al.

Imaging of human brain tumor tissue by near-infrared laser coherence tomography

Acta Neurochirurgica, 151 (2009), pp. 507–517 http://dx.doi.org/10.1007/s00701-009-0248-y

Boppart, 2003

Boppart

Optical coherence tomography: technology and applications for neuroimaging

Psychophysiology, 40 (2003), pp. 529–541 http://dx.doi.org/10.1111/1469-8986.00055

Boppart et al., 1998

Boppart et al.

Optical coherence tomography for neurosurgical imaging of human intracortical melanoma

Neurosurgery, 43 (1998), pp. 834–841 http://dx.doi.org/10.1097/00006123-199810000-00068

Boppart et al., 2004

Boppart et al.

Optical coherence tomography: feasibility for basic research and image-guided surgery of breast cancer

Breast Cancer Research and Treatment, 84 (2004), pp. 85–97

Chen et al., 2007

Chen et al.

Ultrahigh resolution optical coherence tomography of Barrett’s esophagus: preliminary descriptive clinical study correlating images with histology

Endoscopy, 39 (2007), pp. 599–605

Dalimier and Salomon, 2012

Dalimier, Salomon

Full-field optical coherence tomography: a new technology for 3D high-resolution skin imaging

Dermatology, 224 (2012), pp. 84–92 http://dx.doi.org/10.1159/000337423

De Boer et al., 2003

De Boer et al.

Improved signal-to-noise ratio in spectral-domain compared with time-domain optical coherence tomography

Optics Letters, 28 (2003), pp. 2067–2069

Dubois et al., 2002

Dubois et al.

High-resolution full-field optical coherence tomography with a Linnik microscope

Applied Optics, 41 (4) (2002), p. 805

Dubois et al., 2004

Dubois et al.

Ultrahigh-resolution full-field optical coherence tomography

Applied Optics, 43 (14) (2004), p. 2874

Grieve et al., 2004

Grieve et al.

Ocular tissue imaging using ultrahigh-resolution, full-field optical coherence tomography

Investigative Ophthalmology & Visual Science, 45 (2004), pp. 4126-3–4131

Harms et al., 2012

Harms et al.

Multimodal full-field optical coherence tomography on biological tissue: toward all optical digital pathology

Proc. SPIE 2011, Multimodal Biomedical Imaging VII, 8216 (2012)

Hsiung et al., 2007

Hsiung et al.

Benign and malignant lesions in the human breast depicted with ultrahigh resolution and three-dimensional optical coherence tomography

Radiology, 244 (2007), pp. 865–874

Read Full Post »


Author and Curator: Ritu Saxena, Ph.D

Although cancer stem cells constitute only a small percentage of the tumor burden, their self-renewal capacity and possible link with recurrence of cancer post treatment makes them a sought after therapeutic target in cancer. The post on cancer stem cells published on the 22nd of March, 2013, describes the identity of CSCs, their functional characteristics, possible cell of origin and biomarkers. This post focuses on the therapeutic potential of CSCs, their resistance to conventional anti-tumor therapies and current therapeutic targets including biomarkers, signaling pathways and niches.

CSCs Are Resistant to conventional anticancer therapies including chemotherapy, radiotherapy and surgery that are used either alone or in combination. However, these strategies have failed several times to eradicate CSCs resulting in metastasis and relapse, hence, a fatal disease outcome.

The properties of CSCs that contribute to or lead to chemoresistance include:

Quiescent Phenotype

Chemotherapeutic agents target fast-growing cells; however, some CSCs that remain in the dormant or quiescent stage are spared from lethal damage. Later, when the dormant CSCs enter cell cycle, tumor proliferation is stimulated.

Antiapoptosis

Antiapoptotic proteins such as BCL-2 and some self-renewal pathways such as transforming growth factor β, Wnt/ β -catenin or BMI-1 are activated in CSCs. Consequently, DNA damage repair capability of CSCs is enhanced after genotoxic stress or activation of autocrine loops through the production of growth factors like epidermal growth factor (Moserle L, Cancer Lett, 1 Feb 2010;288(1):1-9).

Expression of Drug Efflux Pumps

CSCs express some proteins that have typically been known to contribute to multidrug resistance. The proteins are drug efflux pumps ABCC1, ABCG2 or MDR1. Multidrug resistance-associated proteins (ABCC subfamily) are members of the ATP-binding cassette (ABC) superfamily of transport proteins and act as cellular efflux transporters for a wide variety of substrates, in particular glutathione, glucuronide and sulfate conjugates of diverse compounds.

Radiotherapy is mainly used in breast cancer and glioblastoma multiforme. In glioblastoma multiforme, the properties of CSCs that contribute to radiotherapy resistance is the presence of CD133 marker. CD133+ CSCs preferentially activate DNA damage repair pathway and significantly induced checkpoint kinases that leads to reduced apoptosis in CSCs compared to the CD133- tumor cells (Bao S, Nature, 7 Dec 2006;444(7120):756-60).

Radiotherapy resistance in breast cancer is due to reduced levels of reactive oxygen species in CSCs. In addition, radiation resistance of progenitor cells in an immortalized breast cancer cell line was mediated by the Wnt/β catenin pathway proteins (Diehn M, et al, Nature, 9 Apr 2009;458(7239):780-3; Chen MS, et al, J Cell Sci, 1 Feb 2007;120(Pt 3):468-77).

As mentioned in the previous post on CSCs, CSC targeting therapy could either eliminate CSCs by either killing them after differentiating them from other tumor population, and/or by disrupting their niche. Efficient eradication of CSCs may require the combined ablation of CSCs themselves and their niches. Thus, identification of appropriate and specific markers of CSCs is crucial for targeting them and preventing tumor relapse. Table 1 (adapted from a review article on CSCs by Zhao et al) describes the currently used biomarkers for CSC-targeted therapy (Zhao L, et al, Eur Surg Res, 2012;49(1):8-15).

Table 1

Specific Target Cancer type Marker properties and therapy
Targeting cell markers
CD24+CD44+ESA+ Pancreatic cancer Pancreatic CSCs, elevated during tumorigenesis
CD44+CD24–ESA+ Breast cancer Breast CSCs
EpCAM high CD44+CD166+ Colorectal cancer
CD34+CD38– AML broad use as a target for chemotherapy
CD133+ Prostate cancer and breast cancer 5-transmembrane domain cell surface glycoprotein,also a marker for neuron epithelial, hematopoietic and endothelialprogenitor cells
Stro1+CD105+CD44+ Bone sarcoma
Nodal/activin Knockdown or pharmacological inhibition of its receptorAlk4/7 abrogated self-renewal capacity and in vivo tumorigenicity of CSCs.
Targeting signaling pathways
Hedgehog signaling Upregulated in several cancer types inhibitors: GDC-0449,PF04449913, BMS-833923, IPI-926 and TAK-441
Wnt/β-catenin signaling CML, squamous cell carcinoma Be required for CSC self-renewal and tumor growthinhibitors: PRI-724, WIF-1 and telomerase
Notch signaling Several cancer types An important regulator in normal development, adult stem cell maintenance,and tumorigenesis in multiple organs,inhibitors: RO4929097, BMS-906024, IPI-926 and MK0752
PI3K/Akt/PTEN/mTOR, Several cancer types The pathway is deregulated in many tumors and used to preferentially target CSCsinhibitors: temsirolimus, everolimus FDA-approved therapy for renal cell carcinoma
Targeting CSC Niche
Angiogenesis Niche Colon cancer, breast cancer, NSCLC Inhibitor: bevacizumab results in a disruption of the CSC niche, depleted vasculature and a dramatic reduction in the number of CSCs.
Hypoxia (HIF pathway) Ovarian cancer, lung cancer, cervical cancer Inhibitors: topotecan and digoxin have been approved for ovarian, lung and cervical cancer
Targeting Micro RNA
miR-200 family Inhibits EMT and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2
Let-7 family Regulates BT-IC stem cell-like properties by silencing more than one target
miR-124 Related to neuronal differentiation, targets laminin γ1 and integrin β1.
miR-21 Suppresses the self-renewal of embryonic stem cells

The challenge is to develop an effective treatment regimen that prevents survival, self-renewal and differentiation of CSCs and also disturbs their niche without damaging normal stem cells. In order to evaluate the efficiency of CSC-targeting therapies, in vitro models and mouse xenotransplantation models have been used for preclinical studies. Some potential CSC targeting agents in preclinical stages include notch inhibitors for glioblastoma stem cells and telomerase peptide vaccination after chemoradiotherapy of non-small cell lung cancer stem cells Stem Cells (Hovinga KE, et al, Jun 2010;28(6):1019-29; Serrano D, Mol Cancer, 9 Aug 2011;10:96). In addition, several phase II and phase III trials are currently underway to test CSC-targeting drugs focusing on efficacy and safety of treatment.

Reference:

Bao S, Nature, 7 Dec 2006;444(7120):756-60).

Diehn M, et al, Nature, 9 Apr 2009;458(7239):780-3

Chen MS, et al, J Cell Sci, 1 Feb 2007;120(Pt 3):468-77

Zhao L, et al, Eur Surg Res, 2012;49(1):8-15

Hovinga KE, et al, Jun 2010;28(6):1019-29

Serrano D, Mol Cancer, 9 Aug 2011;10:96

Pharmaceutical Intelligence posts:

https://pharmaceuticalintelligence.com/2013/03/22/in-focus-identity-of-cancer-stem-cells/ Author and curator: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/15/to-die-or-not-to-die-time-and-order-of-combination-drugs-for-triple-negative-breast-cancer-cells-a-systems-level-analysis/ Authors: Anamika Sarkar, PhD and Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/03/07/the-importance-of-cancer-prevention-programs-new-perceptions-for-fighting-cancer/ Author: Ziv Raviv, PhD

https://pharmaceuticalintelligence.com/2013/03/03/treatment-for-metastatic-her2-breast-cancer/ Reporter: Larry H Bernstein, MD

https://pharmaceuticalintelligence.com/2013/03/02/recurrence-risk-for-breast-cancer/ Larry H Bernstein, MD

https://pharmaceuticalintelligence.com/2013/02/14/prostate-cancer-androgen-driven-pathomechanism-in-early-onset-forms-of-the-disease/ Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/15/exploring-the-role-of-vitamin-c-in-cancer-therapy/ Curator: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/01/12/harnessing-personalized-medicine-for-cancer-management-prospects-of-prevention-and-cure-opinions-of-cancer-scientific-leaders-httppharmaceuticalintelligence-com/ Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression/ Author and reporter: Tilda Barliya PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/ Reporter and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/10/17/stomach-cancer-subtypes-methylation-based-identified-by-singapore-led-team/ Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/09/17/natural-agents-for-prostate-cancer-bone-metastasis-treatment/ Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/28/cardiovascular-outcomes-function-of-circulating-endothelial-progenitor-cells-cepcs-exploring-pharmaco-therapy-targeted-at-endogenous-augmentation-of-cepcs/ Aviva Lev-Ari, PhD, RN

 

Read Full Post »


Author and Curator: Ritu Saxena, PhD

Image

What are cancer stem cells?

Cancer is a debilitating disease estimated to be responsible for about 7.6 million deaths in 2008 (Jemal A, et al, CA Cancer J Clin, Mar-Apr 2011;61(2):69-90). Thus, extensive research is underway to deal with the various types of cancer. The concept of cancer stem cells (CSC) has surfaced in in the past decade after identification and characterization of CSC-enriched populations in several different types of cancer (Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8; Reya T, et al, Nature, 1 Nov 2001;414(6859):105-11;  Trumpp A and Wiestler OD, et al, Nat Clin Pract Oncol, Jun 2008;5(6):337-47). Although there has been lot of debate on the cell of origin of CSC, according to the classical concept CSC are defined by their functional properties.

Functional properties of CSC

  • CSCs are at the top of tumor hierarchy. Regenerative tissues follow a hierarchical organization with adult stem cells at the top maintaining tissues and normal adult cells during homeostasis and regeneration during cell loss from injury. Similarly, several tumors follow the hierarchy with CSC at the top. Hierarchical organization has been reported in several cancer types including but not limited to breast cancer, brain cancer, colon cancer, leukemia and pancreatic cancer (Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8; Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8; Singh SK, et al, Nature, 18 Nov 2004;432(7015):396-401; Dalerba P, et al, PNAS USA, 12 Jun 2007;104(24):10158-63; Hermann PC, et al, Cell Stem Cell, 13 Sep 2007;1(3):313-23).
  • CSCs possess unlimited self-renewal capacity similar to that of physiological stem cells and unlike other differentiated cell types within the tumor. Cancer stem cells can also generate non-CSC progeny that is comprised of differentiated cells and forms tumor bulk.
  • Some CSs exhibit quiescent or dormant stage. Although not observed in all CSC types, some CSCs have been found to shuttle between quiescent, slow-cycling, and active states. The CSCs in their dormant and slow-cycling stage are less likely to be affected by conventional anti-tumor therapies which generally target rapidly dividing cells. Dormant stage is exhibited even in adult stem cells and the dormant normal stem cells can regain cell division potential during tissue injury (Wilson A, et al, Cell,  12 Dec 2008;135(6):1118-29). Thus, it has been speculated that dormant CSC might be a reason for tumor relapse even after pathologic complete response is observed post therapy.
  • Some CSCs are resistant to conventional anti-cancer therapies. This leads to accumulation of CSC that might result in relapse after anti-cancer therapy. For instance, Li et al (2008) reported that CSC accumulated in the breast of women with locally advanced tumors after cytotoxic chemotherapy had eliminated the bulk of the tumor cells (Li X,et al, J Natl Cancer Inst, 7 May 2008;100(9):672-9). A similar observation was made by Oravecz-Wilson et al (2009) stating that despite remarkable responses to the tyrosine kinase inhibitor imatinib, CML patients show imatinib refractoriness because leukemia stem cells in CML are resistant tyrosine kinase (Oravecz-Wilson KI, et al, Cancer Cell, 4 Aug 2009;16(2):137-48).
  • The CSC niche. CSC functional traits might be sustained by this microenvironment, termed “niche”. The niche is the environment in which stem cells reside and is responsible for the maintenance of unique stem cell properties such as self-renewal and an undifferentiated state. The heterogeneous populations which constitute a niche include both stem cells and surrounding differentiated cells. The necessary intrinsic pathways that are utilized by this cancer stem cell population to maintain both self-renewal and the ability to differentiate are believed to be a result of the environment where cancer stem cells reside. (Cabarcas SM, et al, Int J Cancer, 15 Nov 2011;129(10):2315-27). For instance, properties of CSC in glioma in a mouse xenograft model were maintained by vascular endothelial cells (Calabrese C, et al, Cancer Cell, Jan 2007;11(1):69-82). Several molecules including interleukin 6 have been observed to play a role in tumor proliferation and hence, participate in maintaining tumorigenic and self-renewal potential of CSC. Moreover, the CSC niche might not only regulate CSCs traits but might also directly provide CSC features to non-CSC population.

What is the origin of CSC?

According to current thinking, CSC result from epithelial-mesenchymal transition (EMT) when cells switch from a polarized epithelial to a non-polarized mesenchymal cell type with stem cell properties, including migratory behavior, self-renewal and generation of differentiated progeny, and reduced responsiveness to conventional cancer therapies (Scheel C and Weinberg RA, Semin Cancer Biol, Oct 2012;22(5-6):396-403; Crews LA and Jamieson CH, Cancer Lett, 17 Aug 2012). Evidence is accumulating that cancers of distinct subtypes within an organ may derive from different ‘cells of origin’. The tumor cell of origin is the cell type from which the disease is derived after it undergoes oncogenic mutation. It might take a series of mutations to achieve the CSC phenotype (Visvader JE, Nature, 20 Jan 2011;469(7330):314-22). Also, CSCs have been reported to originate from stem cells in some cases.

Biomarkers for CSC

CSC targeting therapy could either eliminate CSCs by either killing them after differentiating them from other tumor population, and/or by disrupting their niche. Efficient eradication of CSCs may require the combined ablation of CSCs themselves and their niches. Identifying appropriate biomarkers of CSC is a very important aim for CSCs to be useful as targets of anti-cancer therapies in order to possibly prevent relapse. Using cell surface markers, CSCs have been isolated and purified from cancers of breast, brain, thyroid, cervix, lung, blood (leukemia), skin (melanoma), organs of the gastrointestinal and reproductive tracts, and the retina. The challenge, however, is that CSCs share similar markers with normal cells which makes CSCs targeting difficult as it would harm normal cells in the process. More recently, advanced techniques such as signal sequence trap (SST) PCR screening methods have been developed to identify a leukemia-specific stem cell marker (CD96). After a small subset of human AML cells displayed tumorigenic properties, Leukemia Stem Cells (LSCs) were identified as leukemia cells with CD23+/CD38+ markers. These cells closely resemble hematopeotic stem cells (HSCs) (Bonnet D and Dick JR, Nat Med, Jul 1997;3(7):730-7). In solid tumors, a significant discovery was made when CSCs in breast cancer were identified within the ESA+/CD44+/CD24low-neg population of mammary pleural effusion and tumor samples (Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8).

After these two landmark publications, CSCs were identified in many more solid and hematopoietic human tumors as well. In addition, within a tumor type, CSC-enriched populations display heterogeneity in markers. For example, only 1% of breast cancer cells simultaneously express both reported CSC phenotypes ESA+/CD44+/

CD24low-neg and ALDH-1+ (Ginestier C, et al, Cell Stem Cell, 1 Nov 2007;1(5):555-67). The discrepancy might be due to different techniques used to identify the markers and also a reflection of the molecular heterogeneity within the tumors. Recent advances in genome wide expression profiling studies have led to the identification of different subtypes in a particular type of cancer. Breast cancer was recently classified into different subtypes and this genetic heterogeneity is likely paralleled by a heterogeneous CSC complexity.

Conclusion

A lot of research is currently underway on various aspects of CSCs including biomarker identification, cell of origin, and clinical trials targeting CSC population in cancer. The concept of CSCs has evolved quite a bit since their discovery. Recently, identification of high genetic heterogeneity within a tumor has been in focus and subsequently it has been observed that several CSC clones can coexist and compete with each other within a tumor. Adding complexity to their identity is the fact that CSCs may have unstable phenotypes and genotypes. Taken together, the dynamics associated with CSCs makes it difficult to identify reliable and robust biomarkers and develop efficient targeted therapies. Thus, a major thrust of research should be to focus on the unfolding of the dynamic identity of CSCs in tumor types and at different that might lead to the identification and targeting of highly specific CSCs biomarkers.

Reference

Jemal A, et al, CA Cancer J Clin, Mar-Apr 2011;61(2):69-90

Reya T, et al, Nature, 1 Nov 2001;414(6859):105-11

Trumpp A and Wiestler OD, et al, Nat Clin Pract Oncol, Jun 2008;5(6):337-47

Lapidot T, et al, Nature, 17 Feb 1994;367(6464):645-8

Singh SK, et al, Nature, 18 Nov 2004;432(7015):396-401

Dalerba P, et al, PNAS USA, 12 Jun 2007;104(24):10158-63

Hermann PC, et al, Cell Stem Cell, 13 Sep 2007;1(3):313-23

Wilson A, et al, Cell,  12 Dec 2008;135(6):1118-29

Li X,et al, J Natl Cancer Inst, 7 May 2008;100(9):672-9

Oravecz-Wilson KI, et al, Cancer Cell, 4 Aug 2009;16(2):137-48

Cabarcas SM, et al, Int J Cancer, 15 Nov 2011;129(10):2315-27

Calabrese C, et al, Cancer Cell, Jan 2007;11(1):69-82

Scheel C and Weinberg RA, Semin Cancer Biol, Oct 2012;22(5-6):396-403

Crews LA and Jamieson CH, Cancer Lett, 17 Aug 2012

Visvader JE, Nature, 20 Jan 2011;469(7330):314-22

Bonnet D and Dick JR, Nat Med, Jul 1997;3(7):730-7

Al-Hajj M, et al, PNAS USA, 1 Apr 200;100(7):3983-8

Ginestier C, et al, Cell Stem Cell, 1 Nov 2007;1(5):555-67

Baccelli I and Trumpp AJ, Cell Biol, 6 Aug 2012;198(3):281-93

Zhao L, et al, Eur Surg Res, 2012;49(1):8-15

Pharmaceutical Intelligence posts:

https://pharmaceuticalintelligence.com/2012/08/15/to-die-or-not-to-die-time-and-order-of-combination-drugs-for-triple-negative-breast-cancer-cells-a-systems-level-analysis/

Authors: Anamika Sarkar, PhD and Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/03/07/the-importance-of-cancer-prevention-programs-new-perceptions-for-fighting-cancer/ Author: Ziv Raviv, PhD

https://pharmaceuticalintelligence.com/2013/03/03/treatment-for-metastatic-her2-breast-cancer/ Reporter: Larry H Bernstein, MD

https://pharmaceuticalintelligence.com/2013/03/02/recurrence-risk-for-breast-cancer/

Larry H Bernstein, MD

https://pharmaceuticalintelligence.com/2013/02/14/prostate-cancer-androgen-driven-pathomechanism-in-early-onset-forms-of-the-disease/ Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/15/exploring-the-role-of-vitamin-c-in-cancer-therapy/ Curator: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/01/12/harnessing-personalized-medicine-for-cancer-management-prospects-of-prevention-and-cure-opinions-of-cancer-scientific-leaders-httppharmaceuticalintelligence-com/ Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression/ Author and reporter: Tilda Barliya PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/ Reporter and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/10/17/stomach-cancer-subtypes-methylation-based-identified-by-singapore-led-team/ Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/09/17/natural-agents-for-prostate-cancer-bone-metastasis-treatment/ Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/28/cardiovascular-outcomes-function-of-circulating-endothelial-progenitor-cells-cepcs-exploring-pharmaco-therapy-targeted-at-endogenous-augmentation-of-cepcs/ Aviva Lev-Ari, PhD, RN

Read Full Post »

Older Posts »