Archive for the ‘Genetics & Pharmaceutical’ Category

RNA Testing

Posted in Genetics & Pharmaceutical, Genome Biology, Immunodiagnostics, Immunology, RNA Biology, tagged autoimmune disease, RNA, siagnostic testing on May 18, 2016| Leave a Comment »

RNA Testing

Curator: Larry H. Bernstein, MD, FCAP

LPBI

RNA Testing: A Fast, Accurate Tool for Diagnosing Autoimmune Disease

05/17/2016 – Dr. Chase Spurlock, IQuity CEO and Dr. Thomas Aune, IQuity Chief Science Officer
http://www.dddmag.com/articles/2016/05/rna-testing-fast-accurate-tool-diagnosing-autoimmune-disease

Diagnosing complex autoimmune diseases and related conditions is a challenging endeavor, as these multifaceted conditions often present with non-specific symptoms across several organ systems. Historically, physicians have diagnosed autoimmune disease through detailed physical examination, collection of medical and family history, multiple laboratory tests and monitoring symptoms over time. Current diagnostic algorithms can lead to a protracted and costly diagnostic process of elimination.

However, emerging RNA-based diagnostic technologies can dramatically reduce the time to diagnosis of complex diseases, including autoimmune disease. With the completion of the Human Genome Project and basic research in the field of RNA biology over the past two decades, it is now accepted that the majority of the human genome is transcribed into unique patterns of information that exhibit cell type specificity and are consistent in a variety of chronic conditions, including autoimmune disease. With the invention of new technologies, including next-generation RNA sequencing, we are able to assess global changes in gene expression.

As such, RNA-based tools are now poised to provide physicians with actionable information early in the diagnostic process. While RNA-based testing is a relatively new innovation in the clinical setting and widespread adoption efforts are still in their infancy, the science behind these techniques makes them a reliable method for helping physicians make fast, accurate decisions.

The science behind RNA

Analysis of RNA expression paints a molecular portrait of what’s going on in an individual’s cells at a given point in time. This can provide a picture of disease manifestation. At a high level, current RNA testing involves collecting a patient’s blood sample via standard venipuncture, isolating the RNA and then detecting RNA expression patterns in real time using polymerase chain reaction (PCR). The turning on or off of these expression patterns can serve as an indicator of the presence or absence of disease.

In contrast, DNA tests examine changes in nucleotide sequence to establish a patient’s risk of developing a particular condition. These genotype associations do not always reveal information about disease manifestation. A DNA test, for example, might indicate that a patient is at high risk for an autoimmune disorder, such as rheumatoid arthritis (RA) or multiple sclerosis (MS), but the patient may never develop the condition. Just because the patient has a particular DNA risk marker does not mean he or she actually has—or will have—the disease. Thus, a major limitation with DNA methodology is its inability to reliably forecast active disease. RNA tests are more specific in this regard.

Other types of testing, such as pharmacologic and serologic testing, have also been used to detect autoimmune disorders. However, due to the complexity of these diseases, a single test can only put a physician one small step closer to diagnosis and additional tests are often required. For instance, to accurately identify RA, a clinician may need to perform approximately 10 to 15 different molecular tests over what is often a lengthy time period. If the doctor concludes that a portion of these tests point to the presence of RA, then he or she may be able to make a diagnosis of RA. In some cases, it can take years for the patient to receive a definitive diagnosis. In comparison, new RNA-based diagnostic testing for the same condition offers an accuracy of greater than 90 percent, enabling a higher degree of certainty for establishing the presence or absence of the disease.

The impact on patients, providers and the scientific community

As RNA testing becomes more widespread, its impact on patients, providers and other stakeholders promises to be significant. Once widely adopted, this kind of testing could cut down on the number of tests and amount of the time needed to confirm diagnosis. This, in turn, would enhance the patient experience, as individuals would no longer have to undergo repeated or invasive testing to determine if they have a disease.

Current autoimmune therapies are reasonably effective at slowing disease advancement. Across many autoimmune diseases, the best outcomes are achieved when therapies are initiated early in the disease process. Providing physicians with actionable diagnostic information enables the provider to place patients on the appropriate treatments faster, limiting disease progression and supporting better long-term health outcomes.

RNA testing can improve outcomes for providers by helping to get patients on the path to suitable treatment earlier. This not only facilitates optimal medication performance, but also lessens the cost of diagnosis and management of disease. In turn, this ensures a better quality of life for patients.

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Cyclic Dinucleotides and Histone deacetylase inhibitors

Posted in Anaerobic Glycolysis, Apoptosis, Biological Networks, Cancer and Current Therapeutics, Cell Biology, Signaling & Cell Circuits, Cell death pathways, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Enzyme Induction, Gene Regulation, Genetics & Pharmaceutical, Genomic Expression, Oxidative phosphorylation, Proteolysis, tagged Cancer immunotherapy, CTLA-4, cyclic dinucleotides, Histone deacetylase, Histone Deacetylase Inhibitors (HDAC), PD1, STING on May 12, 2016| Leave a Comment »

Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

https://pharmaceuticalintelligence.com/2016/04/24/new-class-of-immune-system-stimulants-cyclic-di-nucleotides-cdn-shrink-tumors-and-bolster-vaccines-re-arm-the-immune-systems-natural-killer-cells-which-attack-cancer-cells-and-virus-inf/

A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu^1,2, R B Parmigiani^1,2 and P A Marks¹

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD⁺-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005; Rosato and Grant, 2005; Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list). Full table

http://www.nature.com/common/images/table_thumb.gif

In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005; Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005; Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD⁺ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005; Rosato and Grant, 2005; Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004; Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998; Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005; Chavez-Blanco et al., 2005; Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials. Full table

http://www.nature.com/common/images/table_thumb.gif

Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005; Marks and Dokmanovic, 2005;Rosato and Grant, 2005; Bolden et al., 2006; Minucci and Pelicci, 2006; Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005; Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1. Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

http://www.nature.com/onc/journal/v26/n37/images/1210620f1.jpg

HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003; Yoshida et al., 2003; Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC₅₀, at 0.3 m, compared to HDAC3 with an IC₅₀ of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

HDACi induces cell cycle arrest
HDACi activates the extrinsic apoptotic pathways
HDACi activates the intrinsic apoptotic pathways
HDACi induces mitotic cell death
HDACi induces autophagic cell death and senescence
ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
Antitumor effects of HDAC6 inhibition
Activation of protein phosphatase 1
Disruption of the function of chaperonin HSP90
Disruption of the aggresome pathway
HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.

Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004; Bhalla, 2005; Marks and Dokmanovic, 2005; Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References ….

NEWS AND VIEWS Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)

RESEARCH

Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:

The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.

The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology. Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.

The work that has been described covers only cyclic dinucleotides and HDACi’s. Some of the activities described have relevance to microorganisms as well as cancer. As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.

The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.

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Kaiser Permanente collecting patient data for DNA Research Bank

Posted in Electronic Health Record, Epigenetics and Environmental Factors, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology on May 10, 2016| Leave a Comment »

Kaiser Permanente collecting patient data for DNA Research Bank

Reporter: Aviva Lev-Ari, PhD, RN

Kaiser Permanente this week launched a new database that enables researchers to examine participants’ DNA in conjunction with environmental and behavioral health.

Sourced through Scoop.it from: www.healthcareitnews.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Durable responses with checkpoint inhibitor

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Clinical Genomics, Exosomes, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Genomic Expression, Genomic Testing: Methodology for Diagnosis, Genomics Pharmacy, Metabolism, Molecular Genetics & Pharmaceutical, Neuroscience, tagged checkpoint inhibitors, childhood brain malignancy, glioblastoma multiforme, PD-1 inhibitor on May 7, 2016| Leave a Comment »

Durable responses with checkpoint inhibitor

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Immunotherapy Active in ‘Universally Lethal’ Glioblastoma

ACTIVATE CME by Kristin Jenkins

Contributing Writer, MedPage Today

http://www.medpagetoday.com/HematologyOncology/BrainCancer/57641

Two pediatric siblings with recurrent multifocal glioblastoma multiforme (GBM) refractory to current standard therapies exhibited “remarkable and durable” responses to immune checkpoint inhibition with single-agent nivolumab (Opdivo), researchers said.

Following pre-clinical testing in 37 biallelic mismatch repair deficiency (bMMRD) cancers, a regimen of 3 mg/kg nivolumab every 2 weeks resulted in clinically significant responses and a profound radiologic response, Uri Tabori, MD, of The Hospital for Sick Children, Toronto, Ontario, Canada, and colleagues reported in the Journal of Clinical Oncology.

The 6-year-old white female patient and her 3.5-year-old brother resumed normal schooling and daily activities after 9 and 5 months of therapy, respectively, the researchers said.

“This observation is especially encouraging because these children are still clinically stable, whereas most relapsed pediatric GBMs will progress within 1 to 2 months despite salvage treatment, and survival is usually 3 to 6 months post-recurrence. It also highlights the utility of germline predisposition in guiding novel treatment options — in this case, immunotherapy — for cancer treatment.”

Findings from this lab study and small case series report may have implications for GBM as well as for other hypermutant cancers arising from primary (genetic predisposition) or secondary MMRD, the researchers said. “Given the increasing availability of commercial sequencing platforms, analysis of mutation burden and neoantigens can play a role in transforming treatment of these patients.”

Still, they added that these results, while encouraging, need to be validated in multinational prospective clinical trials of these “universally lethal” bMMRD-driven hypermutant cancers.

“Sometimes very small studies can yield meaningful results,” Robert Fenstermaker, MD, of Roswell Park Cancer Institute in Buffalo, N.Y., told MedPage Today via email. “Although anecdotal, the results of this study are quite encouraging because they tend to confirm current theory about immunotherapy for glioblastoma.”

Although these kinds of clinical responses to single-agent drug therapy in GBM are uncommon and the results may not be broadly applicable to all glioblastoma patients, this paper “is of much greater importance than just these few cases,” Fenstermaker emphasized. “The excellent responses in these particular cases suggest that an immune checkpoint inhibitor (nivolumab) may have enabled the immune system to respond fully.”

This “very small case series” report of a “compelling clinical experience” is a “fascinating and beautiful example of how mechanistic insight can be linked to rationally designed clinical applications — in turn, stimulating new downstream ideas,” Stephanie Weiss, MD, a radiation oncologist at Fox Chase Cancer Center in Philadelphia, commented in an email.

“This series also tests ‘proof of principle,’ that bMMRD tumors are hypermutated and associated with a high neoantigen load, and therefore may respond much like other immune checkpoint inhibitor-sensitive tumors. In this sense, the results reveal a tantalizing glimpse into the disease process of at least a subset of GBMs and can guide high-quality study of novel treatment for GBM.”

For the study, Tabori and colleagues performed exome sequencing and neoantigen prediction on 37 bMMRD-associated tumors, including 21 GBMs, and compared them with childhood and adult brain neoplasms.

The bMMRD GBMs were found to be hypermutant and to have an extremely strong neoantigen load — up to 16 times higher than the signature commonly seen in known immune checkpoint inhibitors (P<.001).

The female patient, diagnosed with a left parietal GBM, underwent near-total resection and focal irradiation over 6.5 weeks. After a clinical remission lasting 3 months, surveillance MRI revealed recurrence in the initial tumor bed and a second lesion in the left temporal lobe.

Six months earlier, the index patient’s brother had been diagnosed with a right frontoparietal GBM and treated with surgery, focal irradiation, and temozolomide (Temodal). Ten months after diagnosis, surveillance MRI revealed an asymptomatic diffuse multinodular GBM recurrence.

When given nivolumab as a last-resort therapeutic agent, both children initially experienced serious symptoms that on imaging mimicked tutor progression. After symptomatic management and observation, both stabilized, and follow-up imaging demonstrated significant improvement in tumor-related abnormalities.

Fenstermaker said that important next steps lie ahead, such as combining immune checkpoint inhibitors with specific cancer vaccines designed to immunize patients with glioblastomas other than this rare hypermutated type. “There are a number of prospective vaccines currently in the glioblastoma drug pipeline that would be candidates for this kind of approach,” he told MedPage Today. Examples include SurVaxM, NeoVax, HSPPC-96, and various dendritic cell vaccines.

In addition, newer genomic techniques are being developed that could make it possible to create a personalized profile of the mutant proteins in a given patient’s tumor, he noted. “One can imagine combining such a personalized vaccine against these mutant proteins together with an immune checkpoint inhibitor. Such a combination might result in many more responses like the ones seen in this small study.”

PD-1 Blockade in Tumors with Mismatch-Repair Deficiency

Dung T. Le, Jennifer N. Uram, Hao Wang, Bjarne R. Bartlett, Holly Kemberling, Aleksandra D. Eyring, et al.
http://www.nejm.org/doi/full/10.1056/NEJMoa1500596

BACKGROUND

Somatic mutations have the potential to encode “non-self” immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to mismatch-repair defects may be susceptible to immune checkpoint blockade.

Full Text of Background…

METHODS

We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti–programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair–deficient colorectal cancers, patients with mismatch repair–proficient colorectal cancers, and patients with mismatch repair–deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate.

Full Text of Methods…

RESULTS

The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair–deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair–proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair–deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair–proficient colorectal cancer (hazard ratio for disease progression or death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P=0.05]). Patients with mismatch repair–deficient noncolorectal cancer had responses similar to those of patients with mismatch repair–deficient colorectal cancer (immune-related objective response rate, 71% [5 of 7 patients]; immune-related progression-free survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of 1782 somatic mutations per tumor in mismatch repair–deficient tumors, as compared with 73 in mismatch repair–proficient tumors (P=0.007), and high somatic mutation loads were associated with prolonged progression-free survival (P=0.02).

Full Text of Results…

CONCLUSIONS

This study showed that mismatch-repair status predicted clinical benefit of immune checkpoint blockade with pembrolizumab. (Funded by Johns Hopkins University and others; ClinicalTrials.gov number, NCT01876511.)

Full Text of Discussion…

Read the Full Article…

Immune Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline Biallelic Mismatch Repair Deficiency

Eric Bouffet, Valérie Larouche, Brittany B. Campbell, Daniele Merico, Richard de Borja, et al.
JCO March 21, 2016 http://dx.doi.org:/10.1200/JCO.2016.66.6552

Purpose Recurrent glioblastoma multiforme (GBM) is incurable with current therapies. Biallelic mismatch repair deficiency (bMMRD) is a highly penetrant childhood cancer syndrome often resulting in GBM characterized by a high mutational burden. Evidence suggests that high mutation and neoantigen loads are associated with response to immune checkpoint inhibition.

Patients and Methods We performed exome sequencing and neoantigen prediction on 37 bMMRD cancers and compared them with childhood and adult brain neoplasms. Neoantigen prediction bMMRD GBM was compared with responsive adult cancers from multiple tissues. Two siblings with recurrent multifocal bMMRD GBM were treated with the immune checkpoint inhibitor nivolumab.

Results All malignant tumors (n = 32) were hypermutant. Although bMMRD brain tumors had the highest mutational load because of secondary polymerase mutations (mean, 17,740 ± standard deviation, 7,703), all other high-grade tumors were hypermutant (mean, 1,589 ± standard deviation, 1,043), similar to other cancers that responded favorably to immune checkpoint inhibitors. bMMRD GBM had a significantly higher mutational load than sporadic pediatric and adult gliomas and all other brain tumors (P < .001). bMMRD GBM harbored mean neoantigen loads seven to 16 times higher than those in immunoresponsive melanomas, lung cancers, or microsatellite-unstable GI cancers (P < .001). On the basis of these preclinical data, we treated two bMMRD siblings with recurrent multifocal GBM with the anti–programmed death-1 inhibitor nivolumab, which resulted in clinically significant responses and a profound radiologic response.

Conclusion This report of initial and durable responses of recurrent GBM to immune checkpoint inhibition may have implications for GBM in general and other hypermutant cancers arising from primary (genetic predisposition) or secondary MMRD.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor and the most common cause of death among children with CNS neoplasms.¹ Despite primary management, which consists of surgical resection followed by radiation therapy and chemotherapy, most GBMs will recur, resulting in rapid death. Patients with recurrent disease have a particularly poor prognosis, with a median survival of fewer than 6 months; no effective therapies currently exist.

In contrast to adult CNS malignancies, a significant proportion of childhood brain tumors occur in the context of cancer predisposition syndromes.² Pediatric GBMs are associated with germline mutations in TP53 (Li-Fraumeni syndrome)¹ and the mismatch repair (MMR) genes (biallelic MMR deficiency syndrome [bMMRD]).³ Patients with bMMRD are unique in both the molecular events that lead to GBM formation and opportunities for innovative management of these tumors to possibly improve survival.

bMMRD is caused by homozygous germline mutations in one of the four MMR genes (PMS2, MLH1, MSH2, and MSH6) and is arguably the most penetrant cancer predisposition syndrome, with 100% of biallelic mutation carriers developing cancers in the first two decades of life. These are most commonly malignant gliomas, hematologic malignancies, and GI cancers.^3,4 Understanding the relationship between the bMMRD somatic mutational landscape and tumor biology can lead to development of novel therapies and improved patient outcomes.

bMMRD GBMs harbor the highest mutation load among human cancers.⁵ Combined germline mutations in the MMR genes and somatic mutations in DNA polymerase result in complete ablation of proofreading during DNA replication and underpin this phenomenon. bMMRD GBMs, in contrast to other childhood cancers and adult MMR-proficient gliomas, exhibit a molecular signature characterized by single-nucleotide changes present in exponentially higher numbers. An important characteristic of non-bMMRD cancers exhibiting high mutation loads—subsets of malignant melanomas and lung, bladder, and microsatellite-unstable GI cancers—is responsiveness to immune checkpoint inhibitors.^6–9

Checkpoint inhibitors target the immunomodulatory effect of CTLA-4 (cytotoxic T lymphocyte–associated protein 4) and programmed death-1 (PD-1)/programmed death-ligand 1, restoring effector T-cell function and antitumor activity. Recent reports have shown that patients whose tumors bear a high mutation load and/or definedtumor-associated antigen (neoantigen) signatures derive enhanced clinical benefit from checkpoint inhibitor therapy.¹⁰

Nivolumab is an anti–PD-1–directed immune checkpoint inhibitor approved for use in the treatment of non–small-cell lung cancer¹¹and melanoma and under clinical investigation in multiple adult and pediatric tumors.^12,13 However, this response is currently unknown in bMMRD-associated cancers and the uniformly lethal GBM.

Fig 1.

Fig 1. Clinical and molecular features of the biallelic mismatch repair (MMR) deficiency (bMMRD) family. (A) Pedigree of the family with both bMMRD-affected children (solid square and circle). Both siblings presented with glioblastoma multiforme (GBM), whereas parents remained unaffected, as observed in other bMMRD families. (B) Immunohistochemistry staining of the index patient’s GBM for the four MMR genes: MSH2, MSH6,MLH1, and PMS2. A PMS2-negative stain in both tumor and normal cells prompted subsequent genetic testing that confirmed the diagnosis of bMMRD. NF1, neurofibromatosis type 1. http://jco.ascopubs.org/content/early/2016/03/17/JCO.2016.66.6552/F1.small.gif

To examine whether immune checkpoint inhibitors would be applicable for bMMRD cancers, we surveyed the extent of hypermutation across bMMRD tumors form various tissues. Exome sequencing of 37 cancers collected from the bMMRD consortium revealed that all malignant tumors (n = 32) were hypermutant. Although bMMRD brain tumors had the highest mutational load resulting from secondary polymerase mutations (mean, 17,740 ± standard deviation [SD], 7,703), all other high-grade tumors were hypermutant, harboring more than 100 exonic mutations (mean, 1,589 ± SD, 1,043; Fig 2A). Lower-grade bMMRD tumors (n = 5) did not exhibit hypermutation (mean, 40 ± SD, 18). Importantly, bMMRD GBMs had a significantly higher mutational load than sporadic pediatric and adult gliomas and all other brain tumors (P < .001; Fig 2A). To test the extent to which hypermutation translates to a strong neoantigen signature, a current predictor of response to immune checkpoint inhibition, we performed genome-wide somatic neoepitope analysis using similar algorithms previously used for melanoma, lung, and colon cancers.^9,14,15 For each study, we compared our cohort of tumors with other tumors that were reported to respond to immune checkpoint inhibitors (Fig 2B). Strikingly, bMMRD GBMs had a significantly higher number of predicted neoantigens, whereas other tumors responded with a fraction of the neoantigens found in our patients (P < .001; Fig 2B). The mean neoantigen load was seven to 16 times higher than those of immunoresponsive melanomas, lung cancers, and microsatellite-unstable GI cancers.

Fig 2.

http://jco.ascopubs.org/content/early/2016/03/17/JCO.2016.66.6552/F2.small.gif

Fig 2. Tumor mutation and neoantigen analysis. (A) Boxplot comparing the number of mutations per tumor exome in several biallelic mismatch repair deficiency (bMMRD) cancer types with pediatric and adult brain tumors. (B) Ratio of the number of neoantigens found in immunoresponsive tumors from melanoma (n = 27), lung cancer (n = 14), and colon cancer (n = 7) data sets compared with median number of neoantigens in bMMRD glioblastoma multiforme (GBM; n = 13). ATRT, atypical teratoid rhabdoid tumor; DIPG, diffuse intrinsic pontine glioma; L/L, leukemia/lymphoma; LGG, low-grade glioma; MB, medulloblastoma; PA, pilocytic astrocytoma; PNET, primitive neuroectodermal tumor.

We describe two pediatric patients with recurrent multifocal GBM refractory to current standard therapies who exhibited remarkable and durable responses to immune checkpoint inhibition with single-agent nivolumab. This observation is especially encouraging because these children are still clinically stable, whereas most relapsed pediatric GBMs will progress within 1 to 2 months¹⁹ despite salvage treatment, and survival is usually 3 to 6 months postrecurrence.²⁰ Furthermore, bMMRD GBMs have outcomes similar to those of sporadic childhood GBMs,²¹ and data gathered from the consortium reveal a mean time from relapse to death of 2.6 months in bMMRD GBM. To our knowledge, this is the first report of such a response in childhood or adult GBM. It also highlights the utility of germline predisposition in guiding novel treatment options—in this case, immunotherapy—for cancer treatment. ….

sjwilliamspa

Not sure if the link between PD-L1 response and MMR status is causal in this ase. there are many tumors with MMR and especially all tumors had high degree of MMR. Perhaps they need to look at tumors that have a more stable genome like certain hepatocarcinomas.

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DNA-based nanomotor and chemomechanical crosstalk

Posted in Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Curation, Genetics & Pharmaceutical, Genome Biology, Innovations, ISO 10993 for Product Registration: FDA & CE Mark for Development of Medical Devices and Diagnostics, Metabolomics, Molecular Genetics & Pharmaceutical, Nanotechnology for Drug Delivery, tagged DNA nanomachines, Molecular machines and motors on May 7, 2016| Leave a Comment »

DNA-based nanomotor and chemomechanical crosstalk

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

Nano-walkers take speedy leap forward with first rolling DNA-based motor

By Carol Clark
ESci Commons Dec 1, 2015 http://esciencecommons.blogspot.com/2015/12/nano-walkers-take-speedy-leap-forward.html

“Ours is the first rolling DNA motor, making it far faster and more robust,” says Khalid Salaita, the Emory chemist who led the research. (Photos by Bryan Meltz, Emory Photo/Video.) https://pharmaceuticalintelligence.com/wp-content/uploads/2016/05/c4943-khalid_salaita.jpg

hysical chemists have devised a rolling DNA-based motor that’s 1,000 times faster than any other synthetic DNA motor, giving it potential for real-world applications, such as disease diagnostics. Nature Nanotechnology is publishing the finding.

“Unlike other synthetic DNA-based motors, which use legs to ‘walk’ like tiny robots, ours is the first rolling DNA motor, making it far faster and more robust,” says Khalid Salaita, the Emory University chemist who led the research. “It’s like the biological equivalent of the invention of the wheel for the field of DNA machines.”

The speed of the new DNA-based motor, which is powered by ribonuclease H, means a simple smart phone microscope can capture its motion through video. The researchers have filed an invention disclosure patent for the concept of using the particle motion of their rolling molecular motor as a sensor for everything from a single DNA mutation in a biological sample to heavy metals in water.

“Our method offers a way of doing low-cost, low-tech diagnostics in settings with limited resources,” Salaita says.

The field of synthetic DNA-based motors, also known as nano-walkers, is about 15 years old. Researchers are striving to duplicate the action of nature’s nano-walkers. Myosin, for example, are tiny biological mechanisms that “walk” on filaments to carry nutrients throughout the human body.

“It’s the ultimate in science fiction,” Salaita says of the quest to create tiny robots, or nano-bots, that could be programmed to do your bidding. “People have dreamed of sending in nano-bots to deliver drugs or to repair problems in the human body.”

So far, however, mankind’s efforts have fallen far short of nature’s myosin, which speeds effortlessly about its biological errands. “The ability of myosin to convert chemical energy into mechanical energy is astounding,” Salaita says. “They are the most efficient motors we know of today.”

Some synthetic nano-walkers move on two legs. They are essentially enzymes made of DNA, powered by the catalyst RNA. These nano-walkers tend to be extremely unstable, due to the high levels of Brownian motion at the nano-scale. Other versions with four, and even six, legs have proved more stable, but much slower. In fact, their pace is glacial: A four-legged DNA-based motor would need about 20 years to move one centimeter.

Kevin Yehl, a post-doctoral fellow in the Salaita lab, had the idea of constructing a DNA-based motor using a micron-sized glass sphere. Hundreds of DNA strands, or “legs,” are allowed to bind to the sphere. These DNA legs are placed on a glass slide coated with the reactant: RNA.

The DNA legs are drawn to the RNA, but as soon as they set foot on it they destroy it through the activity of an enzyme called RNase H. As the legs bind and then release from the substrate, they guide the sphere along, allowing more of the DNA legs to keep binding and pulling.

“It’s called a burnt-bridge mechanism,” Salaita explains. “Wherever the DNA legs step, they trample and destroy the reactant. They have to keep moving and step where they haven’t stepped in order to find more reactant.”

The combination of the rolling motion, and the speed of the RNase H enzyme on a substrate, gives the new DNA motor its stability and speed.

“Our DNA-based motor can travel one centimeter in seven days, instead of 20 years, making it 1,000 times faster than the older versions,” Salaita says. “In fact, nature’s myosin motors are only 10 times faster than ours, and it took them billions of years to evolve.”

http://2.bp.blogspot.com/-tB7Dtk9txtY/Vl3R_IInh3I/AAAAAAAAK7g/Kf3lSVSHzr8/s400/smart-phone_setup.jpg

Emory post-doctoral fellow Kevin Yehl sets up a smart-phone microscope to get a readout for the particle motion of the rolling DNA-based motor.

The researchers demonstrated that their rolling motors can be used to detect a single DNA mutation by measuring particle displacement. They simply glued lenses from two inexpensive laser pointers to the camera of a smart phone to turn the phone into a microscope and capture videos of the particle motion.

“Using a smart phone, we can get a readout for anything that’s interfering with the enzyme-substrate reaction, because that will change the speed of the particle,” Salaita says. “For instance, we can detect a single mutation in a DNA strand.”

This simple, low-tech method could come in handy for doing diagnostic sensing of biological samples in the field, or anywhere with limited resources.

The proof that the motors roll came by accident, Salaita adds. During their experiments, two of the glass spheres occasionally became stuck together, or dimerized. Instead of making a wandering trail, they left a pair of straight, parallel tracks across the substrate, like a lawn mower cutting grass. “It’s the first example of a synthetic molecular motor that goes in a straight line without a track or a magnetic field to guide it,” Salaita says.

In addition to Salaita and Yehl, the co-authors on the Nature Nanotechnology paper include Emory researchers Skanda Vivek, Yang Liu, Yun Zhang, Megzhen Fan, Eric Weeks and Andrew Mugler (who is now at Purdue University).

High-speed DNA-based rolling motors powered by RNase H

Kevin Yehl, Andrew Mugler, Skanda Vivek, Yang Liu, Yun Zhang, Mengzhen Fan, Eric R. Weeks & Khalid Salaita
Nature Nanotechnology 11;184–190 (30 Nov 2016) doi:10.1038/nnano.2015.259

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next-generation sensors, drug-delivery platforms and biological computing. Despite their exquisite programmability, DNA-based walkers are challenging to work with because of their low fidelity and slow rates (∼1 nm min^–1). Here we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three orders of magnitude greater than the maximum for conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridize to a surface modified with complementary RNA; the motion is achieved through the addition of RNase H, which selectively hydrolyses the hybridized RNA. The spherical motors can move in a self-avoiding manner, and anisotropic particles, such as dimerized or rod-shaped particles, can travel linearly without a track or external force. We also show that the motors can be used to detect single nucleotide polymorphism by measuring particle displacement using a smartphone camera.

http://www.nature.com/nnano/journal/v11/n2/carousel/nnano.2015.259-f1.jpg

http://www.nature.com/nnano/journal/v11/n2/carousel/nnano.2015.259-f2.jpg

http://www.nature.com/nnano/journal/v11/n2/full/nnano.2015.259.html

T cells use ‘handshakes’ to sort friends from foes

http://esciencecommons.blogspot.ca/2016/05/t-cells-use-handshakes-to-sort-friends.html

A 3-D rendering of a fluorescence image mapping the piconewton forces applied by T cells. The height and color indicates the magnitude of the applied force. (Microscopy image by Yang Liu.)

By Carol Clark

T cells, the security guards of the immune system, use a kind of mechanical “handshake” to test whether a cell they encounter is a friend or foe, a new study finds.

The Proceedings of the National Academy of Sciences (PNAS) published the study, led by Khalid Salaita, a physical chemist at Emory University who specializes in the mechanical forces of cellular processes.

“We’ve provided the first direct evidence that a T cell gives precise mechanical tugs to other cells,” Salaita says. “And we’ve shown that these tugs are central to a T cell’s process of deciding whether to mount an immune response. A tug that releases easily, similar to a casual handshake, signals a friend. A stronger grip indicates a foe.”

Salaita, from Emory’s Department of Chemistry, collaborated on the research with Brian Evavold in the Emory School of Medicine’s Department of Microbiology and Immunology.

T cells continuously patrol through the body in search of foreign invaders. They have molecules known as T-cell receptors (TCR) that can recognize specific antigenic peptides on the surface of a pathogenic or cancerous cell. When a T cell detects an antigen-presenting cell (APC), its TCR connects to a ligand, or binding molecule, of the APC. If the T cell determines the ligand is foreign, it becomes activated and starts pumping calcium. The calcium is part of a signaling chain that recruits other cells to come and help mount an immune response.

Scientists have known about this process for decades, but they have not fully understood how the T cell distinguishes small modifications to the antigenic ligand and how it decides to respond to it. “If you view this T cell response purely as a chemical process, it does not fully explain the remarkable specifity of the binding,” Salaita says. “When you take the two components – the TCR and the ligand on the surface of cells – and just let them chemically bind in a solution, for example, you can’t predict what will trigger a strong or a weak immune response.”

The researchers hypothesized that mechanical strain might also play a role in a T cell response, since the T cell continues to move even as it locks into a bind with an antigenic ligand.

To test this idea, the Salaita lab developed DNA-based gold nanoparticle tension sensors that light up, or fluoresce, in response to a miniscule mechanical force of a piconewton – about one million-millionth the weight of an apple.

The researchers designed experiments using T cells from a mouse and allowed them to test ligands containing eight amino acid peptides that had slight mutations.

“We swapped out the fourth amino acid position to create really subtle chemical changes in the ligand that would be very difficult to distinguish without a mechanical component,” Salaita says.

Some of the mutated ligands were given a firmer anchor to give them a tighter “grip” to the moving TCR.

Through the experiments, captured on microscopy video, the researchers were able to see, record and measure the responses of the T cells as they moved across the ligands.

“As a T cell moves across a cell’s surface and encounters a ligand, it pulls on it,” Salaita explains. “It doesn’t pull very hard, it’s a very precise and tiny tug that is not sustained. The T cell pulls and stops, pulls and stops, all across the surface. It’s like the T cell is doing a mechanical test of the ligand.”

During the experiments, the T cells did not activate fully when they encountered ligands with weak anchors. In contrast, when a T cell encountered a ligand with a firm anchor, the T cell became activated, showing that it experienced a piconewton level of resistance.

The amount of force that was applied by the T cell was mapped by using tension probes of different stiffness. Probes that responded to 19 piconewtons did not fluoresce, while softer, 12-piconewton probes produced high signal.

Following the fluorescence of the probe, the T cells switched on their calcium pumps and increased the calcium concentration within the cell, indicating that the T cell is mounting an immune response.

“We were able to map out the order of the cascade of chemical and mechanical reactions,” Salaita says. “First, the T cell uses a very specific and finely tuned mechanical tug to distinguish friend from foe. And when it senses a precise, piconewton level of force in response to that tug, the T cell realizes that it has encountered a foreign body and gives the signal for attack.”

The discovery could help in the search for treatments of auto-immune diseases and the development of immune therapies for cancer.

“Cancer cells have an extra molecule that can make T cell security guards ‘drunk’ or ‘sleepy’ so that they are not able to function properly,” Salaita says. “Learning more about the mechanical forces involved in an effective immune response may help us develop ways to evade this defense system of cancer cells.”

Co-authors on the study include Yang Liu, Victor Pui-Yan Ma, Kornelia Galior and Zheng Liu (from the Salaita lab); and Lori Blanchfield and Rakieb Andargachew (from the Evavold lab).

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity

Yang Liu^a, Lori Blanchfield^b, Victor Pui-Yan Ma^a, Rakieb Andargachew^b, Kornelia Galior^a, Zheng Liu^a, Brian Evavold^b, and Khalid Salaita^a,^c,^{1

PNAS May 2016; http://dx.doi.org:/10.1073/pnas.1600163113}

Significance

T cells protect the body against pathogens and cancer by recognizing specific foreign peptides on the cell surface. Because antigen recognition occurs at the junction between a migrating T cell and an antigen-presenting cell (APC), it is likely that cellular forces are generated and transmitted through T-cell receptor (TCR)-ligand bonds. Here we develop a DNA-based nanoparticle tension sensor producing the first molecular maps of TCR-ligand forces during T cell activation. We find that TCR forces are orchestrated in space and time, requiring the participation of CD8 coreceptor and adhesion molecules. Loss or damping of TCR forces results in weakened antigen discrimination, showing that T cells harness mechanics to optimize the specificity of response to ligand.

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12–19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically “filtered” to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

The two articles above are connected in an interesting way by the fact that cellular forces are generated and transmitted through T-cell receptor (TCR)-ligand bonds. The T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). The movement detected by the fluorescent sensor may be based on only a single amino acid at the cell surface ligand. The result is chemomechanical crosstalk between TCR and LFA-1 receptor signaling

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Gene Editing with CRISPR gets Crisper

Posted in Artificial Intelligence - Breakthroughs in Theories and Technologies, Artificial Intelligence - General, BioIT: BioInformatics, Biological Networks, Gene Regulation and Evolution, BioTechnology - Venture Creation, Cancer and Current Therapeutics, Cancer Informatics, Cancer Screening, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, CRISPR/Cas9 & Gene Editing, Diagnostics and Lab Tests, Disease Biology, Small Molecules in Development of Therapeutic Drugs, DNA repair, FDA Regulatory Affairs, Gene Regulation, Genetics & Pharmaceutical, Genomic Expression, Genomic Testing: Methodology for Diagnosis, Genomics Pharmacy, Innovations, Loss of function gene, Mutant Gene Expression, Nanotechnology for Drug Delivery, Next Generation Sequencing (NGS), Oligonucleotide Therapeutics & Delivery, tagged causal variants underlying trait differences, CRISPR-based gene editing, Drug discovery, gene mapping and CRISPR/Cas, genetic markers, mapping, mapping panels, phenotype, sgRNA, systems biology, targeted recombination events, yeast on May 3, 2016| Leave a Comment »

Gene Editing with CRISPR gets Crisper

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

CRISPR Moves from Butchery to Surgery

More Genomes Are Going Under the CRISPR Knife, So Surgical Standards Are Rising

Meghaan M. Ferreira, Ph.D.

http://www.genengnews.com/gen-articles/crispr-moves-from-butchery-to-surgery/5759/

http://www.genengnews.com/Media/images/Article/thumb_GE_Dharmacon_cas9tracrrna1332182551.jpg

The Dharmacon subsidary of GE Healthcare provides the Edit-R Lentiviral Gene Engineering platform. It is based on the natural S. pyrogenes system, but unlike that system, which uses a single guide RNA (sgRNA), the platform uses two component RNAs, a gene-specific CRISPR RNA (crRNA) and a universal trans-activating crRNA (tracrRNA). Once hybridized to the universal tracrRNA (blue), the crRNA (green) directs the Cas9 nuclease to a specific genomic region to induce a double- strand break.

Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.

In the naturally occurring system, CRISPR-Cas9 works like a self-vaccination in the bacterial immune system by targeting and cleaving viral DNA sequences stored from previous encounters with invading phages. The endogenous system uses two RNA elements, CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), which come together and guide the Cas9 nuclease to the target DNA.

Early publications that demonstrated CRISPR gene editing in mammalian cells combined the crRNA and tracrRNA sequences to form one long transcript called asingle-guide RNA (sgRNA). However, an alternative approach is being explored by scientists at the Dharmacon subsidiary of GE Healthcare. These scientists have a system that mimics the endogenous system through a synthetic two-component approach thatpreserves individual crRNA and tracrRNA. The tracrRNA is universal to any gene target or species; the crRNA contains the information needed to target the gene of interest.

Predesigned Guide RNAs

In contrast to sgRNAs, which are generated through either in vitro transcription of a DNA template or a plasmid-based expression system, synthetic crRNA and tracrRNA eliminate the need for additional cloning and purification steps. The efficacy of guide RNA (gRNA), whether delivered as a sgRNA or individual crRNA and tracrRNA, depends not only on DNA binding, but also on the generation of an indel that will deliver the coup de grâce to gene function.

“Almost all of the gRNAs were able to create a break in genomic DNA,” said Louise Baskin, senior product manager at Dharmacon. “But there was a very wide range in efficiency and in creating functional protein knock-outs.”

To remove the guesswork from gRNA design, Dharmacon developed an algorithm to predict gene knockout efficiency using wet-lab data. They also incorporated specificity as a component of their algorithm, using a much more comprehensive alignment tool to predict potential off-target effects caused by mismatches and bulges often missed by other alignment tools. Customers can enter their target gene to access predesigned gRNAs as either two-component RNAs or lentiviral sgRNA vectors for multiple applications.

“We put time and effort into our algorithm to ensure that our guide RNAs are not only functional but also highly specific,” asserts Baskin. “As a result, customers don’t have to do any design work.”

Donor DNA Formats

http://www.genengnews.com/Media/images/Article/thumb_MilliporeSigma_CRISPR3120824917.jpg

MilliporeSigma’s CRISPR Epigenetic Activator is based on fusion of a nuclease-deficient Cas9 (dCas9) to the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300. This technology allows researchers to target specific DNA regions or gene sequences. Researchers can localize epigenetic changes to their target of interest and see the effects of those changes in gene expression.

Knockout experiments are a powerful tool for analyzing gene function. However, for researchers who want to introduce DNA into the genome, guide design, donor DNA selection, and Cas9 activity are paramount to successful DNA integration.MilliporeSigma offers two formats for donor DNA: double-stranded DNA (dsDNA) plasmids and single-stranded DNA (ssDNA) oligonucleotides. The most appropriate format depends on cell type and length of the donor DNA. “There are some cell types that have immune responses to dsDNA,” said Gregory Davis, Ph.D., R&D manager, MilliporeSigma.

The ssDNA format can save researchers time and money, but it has a limited carrying capacity of approximately 120 base pairs.In addition to selecting an appropriate donor DNA format, controlling where, how, and when the Cas9 enzyme cuts can affect gene-editing efficiency. Scientists are playing tug-of-war, trying to pull cells toward the preferred homology-directed repair (HDR) and away from the less favored nonhomologous end joining (NHEJ) repair mechanism.One method to achieve this modifies the Cas9 enzyme to generate a nickase that cuts only one DNA strand instead of creating a double-strand break. Accordingly, MilliporeSigma has created a Cas9 paired-nickase system that promotes HDR, while also limiting off-target effects and increasing the number of sequences available for site-dependent gene modifications, such as disease-associated single nucleotide polymorphisms (SNPs).“The best thing you can do is to cut as close to the SNP as possible,” advised Dr. Davis. “As you move the double-stranded break away from the site of mutation you get an exponential drop in the frequency of recombination.”

Ribonucleo-protein Complexes

Another strategy to improve gene-editing efficiency, developed by Thermo Fisher, involves combining purified Cas9 protein with gRNA to generate a stable ribonucleoprotein (RNP) complex. In contrast to plasmid- or mRNA-based formats, which require transcription and/or translation, the Cas9 RNP complex cuts DNA immediately after entering the cell. Rapid clearance of the complex from the cell helps to minimize off-target effects, and, unlike a viral vector, the transient complex does not introduce foreign DNA sequences into the genome.

To deliver their Cas9 RNP complex to cells, Thermo Fisher has developed a lipofectamine transfection reagent called CRISPRMAX. “We went back to the drawing board with our delivery, screened a bunch of components, and got a brand-new, fully optimized lipid nanoparticle formulation,” explained Jon Chesnut, Ph.D., the company’s senior director of synthetic biology R&D. “The formulation is specifically designed for delivering the RNP to cells more efficiently.”

Besides the reagent and the formulation, Thermo Fisher has also developed a range of gene-editing tools. For example, it has introduced the Neon^® transfection system for delivering DNA, RNA, or protein into cells via electroporation. Dr. Chesnut emphasized the company’s focus on simplifying complex workflows by optimizing protocols and pairing everything with the appropriate up- and downstream reagents.

From Mammalian Cells to Microbes

One of the first sources of CRISPR technology was the Feng Zhang laboratory at the Broad Institute, which counted among its first licensees a company called GenScript. This company offers a gene-editing service called GenCRISPR™ to establish mammalian cell lines with CRISPR-derived gene knockouts.

“There are a lot of challenges with mammalian cells, and each cell line has its own set of issues,” said Laura Geuss, a marketing specialist at GenScript. “We try to offer a variety of packages that can help customers who have difficult-to-work-with cells.” These packages include both viral-based and transient transfection techniques.

However, the most distinctive service offered by GenScript is its microbial genome-editing service for bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). The company’s strategy for gene editing in bacteria can enable seamless knockins, knockouts, or gene replacements by combining CRISPR with lambda red recombineering. Traditionally one of the most effective methods for gene editing in microbes, recombineering allows editing without restriction enzymes through in vivo homologous recombination mediated by a phage-based recombination system such as lambda red.

On its own, lambda red technology cannot target multiple genes, but when paired with CRISPR, it allows the editing of multiple genes with greater efficiency than is possible with CRISPR alone, as the lambda red proteins help repair double-strand breaks in E. coli. The ability to knockout different gene combinations makes Genscript’s microbial editing service particularly well suited for the optimization of metabolic pathways.

Pooled and Arrayed Library Strategies

Scientists are using CRISPR technology for applications such as metabolic engineering and drug development. Yet another application area benefitting from CRISPR technology is cancer research. Here, the use of pooled CRISPR libraries is becoming commonplace. Pooled CRISPR libraries can help detect mutations that affect drug resistance, and they can aid in patient stratification and clinical trial design.

Pooled screening uses proliferation or viability as a phenotype to assess how genetic alterations, resulting from the application of a pooled CRISPR library, affect cell growth and death in the presence of a therapeutic compound. The enrichment or depletion of different gRNA populations is quantified using deep sequencing to identify the genomic edits that result in changes to cell viability.

MilliporeSigma provides pooled CRISPR libraries ranging from the whole human genome to smaller custom pools for these gene-function experiments. For pharmaceutical and biotech companies, Horizon Discovery offers a pooled screening service, ResponderSCREEN, which provides a whole-genome pooled screen to identify genes that confer sensitivity or resistance to a compound. This service is comprehensive, taking clients from experimental design all the way through to suggestions for follow-up studies.

Horizon Discovery maintains a Research Biotech business unit that is focused on target discovery and enabling translational medicine in oncology. “Our internal backbone gives us the ability to provide expert advice demonstrated by results,” said Jon Moore, Ph.D., the company’s CSO.

In contrast to a pooled screen, where thousands of gRNA are combined in one tube, an arrayed screen applies one gRNA per well, removing the need for deep sequencing and broadening the options for different endpoint assays. To establish and distribute a whole-genome arrayed lentiviral CRISPR library, MilliporeSigma partnered with the Wellcome Trust Sanger Institute. “This is the first and only arrayed CRISPR library in the world,” declared Shawn Shafer, Ph.D., functional genomics market segment manager, MilliporeSigma. “We were really proud to partner with Sanger on this.”

Pooled and arrayed screens are powerful tools for studying gene function. The appropriate platform for an experiment, however, will be determined by the desired endpoint assay.

Detection and Quantification of Edits

http://www.genengnews.com/Media/images/Article/BioRad_QX200_System4276117210.jpg

The QX200 Droplet Digital PCR System from Bio-Rad Laboratories can provide researchers with an absolute measure of target DNA molecules for EvaGreen or probe-based digital PCR applications. The system, which can provide rapid, low-cost, ultra-sensitive quantification of both NHEJ- and HDR-editing events, consists of two instruments, the QX200 Droplet Generator and the QX200 Droplet Reader, and their associated consumables.

Finally, one last challenge for CRISPR lies in the detection and quantification of changes made to the genome post-editing. Conventional methods for detecting these alterations include gel methods and next-generation sequencing. While gel methods lack sensitivity and scalability, next-generation sequencing is costly and requires intensive bioinformatics.

To address this gap, Bio-Rad Laboratories developed a set of assay strategies to enable sensitive and precise edit detection with its Droplet Digital PCR (ddPCR) technology. The platform is designed to enable absolute quantification of nucleic acids with high sensitivity, high precision, and short turnaround time through massive droplet partitioning of samples.

Using a validated assay, a typical ddPCR experiment takes about five to six hours to complete. The ddPCR platform enables detection of rare mutations, and publications have reported detection of precise edits at a frequency of <0.05%, and of NHEJ-derived indels at a frequency as low as 0.1%. In addition to quantifying precise edits, indels, and computationally predicted off-target mutations, ddPCR can also be used to characterize the consequences of edits at the RNA level.

According to a recently published Science paper, the laboratory of Charles A. Gersbach, Ph.D., at Duke University used ddPCR in a study of muscle function in a mouse model of Duchenne muscular dystrophy. Specifically, ddPCR was used to assess the efficiency of CRISPR-Cas9 in removing the mutated exon 23 from the dystrophin gene. (Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene and significant enhancement of muscle force.)

Quantitative ddPCR showed that exon 23 was deleted in ~2% of all alleles from the whole-muscle lysate. Further ddPCR studies found that 59% of mRNA transcripts reflected the deletion.

“There’s an overarching idea that the genome-editing field is moving extremely quickly, and for good reason,” asserted Jennifer Berman, Ph.D., staff scientist, Bio-Rad Laboratories. “There’s a lot of exciting work to be done, but detection and quantification of edits can be a bottleneck for researchers.”

The gene-editing field is moving quickly, and new innovations are finding their way into the laboratory as researchers lay the foundation for precise, well-controlled gene editing with CRISPR.

Are Current Cancer Drug Discovery Methods Flawed?

GEN May 3, 2016 http://www.genengnews.com/gen-news-highlights/are-current-cancer-drug-discovery-methods-flawed/81252682/

Researchers utilized a systems biology approach to develop new methods to assess drug sensitivity in cells. [The Institute for Systems Biology]

Understanding how cells respond and proliferate in the presence of anticancer compounds has been the foundation of drug discovery ideology for decades. Now, a new study from scientists at Vanderbilt University casts significant suspicion on the primary method used to test compounds for anticancer activity in cells—instilling doubt on methods employed by the entire scientific enterprise and pharmaceutical industry to discover new cancer drugs.

“More than 90% of candidate cancer drugs fail in late-stage clinical trials, costing hundreds of millions of dollars,” explained co-senior author Vito Quaranta, M.D., director of the Quantitative Systems Biology Center at Vanderbilt. “The flawed in vitro drug discovery metric may not be the only responsible factor, but it may be worth pursuing an estimate of its impact.”

The Vanderbilt investigators have developed what they believe to be a new metric for evaluating a compound’s effect on cell proliferation—called the DIP (drug-induced proliferation) rate—that overcomes the flawed bias in the traditional method.

The findings from this study were published recently in Nature Methods in an article entitled “An Unbiased Metric of Antiproliferative Drug Effect In Vitro.”

For more than three decades, researchers have evaluated the ability of a compound to kill cells by adding the compound in vitro and counting how many cells are alive after 72 hours. Yet, proliferation assays that measure cell number at a single time point don’t take into account the bias introduced by exponential cell proliferation, even in the presence of the drug.

“Cells are not uniform, they all proliferate exponentially, but at different rates,” Dr. Quaranta noted. “At 72 hours, some cells will have doubled three times and others will not have doubled at all.”

Dr. Quaranta added that drugs don’t all behave the same way on every cell line—for example, a drug might have an immediate effect on one cell line and a delayed effect on another.

The research team decided to take a systems biology approach, a mixture of experimentation and mathematical modeling, to demonstrate the time-dependent bias in static proliferation assays and to develop the time-independent DIP rate metric.

“Systems biology is what really makes the difference here,” Dr. Quaranta remarked. “It’s about understanding cells—and life—as dynamic systems.”This new study is of particular importance in light of recent international efforts to generate data sets that include the responses of thousands of cell lines to hundreds of compounds. Using the

Cancer Cell Line Encyclopedia (CCLE) and
Genomics of Drug Sensitivity in Cancer (GDSC) databases

will allow drug discovery scientists to include drug response data along with genomic and proteomic data that detail each cell line’s molecular makeup.

“The idea is to look for statistical correlations—these particular cell lines with this particular makeup are sensitive to these types of compounds—to use these large databases as discovery tools for new therapeutic targets in cancer,” Dr. Quaranta stated. “If the metric by which you’ve evaluated the drug sensitivity of the cells is wrong, your statistical correlations are basically no good.”

The Vanderbilt team evaluated the responses from four different melanoma cell lines to the drug vemurafenib, currently used to treat melanoma, with the standard metric—used for the CCLE and GDSC databases—and with the DIP rate. In one cell line, they found a glaring disagreement between the two metrics.

“The static metric says that the cell line is very sensitive to vemurafenib. However, our analysis shows this is not the case,” said co-lead study author Leonard Harris, Ph.D., a systems biology postdoctoral fellow at Vanderbilt. “A brief period of drug sensitivity, quickly followed by rebound, fools the static metric, but not the DIP rate.”

Dr. Quaranta added that the findings “suggest we should expect melanoma tumors treated with this drug to come back, and that’s what has happened, puzzling investigators. DIP rate analyses may help solve this conundrum, leading to better treatment strategies.”

The researchers noted that using the DIP rate is possible because of advances in automation, robotics, microscopy, and image processing. Moreover, the DIP rate metric offers another advantage—it can reveal which drugs are truly cytotoxic (cell killing), rather than merely cytostatic (cell growth inhibiting). Although cytostatic drugs may initially have promising therapeutic effects, they may leave tumor cells alive that then have the potential to cause the cancer to recur.

The Vanderbilt team is currently in the process of identifying commercial entities that can further refine the software and make it widely available to the research community to inform drug discovery.

An unbiased metric of antiproliferative drug effect in vitro

Leonard A Harris, Peter L Frick, Shawn P Garbett, Keisha N Hardeman, B Bishal Paudel, Carlos F Lopez, Vito Quaranta & Darren R Tyson
Nature Methods 2 May (2016) doi:10.1038/nmeth.3852

In vitro cell proliferation assays are widely used in pharmacology, molecular biology, and drug discovery. Using theoretical modeling and experimentation, we show that current metrics of antiproliferative small molecule effect suffer from time-dependent bias, leading to inaccurate assessments of parameters such as drug potency and efficacy. We propose the drug-induced proliferation (DIP) rate, the slope of the line on a plot of cell population doublings versus time, as an alternative, time-independent metric.

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Mapping Traits to Genes with CRISPR

Researchers develop a technique to direct chromosome recombination with CRISPR/Cas9, allowing high-resolution genetic mapping of phenotypic traits in yeast.

By Catherine Offord | May 5, 2016

http://www.the-scientist.com/?articles.view/articleNo/46029/title/Mapping-Traits-to-Genes-with-CRISPR

http://www.the-scientist.com/images/News/May2016/sciencefigure.jpg

Researchers used CRISPR/Cas9 to make a targeted double-strand break (DSB) in one arm of a yeast chromosome labeled with a green fluorescent protein (GFP) gene. A within-cell mechanism called homologous repair (HR) mends the broken arm using its homolog, resulting in a recombined region from the site of the break to the chromosome tip. When this cell divides by mitosis, each daughter cell will contain a homozygous section in an outcome known as “loss of heterozygosity” (LOH). One of the daughter cells is detectable because, due to complete loss of the GFP gene, it will no longer be fluorescent.REPRINTED WITH PERMISSION FROM M.J. SADHU ET AL., SCIENCE

When mapping phenotypic traits to specific loci, scientists typically rely on the natural recombination of chromosomes during meiotic cell division in order to infer the positions of responsible genes. But recombination events vary with species and chromosome region, giving researchers little control over which areas of the genome are shuffled. Now, a team at the University of California, Los Angeles (UCLA), has found a way around these problems by using CRISPR/Cas9 to direct targeted recombination events during mitotic cell division in yeast. The team described its technique today (May 5) in Science.

“Current methods rely on events that happen naturally during meiosis,” explained study coauthor Leonid Kruglyak of UCLA. “Whatever rate those events occur at, you’re kind of stuck with. Our idea was that using CRISPR, we can generate those events at will, exactly where we want them, in large numbers, and in a way that’s easy for us to pull out the cells in which they happened.”

Generally, researchers use coinheritance of a trait of interest with specific genetic markers—whose positions are known—to figure out what part of the genome is responsible for a given phenotype. But the procedure often requires impractically large numbers of progeny or generations to observe the few cases in which coinheritance happens to be disrupted informatively. What’s more, the resolution of mapping is limited by the length of the smallest sequence shuffled by recombination—and that sequence could include several genes or gene variants.

“Once you get down to that minimal region, you’re done,” said Kruglyak. “You need to switch to other methods to test every gene and every variant in that region, and that can be anywhere from challenging to impossible.”

But programmable, DNA-cutting champion CRISPR/Cas9 offered an alternative. During mitotic—rather than meiotic—cell division, rare, double-strand breaks in one arm of a chromosome preparing to split are sometimes repaired by a mechanism called homologous recombination. This mechanism uses the other chromosome in the homologous pair to replace the sequence from the break down to the end of the broken arm. Normally, such mitotic recombination happens so rarely as to be impractical for mapping purposes. With CRISPR/Cas9, however, the researchers found that they could direct double-strand breaks to any locus along a chromosome of interest (provided it was heterozygous—to ensure that only one of the chromosomes would be cut), thus controlling the sites of recombination.

Combining this technique with a signal of recombination success, such as a green fluorescent protein (GFP) gene at the tip of one chromosome in the pair, allowed the researchers to pick out cells in which recombination had occurred: if the technique failed, both daughter cells produced by mitotic division would be heterozygous, with one copy of the signal gene each. But if it succeeded, one cell would end up with two copies, and the other cell with none—an outcome called loss of heterozygosity.

“If we get loss of heterozygosity . . . half the cells derived after that loss of heterozygosity event won’t have GFP anymore,” study coauthor Meru Sadhu of UCLA explained. “We search for these cells that don’t have GFP out of the general population of cells.” If these non-fluorescent cells with loss of heterozygosity have the same phenotype as the parent for a trait of interest, then CRISPR/Cas9-targeted recombination missed the responsible gene. If the phenotype is affected, however, then the trait must be linked to a locus in the recombined, now-homozygous region, somewhere between the cut site and the GFP gene.

By systematically making cuts using CRISPR/Cas9 along chromosomes in a hybrid, diploid strain ofSaccharomyces cerevisiae yeast, picking out non-fluorescent cells, and then observing the phenotype, the UCLA team demonstrated that it could rapidly identify the phenotypic contribution of specific gene variants. “We can simply walk along the chromosome and at every [variant] position we can ask, does it matter for the trait we’re studying?” explained Kruglyak.

For example, the team showed that manganese sensitivity—a well-defined phenotypic trait in lab yeast—could be pinpointed using this method to a single nucleotide polymorphism (SNP) in a gene encoding the Pmr1 protein (a manganese transporter).

Jason Moffat, a molecular geneticist at the University of Toronto who was not involved in the work, toldThe Scientist that researchers had “dreamed about” exploiting these sorts of mechanisms for mapping purposes, but without CRISPR, such techniques were previously out of reach. Until now, “it hasn’t been so easy to actually make double-stranded breaks on one copy of a pair of chromosomes, and then follow loss of heterozygosity in mitosis,” he said, adding that he hopes to see the approach translated into human cell lines.

Applying the technique beyond yeast will be important, agreed cell and developmental biologist Ethan Bier of the University of California, San Diego, because chromosomal repair varies among organisms. “In yeast, they absolutely demonstrate the power of [this method],” he said. “We’ll just have to see how the technology develops in other systems that are going to be far less suited to the technology than yeast. . . . I would like to see it implemented in another system to show that they can get the same oomph out of it in, say, mammalian somatic cells.”

Kruglyak told The Scientist that work in higher organisms, though planned, is still in early stages; currently, his team is working to apply the technique to map loci responsible for trait differences between—rather than within—yeast species.

“We have a much poorer understanding of the differences across species,” Sadhu explained. “Except for a few specific examples, we’re pretty much in the dark there.”

M.J. Sadhu, “CRISPR-directed mitotic recombination enables genetic mapping without crosses,” Science, doi:10.1126/science.aaf5124, 2016.

CRISPR-directed mitotic recombination enables genetic mapping without crosses

, Joshua S Bloom, , Leonid Kruglyak

http://biorxiv.org/content/biorxiv/early/2016/02/19/040428.full.pdf doi: http://dx.doi.org/10.1101/040428

Linkage and association studies have mapped thousands of genomic regions that contribute to phenotypic variation, but narrowing these regions to the underlying causal genes and variants has proven much more challenging. Resolution of genetic mapping is limited by the recombination rate. We developed a method that uses CRISPR to build mapping panels with targeted recombination events. We tested the method by generating a panel with recombination events spaced along a yeast chromosome arm, mapping trait variation, and then targeting a high density of recombination events to the region of interest. Using this approach, we fine-mapped manganese sensitivity to a single polymorphism in the transporter Pmr1. Targeting recombination events to regions of interest allows us to rapidly and systematically identify causal variants underlying trait differences.

The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.

Meru Sadhu • 2 months ago

Thank you, David, for the kind words and comments. We agree that the most immediate applications of the CRISPR-based recombination mapping will be in unicellular organisms and cell culture. We also think the method holds a lot of promise for research in multicellular organisms, although we did not mean to imply that it “will be an efficient mapping method for all multicellular organisms”. Every organism will have its own set of constraints as well as experimental tools that will be relevant when adapting a new technique. To best help experts working on these organisms, here are our thoughts on your questions.

You asked about mutagenesis during recombination. We Sanger sequenced 72 of our LOH lines at the recombination site and did not observe any mutations, as described in the supplementary materials. We expect the absence of mutagenesis is because we targeted heterozygous sites where the untargeted allele did not have a usable PAM site; thus, following LOH, the targeted site is no longer present and cutting stops. In your experiments you targeted sites that were homozygous; thus, following recombination, the CRISPR target site persisted, and continued cutting ultimately led to repair by NHEJ and mutagenesis.

As to the more general question of the optimal mapping strategies in different organisms, they will depend on the ease of generating and screening for editing events, the cost and logistics of maintaining and typing many lines, and generation time, among other factors. It sounds like in Drosophila today, your related approach of generating markers with CRISPR, and then enriching for natural recombination events that separate them, is preferable. In yeast, we’ve found the opposite to be the case. As you note, even in Drosophila, our approach may be preferable for regions with low or highly non-uniform recombination rates.

Finally, mapping in sterile interspecies hybrids should be straightforward for unicellular hybrids (of which there are many examples) and for cells cultured from hybrid animals or plants. For studies in hybrid multicellular organisms, we agree that driving mitotic recombination in the early embryo may be the most promising approach. Chimeric individuals with mitotic clones will be sufficient for many traits. Depending on the system, it may in fact be possible to generate diploid individuals with uniform LOH genotype, but this is certainly beyond the scope of our paper. The calculation of the number of lines assumes that the mapping is done in a single step; as you note in your earlier comment, mapping sequentially can reduce this number dramatically.

David Stern 3 months ago

This is a lovely method and should find wide applicability in many settings, especially for microorganisms and cell lines. However, it is not clear that this approach will be, as implied by the discussion, an efficient mapping method for all multicellular organisms. I have performed similar experiments in Drosophila, focused on meiotic recombination, on a much smaller scale, and found that CRISPR-Cas9 can indeed generate targeted recombination at gRNA target sites. In every case I tested, I found that the recombination event was associated with a deletion at the gRNA site, which is probably unimportant for most mapping efforts, but may be a concern in some specific cases, for example for clinical applications. It would be interesting to know how often mutations occurred at the targeted gRNA site in this study.

The wider issue, however, is whether CRISPR-mediated recombination will be more efficient than other methods of mapping. After careful consideration of all the costs and the time involved in each of the steps for Drosophila, we have decided that targeted meiotic recombination using flanking visible markers will be, in most cases, considerably more efficient than CRISPR-mediated recombination. This is mainly due to the large expense of injecting embryos and the extensive effort and time required to screen injected animals for appropriate events. It is both cheaper and faster to generate markers (with CRISPR) and then perform a large meiotic recombination mapping experiment than it would be to generate the lines required for CRISPR-mediated recombination mapping. It is possible to dramatically reduce costs by, for example, mapping sequentially at finer resolution. But this approach would require much more time than marker-assisted mapping. If someone develops a rapid and cheap method of reliably introducing DNA into Drosophila embryos, then this calculus might change.

However, it is possible to imagine situations where CRISPR-mediated mapping would be preferable, even for Drosophila. For example, some genomic regions display extremely low or highly non-uniform recombination rates. It is possible that CRISPR-mediated mapping could provide a reasonable approach to fine mapping genes in these regions.

The authors also propose the exciting possibility that CRISPR-mediated loss of heterozygosity could be used to map traits in sterile species hybrids. It is not entirely obvious to me how this experiment would proceed and I hope the authors can illuminate me. If we imagine driving a recombination event in the early embryo (with maternal Cas9 from one parent and gRNA from a second parent), then at best we would end up with chimeric individuals carrying mitotic clones. I don’t think one could generate diploid animals where all cells carried the same loss of heterozygosity event. Even if we could, this experiment would require construction of a substantial number of stable transgenic lines expressing gRNAs. Mapping an ~20Mbp chromosome arm to ~10kb would require on the order of two-thousand transgenic lines. Not an undertaking to be taken lightly. It is already possible to perform similar tests (hemizygosity tests) using D. melanogaster deficiency lines in crosses with D. simulans, so perhaps CRISPR-mediated LOH could complement these deficiency screens for fine mapping efforts. But, at the moment, it is not clear to me how to do the experiment.

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Clinical Biomarkers Overview

Posted in Cell Biology, Chemical Genetics, Clinical Genomics, Disease Biology, Epigenetics and Environmental Factors, Gene Regulation, Genetics & Pharmaceutical, tagged biomarkers, cancer signatures, diagnostics, DNA sequence, double-strand DNA, Methylation on May 2, 2016| Leave a Comment »

Clinical Biomarkers Overview

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Paving the Road for Clinical Biomarkers

Where Trackless Terrain Once Challenged Biomarker Development, Clearer Paths Are Emerging

Kate Marusina, Ph.D.

http://www.genengnews.com/gen-articles/paving-the-road-for-clinical-biomarkers/5757/

http://www.genengnews.com/Media/images/Article/thumb_ArcherDX_AnalyticalSensitivity2362411344.jpg

Fusion detection can be carried out with traditional opposing primer-based library preparation methods, which require target- and fusion-specific primers that define the region to be sequenced. With these methods, primers are needed that flank the target region and the fusion partner, so only known fusions can be detected. An alternative method, ArcherDX’ Anchored Multiplex PCR (AMP), can be used to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific unidirectional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification.

In time, the narrow, tortuous paths followed by pioneers become wider and straighter, whether the pioneers are looking to settle new land or bring new biomarkers to the clinic.

In the case of biomarkers, we’re still at the stage where pioneers need to consult guides and outfitters or, in modern parlance, consultants and technology providers. These hardy souls tend to congregate at events like the Biomarker Conference, which was held recently in San Diego.

At this event, biomarker experts discussed ways to avoid unfortunate detours on the trail from discovery and development to clinical application and regulatory approval. Of particular interest were topics such as the identification of accurate biomarkers, the explication of disease mechanisms, the stratification of patient groups, and the development of standard protocols and assay platforms. In each of these areas, presenters reported progress.

Another crucial subject is the integration of techniques such as next-generation sequencing (NGS). This particular technique has been instrumental in advancing clinical cancer genomics and continues to be the most feasible way of simultaneously interrogating multiple genes for driver mutations.

Enriching nucleic acid libraries for target genes of interest prior to NGS greatly enhances the sensitivity of
detecting mutations, as the enriched regions are sequenced multiple times. This is particularly useful when analyzing clinical samples, which generate low amounts of poor-quality nucleic acids.

However, NGS has been limited in its ability to identify gene fusions and translocations, which underlie oncogenesis in a variety of cancers. “These challenges are largely related to the enrichment chemistry used to produce sequencing libraries,” commented Joshua Stahl, chief scientific officer and general manager, ArcherDX.

Most target-enrichment strategies require prior knowledge of both ends of the target region to be sequenced. Therefore, only gene fusions with known partners can be amplified for downstream NGS assays.

Archer’s Anchored Multiplex PCR (AMP™) technology overcomes this limitation, as it can enrich for novel fusions, while only requiring knowledge of one end of the fusion pair. At the heart of the AMP chemistry are unique Molecular Barcode (MBC) adapters, ligated to the 5′ ends of DNA fragments prior to amplification. The MBCs contain universal primer binding sites for PCR and a molecular barcode for identifying unique molecules. When combined with 3′ gene-specific primers, MBCs enable amplification of target regions with unknown 5′ ends.

“AMP is ideal for identifying gene fusions and other driver mutations from FFPE samples,” asserted Mr. Stahl. “Its robust utility was demonstrated for detection of gene fusions, point mutations, insertions, deletions, and copy number changes from low amounts of clinical formalin-fixed, paraffin-embedded (FFPE) RNA and DNA samples.

“Tagging each molecule of input nucleic acid with a unique molecular barcode allows for de-duplication, error correction, and quantitative analysis, resulting in high sequencing consensus. With its low error rate and low limits of detection, AMP is revolutionizing the field of cancer genomics.”

In a proof-of-concept study, a single-tube 23-plex panel was designed to amplify the kinase domains of ALK, RET, ROS1, and MUSK genes by AMP. This enrichment strategy enabled identification of gene fusions with multiple partners and alternative splicing events in lung cancer, thyroid cancer, and glioblastoma specimens by NGS.

Ignyta, a precision medicine company, adopted Archer’s AMP technology in Trailblaze Pharos™, a multiplex assay employed in their STARTRK-2 trial for identifying actionable NTRK, ROS1, and ALK gene rearrangements in solid tumors that can be treated with the novel kinase inhibitor, entrectinib. “Gene fusions are incredibly important in personalized medicine right now,” stated Mr. Stahl. “Archer’s FusionPlex assays are quickly becoming the new gold standard.”

Reading Cancer Signatures

This image, from the Massachusetts General Hospital Cancer Center, shows multicolor fluorescence in situ hybridization (FISH) analysis of cells from a patient with esophagogastric cancer. Remarkably, the FISH analysis revealed that co-amplification of the MET gene (red signal) and the EGFR gene (green signal) existed simultaneously in the same tumor cells. A chromosome 7 control probe is shown in blue.
“Each year 23,000 kidneys are transplanted, and over 175,000 kidney transplants are functional today,” noted Daniel R. Salomon, M.D., medical program director, Scripps Center for Organ Transplantation, Scripps Research Institute. “However, in just 5 years, 3 out of every 10 patients will be back on dialysis, and in 15 years, at least 75% of all patients will lose their kidney grafts.“Tumor biomarkers are critical for predicting and following patient responses to today’s cancer therapies,” said Darrell Borger, Ph.D., co-director of the Translational Research Laboratory and director of the Biomarker Laboratory, Massachussetts General Hospital (MGH) Cancer Center, Harvard Medical School. “If we understand what drives the malignancy in any given patient, we are able to match existing therapies to the patient’s genotype.”

Over the last decade, the Biomarker/Translational Research Laboratory has focused on developing clinical genotyping and fluorescent in situ hybridization (FISH) assays for rapid personalized genomic testing.

“Initially, we analyzed the most prevalent hotspot mutations, about 160 in 25 cancer genes,” continued Dr. Borger. “However, this approach revealed mutations in only half of our patients. With the advent of NGS, we are able to sequence 190 exons in 39 cancer genes and obtain significantly richer genetic fingerprints, finding genetic aberrations in 92% of our cancer patients.”

Using multiplexed approaches, Dr. Borger’s team within the larger Center for Integrated Diagnostics (CID) program at MGH has established high-throughput genotyping service as an important component of routine care. While only a few susceptible molecular alterations may currently have a corresponding drug, the NGS-driven analysis may supply new information for inclusion of patients into ongoing clinical trials, or bank the result for future research and development.

“A significant impediment to discovery of clinically relevant genomic signatures is our current inability to interconnect the data,” explained Dr. Borger. “On the local level, we are striving to compile the data from clinical observations, including responses to therapy and genotyping. Globally, it is imperative that comprehensive public databases become available to the research community.”

Tumor profiling at MGH have already yielded significant discoveries. Dr. Borger’s lab, in collaboration with oncologists at the MGH Cancer Center, found significant correlations between mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2) and certain types of cancers, such as cholangiocarcinoma and acute myelogenous leukemia (AML).

Historically, cancer signatures largely focus on signaling proteins. Discovery of a correlative metabolic enzyme offered a promise of diagnostics based on metabolic byproducts that may be easily identified in blood. Indeed, the metabolite 2-hydroxyglutarate accumulates to high levels in the tissues of patients carrying IDH1 and IDH2 mutations. They have reported that circulating 2-hydroxyglutarate as measured in the blood correlates with tumor burden, and could serve as an important surrogate marker of treatment response.

Tuning Immunosuppression, Preventing Chronic Rejection

“We believe that this is caused by chronic immune-mediated rejection. Failure of effective immunosuppression reduces functional life of these patients and adds in $9–13 billion in yearly healthcare costs.” Dr. Salomon emphasized that ineffective use of immunosuppressive drugs is partially due to the lack of an objective biomarker which could provide decision support for just-in-time adjustment in therapeutic regimens.

“Our research aims to provide that objective measure to clinicians,” explained Dr. Salomon.

To date, kidney transplant biopsies remain the gold standard, even though they are not suitable for continuous monitoring and have both costs and risks. Dr. Salomon’s team developed a minimally invasive diagnostic approach based on unbiased whole-genome expression profiling of blood samples. Using Affymetrix Human Genome U133 Plus 2.0 Gene Chips, the team analyzed 275 bloodsamples of kidney transplant patients with biopsy-proved acute rejection, acute dysfunction without rejection and transplant excellent phenotype.

The data was passed through several machine-learning algorithms to identify a group of about 250 classifiers that predict subacute or acute rejection with 80% accuracy. This signature is locked while the team continues to expand the core dataset aiming to reach a thousand samples by the end of this year.

“As opposed to classical approaches to biomarker discoveries limited to just a few classifiers, our methodology provides for the first use of unbiased whole-genome profiling in the identification of multiple molecular predictors,” declared Dr. Salomon. “We can use this molecular diagnostic strategy to reveal a subacute rejection prior to significant tissue injury leading to transplant dysfunction. Continuous monitoring would inform physicians on the balance between over-suppression and effective/optimal therapy.”

Dr. Salomon is a chief scientific advisor for Transplant Genomics (TGI), a start-up company created to translate the blood-based molecular diagnostics into clinical tests. In late 2016, TGI will begin providing its TruGraf blood tests for kidney transplant recipients for use by four to six U.S. transplant centers through an early-access program (EAP).

Additional tests designed to be used serially to diagnose and treat subclinical episodes of rejection including biopsy gene profiling are in the final stages of development. Validation and will be made available through the EAP in the upcoming months.

http://www.genengnews.com/Media/images/Article/BioAgilytix_MultiMuscleAnalysis5413927931.jpg

BioAgilytix’ MultiMuscle Analysis is a process that can split sample analysis into multiple parallel tracks to minimize antibody cross-reactivity and allow for use of the best-fit platform or kit for each biomarker analysis. The process may require only one tube of sample with only one F/T cycle.

Focusing on Large Molecules

BioAgilytix, a specialized bioanalytical laboratory, is a global leader in large molecule bioanalysis. The company’s business encompasses pharmacokinetic/pharmacodynamic (PK/PD) studies of large biomolecules, in addition to immunogenicity, biomarkers, and cell-based assays. In less than 10 years,BioAgilytix has grown from a start-up to an international powerhouse with over 100 employees—more than half possessing advanced scientific degrees—because of its team’s expertise in the complexities of large molecule drug development.

“In contrast to small molecule analysis, which has become more of a commodity due to its semiautomated and process-oriented nature, large molecule analysis is inherently challenging,” said Afshin Safavi, Ph.D., founder and chief science officer of BioAgilytix. “In large molecule bioanalysis, we rely heavily on analytical reagents, such as antibodies and recombinant proteins, which are known to show considerable variability from lot to lot.

“Therefore, designing an effective analytical process for large biomolecules requires scientific personnel with years of experience. It also requires careful management of critical reagents, and a deep understanding of the capabilities and limitations of the platforms selected for use.”

Dr. Safavi explains that the biomarker field has been trending away from a gunshot approach traditionally favored by large pharma to more focused analyses of a few key biomarkers.

“Unlike several years ago, most biotech and pharma companies now perform careful due diligence and literature research before approaching us, to narrow down their investigation to just a handful of biomarkers,” he explained. Limited samples may drive the desire to multiplex as many biomarkers as possible, but a multiplex approach may often result in low quality data due to reagent cross-reactivity.

A recent process innovation developed by BioAgilytix, called MultiMuscle Analysis™, uses a customized parallel process to drastically reduce analytical process time and increase data quality. MultiMuscle Analysis splits the sample analysis into multiple parallel tracks, each performed on specialized equipment by scientists experienced in that particular platform.

“Say, for example, a customer requests measurements of 10 biomarkers,” ventured Dr. Safavi. “If we know some of the antibodies may cross-react, then we may, for example, end up with one heptaplex and three as uniplexes, all done in parallel.”

Using this approach, BioAgilytix is able to perform large biomarker analyses on a very large number of samples in near real-time. “We now receive samples from over 20 countries,” Dr. Safavi stated. “We have used the MultiMuscle approach successfully over and over.”

Feature ArticlesMore » May 1, 2016 (Vol. 36, No. 9)

Paving the Road for Clinical Biomarkers

Where Trackless Terrain Once Challenged Biomarker Development, Clearer Paths Are Emerging

Kate Marusina, Ph.D.

Focusing on Large Molecules

BioAgilytix, a specialized bioanalytical laboratory, is a global leader in large molecule bioanalysis. The company’s business encompasses pharmacokinetic/pharmacodynamic (PK/PD) studies of large biomolecules, in addition to immunogenicity, biomarkers, and cell-based assays. In less than 10 years, BioAgilytix has grown from a start-up to an international powerhouse with over 100 employees—more than half possessing advanced scientific degrees—because of its team’s expertise in the complexities of large molecule drug development.

Dr. Safavi explains that the biomarker field has been trending away from a gunshot approach traditionally favored by large pharma to more focused analyses of a few key biomarkers.

Predicting Clotting or Hemorrhaging

Venous thromboembolism (VTE) is a disease that includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). It is a common, lethal disorder, symptoms of which are often overlooked. VTE is the third most common cardiovascular illness after acute coronary syndrome and stroke.

Venous thrombi, composed predominately of red blood cells bound together by fibrin, form in sites of vessel damage and areas of stagnant blood flow. Once VTE is diagnosed, anticoagulation therapy is indicated.

A novel anticoagulant that reversibly and directly inhibits factor Xa, a key factor in the coagulation system, has been developed by Daiichi Sankyo. “Once on the path of development of an anticoagulant, we recognized the lack of a rapid and sensitive coagulation test that would not be affected by blood traces of anticoagulant therapies,” said Michele Mercuri, M.D., Ph.D., the company’s senior vice president. “An improved diagnostic test would speed up recognition and treatment of thrombosis, and would aid in development of reversing agents that reduce the effect of anticoagulant therapies when needed.”

When Daiichi Sankyo entered in collaboration with Perosphere to develop a novel broad-spectrum reversing agent, the company also supported development of a point-of-care coagulometer (still under development), a hand-held device designed for broad-spectrum monitoring of the activity of anticoagulants and their corresponding reversing agents, across drug classes. A single test requires only 10 µL of fresh or citrated whole blood from a venous draw or finger stick. It optically measures clotting starting with Factor XII activation to fibrin assembly.

Dr. Mercuri explains that none of the existing tests are able to predict whether a patient is at risk for either clotting or hemorrhaging. “Together with Prof. Zahi Fayad’s Team from the Icahn School of Medicine at Mt Sinai, we used magnetic resonance imaging with the gadolinium-based contrast reagent to detect the venous thrombi and follow their dissolution with edoxaban treatment,” reported Dr. Mercuri.

This study, the edoxaban Thrombus Reduction Imaging Study (eTRIS), was focused on developing and validating a magnetic resonance venography (MRV) image acquisition and analysis protocol for the quantification of thrombus volume in deep vein thrombosis. The multicenter study demonstrated excellent reproducibility of analysis of quantifying thrombus volume.

Sequence and Epigenetic Factors Determine Overall DNA Structure

Researchers at Ulsan National Institute of Science and Technology (UNIST) in South Korea found that DNA molecules directly interact with one another in ways that are dependent on the sequence of the DNA and epigenetic factors.

The researchers found evidence for sequence-dependent attractive interactions between double-stranded DNA molecules that neither involve intermolecular strand exchange nor are mediated by DNA-binding proteins.

“DNA molecules tend to repel each other in water, but in the presence of special types of cations, they can attract each other just like nuclei pulling each other by sharing electrons in between,” explained lead study author Hajin Kim, Ph.D., assistant professor of biophysics at UNIST. “Our study suggests that the attractive force strongly depends on the nucleic acid sequence and also the epigenetic modifications.”

The investigators used atomic-level simulations to measure forces between double-stranded DNA helices, proposing that the distribution of methyl groups on DNA were the key to regulating this sequence-dependent attraction.

The findings from this study were published recently in Nature Communications through an article entitled “Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.”

The researchers surmised that direct DNA-DNA interactions could play a central role in how chromosomes are organized and packaged, determining the ultimate fate of many cell types.

Dr. Kim concluded by stating that “in our lab, we try to unravel the mysteries within human cells based on the principles of physics and the mechanisms of biology—seeking for ways to prevent chronic illnesses and diseases associated with aging.”

Searches Related to Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Jejoong Yoo, Hajin Kim, Aleksei Aksimentiev & Taekjip Ha

Nature Communications 22 Mar 2016; 7(11045) http://dx.doi.org:/10.1038/ncomms11045

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Formation of a DNA double helix occurs through Watson–Crick pairing mediated by the complementary hydrogen bond patterns of the two DNA strands and base stacking. Interactions between double-stranded (ds)DNA molecules in typical experimental conditions containing mono- and divalent cations are repulsive¹, but can turn attractive in the presence of high-valence cations². Theoretical studies have identified the ion–ion correlation effect as a possible microscopic mechanism of the DNA condensation phenomena³^,⁴^,⁵. Theoretical investigations have also suggested that sequence-specific attractive forces might exist between two homologous fragments of dsDNA⁶, and this ‘homology recognition’ hypothesis was supported by in vitro atomic force microscopy⁷ and in vivo point mutation assays⁸. However, the systems used in these measurements were too complex to rule out other possible causes such as Watson–Crick strand exchange between partially melted DNA or protein-mediated association of DNA.

Here we present direct evidence for sequence-dependent attractive interactions between dsDNA molecules that neither involve intermolecular strand exchange nor are mediated by proteins. Further, we find that the sequence-dependent attraction is controlled not by homology—contradictory to the ‘homology recognition’ hypothesis⁶—but by a methylation pattern. Unlike the previous in vitro study that used monovalent (Na⁺) or divalent (Mg²⁺) cations⁷, we presumed that for the sequence-dependent attractive interactions to operate polyamines would have to be present. Polyamine is a biological polycation present at a millimolar concentration in most eukaryotic cells and essential for cell growth and proliferation⁹^,¹⁰. Polyamines are also known to condense DNA in a concentration-dependent manner²^,¹¹. In this study, we use spermine⁴⁺(Sm⁴⁺) that contains four positively charged amine groups per molecule.

Methylation determines the strength of DNA–DNA attraction

Analysis of the MD simulations revealed the molecular mechanism of the polyamine-mediated sequence-dependent attraction (Fig. 2). In the case of the AT-rich fragments, the bulky methyl group of thymine base blocks Sm⁴⁺ binding to the N7 nitrogen atom of adenine, which is the cation-binding hotspot²¹^,²². As a result, Sm⁴⁺ is not found in the major grooves of the AT-rich duplexes and resides mostly near the DNA backbone (Fig. 2a,d). Such relocated Sm⁴⁺ molecules bridge the two DNA duplexes better, accounting for the stronger attraction¹⁶^,²³^,²⁴^,²⁵. In contrast, significant amount of Sm⁴⁺ is adsorbed to the major groove of the GC-rich helices that lacks cation-blocking methyl group (Fig. 2b,e).

Figure 2: Molecular mechanism of polyamine-mediated DNA sequence recognition.

(a–c) Representative configurations of Sm⁴⁺ molecules at the DNA–DNA distance of 28 Å for the (AT)₁₀–(AT)₁₀ (a), (GC)₁₀–(GC)₁₀ (b) and (GmC)₁₀–(GmC)₁₀ (c) DNA pairs. The backbone and bases of DNA are shown as ribbon and molecular bond, respectively; Sm⁴⁺ molecules are shown as molecular bonds. Spheres indicate the location of the N7 atoms and the methyl groups. (d–f) The average distributions of cations for the three sequence pairs featured in a–c. Top: density of Sm⁴⁺ nitrogen atoms (d=28 Å) averaged over the corresponding MD trajectory and the z axis. White circles (20 Å in diameter) indicate the location of the DNA helices. Bottom: the average density of Sm⁴⁺ nitrogen (blue), DNA phosphate (black) and sodium (red) atoms projected onto the DNA–DNA distance axis (x axis). The plot was obtained by averaging the corresponding heat map data over y=[−10, 10] Å. See Supplementary Figs 4 and 5 for the cation distributions at d=30, 32, 34 and 36 Å.

Full size image (182 KB)

Genome-wide investigations of chromosome conformations using the Hi–C technique revealed that AT-rich loci form tight clusters in human nucleus²⁷^,²⁸. Gene or chromosome inactivation is often accompanied by increased methylation of DNA²⁹ and compaction of facultative heterochromatin regions³⁰. The consistency between those phenomena and our findings suggest the possibility that the polyamine-mediated sequence-dependent DNA–DNA interaction might play a role in chromosome folding and epigenetic regulation of gene expression.

Phenotypic and Biomarker-based Drug Discovery

Organizers: Michael Foley (Tri-Institutional Therapeutics Discovery Institute), Ralph Garippa (Memorial Sloan-Kettering Cancer Center), David Mark (F. Hoffmann-La Roche), Lorenz Mayr (Astra Zeneca), John Moffat (Genentech), Marco Prunotto (F. Hoffmann-La Roche), and Sonya Dougal (The New York Academy of Sciences)Presented by the Biochemical Pharmacology Discussion Group

Reported by Robert Frawley | Posted January 12, 2016

http://www.nyas.org/image.axd?id=7fb0ef88-6198-465b-9f1f-52ec1e0b0b3d&t=635616760148070000

Overview

There are two major methods for designing pharmaceutical drugs. In traditional drug discovery (TDD), or empiric design, researchers target a particular domain or protein after working to understand its mechanisms and molecular biology. In phenotypic drug discovery (PDD), many different compounds are tested on a system until one results in an observable phenotype of success, and the compounds’ mechanisms of action are not considered. The Phenotypic and Biomarker-based Drug Discovery symposium, presented by the Academy’s Biochemical Pharmacology Discussion Group on October 27, 2015, featured current work in PDD and highlighted the need to bridge commercial and academic research to improve phenotypic drug design.

Phenotypic drug discovery—screening of thousands of substances for functional cellular outputs such as gene expression, growth arrest, and cancer cell death—has led to the development of more commercial drugs than TDD, the more common method of discovery. Indeed, as Jonathan A. Lee of Eli Lilly noted, spending on TDD is out of sync with the rate of new drugs reaching approval; the number of new drugs per billion dollars spent dropped sharply in the last few decades. He argued that the need for functionally validated drugs could be met through a renewed focus on PDD.

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Bruce A. Posner started the morning session with a discussion of a phenotypic screen conducted at the University of Texas Southwestern Medical Center which identified two chemical scaffolds that are effective in killing non-small cell lung cancer (NSCLC) cells but are harmless to the non-cancer cells tested. In further studies, the group showed that an optimized analog of one scaffold arrested tumor growth in a mouse xenograft model of NSCLC. Both chemical scaffolds appear to work through a novel mechanism targeting stearoyl-CoA desaturase (SCD), which is known to be important in unsaturated fatty acid synthesis. These compounds were found to be specific, effective, and potent in NSCLC cell lines that express elevated levels of Cyp4F11 and/or related Cyp family members. The group also showed that these scaffolds function as prodrugs that are activated only in cancer cells expressing these Cyp isoforms and that the Cyps produce metabolites of the prodrug that bring about cancer-specific cell toxicity. The group is working to improve these scaffolds and to develop a putative biomarker based on Cyp expression.

The Broad Institute’s LINCS (Library of Network-based Cellular Signatures) database is designed to keep track of small-molecule therapeutics, collecting data on cellular responses to “perturbagens” (drugs, factors, and others stimuli). Data are generated using the L1000 assay, which assesses the expression of 1000 genes known to explain 80% of genetic variation in assayed cell lines. Aravind Subramanian explained that the technique can identify the majority of drug effects for a fraction of the cost of RNA sequencing. Although it examines only a subset of molecules and relies on measuring genetic responses, the technique can help predict the likelihood that new compounds will elicit desired effects.

Martin Main of AstraZeneca described phenotypic drug discovery at AstraZeneca. The company’s model for discovery is to check phenotypic markers at every step, as drugs are moved from cell lines to patients. Main’s team identified a molecule that enhances the regenerative function of cardiac myocytes after infarction. Using cells from several donors, the team validated a promising compound that increases proliferation of cardiac myocytes and drives epicardium-derived progenitor cells to assume a myocyte lineage. In another discovery, the team used islet β-cell regeneration as the phenotype, discovering a compound the researchers believe will reach clinical trials for type 2 diabetes.

Andras J. Bauer of Boehringer Ingelheim discussed a method to increase predictive strength in compound selection before phenotypic screening. By cataloging the structures of known target–reference compound binding pairs, the team can compare those structures to untested compounds, and then assess only the most promising compounds. The THICK (Target Hypothesis Information from Curated Knowledge bases) database gives interaction-probability scores to untested compounds on the basis of structure. Bauer also described a method to verify target–compound interaction without labeling the molecules, in which phenotypic results were verified with mass spectrometry.

In the afternoon session, Myles Fennell of Memorial Sloan-Kettering Cancer Center described his work testing small interfering RNA (siRNA) libraries to find siRNAs that alter macropinocytosis (MP), cell-surface ruffling that is seen in prostate cancer cells. The surface phenotype allows TMR-dextran uptake, which the researchers measured in the screen. MP is driven by RAS (a commonly affected gene family in cancers) and the pathways are already popular drug targets. The researchers tested two libraries of siRNAs, which block translation of specific proteins, using TMR as a marker to report MP severity, as well as sensitive single-cell assays to determine siRNA efficacy. The team identified promising target sequences and used a data-analysis pipeline called KNIME to define several hits, which the researchers are pursuing in therapeutic development.

http://www.nyas.org/image.axd?id=0b4496f6-28fb-435c-bd11-06b4d31fc0ad&t=635863102714400000

TMR-dextran is able to work into cells undergoing macropinocytosis and thus these cells can be separated by phenotype as seen in the controls above. (Image courtesy of Myles Fennell)

Giulio Superti-Furga of the Austrian Academy of Sciences is a proponent of understanding the mechanisms of action (MOA) of candidate drugs. He began by explaining that the genome is an incomplete indicator of disease; epigenetics, altered protein function, metabolism, and other factors are also important. He then introduced pharmacoscopy and the “thermal shiftome” as methods to phenotypically screen compounds. Pharmacoscopy uses high-power automated microscopy to describe how compounds affect cell populations by using specific stains for different cell types; a computer then counts the cells expressing each stain, yielding results similar to those obtained via fluorescence-activated cell sorting but generated through an automated process. The thermal shiftome catalogs changes in thermal stability after protein binding in known reactions and is used to characterize the stability of new reactions. Superti-Furga offered a perspective that tempered the enthusiasm for pure PDD and advocated a mechanistic approach to drug discovery.

Michael R. Jackson, at one of the largest academic screening facilities, the Sanford Burnham Prebys Medical Discovery Institute, led a reexamination of drug screens performed by pharmaceutical companies. His team conducted millions of assays and accumulated a large data library with few new hits. However, the researchers were able to closely characterize the chemistry of one hit, an undisclosed interaction, and Jackson’s group is proceeding to develop a drug to modulate nuclear receptor signaling. The researchers also have a procedure that can screen for the differentiation of human induced pluripotent stem cells (iPSCs) into neurons for potential neuro-regenerative therapies. They developed high-throughput morphology, endpoint-measurement, and proliferation assays that generate tightly clustered, repeatable data. The team has produced consistent results screening 10 immune modulators and various cytokines to assess the reactivity and stability of the cells, providing reliable compound characterization. This success in human cells shows that a disease-relevant patient-derived screening platform to characterize differentiation and immune response is possible with robust assays.

In the next set of talks, Friedrich Metzger and Susanne Swalley described the parallel work of Hoffmann-La Roche and Novartis, respectively, toward treating spinal muscular atrophy (SMA). A devastating disease that leads to loss of motor function and affects motor nerve cells in the spinal cord, SMA presents a unique drug development opportunity. The condition is caused by the loss of function of a single gene product called survival of motor neuron (SMN1). Humans encode an unstable gene product, called SMN2, which is nearly homologous to SMN1.

Metzger explained that the inactive SMN2 variant is largely the same as active SMN1 but, missing exon 7, cannot compensate in its absence. The group from Hoffmann-La Roche aimed to stabilize SMN2 by promoting the inclusion of exon 7. The researchers conducted a phenotypic screen seeking a compound that could change the splicing in patient fibroblasts in vitro and produce a stable, functional SMN2 protein including exon 7. In studies with an SMN2Δ7 mouse model (lacking exon 7), mice drugged with the compound experienced full phenotypic rescue. The compound has been shown to induce alternative splicing of SMN2 to include exon 7 in healthy human volunteers; it was well tolerated and is moving to human patient trials.

Swalley discussed the target identification and MOA of the Novartis compound. After a screening process similar to Roche’s, Novartis moved its compound into animal models while also beginning parallel experimentation to find out why it worked. The group found that U1-snRNP, a spliceosome component required for the splicing process, is bound at two essential nucleotides by the compound. In the SMN2Δ7 mice, the compound improved survival and rescued full SMN2 protein expression. The Novartis compound stabilizes the appropriate spliceosome components to produce SMN2 with exon 7 intact. This novel mechanism demonstrates that a sequence-selective small molecule therapy can alter splicing activity to treat SMA. Together these talks demonstrated the power of PDD and the importance of validating drug mechanisms.

The final talk of the day was given by Hoffmann-La Roche’s Jitao David Zhang, who suggested that pathway reporter genes, which are only modulated when a specific signaling pathway is activated or inhibited, can be used as phenotypic readouts. It is known that gene expression data can predict cell phenotype. Using transcriptomics as a surrogate for downstream phenotypes, for example by using expression data from a gene subset to predict outcomes, would save time and effort. In an iPSC cardiomyocyte model of diabetic stress, machine learning (guided by pathway information) characterizes the response of the iPSCs to a library of compounds, highlighting compounds and pathways worthy of further investigation. This new platform for molecular phenotyping using pathway reporter genes, sequencing, and early analysis speeds compound characterization.

Use the tabs above to find multimedia from this event.

Presentations available from:
Andras J. Bauer, PhD, PharmD (Boehringer Ingelheim)
Myles Fennell, PhD (Memorial Sloan-Kettering Cancer Center)
Jonathan A. Lee, PhD (Eli Lilly)
Martin Main, PhD (AstraZeneca)
Yao Shen, PhD (Columbia University)
Susanne Swalley, PhD (Novartis Institutes for BioMedical Research)
Jitao David Zhang, PhD (F. Hoffmann-La Roche)

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What about PDL-1 in oncotherapy diagnostics for NSCLC?

Posted in Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Cancer and Current Therapeutics, Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Clinical Diagnostics, Conference Coverage with Social Media, Diagnostics and Lab Tests, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Gene Regulation, Genetics & Pharmaceutical, Human aging, Human Immune System in Health and in Disease, Immunodiagnostics, Lung and pulmonology, tagged diagnostic PDL1 tests, harmonization, Lung cancer, NSCLC, PD-1/PDL1, PDL-1 biomarker, tumor biopsy on May 2, 2016| Leave a Comment »

What about PDL-1 in oncotherapy diagnostics for NSCLC?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

UPDATED 5/15/2019

Questions on PD-L1 Diagnostics for Immunotherapy in NSCLC
Alexander M. Castellino, PhD
http://www.medscape.com/viewarticle/862275

Coverage from the

American Association for Cancer Research (AACR) 2016 Annual Meeting

Two immunotherapies that target the cell programmed death (PD) pathway are now available, and both nivolumab (Opdivo, Bristol-Myers Squibb Company) and pembrolizumab (Keytruda, Merck Sharp & Dohme Corp) are approved for treating advanced, refractory, non–small cell lung cancer (NSCLC). Across several studies in patients with NSCLC, response to these agents has been correlated with PD-L1 staining, which determines PD-L1 levels in the tumor tissue. How do the available assays for PD-L1 compare?

The linear correlation between three commercially available assays is good across a range of cutoff points, concluded a presentation at the 2016 American Association for Clinical Research Annual Meeting.

Cutoffs are defined as the percentage of cells expressing PD-L1 when analyzed histochemically. “The dataset builds confidence that the assays may be used according to the cutoff clinically validated for the drug in question,” Marianne J. Radcliffe, MD, diagnostic associate director at AstraZeneca, toldMedscape Medical News.

“The correlation is good between the assays across the range examined,” she added.

However, a recently published study showed a high rate of discordance between another set of PD-L1 assays that were tested.

Dr Marianne Radcliffe

“Different diagnostic tests yield different results, depending on the cutoff for each assay. We need to harmonize the assays so clinicians are talking about the same thing,” Brendon Stiles, MD, associate professor of cardiothoracic surgery at Weill Cornell Medicine and New York-Presbyterian Hospital, New York City, told Medscape Medical News.

For Dr Stiles, these studies raise the issue that it is difficult to compare results of diagnostic testing across the different drugs and even with the same drug that are derived from different assays. “More importantly, it raises confusion in clinical practice when a patient’s sample stains positive for PD-L1 with one assay and negative with another,” he said.

“The commercial strategy for developing companion diagnostics for each drug is not in the best interests of the patients. It generates confusion among both clinicians and patients,” Dr Stiles commented. “We need to know if these assays can be used interchangeably,” he said.

As new agents come into the clinic, Dr Stiles believes there should be a universal yes-or-no answer, so that clinicians can use the assay to help decide on the use of immunotherapy.

Three Assays Tested

The study presented by Dr Radcliffe and colleagues investigated three commercially available assays, Ventana SP263, Dako 22C3, and Dako 28-8, with regard to how they compare at different cutoffs. Different studies use different cutoffs to express positivity.

Ventana SP263 was developed as a companion diagnostic for durvalumab (under development by AstraZeneca) using a rabbit monoclonal antibody. Positivity is defined as ≥25% staining of tumor cells.

Dako 22C3 was developed, and is approved, as a companion diagnostic for pembrolizumab. It uses a mouse monoclonal antibody. Positivity is defined as ≥1% and ≥50% staining of tumor cells.

Dako 28-8 was developed as a companion diagnostic for nivolumab and uses a rabbit monoclonal antibody (different from the one used in the Ventana SP263). In clinical practice, this assay is used as a complementary diagnostic for nivolumab, but the drug is approved for use regardless of PD-L1 expression. Positivity is defined as ≥1%, ≥5%, or ≥10% staining of tumor cells.

Ventana SP142 was not included in the study because it is not commercially available, Dr Ratcliffe indicated.

The three assays were used on consecutive sections of 500 archival NSCLC tumor samples obtained from commercial vendors. A single pathologist trained by the manufacturer read all samples in batches on an assay-by-assay basis. Samples were assessed per package inserts provided by Ventana and Dako in a Clinical Laboratory Improvement Amendments program-certified laboratory.

Dr Ratcliffe indicated that between reads of samples from the same patient, there was a washout period for the pathologist to remove bias.

The NSCLC samples included patients with stage I (38%), II (39%), III (20%), and IV (<1%) disease. Histologies included nonsquamous (54%) and squamous (43%) cancers.

All three PD-L1 assays showed similar patterns of staining in the range of 0% to 100%, Dr Ratcliffe indicated.

The correlation between any two of the assays was determined from tumor cell membrane staining. The correlation was linear with Spearman correlation of 0.911 for Ventana SP263 vs Dako 22C3; 0.935 for Ventana SP263 vs Dako 28-8; and 0.954 for Dako 28-8 vs Dako 22C3.

“With an overall predictive value of >90%, the assays have closely aligned dynamic ranges, but more work is needed,” Dr Ratcliffe said. “In general, scoring of immunohistochemical assays can be more variable between 1% and 10%, and we plan to look at this in more detail,” she said. These samples need to be reviewed by an independent pathologist, she added.

Dr Radcliffe said that currently, “Direct clinical efficacy data supporting a specific diagnostic test should still be considered as the highest standard of proof for diagnostic clinical utility.”

Why Correlations Are Needed

Pembrolizumab is approved for use only in patients with PD-L1-positive, previously treated NSCLC. A similar patient profile is being considered for nivolumab, for which testing for PD-L1 expression is not required.

For new PD-immunotherapy agents in clinical development, it is not clear whether PD-L1 testing will be mandated.

However, in clinical practice, it is clear that some patients respond to therapy, even if they are PD-L1 negative, as defined from the study. “Is it a failure of the assay, tumor heterogeneity, or is there another time point when PD-L1 expression is turned on?” Dr Stiles asked.

Dr Stiles also pointed out that a recent publication from Yale researchers showed a high a rate of discordance. In this study, PD-L1 expression was determined using two rabbit monoclonal antibodies. Both of these were different from the ones used in the Ventana SP263 and Dako 28-8 assays.

In this study, whole-tissue sections from 49 NSCLC samples were used, and a corresponding tissue microarray was also used with the same 49 samples. Researchers showed that in 49 NSCLC tissue samples, there was intra-assay variability, with results showing fair to poor concordance with the two antibodies. “Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%.

“Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way,” the study authors conclude.

The Blueprint Proposal

Coincidentally, a blueprint proposal was announced here at the AACR meeting at a workshop entitled FDA-AACR-ASCO Complexities in Personalized Medicine: Harmonizing Companion Diagnostics across a Class of Targeted Therapies.

The blueprint proposal was developed by four pharmaceutical giants (Bristol-Myers Squibb Company, Merck & Co, Inc, AstraZeneca PLC, and Genentech, Inc) and two diagnostic companies (Agilent Technologies, Inc/Dako Corp and Roche/Ventana Medical Systems, Inc).

In this proposal, the development of an evidence base for PD-1/PD-L1 companion diagnostic characterization for NSCLC would be built into studies conducted in the preapproval stage. Once the tests are approved, the information will lay the foundation for postapproval studies to inform stakeholders (eg, patients, physicians, pathologists) on how the test results can best be used to make treatment decisions.

The blueprint proposal is available online.

Dr Ratcliffe is an employee and shareholder of AstraZeneca. Dr Stiles has disclosed no relevant financial relationships.

American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract LB-094, presented April 18, 2016.

Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non–Small-Cell Lung Cancer

Joseph McLaughlin, ^1,2; Gang Han, ³; Kurt A. Schalper, ²; …., Roy Herbst, ¹; Patricia LoRusso, ¹; David L. Rimm, ²

JAMA Oncol. 2016;2(1):46-54. http://dx.doi.org:/10.1001/jamaoncol.2015.3638.

Importance Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non–small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression.

Objective To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies.

Design, Setting, and Participants PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non–small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined.

Main Outcomes and Measures PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.

Results Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02).

Conclusions and Relevance Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

Invited Commentary
PD-L1 Expression as a Predictive Biomarker: Is Absence of Proof the Same as Proof of Absence?
JAMA Oncol. 2016;2(1):54-55. http://dx.doi.org:/10.1001/jamaoncol.2015.3782.

Viewpoint
PD-L1 Testing in Cancer: Challenges in Companion Diagnostic Development
JAMA Oncol. 2016;2(1):15-16. http://dx.doi.org:/10.1001/jamaoncol.2015.4685.

Prognostic and Predictive Value in KRASin Non–Small-Cell Lung Cancer: A Review

JAMA Oncol. Published online April 21, 2016.;():. http://dx.doi.org:/10.1001/jamaoncol.2016.0405.

Association Between Younger Age and Targetable Genomic Alterations and Prognosis in Non–Small-Cell Lung Cancer

JAMA Oncol. 2016;2(3):313-320. http://dx.doi.org:/10.1001/jamaoncol.2015.4482.

Clinical Application of Liquid Biopsies

JAMA Oncol. Published online April 07, 2016.;():. http://dx.doi.org:/10.1001/jamaoncol.2016.0240.

A Blueprint Proposal for Companion Diagnostic Comparability

A highlight of the FDA-AACR-ASCO Complexities in Personalized Medicine: Harmonizing Companion Diagnostics across a Class of Targeted Therapies workshop was the unveiling of a blueprint proposal developed by four pharmaceutical companies (Bristol-Myers Squibb Company, Merck & Co. Inc., AstraZeneca PLC, and Genentech, Inc.) and two diagnostic companies (Agilent Technologies, Inc./Dako Corp and Roche/Ventana Medical Systems, Inc.). The proposed study would help build an evidence base for PD-1/PD-L1 companion diagnostic characterization for non-small cell lung cancer in the pre-approval stage, such that once the tests are approved, the information generated can lay the groundwork for post-approval studies that will help inform patients, physicians, pathologists, and others on how best to use the test results to determine treatment decisions.

Read the Blueprint Proposal.

During the workshop the blueprint development team solicited input and feedback from interested parties. Read the feedback on the blueprint from

The blueprint team has considered the feedback received. Read the team’s combined response to both groups.Back to the Complexities in Personalized Medicine Workshop page.

A Blueprint Proposal for Companion Diagnostic Comparability
http://www.aacr.org/AdvocacyPolicy/GovernmentAffairs/Documents/FDA-AACR-ASCO-Complexities-in-Personalized-Medicine-Blueprint-Proposal.pdf

Introduction We are in an era of rapid incorporation of basic scientific discoveries into the drug development pipeline. Currently, numerous sponsors are developing therapeutic products that may use similar or identical biomarkers for therapeutic selection, measured or detected by an in vitro companion diagnostic device. The current practice is to independently develop a companion diagnostic for each therapeutic. Thus, the matrix of therapeutics and companion diagnostics, if each therapeutic were approved in conjunction with a companion diagnostic, may present a complex challenge for testing and decision making in the clinic, potentially putting patients at risk if inappropriate diagnostic tests were used to make treatment decisions. To address this challenge, there is a desire to understand assay comparability and/or standardize analytical and clinical performance characteristics supporting claims that are shared across companion diagnostic devices. Pathologists and oncologists also need clarity on how to interpret test results to inform downstream treatment options for their patients.

Clearly using each of the companion diagnostics to select one of the several available targeted therapies in the same class is not practical and may be impossible. Likewise, having a single test or assay as a sole companion test for all of the multiple therapeutic options within a class is also impractical since the individual therapies have differing modes of action, intended use populations, specificities, safety and efficacy outcomes. Thus, a single assay or test may not adequately capture the appropriate patient population that may benefit (or not) from each individual therapeutic option within a class of therapies. Furthermore, aligning multiple sponsors’ study designs and timelines in order that they all adopt a single companion test may inadvertently slow down development of critical therapeutic products and delay patient access to these life-saving products.

Any solution to this challenge will be multifaceted and will, by necessity, involve multiple stakeholders. Thus, the US Food and Drug Administration (FDA), the American Association for Cancer Research (AACR) and American Society of Clinical Oncology (ASCO) convened a workshop titled “Complexities in Personalized Medicine: Harmonizing Companion Diagnostics Across a Class of Targeted Therapies” to draw out and assess possible solutions. Recognizing that the complex scientific, regulatory and market forces at play here require a collaborative effort, an industry workgroup volunteered to develop a blueprint proposal of potential solutions using nonsmall cell lung cancer (NSCLC) as the use case indication.

Goal and Scope of Blueprint The imminent arrival to the market of multiple PD1 / PD-L1 compounds and the possibility of one or more associated companion diagnostics is unprecedented in the field of oncology. Some may assume that since these products target the same biological pathway, they are interchangeable; however, each PD1/PD-L1 compound is unique with respect to its clinical pharmacology and each compound is being developed in the context of a unique biological scientific hypothesis and registration strategy. Similarly, each companion diagnostic has been optimized within the individual therapeutic development programs to meet specific development goals, e.g., 1) validation for patient selection, 2) subgroup analysis as a prognostic variable, or 3) enrichment.

Further, each companion diagnostic test is optimized for its specific therapy and with its own unique performance characteristics and scoring/interpretation guidelines.

The blueprint development group recognizes that to assume that any one of the available tests could be used for guiding the treatment decision with any one or all of the drugs available in this class presents a potential risk to patients that must be addressed.

The goal of this proposal is to agree and deliver, via cross industry collaboration, a package of information /data upon which analytic comparison of the various diagnostic assays may be conducted, potentially paving the way for post-market standardization and/or practice guideline development as appropriate.

A comparative study of PD-L1 diagnostic assays and the classification of patients as PD-L1 positive and PD-L1 negative


Presentation Time:	Monday, Apr 18, 2016, 8:00 AM -12:00 PM
Location:	Section 10
Poster Board Number:	18
Author Block:	Marianne J. Ratcliffe¹, Alan Sharpe², Anita Midha¹, Craig Barker², Paul Scorer², Jill Walker². ¹AstraZeneca, Alderley Park, United Kingdom; ²AstraZeneca, Cambridge, United Kingdom
Abstract Body:	Background: PD-1/PD-L1 directed antibodies are emerging as effective therapeutics in multiple oncology settings. Keynote 001 and Checkmate 057 have shown more frequent response to PD-1 targeted therapies in NSCLC patients with high tumour PD-L1 expression than patients with low or no PD-L1 expression. Multiple diagnostic PD-L1 tests are available using different antibody clones, different staining protocols and diverse scoring algorithms. It is vital to compare these assays to allow appropriate interpretation of clinical outcomes. Such understanding will promote harmonization of PD-L1 testing in clinical practice. Methods: Approximately 500 tumour biopsy samples from NSCLC patients, including squamous and non-squamous histologies, will be assessed using three leading PD-L1 diagnostics assays. PD-L1 assessment by the Ventana SP263 assay that is currently being used in Durvalumab clinical trials (positivity cut off: ≥25% tumour cells with membrane staining) will be compared with the Dako 28-8 assay (used in the Nivolumab Checkmate 057 trial at the 1%, 5% and 10% tumour membrane positivity cut offs), and the Dako 22C3 assay (used in the Pembrolizumab Keynote 001 trial) at the 1% and 50% cut offs). Results: Preliminary data from 81 non-squamous patients indicated good concordance between the Ventana SP263 and Dako 28-8 assays. Optimal overall percent agreement (OPA) was observed between Dako 28-8 at the 10% cut off and the Ventana SP263 assay (OPA; 96%, Positive percent agreement (PPA); 91%, Negative percent agreement (NPA); 98%), where the Ventana SP263 assay was set as the reference. Data on the full cohort will be presented for all three assays, and a lower 95% confidence interval calculated using the Clopper-Pearson method. Conclusions: This study indicates that the patient population defined by Ventana SP263 at the 25% cut off is similar to that identified by the Dako-28-8 assay at the 10% tumour membrane cut off. This, together with data on the 22C3 assay, will enable cross comparison of studies using different PD-L1 tests, and widen options for harmonization of PD-L1 diagnostic testing.

http://www.abstractsonline.com/Plan/ViewAbstract.aspx

Table 1
	Reference: Ventana SP-263 (≥25% tumour membrane staining)
Dako 28-8 assay cut off	PPA (%)	NPA (%)	OPA (%)
>1%	58	100	81
>5%	72	100	90
>10%	91	98	96

UPDATED 5/19/2019

Incidence of Adverse Events for PD-1/PD-L1 Inhibitors Underscores Toxicity Risk

https://www.cancernetwork.com/immuno-oncology/incidence-adverse-events-pd-1pd-l1-inhibitors-underscores-toxicity-risk

Christina Bennett, MS

May 7, 2019

Immuno Oncology, News

Approximately two-thirds of cancer patients who received a programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitor in clinical trials experienced treatment-related adverse events, according to a systematic review and meta-analysis recently published in JAMA Oncology. The study findings may facilitate discussions with cancer patients who are considering PD-1 or PD-L1 therapy.

“The vast majority of patients with advanced cancer want to be on the [PD-1 or PD-L1] therapy,” Eric H. Bernicker, MD, a thoracic medical oncologist with Houston Methodist Cancer Center, told Cancer Network. Not involved in the current study, Bernicker explained that patients perceive these therapies to have “very different” side effects and risks from chemotherapy.

While they do, Bernicker explained, it’s important to underscore, which this study does, that these are not “completely innocuous” therapies. The study findings allow physicians to give numbers to patients and families when counseling them about the risks involved, he said.

The systematic review and meta-analysis is based on data from 125 clinical trials and 20,128 participants. Clinical trials were identified by systematically searching for published clinical trials that evaluated single-agent PD-1 and PD-L1 inhibitors and reported treatment-related adverse events in PubMed, Web of Science, Embase, and Scopus. The majority of trials evaluated nivolumab (n = 46) or pembrolizumab (n = 49), and the most common cancer types were lung cancer (n = 26), genitourinary cancer (n = 22), melanoma (n = 16), and gastrointestinal cancer (n = 14).

In all, 66.0% of clinical trial participants reported at least 1 adverse event of any grade, and 14.0% reported at least 1 grade 3 or higher adverse event. The most frequently reported adverse events of any grade were fatigue (18.26%), pruritus (10.61%), and diarrhea (9.47%). As for grade 3 or higher events, the most commonly reported were fatigue (0.89%), anemia (0.78%), and aspartate aminotransferase (AST) increase (0.75%).

Frequently reported immune-related adverse events of any grade included diarrhea (9.47%), AST increase (3.39%), vitiligo (3.26%), alanine aminotransferase (ALT) increase (3.14%), pneumonitis (2.79%), and colitis (1.24%). Grade 3 or higher immune-related adverse events included AST increase (0.75%), ALT increase (0.70%), pneumonitis (0.67%), diarrhea (0.59%), and colitis (0.47%).

If present, certain adverse events had increased likelihood of being grade 3 or higher, including hepatitis (risk ratio [RR], 50.59%), pneumonitis (RR, 24.01%), type 1 diabetes (RR, 41.86%), and colitis (RR, 37.90%).

“In terms of the rough percentage of side effects and the breadth of the side effects, this is pretty much what most of us see in the clinic,” Bernicker said, noting that none of the findings were particularly surprising.

Although no differences in adverse event incidence were found across different cancer types, differences were found between PD-1 and PD-L1 inhibitors in a subgroup analysis. Overall, compared with PD-L1 inhibitors, PD-1 inhibitors had a higher mean incidence of grade 3 or higher events (odds ratio [OR], 1.58; 95% CI, 1.00–2.54). Specifically, nivolumab had a higher mean incidence of grade 3 or higher events (OR, 1.81; 95% CI, 1.04–3.01) compared with PD-L1 inhibitors.

Bernicker commented that these incidence differences on the basis of drug type were “intriguing” but not clinically useful, given that PD-1 and PD-L1 inhibitors are not interchangeable. He said the finding “needs to be further looked at.”

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New e-Book TITLES forthcoming on Amazon.com in 2017-2018 from LPBI Group’s BioMed e-Series – Forthcoming COVER PAGES

Posted in 3D Printing for Medical Application, Academic Publishing, Cancer - General, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiac and Cardiovascular Surgical Procedures, Computational Biology/Systems and Bioinformatics, CRISPR/Cas9 & Gene Editing, Frontiers in Cardiology and Cardiovascular Disorders, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Genomic Testing: Methodology for Diagnosis, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease on April 24, 2016| Leave a Comment »

New e-Book TITLES forthcoming on Amazon.com in 2017-2018 from LPBI Group’s BioMed e-Series – Forthcoming COVER PAGES

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Editors: Dr. Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Scheduled for publication in 2017

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FDA approves blood-based colorectal screen

Posted in Biomarkers & Medical Diagnostics, Gastroenterology, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Genomic Testing: Methodology for Diagnosis, Proteins, Proteomics, tagged biomarker/companion diagnostics, Blood test, Cancer screening, Colorectal cancer, septin 9 on April 21, 2016| Leave a Comment »

FDA approves blood-based colorectal screen

Larry H. Bernstein, MD, FCAP, Curator

LPBI

FDA Clears First Blood-Based Colorectal Cancer Screening Test.
Lab Soft News by Bruce Friedman
https://www.linkedin.com/pulse/fda-clears-first-blood-based-colorectal-cancer-screening-joseph-colao

In the upcoming months, there surely will be an increasing number of blood-based genomic cancer tests approved by the FDA. This specific test market is just too attractive. In a recent note, I discussed some of these testing initiatives from the perspective of companion diagnostics (see: An Expanding Definition for Companion Diagnostics). This form of testing in used in collaboration with cancer therapy to select the right drug or monitor the effectiveness of drug therapy. Obviously and of equal importance are biomarkers intended for cancer screening. A recent article reported that the FDA has cleared the first blood-based screening test for colorectal cancer (see: FDA Clears First Blood-Based Colorectal Cancer Screening Test), Below is an excerpt from it:

The first blood-based colorectal cancer (CRC) screening test, Epi proColon...has been approved by the US Food and Drug Administration (FDA)….The Epi proColon test is a qualitative in vitro diagnostic test for detecting methylated Septin9 DNA, which has been associated with the occurrence of CRC, in plasma obtained from whole-blood specimens. It is indicated for use in average-risk patients who have chosen not to undergo other screening methods, such as colonoscopy or stool-based tests.The test was recommended for FDA approval in 2014 by the Molecular and Clinical Genetics Panel of the FDA’s Medical Devices Advisory Committee, but some of the experts were not convinced….The agency approved the Epi proColon test for CRC screening in average-risk patients (as defined by current screening guidelines) who choose not to be screened by colonoscopy or a stool-based FIT [fecal immunochemical test for occult blood in the stool].The Epi proColon blood test for CRC screening can be performed during routine office visits. It requires no dietary restrictions or alterations in medication use. The blood sample is analyzed by a local or regional diagnostic laboratory….The company will initiate a postapproval study to show the long-term benefit of blood-based CRC screening using Epi proColon, as required by the FDA.

Here’s more information about Septin9 DNA (see: Plasma methylated septin 9: a colorectal cancer screening marker):

The biomarker septin 9 has been found to be hypermethylated in nearly 100% of tissue neoplasia specimens and detected in circulating DNA fractions of CRC patients. A commercially available assay for septin 9 has been developed with moderate sensitivity (∼70%) and specificity (∼90%) and a second generation assay, Epi proColon 2.0 (Epigenomics AG), shows increased sensitivity (∼92%).The performance of the assay proved to be independent of tumor site and reaches a high sensitivity of 77%, even in early cancer stages (I and II). Furthermore, septin 9 was recently used in follow-up studies for detection of early recurrence of CRC.

There is clearly a need for a blood-based biomarker for colorectal cancer screening. Patients tend to dislike the home stool collection that is required for fecal immunochemical tests for occult blood in the stool (FIT). Moreover, testing for blood in the stool offers a somewhat crude substitute for the identification of reliable cancer biomarkers in the blood. It must be noted, however, that some of the FDA experts in 2014 were not convinced that the septic 9 biomarker offered advantages over FIT.

I am not sure that septin 9 will be the final and most efficient biomarker for CRC but I am sure of two things. The first is that there will eventually be a high-specificity, high-sensitivity blood test for CRC. The second is that probably tens of billions of dollars would be saved by the elimination of screening colonoscopies for CRC by such a test. I found an article dating way back to 2002 about the number of screening endoscopies performed in the U.S. but the numbers are sill impressive (see: How many endoscopies are performed for colorectal cancer screening?) Here is a quote from it: “Approximately 2.8 million flexible sigmoidoscopes and 14.2 million colonoscopies were estimated to have been performed in 2002.” Needless to say, many gastroenterologists and radiologists may be hoping that such a lab test does not reach the market soon.

Septin-9 is a protein that in humans is encoded by the SEPT9 gene.^[1]^[2]^[3

SEPT9 has been shown to interact with SEPT2^[4] and SEPT7.^[4]

Along with AHNAK, eIF4E and S100A11, SEPT9 has been shown to be essential for pseudopod protrusion, tumor cell migration and invasion.^[5]

The v2 region of the SEPT9 promoter has been shown to be methylated in colorectal cancer tissue compared with normal colonic mucosa.^[6] Using highly sensitive real time PCR assays, methylated SEPT9 was detected in the blood of colorectal cancer patients. This alternate methylation pattern in cancer samples is suggestive of an aberrant activation or repression of the gene compared to normal tissue samples.^[7]^[8]

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Kaiser Permanente collecting patient data for DNA Research Bank

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BACKGROUND

METHODS

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Gene Editing with CRISPR gets Crisper

Predesigned Guide RNAs

Donor DNA Formats

Ribonucleo-protein Complexes

Are Current Cancer Drug Discovery Methods Flawed?

An unbiased metric of antiproliferative drug effect in vitro

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Overview

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A Blueprint Proposal for Companion Diagnostic Comparability

Incidence of Adverse Events for PD-1/PD-L1 Inhibitors Underscores Toxicity Risk

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New e-Book TITLES forthcoming on Amazon.com in 2017-2018 from LPBI Group’s BioMed e-Series – Forthcoming COVER PAGES

BioMed e-Series

WE ARE ON AMAZON.COM

FORTHCOMING

CANCER – Volume Two on Amazon.com

GENOMICS – Volume Two – 2018

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