Foundation Medicine and the Big Barrier to Cancer Genomic Sequencing

[gdpawel]

According to Dr. Eric Topol, Director, Scripps Translational Science Institute, recent studies have highlighted the potential value of whole genome or exome sequencing to precisely guide therapy for patients with cancer. However, almost all samples today go into formalin-fixed, paraffin embedded (FFPE) blocks, which alters the DNA and makes sequencing quite compromised and difficult.

He told Medscape Connect that the company, Foundation Medicine, which works with formalin-fixed paraffin-embedded (FFPE) blocks, and gets about 250-300 genes, the exons or coding elements in those genes, and reads out any potential links to drugs. But the rate-liimiting step appears to be getting something beyond these paraffin blocks. This is, we could do better if we could use either fresh formalin-fixed or frozen tissue samples from a biopsy or surgical specimen.

Topol says the problem is that pathologists are seemingly quite ritualistic. They don't want to go to frozen samples, which would be the best for whole genome sequencing. We're just at the cusp of getting started with this type of limited, not even full exome sequencing, just a few hundred genes, but that isn't enough.

Rencent papers in multiple journals in Nature, Science, Nature Genetics and Cell have shown that with hundreds of tumor samples fully sequenced, no two cancers are the same and a lot of the action is not in the coding elements of the genes per se. Whole genome sequencing certainly appears to be an ideal path to pursue, but we can't do it with the fixed problems that we have with the way samples are handled today.

Topol thinks that maybe we could get fresh formalin-fixed samples, as those appear to be well-suited to whole genome sequencing, although this is still a somewhat bootstrapped situation, like the paraffin-embedded samples. It appears that the long those samples are embedded, the harder it is to get a reasonable sequence beyond very targeted regions.

There are no two cancer tissues that are the same on a molecular basis. There's quite a bit of heterogeneity within the samples and multiple sequencing could account for that. And we also want to anticipate recurrence, match up the right driver mutations and the backseat passenger mutations, whether or not there's needed immunotherapy; all those things that could be done if we could get the right information from the get go.

So Dr. Topol asks this: How are we going to move to a world with a clinic of the future where patients with cancer can get whole genome sequencing rapidly? That is, to have annotation and interpretation of the genome with a day, and have your therapy precisely guided genomically?

[gdpawel]

Researchers have realized that cancer biology is driven by signaling pathways. Cells speak to each other and the messages they send are interpreted via intracellular pathways known as signal transduction. Many of these pathways are activated or deactivated by phosphorylations on select cellular proteins.

Sequencing the genome of cancer cells is explicitly based upon the assumption that the pathways - network of genes - of tumor cells can be known in sufficient detail to control cancer. Each cancer cell can be different and the cancer cells that are present change and evolve with time.

Although the theory behind inhibitor targeted therapy is appealing, the reality is more complex. Cancer cells often have many mutations in many different pathways, so even if one route is shut down by a targted treatment, the cancer cell may be able to use other routes.

In other words, cancer cells have "backup systems" that allow them to survive. The result is that the drug does not affect the tumor as expected. The cancer state is typically characterized by a signaling process that is unregulated and in a continuous state of activation.

In chemotherapy selection, molecular profiling examines a single process within the cell or a relatively small number of processes. All a gene mutation study can tell is whether or not the cells are potentially susceptible to a mechanism of attack. The aim is to tell if there is a theoretical predisposition to drug response.

It doesn't tell you the effectiveness of one drug (or combination) or any other drug which may target this in the individual. There are many pathways to altered cellular function. Functional Profiling measures the end result of pathway activation or deactivation to predict whether patients will actually respond (clinical responders).

It measures what happens at the end, rather than the status of the individual pathway, by assessing the activity of a drug (or combinations) upon combined effect of all cellular processes, using combined metabolic and morphologic endpoints, at the cell population level, measuring the interaction of the entire genome.

Translational science: past, present, and future

http://www.biotechniques.com/multime...49_O_3671a.pdf

Note: Foundation Medicine is not any different than Caris Diagnostics in Phoenix (now Miraca Life Sciences), beyond testing for standard pathology "targets" such as ER, PR, Her2, EGFR mutations, KRAS, BRAF. They aren't worth much for the sorts of chemotherapy which is used in 95% of all cancers and useless with respect to drug combinations. While fresh tissue is very dear and hard to come by, function trumps structure, in terms of potency and robustness of information provided than using archival paraffin blocks.

[gdpawel]

Needle biopsies assay on 33 pretherapy primaries and 17 mBCs. NGS of 3,230 exons in 182 cancer-related genes and 37 introns in 14 genes. 117 "potentially" targetable driver mutations were identified (mean: 2.3 in primary tumors, 2.8 in mBCs); however, further research is needed.

According to Dr. Neil Love of Research To Practice, one of several presentations at the ASCO trade show was in a variety of solid tumors on the now commercially available FoundationOne assay. This study of 50 patients with breast cancer — like similar reports in lung, prostate and colorectal cancer — documented that fine needle biopsies provided enough tissue to adequately perform the test. Even more relevant was that multiple potentially “targetable” mutations were found in all these specimens. Although the authors suggest that some of these targets may correlate with benefit from approved agents (eg, crizotinib for ALK translocations), and it might be tempting to consider ordering the assay for patients who have run out of conventional options, this concept has not yet been tested. In that regard, it is worth reflecting on the wholly disappointing experience with BRAF inhibitors for patients with V600E mutation-positive colorectal cancer and appreciating that the term “targetable” is highly theoretical at this point in time.

Use of next-generation sequencing (NGS) to detect high frequency of targetable alterations in primary and metastatic breast cancer (MBC).

Sub-category:
Genomic and Epigenomic Biomarkers

Category:
Tumor Biology

Meeting:
2012 ASCO Annual Meeting

Session Type and Session Title:
General Poster Session, Tumor Biology

Abstract No:
10559

Citation:
J Clin Oncol 30, 2012 (suppl; abstr 10559)

Author(s):

Lajos Pusztai, Roman Yelensky, Bailiang Wang, Rony Avritscher, William Fraser Symmans, Doron Lipson, Gary A. Palmer, Stacy L. Moulder, Philip Stephens, Yun Wu, Maureen T. Cronin; University of Texas M. D. Anderson Cancer Center, Houston, TX; Foundation Medicine, Cambridge, MA; University of Texas, Houston, TX; Department of Radiology, University of Texas M. D. Anderson Cancer Center, Houston, TX

Abstract:

Background:

The aim of this study was to assess the frequency of genomic alterations in breast cancer potentially treatable with approved targeted agents or investigational drugs in clinical trials. NGS was performed in a CLIA setting (Foundation Medicine).

Methods:

DNA was extracted from needle biopsies of 33 pre-therapy primary and 17 MBCs (mean age 52 yrs; 58% ER+, 20% HER2+, 30% triple negative) obtained prospectively for biomarker discovery and preserved in RNAlater. Patients with MBC received an average of 7 drugs (range 5-17) including adjuvant therapy before biopsy for this research; 13 biopsies were from soft tissues, 3 from liver and 1 from bone. Sequencing was targeted to 3230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer. Average median depth was >1200x.

Results:

All biopsies yielded sufficient DNA. NGS revealed a total of 117 known driver mutations across 36 genes (per-tumor average=2.5, range 1-6), including 37 base substitutions (32%), 28 indels (24%), 42 amplifications (36%) and 10 homozygous deletions (9%). NGS identified HER2 gene amplification in 6/7 cases scored HER2+ by FISH. The average number of functionally important alterations was surprisingly similar, 2.3 in primaries vs 2.8 in heavily treated MBCs (p=0.32). Remarkably, 25/33 (76%) of primary and 14/17 (82%) of MBCs had at least 1 genomic alteration targetable with an FDA approved drug or novel agent in clinical trials. These included: ERBB2 alterations (n=9), PIK3CA mutations (n=8), NF1 mutations (n=4, candidate for PI3K/MEK inhibitors), AKT1-3 mutations (n=5, PI3K inhibitors), BRCA1/2, (n=6, PARP inhibitors), and CCND2 (n=3)/CDKN2A (n=3) mutations (CDK inhibitors). Numerous other alterations with less apparent therapeutic implications were also observed.

Conclusions:

Comprehensive NGS profiling in breast cancer needle biopsies showed high frequency of genomic alterations linked to a clinical treatment option or clinical trials of targeted therapies. These results demonstrate it is feasible to use NGS to guide targeted therapy. Prospective testing of the diagnostic/predictive value of this patient selection approach is currently under way.

[gdpawel]

Accuracy and clinical utility of in vitro cytometric profiling to personalize chemotherapy: Preliminary findings of a systematic review and meta-analysis.

Sub-category: Molecular Diagnostics and Imaging

Category: Tumor Biology

Meeting: 2013 ASCO Annual Meeting

Abstract No: e22188

Citation: J Clin Oncol 31, 2013 (suppl; abstr e22188)

Author(s): Christian Apfel, Kimberly Souza, Cyrill Hornuss, Larry Weisenthal, Robert Alan Nagourney; SageMedic, Inc, Larkspur, CA; Ludwig Maximilians University of Munich, Munich, Germany; Weisenthal Cancer Group, Huntington Beach, CA; Rational Therapeutics, Long Beach, CA

Abstract:

Background:

Cytometric analysis, or in-vitro functional profiling, has been developed as a method to predict tumor response to different drugs with the premise to personalize chemotherapy and improve patient outcomes.

Methods:

We performed a systematic review and a meta-analysis a) of correlative studies using cytometric profiling that reported diagnostic accuracy (sensitivity and specificity) and b) of effectiveness studies comparing patient outcomes when allocated to treatment guided by a cytometric assay versus population-based standard of care. We used Meta-DiSc software to find pooled sensitivity and specificity and analyze the summary receiver operating characteristic (sROC) curve and used Review Manager 5.1 to generate forest plots on overall tumor response (50% or greater decrease in tumor diameter) and on 1-year overall survival.

Results:

We included 28 mostly retrospective trials (n=664) reporting accuracy data and 15 prospective trials (n=1917) reporting therapeutic efficacy data. The accuracy of correlative study revealed an overall sensitivity of 0.922 (95% confidence interval 0.888 to 0.948), specificity of 0.724 (95% CI 0.669 to 0.774) and an area under the sROC curve of 0.893 (SE=0.023, p<0.001). Studies comparing the clinical utility revealed a two-fold overall tumor response for an assay-guided therapy versus standard of care therapy (odds ratio 2.04, 95% CI 1.62 to 2.57, p<0.001). Similarly, patients who received assay-guided therapy compared to those who received standard of care or physician’s choice had a significantly higher 1-year survival rate (OR 1.44, 95% CI 1.06 to 1.95, p=0.02).

Conclusions:

Despite various limitations of individual studies, the aggregate and fairly consistent evidence of these data suggests cytometric profiling to be accurate, to improve overall tumor response, and to increase 1-year patient survival. Given the enormous potential for our society, a well-designed and sufficiently-powered randomized controlled trial is urgently needed to validate these results.

http://abstracts2.asco.org/AbstView_132_118466.html

[gdpawel]

Robert A. Nagourney, M.D.

During the 1960s, 70s and into the 90s, a field of investigation arose that examined buyer’s practices when it came to the consumption of goods and services. Algorithms were developed to interrogate consumer choice. One such treatise was reported in 1994 (Carson, RT et al, Experimental Analysis of Choice, Marketing Letters 1994). What these researchers explored were the motivations and forces that drove consumption. When choices are offered, decisions are driven by such factors as complexity and utility. Complexity demands personal expertise or failing that, input from experts, while utility places a value on the good or service.

A recent report from a small biotechnology company called Foundation Medicine has brought this field of endeavor to mind. It seems that this group will be offering DNA sequencing to select chemotherapy drugs. This service, currently priced at $5,800, will focus upon a small cassette of genes that they described as “key” in tumor growth. Based on their technology they have already raised $33.5 million from the likes of Third Rock, Google and Kleiner Perkins Caulfield & Byers, venture capital sources. The CEO of Foundation substantiates the approach by pointing out that fully 150 people have already used their services. One hundred and fifty!

It seems from this report that our colleagues in the field of molecular profiling have studied the dictates of “Experimental Analysis of Choice” to a “T.” What we have is the perfect storm of medical marketing.

First, the technology is so complex as to be beyond the ken of both patients and physicians alike. Thus, expertise is required and that expertise is provided by those engaged in the field. Second, the utility of drug selection is beyond reproach. Who in their right mind wouldn’t want to receive a drug with a higher likelihood of a response when we consider the toxicities and costs, as well as the consequences of the wrong treatment? Dazzled by the prospect of curative outcomes, patients will, no doubt, be lining up around the block.

But, let’s deconstruct what this report is actually telling us. First, a scientifically interesting technology has been brought to the market. Second, it exists to meet an unmet need. So far, so good. What is lacking, however, is evidence. Not necessarily evidence in the rarefied Cochrane sense of idealized survival curves, nor even Level II evidence, but any evidence at all. Like whirling dervishes, patients and their physicians are drawn into a trancelike state, when terms like NextGen sequencing, SNP analysis and splice variants are bandied about.

Despite the enthusiastic reception by investors, I fear a lack of competent due diligence. To wit, a recent article in Biotechniques, “Will the Real Cancer Cell Please Stand Up,” comes to mind. It seems that cancer cells are not individual entities but networks. A harmonic oscillation develops between tumor, stroma, vasculature and cytokines. In this mix, the cancer cell is but one piece of the puzzle.

Indeed, according to recent work from Baylor, some of the tumor promotion signals in the form of small interfering RNAs, may arise not from the cancer cells, but instead from the surrounding stroma. How then, will even the most punctiliously perfect genomic analyses of a cancer cells play out in the real world of human tumor biology and clinical response prediction? Not very well I fear. But then again such a discussion would require data on the predictive validity of the method, something that appears to be sorely lacking.

Will today’s gene profile companies prove to be the biotech Facebook IPOs of tomorrow?

[gdpawel]

There is the issue about cell-lines vs fresh cells. Cell-lines have always played, and continue to play, an important role in drug screening and drug development.

The problem is that cell-lines do not predict for disease or patient specific drug effects. If you can kill cancer cell-lines with a given drug, it doesn’t tell you anything about how the drug will work in real world, clinical cancer (real-world conditions). But you can learn certain things about general drug biology through the study of cell-lines.

As a general rule, studies from established cell-lines (tumor cells that are cultured and maniplated so that they continue to divide) have proved worthless as models to predict the activity of drugs in cancer. They are more misleading than helpful. An established cell-line is not reflective of the behavior of the fresh tumor samples (live samples derived from tumors) in primary culture, much less in the patient.

Established cell-lines have been a huge disappointment over the decades, with respect to their ability to correctly model the disease-specific activity of new drugs. What works in cell-lines do not often translate into human beings. You get different results when you test passaged cells compared to primary, fresh tumor.

Research on cell-lines is cheap compared to clinical trials on humans. One gets more accurate information when using intact RNA isolated from “fresh” tissue than from using degraded RNA, which is present in paraffin-fixed tissue.

My question would be, do you want to utilize your tissue specimen for “drug selection” against “your” individual cancer cells or for mutation identification, to see if you are “potentially” susceptible to a certain mechanism of attack?

Cell Lines vs Fresh Cells

http://cancerfocus.org/forum/showthread.php?t=3702