Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Interpreting Mutations Piled Higher and Deeper

Clinical actionability, or the determination of whether clinical action should be taken based on heterogeneous information generated by cancer genomic analysis, remains a key challenge for scientists and clinicians.

Rodrigo Dienstmann, M.D., et al., writing in Molecular Oncology in 2014 (“Standardized decision support in next generation sequencing reports of somatic cancer variants”), noted that hundreds to thousands of somatic mutations exist in each cancer genome.  Many of these variants have well-established biological and clinical relevance and are putative targets of molecular therapy. But the author said most variants have no known significance, greatly complicating clinical decision making.

While investigators say that it remains unclear how and to what extent these variants may affect clinical decision making, work continues in multiple laboratories to develop platforms to identify potentially actionable DNA alterations in tumor samples. These laboratories, usually at major cancer clinical centers, have devoted significant resources to optimizing sophisticated deep-sequencing platforms and bioinformatics power to connect the dots between genomic variations and potential actionability.

Andrea Desmond and colleagues at the Massachusetts General Hospital, Harvard, Stanford, and Beth Israel Deaconess Hospitals investigated how often multigene panel testing would identify clinically actionable mutations among patients  tested for, but lacking BRCA1 and BRCA 2 mutations.

Depending on whether participants were enrolled at Mass General or Stanford, the investigators tested their tumor samples with either Myriad Genetics’ MyRisk test, a 25-gene panel, or with Invitae’s 29-gene Hereditary Cancer Syndromes test through academic collaborations with the companies.

Reporting in Clinical Oncology in October 2015 (“Clinical Actionability of Multigene Panel Testing for Hereditary Breast and Ovarian Cancer Risk Assessment”), the researchers said their study results suggest that mutations discovered using multigene panel sequencing may alter cancer prevention and management strategies offered to a “significant subset” of individuals thought to be at risk of hereditary breast or ovarian cancer (HBOC) but lacking mutations in BRCA1 or BRCA2 genes.

Of 1,046 study participants, 40 BRCA1/2-negative patients harbored deleterious mutations, most commonly in moderate-risk breast and ovarian cancer genes CHEK2, ATM, and PALB2 and Lynch syndrome genes. Among these, and an additional 23 mutation-positive individuals enrolled from participating clinics, most of the mutations (92%) reflected the spectrum of cancer(s) observed in the patient or family, suggesting that these results are clinically significant.

The authors concluded that in a clinically representative cohort, multigene panel testing for HBOC risk assessment yielded findings that could change clinical management for substantially more patients than does BRCA1 and BRCA 2 testing alone. Multigene testing in this setting, they say, will likely alter near-term cancer risk assessment and management recommendations for mutation-affected individuals across a broad spectrum of cancer predisposition genes, showing that positive panel test results warrant consideration of a change in management for the patient more often than is true for just BRCA1/2 testing.

Scientists at The University of Texas MD Anderson Cancer Center reported that they have established a deep targeted sequencing platform, T200, comprising 4,874 exons encoding 938,607 bases to identify potentially actionable DNA alterations in tumor samples. They note that the relatively low average coverage of whole genome sequencing and whole exome sequencing as currently implemented in most sequencing laboratories at a depth of 100–250x may have limited ability to cost-effectively detect aberrations present in tumor cell subpopulations.

Writing in the March 2015 edition of Clinical Chemistry (“Clinical Actionability Enhanced through Deep Targeted Sequencing of Solid Tumors“), Ken Chen, Ph.D., et al. described results from the assay of 515 formalin-fixed paraffin-embedded tumor samples and matched germline DNA (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer-related genes, a report that included mutations, indels, and copy number data.

The investigators said they had obtained a thousand-fold mean sequencing depth and identified 4,794 nonsynonymous mutations in the samples analyzed, of which 15.2% were present at <10% allele frequency. Most of these low-level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low-level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) otherwise would have been unactionable.

Their results, the researchers said, were as accurate as a commercially available CLIA-compliant hotspot panel, but allowed the detection of a higher number of mutations in actionable genes. This reveals the critical importance of acquiring and utilizing high sequencing depth in profiling clinical tumor samples and presents a very useful platform for implementing routine sequencing in cancer care institutions.

Kenna Mills Shaw, Ph.D., executive director of Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy (IPCT) at MD Anderson, said her team worked with a patient population with mutations that did not have a “standard of care” assay ordered. “If you don’t sequence to a depth of at least 500 you can miss rare mutations in tumors, including emerging mutations that may be indicative of resistance,” Dr. Shaw explained. “Ultimately, these results would have to be validated in a CLIA assay before they could be actionable. For patients that could benefit, for example, for clinical trial enrollment, we did confirm the research result in a CLIA laboratory.”

The investigators say that their study results show the critical importance of high sequencing depth is profiling clinical tumor samples. Although the results were obtained in a preclinical research environment through an IRB-approved protocol, they were provided to treating physicians who could order additional CLIA-compliant assays to confirm alterations for clinical decision making.

Similarly to MD Anderson, New York’s Memorial Sloan Kettering Cancer Center (MSKCC) uses a consensus-based approach for reviewing potential actionability for genomic findings.

Mark E. Robson, M.D., clinic director of Clinical Genetics Service of MSKCC, speaking at a roundtable discussion, “Translating Genomic-Based Research for Health,” convened by the Institute of Medicine in 2014, explained that at MSKCC a multidisciplinary panel of individuals with expertise in basic science, drug development, clinical trial design, assay development and interpretation, and computational biology and biostatistics does a case-by-case evaluation of the evidence. For a tumor-specific driver mutation, he said, actionability relates to whether a targeted therapeutic is indicated and available, while for a deleterious germline mutation, actionability relates to mutation penetrance and the efficacy of available preventive medical interventions.

Incidental germline findings from tumor profiling are reported only in discovery studies with approval from the institutional review board and only with proper consent. The actionability criteria remain “a bit fluid,” Dr. Robson said, and discussions have centered on what level of risk justifies contact.

Dr. Robson commented in his article, “Multigene Panel Testing: Planning the Next Generation of Research Studies in Clinical Cancer Genetics,” in 2014 in the Journal of Clinical Oncology, that the rapid pace of technological innovation has “driven multiplex panel testing into the clinic, perhaps a bit before we have built a responsible framework to accommodate it.”

Now next-generation sequencing is creating a massive experiment in whether knowing the pattern of genomic aberrations will allow therapies to be targeted more effectively. “Although everybody is very enthusiastic about it,” Dr. Robson said, “whether or not we are going to be able to achieve better outcomes on a global scale throughout the cancer population still remains to be seen.”

According to Dr. Robson’s paper, most scientists agree that well-designed clinical trials, preferably randomized, are needed to assess whether panel testing arrives at a diagnosis faster than phenotype-directed testing, whether panel testing improves overall health outcomes compared with phenotype-directed testing, and whether up-front panel testing is as cost-effective as a general strategy compared with phenotype-directed testing.










































 

This article was originally published in the March 2016 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to www.clinicalomics.com.

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