Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine

Translating whole exome sequencing (WES) for prospective clinical use may impact the care of cancer patients; however, multiple innovations are necessary for clinical implementation. These include: (1) rapid and robust WES from formalin-fixed paraffin embedded (FFPE) tumor tissue, (2) analytical output similar to data from frozen samples, and (3) clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival FFPE tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a “long tail” of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15/16 patients. In one patient, previously undetected findings guided clinical trial enrollment leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine

Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine Corresponding Authors

. "Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine." Nature medicine 20 Disclosures: Dr. Garraway and Dr. Wagle are equity holders and consultants in Foundation Medicine. Dr. Garraway is a consultant to Novartis, Millenium/Takeda, and Boehringer Ingelheim, and a recipient of a grant from Novartis. Dr. Getz is a consultant to The Kew Group. Dr. Barbie is a consultant to N-of-1. NIH Public Acces

Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine. Massively parallel sequencing approaches such as WES have elucidated the landscape of genetic alterations in many tumor types and revealed biological insights relevant to clinical contexts 1 . The increased practical availability and decreased cost of tumor genomic profiling has generated opportunities to test the 'precision medicine' hypothesis in clinical oncology 2 . In principle, knowledge of alterations in the coding regions of all genes may inform immediate treatment choices and further therapeutic discovery efforts 3 . Most prospective clinical genotyping efforts have used 'hotspot' genotyping 4-6 or targeted sequencing panels of clinically relevant genes using either fresh frozen or formalin-fixed, paraffin-embedded (FFPE) tissue Here, we describe an approach to generate high-quality WES data from archival tumor material and validate WES data from FFPE tumor samples with corresponding WES data from frozen samples. We also present a heuristic algorithm that interprets the resulting data for clinical oncologists and establish the clinical applicability of this interpretation algorithm in a retrospective cohort of 511 cases. Prospective application of this platform in patients with a range of tumor types indicates that this approach can be used for both biological discovery and clinical trial enrollment. This approach may therefore facilitate widespread application of WES for precision cancer medicine studies. RESULTS WES of FFPE samples To produce WES data for clinical use, robust sequencing data must frequently be generated from small quantities of archival FFPE tissue. To test this, we extracted DNA from 99 FFPE samples using the FFP

Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine. Massively parallel sequencing approaches such as WES have elucidated the landscape of genetic alterations in many tumor types and revealed biological insights relevant to clinical contexts 1 . The increased practical availability and decreased cost of tumor genomic profiling has generated opportunities to test the 'precision medicine' hypothesis in clinical oncology 2 . In principle, knowledge of alterations in the coding regions of all genes may inform immediate treatment choices and further therapeutic discovery efforts 3 . Most prospective clinical genotyping efforts have used 'hotspot' genotyping 4-6 or targeted sequencing panels of clinically relevant genes using either fresh frozen or formalin-fixed, paraffin-embedded (FFPE) tissue Here, we describe an approach to generate high-quality WES data from archival tumor material and validate WES data from FFPE tumor samples with corresponding WES data from frozen samples. We also present a heuristic algorithm that interprets the resulting data for clinical oncologists and establish the clinical applicability of this interpretation algorithm in a retrospective cohort of 511 cases. Prospective application of this platform in patients with a range of tumor types indicates that this approach can be used for both biological discovery and clinical trial enrollment. This approach may therefore facilitate widespread application of WES for precision cancer medicine studies. RESULTS WES of FFPE samples To produce WES data for clinical use, robust sequencing data must frequently be generated from small quantities of archival FFPE tissue. To test this, we extracted DNA from 99 FFPE samples using the FFP

The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset

The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway[subscript 1, 2]. BRAF(V600E) mutations are also present in benign melanocytic naevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes[subscript 4, 5]. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis

Management of coronary disease in patients with advanced kidney disease

BACKGROUND Clinical trials that have assessed the effect of revascularization in patients with stable coronary disease have routinely excluded those with advanced chronic kidney disease. METHODS We randomly assigned 777 patients with advanced kidney disease and moderate or severe ischemia on stress testing to be treated with an initial invasive strategy consisting of coronary angiography and revascularization (if appropriate) added to medical therapy or an initial conservative strategy consisting of medical therapy alone and angiography reserved for those in whom medical therapy had failed. The primary outcome was a composite of death or nonfatal myocardial infarction. A key secondary outcome was a composite of death, nonfatal myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. RESULTS At a median follow-up of 2.2 years, a primary outcome event had occurred in 123 patients in the invasive-strategy group and in 129 patients in the conservative-strategy group (estimated 3-year event rate, 36.4% vs. 36.7%; adjusted hazard ratio, 1.01; 95% confidence interval [CI], 0.79 to 1.29; P=0.95). Results for the key secondary outcome were similar (38.5% vs. 39.7%; hazard ratio, 1.01; 95% CI, 0.79 to 1.29). The invasive strategy was associated with a higher incidence of stroke than the conservative strategy (hazard ratio, 3.76; 95% CI, 1.52 to 9.32; P=0.004) and with a higher incidence of death or initiation of dialysis (hazard ratio, 1.48; 95% CI, 1.04 to 2.11; P=0.03). CONCLUSIONS Among patients with stable coronary disease, advanced chronic kidney disease, and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of death or nonfatal myocardial infarction

Health status after invasive or conservative care in coronary and advanced kidney disease

BACKGROUND In the ISCHEMIA-CKD trial, the primary analysis showed no significant difference in the risk of death or myocardial infarction with initial angiography and revascularization plus guideline-based medical therapy (invasive strategy) as compared with guideline-based medical therapy alone (conservative strategy) in participants with stable ischemic heart disease, moderate or severe ischemia, and advanced chronic kidney disease (an estimated glomerular filtration rate of <30 ml per minute per 1.73 m2 or receipt of dialysis). A secondary objective of the trial was to assess angina-related health status. METHODS We assessed health status with the Seattle Angina Questionnaire (SAQ) before randomization and at 1.5, 3, and 6 months and every 6 months thereafter. The primary outcome of this analysis was the SAQ Summary score (ranging from 0 to 100, with higher scores indicating less frequent angina and better function and quality of life). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate the treatment effect with the invasive strategy. RESULTS Health status was assessed in 705 of 777 participants. Nearly half the participants (49%) had had no angina during the month before randomization. At 3 months, the estimated mean difference between the invasive-strategy group and the conservative-strategy group in the SAQ Summary score was 2.1 points (95% credible interval, 120.4 to 4.6), a result that favored the invasive strategy. The mean difference in score at 3 months was largest among participants with daily or weekly angina at baseline (10.1 points; 95% credible interval, 0.0 to 19.9), smaller among those with monthly angina at baseline (2.2 points; 95% credible interval, 122.0 to 6.2), and nearly absent among those without angina at baseline (0.6 points; 95% credible interval, 121.9 to 3.3). By 6 months, the between-group difference in the overall trial population was attenuated (0.5 points; 95% credible interval, 122.2 to 3.4). CONCLUSIONS Participants with stable ischemic heart disease, moderate or severe ischemia, and advanced chronic kidney disease did not have substantial or sustained benefits with regard to angina-related health status with an initially invasive strategy as compared with a conservative strategy