Michael F. Berger

Phase II Trial of Sorafenib in Patients with Chemotherapy Refractory Metastatic Esophageal and Gastroesophageal (GE) Junction Cancer

Purpose Vascular endothelial growth factor receptor (VEGFR2) directed therapies result in a modest survival benefit for patients with advanced esophageal and gastroesophageal (GE) junction cancer. Platelet-derived growth factor receptor (PDGFR) may contribute to escape from VEGFR2 inhibition. We evaluated the efficacy of sorafenib, a broad spectrum tyrosine kinase inhibitor targeting VEGFR2 and PDGFR as well as RET and RAF1, in patients with metastatic chemotherapy refractory esophageal and GE junction cancer. Patients and Methods This phase II trial of sorafenib 400 mg twice daily enrolled chemotherapy refractory patients with metastatic esophageal and GE junction cancer with primary endpoint of progression-free survival (PFS) rate at two months. Secondary endpoints included overall survival, objective response rate and toxicity. Results Among 34 patients, 8 week Kaplan-Meier estimated PFS was 61% (90%CI 45 to 73%). Median PFS is 3.6 months (95% CI 1.8 to 3.9 months), with median overall survival OS 9.7 months (95% CI 5.9 to 11.6 months). Grade 3 toxicities were uncommon and included hand foot skin reaction, rash, dehydration and fatigue. One patient (3%) with ongoing complete response and remains on trial for over 5 years. Whole exome sequencing of this tumor revealed mutations in many cancer-associated genes including ARID1A, PIK3CA, and TP53, and focal amplifications of HMGA2 and MET. Conclusion Sorafenib therapy results in disease stabilization and encouraging PFS in patients with refractory esophageal and GE junction cancer. Trial Registration ClinicalTrials.gov NCT00917462

Somatic mutations in leukocytes infiltrating primary breast cancers

Background:Malignant transformation requires the interaction of cancer cells with their microenvironment, including infiltrating leukocytes. However, somatic mutational studies have focused on alterations in cancer cells, assuming that the microenvironment is genetically normal. Because we hypothesized that this might not be a valid assumption, we performed exome sequencing and targeted sequencing to investigate for the presence of pathogenic mutations in tumor-associated leukocytes in breast cancers.Methods:We used targeted sequencing and exome sequencing to evaluate the presence of mutations in sorted tumor-infiltrating CD45-positive cells from primary untreated breast cancers. We used high-depth sequencing to determine the presence/absence of the mutations we identified in breast cancer-infiltrating leukocytes in purified tumor cells and in circulating blood cells.Results:Capture-based sequencing of 15 paired tumor-infiltrating leukocytes and matched germline DNA identified variants in known cancer genes in all 15 primary breast cancer patients in our cohort. We validated the presence of mutations identified by targeted sequencing in infiltrating leukocytes through orthogonal exome sequencing. Ten patients harbored alterations previously reported as somatically acquired variants, including in known leukemia genes (DNTM3A, TET2, and BCOR). One of the mutations observed in the tumor-infiltrating leukocytes was also detected in the circulating leukocytes of the same patients at a lower allele frequency than observed in the tumor-infiltrating cells.Conclusions:Here we show that somatic mutations, including mutations in known cancer genes, are present in the leukocytes infiltrating a subset of primary breast cancers. This observation allows for the possibility that the cancer cells interact with mutant infiltrating leukocytes, which has many potential clinical implications.

PTEN Loss-of-Function Alterations Are Associated With Intrinsic Resistance to BRAF Inhibitors in Metastatic Melanoma

PurposenThe clinical use of BRAF inhibitors in patients with melanoma is limited by intrinsic and acquired resistance. We asked whether next-generation sequencing of pretreatment tumors could identify coaltered genes that predict for intrinsic resistance to BRAF inhibitor therapy in patients with melanoma as a prelude to rational combination strategies.nnnPatients and MethodsnWe analyzed 66 tumors from patients with metastatic BRAF-mutant melanoma collected before treatment with BRAF inhibitors. Tumors were analyzed for > 250 cancer-associated genes using a capture-based next-generation sequencing platform. Antitumor responses were correlated with clinical features and genomic profiles with the goal of identifying a molecular signature predictive of intrinsic resistance to RAF pathway inhibition.nnnResultsnAmong the 66 patients analyzed, 11 received a combination of BRAF and MEK inhibitors for the treatment of melanoma. Among the 55 patients treated with BRAF inhibitor monotherapy, objective responses, as assessed by Response Evaluation Criteria in Solid Tumors (RECIST), were observed in 30 patients (55%), with five (9%) achieving a complete response. We identified a significant association between alterations in PTEN that would be predicted to result in loss of function and reduced progression-free survival, overall survival, and response grade, a metric that combines tumor regression and duration of treatment response. Patients with melanoma who achieved an excellent response grade were more likely to have an elevated BRAF-mutant allele fraction.nnnConclusionnThese results provide a rationale for cotargeting BRAF and the PI3K/AKT pathway in patients with BRAF-mutant melanoma when tumors have concurrent loss-of-function mutations in PTEN. Future studies should explore whether gain of the mutant BRAF allele and/or loss of the wild-type allele is a predictive marker of BRAFi sensitivity.

Plasma DNA-Based Molecular Diagnosis, Prognostication, and Monitoring of Patients With EWSR1 Fusion-Positive Sarcomas

PurposenEwing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized by EWSR1 gene fusions. As pathognomonic genomic events in these respective tumor types, EWSR1 fusions represent robust potential biomarkers for disease monitoring.nnnPatients and MethodsnTo investigate the feasibility of identifying EWSR1 fusions in plasma derived cell-free DNA (cfDNA) from ES and DSRCT patients, we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease. The first approach involved identification of patient-specific genomic EWSR1 fusion breakpoints in formalin-fixed, paraffin-embedded tumor DNA using a broad, hybridization capture-based next generation sequencing (NGS) panel, followed by design of patient-specific droplet digital PCR (ddPCR) assays for plasma cfDNA interrogation . The second approach employed a disease-tailored targeted hybridization capture-based NGS panel applied directly to cfDNA which included EWSR1 as well as several other genes with potential prognostic utility.nnnResultsnEWSR1 fusions were identified in 11/11 (100%) ES and 5/6 (83%) DSRCT samples by ddPCR, while 10/11 (91%) and 4/6 (67%) were identified by NGS. The ddPCR approach had higher sensitivity, ranging between 0.009-0.018% sensitivity. However, the hybrid capture-based NGS assay identified the precise fusion breakpoints in the majority of cfDNA samples, as well as mutations in TP53 and STAG2, two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor sequencing results.nnnConclusionnThese results provide a compelling rationale for an integrated approach utilizing both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

Real-Time Genomic Characterization of Metastatic Pancreatic Neuroendocrine Tumors Has Prognostic Implications and Identifies Potential Germline Actionability

PurposenWe assessed the usefulness of real-time molecular profiling through next-generation sequencing (NGS) in predicting the tumor biology of advanced pancreatic neuroendocrine tumors (panNETs) and in characterizing genomic evolution.nnnMethodsnPatients with metastatic panNETs were recruited in the routine clinical practice setting (between May 2014 and March 2017) for prospective NGS of their tumors as well as for germline analysis using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing platform. When possible, NGS was performed at multiple time points.nnnResultsnNGS was performed in 96 tumor samples from 80 patients. Somatic alterations were identified in 76 of 80 patients (95%). The most commonly altered genes were MEN1 (56%), DAXX (40%), ATRX (25%), and TSC2 (25%). Alterations could be defined in pathways that included chromatin remodeling factors, histone methyltransferases, and mammalian target of rapamycin pathway genes. Somatic loss of heterozygosity was particularly prevalent (50 of 96 samples [52%]), and the presence of loss of heterozygosity resulted in inferior overall survival (P < .01). Sequencing of pre- and post-treatment samples revealed tumor-grade progression; clonal evolution patterns were also seen (molecular resistance mechanisms and chemotherapy-associated mutagenesis). Germline genetic analysis identified clinically actionable pathogenic or likely pathogenic variants in 14 of 88 patients (16%), including mutations in high-penetrance cancer susceptibility genes (MEN1, TSC2, and VHL).nnnConclusionnA clinical NGS platform reveals pertubations of biologic pathways in metastatic panNETs that may inform prognosis and direct therapies. Repeat sequencing at disease progression reveals increasing tumor grade and genetic evolution, demonstrating that panNETs adopt a more aggressive behavior through time and therapies. In addition to frequent somatic mutations in MEN1 and TSC2, germline mutations in these same genes underlie susceptibility to panNETs and highlight the need to re-evaluate whether germline genetic analysis should be performed for all patients with panNETs.

Abstract CN02-03: Targeted multiplexed DNA sequencing for biomarker discovery and clinical diagnosis.

Massively parallel sequencing technology has catalyzed the discovery of genetic alterations that drive tumor initiation and progression. Efforts to comprehensively characterize the genomes of the most common tumor types have revealed recurrent alterations, many of which may be suppressed by targeted therapies. Moreover, through retrospective analysis of clinically annotated tumor specimens, one can identify genomic biomarkers that correlate with outcomes and therapeutic response. Profiling multiple tumors from individual patients at different sites or time points can reveal factors that influence clonal evolution and the onset of drug resistance. Finally, the prospective analysis of tumors in clinical laboratories can meaningfully influence the routine diagnosis and treatment of cancer patients. This promises to improve treatment decisions and to pre-identify patients eligible for future clinical trials of targeted therapies. Clinical labs have traditionally relied upon low-throughput capillary DNA sequencing and multiplexed base-pair genotyping (e.g. Sequenom). Massively parallel sequencing offers several key advantages over conventional mutation profiling strategies: 1) many more genes can be interrogated simultaneously; 2) entire exons can be sequenced, rather than pre-specified mutational hotspots, enabling complete characterization of tumor suppressor genes where mutations tend to be scattered; 3) structural aberrations such as gene amplification, deletions, and rearrangements can be simultaneously monitored; and 4) mutations at low allelic frequencies in heterogeneous tumors are more readily detected with high sensitivity. For increasingly lower costs, one can interrogate all clinically relevant genes for mutations, copy number alterations, and structural rearrangements in formalin-fixed paraffin embedded tumor tissue. We have developed a multiplexed massively parallel sequencing assay utilizing solution-phase exon capture of >300 cancer associated genes, which we have deployed both retrospectively and prospectively to profile more than 1,000 patients in a research setting. I will describe examples in which our group has identified genomic biomarkers predictive of drug response and resistance in a variety of tumor types. Additionally, I will discuss our progress in adapting this platform for clinical sequencing, compliant with federal and New York state regulations. Finally, I will describe general challenges in the establishment of clinical sequencing workflows involving bioinformatics, scalability, clinical interpretation, reporting, regulatory compliance, reimbursement, and ethics.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):CN02-03.nnCitation Format: Michael F. Berger. Targeted multiplexed DNA sequencing for biomarker discovery and clinical diagnosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN02-03.

Abstract 392: Delineating genomic heterogeneity in paired primary and metastatic colorectal cancer by massively parallel sequencing.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnColorectal cancer (CRC) is the third most common cause of cancer and cancer death for men and women in the United States. While the genetic steps necessary for carcinogenesis are well-defined, the subsequent genetic alterations driving tumor evolution to metastasis are not well characterized. We have performed deep sequencing of 230 key cancer-associated genes in 60 patient-matched primary tumor, metastatic tumor, and normal samples from CRC patients. Genes were selected on the basis of recurrent and/or “actionable” mutations reported in human tumors. Our custom captured-based sequencing assay, termed IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), provided a median sequence coverage of 644-fold, allowing for the identification of low frequency genetic events involving target genes. Somatic mutations, insertions/deletions (indels), and copy number alterations were identified and compared in both primary and metastatic samples. The most frequently mutated genes have all been previously implicated in CRC: APC, TP53, KRAS, PIK3CA, and SMAD4. Most genetic events were identical between primary and metastatic tumors; however, we identified particular alterations specific to the metastatic samples. The majority of these alterations were members of two important proliferative pathways, further lending credence that they might play an important role in metastatic progression. For patients with metastasis-specific mutations, we have sampled multiple microdissected regions from primary tumors to quantify the extent of intratumor heterogeneity and ascertain whether these differences are caused by sampling biases. The identification of these genetic variations between primary and metastatic tumors could have significant impact on biomarker analysis as well as treatment decisions for CRC patients.nnCitation Format: Angela Rose Brannon, Efsevia Vakiani, Sasinya N. Scott, Brooke Sylvester, Krishan Kania, Agnes Viale, Nancy Kemeny, Martin Weiser, David B. Solit, Michael F. Berger. Delineating genomic heterogeneity in paired primary and metastatic colorectal cancer by massively parallel sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 392. doi:10.1158/1538-7445.AM2013-392

Abstract 965: Massively parallel sequencing of cancer FFPE specimens matches diagnostic accuracy of methods in current clinical use and reveals additional actionable mutations

As genomic data accumulate at an ever-increasing rate, it is becoming evident that a comprehensive description of molecular aberrations that define individual patients’ tumors will prove useful to determine optimal therapeutic strategies. Several platforms are currently in use for clinical molecular diagnostics testing, including PCR, Sanger sequencing, restriction fragment length polymorphism analysis, and mass spectrometric genotyping (Sequenom). Massively parallel sequencing (MPS) technology has the potential to expand upon Sequenom genotyping because it allows for the identification of mutations across entire exons (rather than single base pairs) as well as copy gains, losses and gene fusions. However, for MPS to be considered a viable clinical strategy, it must first be shown to be compatible with formalin-fixed, paraffin embedded (FFPE) tissue across a range of tumor types, sizes, and cellularities. Also, it must exhibit high concordance with mutation profiles derived using the current best diagnostic methods available. To explore the clinical utility of MPS, we selected 71 surgically resected FFPE tumors that had previously been tested for approximately 100 oncogenic mutations in 8 oncogenes by Sequenom genotyping and subjected them to targeted DNA sequencing of 189 cancer-related genes. DNA was extracted from four 10-micron unstained sections from the diagnostic FFPE block (yielding a minimum of 250 nanograms per case), followed by sequencing library construction and hybridization-based capture of 3230 exons and 37 intronic intervals. Deep sequencing was performed, yielding an average coverage of >750X for uniquely-mapping reads. Sequence data were analyzed for single nucleotide variants and small insertions and deletions. High concordance was noted between Sequenom and MPS: 62 and 65 mutations were called by the two technologies, respectively, at mutually tested sites, with 60 mutation calls in common. Notably, mutant allele frequencies in these concordant calls ranged as low as 4% by MPS, highlighting the sensitivity of detection enabled by both approaches. The few discordant mutation calls exhibited no or weak evidence in the other dataset, possibly due to local tumor heterogeneity. Further, MPS revealed 73 sequence variants at additional sites of known recurrent somatic mutations and 30 loss-of-function variants in key tumor suppressor genes not tested by Sequenom. Many of these variants represent plausibly actionable mutations that could influence treatment decisions. Thus, we conclude that: (1) MPS exhibits high concordance with current best methods and is a viable strategy for clinical diagnostics, and (2) MPS captures additional variants not typically interrogated in the current clinical setting but with potential implications for the selection of approved and/or experimental targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 965. doi:1538-7445.AM2012-965