Helen H. Won

Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle

BACKGROUND Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy. PATIENT AND METHODS A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor. RESULTS Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography. CONCLUSIONS This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity. REGISTERED CLINICAL TRIAL: www.clinicaltrials.gov, NCT01090960.BACKGROUND Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy. PATIENT AND METHODS A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor. RESULTS Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography. CONCLUSIONS This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity. REGISTERED CLINICAL TRIAL www.clinicaltrials.gov, NCT01090960.

ESR1 ligand-binding domain mutations in hormone-resistant breast cancer

Seventy percent of breast cancers express estrogen receptor (ER), and most of these are sensitive to ER inhibition. However, many such tumors for unknown reasons become refractory to inhibition of estrogen action in the metastatic setting. We conducted a comprehensive genetic analysis of two independent cohorts of metastatic ER-positive breast tumors and identified mutations in ESR1 affecting the ligand-binding domain (LBD) in 14 of 80 cases. These included highly recurrent mutations encoding p.Tyr537Ser, p.Tyr537Asn and p.Asp538Gly alterations. Molecular dynamics simulations suggest that the structures of the Tyr537Ser and Asp538Gly mutants involve hydrogen bonding of the mutant amino acids with Asp351, thus favoring the agonist conformation of the receptor. Consistent with this model, mutant receptors drive ER-dependent transcription and proliferation in the absence of hormone and reduce the efficacy of ER antagonists. These data implicate LBD-mutant forms of ER in mediating clinical resistance to hormonal therapy and suggest that more potent ER antagonists may be of substantial therapeutic benefit.

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.

Loss of NF1 in Cutaneous Melanoma Is Associated with RAS Activation and MEK Dependence

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition.

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor

Precision medicines exert selective pressure on tumor cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next generation therapies to treat the evolving cancer. The PIK3CA/AKT/mTOR pathway is one of the most commonly activated pathways in human cancers1, which has led to the development of small molecule inhibitors that target various nodes in the pathway. Among these agents, first generation mTOR inhibitors (rapalogs) have caused responses in so-called “N-of-1” cases and second generation mTOR kinase inhibitors (TORKi) are currently in clinical trials2–4. We sought to delineate the likely resistance mechanisms to existing mTOR inhibitors as a guide for next generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active site mutation interfering with drug binding. Indeed, the identical drug resistant mutations have been also identified in drug-naïve patients4, suggesting that tumors with activating mTOR mutations will be intrinsically resistant to second generation mTOR inhibitors. Here, we report the development of a new class of mTOR inhibitors which overcomes resistance to existing first and second generation inhibitors. The third generation mTOR inhibitor exploits the unique juxtaposition of two drug binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cancer: recognition of the syndrome by pathologic features and the utility of detecting aberrant succination by immunohistochemistry.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome is an autosomal dominant disorder in which germline mutations of fumarate hydratase (FH) gene confer an increased risk of cutaneous and uterine leiomyomas and renal cancer. HLRCC-associated renal cancer is highly aggressive and frequently presents as a solitary mass. We reviewed the clinicopathologic features of 9 patients with renal tumors presenting as sporadic cases but who were later proven to have FH germline mutations. Histologically, all tumors showed mixed architectural patterns, with papillary as the dominant pattern in only 3 cases. Besides papillary, tubular, tubulopapillary, solid, and cystic elements, 6 of 9 tumors contained collecting duct carcinoma–like areas with infiltrating tubules, nests, or individual cells surrounded by desmoplastic stroma. Prominent tubulocystic carcinoma–like component and sarcomatoid differentiation were identified. Although all tumors exhibited the proposed hallmark of HLRCC (large eosinophilic nucleolus surrounded by a clear halo), this feature was often not uniformly present throughout the tumor. Prior studies have shown that a high level of fumarate accumulated in HLRCC tumor cells causes aberrant succination of cellular proteins by forming a stable chemical modification, S-(2-succino)-cysteine (2SC), which can be detected by immunohistochemistry. We thus explored the utility of detecting 2SC by immunohistochemistry in the differential diagnosis of HLRCC tumors and other high-grade renal tumors and investigated the correlation between 2SC staining and FH molecular alterations. All confirmed HLRCC tumors demonstrated diffuse and strong nuclear and cytoplasmic 2SC staining, whereas all clear cell (184/184, 100%), most high-grade unclassified (93/97, 96%), and the large majority of “type 2” papillary (35/45, 78%) renal cell carcinoma cases showed no 2SC immunoreactivity. A subset of papillary (22%) and rare unclassified (4%) tumors showed patchy or diffuse cytoplasmic staining without nuclear labeling, unlike the pattern seen with confirmed HLRCC tumors. Sequencing revealed no germline or somatic FH alterations in 14 tumors that either exhibited only cytoplasmic 2SC staining (n=5) or were negative for 2SC (n=9), despite their HLRCC-like morphologic features. Our results emphasize the pivotal role of pathologic examination in the diagnosis of HLRCC patients and indicate immunohistochemical detection of 2SC as a useful ancillary tool in the differentiation of HLRCC renal tumors from other high-grade renal cell carcinomas.

A Phase Ib Study of Alpelisib (BYL719), a PI3Kα-Specific Inhibitor, with Letrozole in ER+/HER2− Metastatic Breast Cancer

Purpose: Alpelisib, a selective oral inhibitor of the class I PI3K catalytic subunit p110α, has shown synergistic antitumor activity with endocrine therapy against ER+/PIK3CA-mutated breast cancer cells. This phase Ib study evaluated alpelisib plus letrozoles safety, tolerability, and preliminary activity in patients with metastatic ER+ breast cancer refractory to endocrine therapy. Experimental Design: Twenty-six patients received letrozole and alpelisib daily. Outcomes were assessed by standard solid-tumor phase I methods. Tumor blocks were collected for DNA extraction and next-generation sequencing. Results: Alpelisibs maximum-tolerated dose (MTD) in combination with letrozole was 300 mg/d. Common drug-related adverse events included hyperglycemia, nausea, fatigue, diarrhea, and rash with dose-limiting toxicity occurring at 350 mg/d of alpelisib. The clinical benefit rate (lack of progression ≥6 months) was 35% (44% in patients with PIK3CA-mutated and 20% in PIK3CA wild-type tumors; 95% CI, 17%–56%), including five objective responses. Of eight patients remaining on treatment ≥12 months, six had tumors with a PIK3CA mutation. Among evaluable tumors, those with FGFR1/2 amplification and KRAS and TP53 mutations did not derive clinical benefit. Overexpression of FGFR1 in ER+/PIK3CA mutant breast cancer cells attenuated the response to alpelisib in vitro. Conclusions: The combination of letrozole and alpelisib was safe, with reversible toxicities. Clinical activity was observed independently of PIK3CA mutation status, although clinical benefit was seen in a higher proportion of patients with PIK3CA-mutated tumors. Phase II and III trials of alpelisib and endocrine therapy in patients with ER+ breast cancer are ongoing. Clin Cancer Res; 23(1); 26–34. ©2016 AACR.

Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine Carcinoma Reveals Small Cell Carcinoma–like and Non–Small Cell Carcinoma–like Subsets

Purpose: Pulmonary large cell neuroendocrine carcinoma (LCNEC) is a highly aggressive neoplasm, whose biologic relationship to small cell lung carcinoma (SCLC) versus non-SCLC (NSCLC) remains unclear, contributing to uncertainty regarding optimal clinical management. To clarify these relationships, we analyzed genomic alterations in LCNEC compared with other major lung carcinoma types. Experimental Design: LCNEC (n = 45) tumor/normal pairs underwent targeted next-generation sequencing of 241 cancer genes by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform and comprehensive histologic, immunohistochemical, and clinical analysis. Genomic data were compared with MSK-IMPACT analysis of other lung carcinoma histologies (n = 242). Results: Commonly altered genes in LCNEC included TP53 (78%), RB1 (38%), STK11 (33%), KEAP1 (31%), and KRAS (22%). Genomic profiles segregated LCNEC into 2 major and 1 minor subsets: SCLC-like (n = 18), characterized by TP53+RB1 co-mutation/loss and other SCLC-type alterations, including MYCL amplification; NSCLC-like (n = 25), characterized by the lack of coaltered TP53+RB1 and nearly universal occurrence of NSCLC-type mutations (STK11, KRAS, and KEAP1); and carcinoid-like (n = 2), characterized by MEN1 mutations and low mutation burden. SCLC-like and NSCLC-like subsets revealed several clinicopathologic differences, including higher proliferative activity in SCLC-like tumors (P < 0.0001) and exclusive adenocarcinoma-type differentiation marker expression in NSCLC-like tumors (P = 0.005). While exhibiting predominant similarity with lung adenocarcinoma, NSCLC-like LCNEC harbored several distinctive genomic alterations, including more frequent mutations in NOTCH family genes (28%), implicated as key regulators of neuroendocrine differentiation. Conclusions: LCNEC is a biologically heterogeneous group of tumors, comprising distinct subsets with genomic signatures of SCLC, NSCLC (predominantly adenocarcinoma), and rarely, highly proliferative carcinoids. Recognition of these subsets may inform the classification and management of LCNEC patients. Clin Cancer Res; 22(14); 3618–29. ©2016 AACR.