Siraj M. Ali

Activation of MET via Diverse Exon 14 Splicing Alterations Occurs in Multiple Tumor Types and Confers Clinical Sensitivity to MET Inhibitors

UNLABELLED Focal amplification and activating point mutation of the MET gene are well-characterized oncogenic drivers that confer susceptibility to targeted MET inhibitors. Recurrent somatic splice site alterations at MET exon 14 (METex14) that result in exon skipping and MET activation have been characterized, but their full diversity and prevalence across tumor types are unknown. Here, we report analysis of tumor genomic profiles from 38,028 patients to identify 221 cases with METex14 mutations (0.6%), including 126 distinct sequence variants. METex14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%). Further in vitro studies demonstrate sensitivity to MET inhibitors in cells harboring METex14 alterations. We also report three new patient cases with METex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of METex14 mutations indicates that diagnostic testing via comprehensive genomic profiling is necessary for detection in a clinical setting. SIGNIFICANCE Here we report the identification of diverse exon 14 splice site alterations in MET that result in constitutive activity of this receptor and oncogenic transformation in vitro. Patients whose tumors harbored these alterations derived meaningful clinical benefit from MET inhibitors. Collectively, these data support the role of METex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefit from MET inhibitors.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

UNLABELLED Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. SIGNIFICANCE We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

New Routes to Targeted Therapy of Intrahepatic Cholangiocarcinomas Revealed by Next-Generation Sequencing

BACKGROUND Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer that is rarely curable by surgery and is rapidly increasing in incidence. Relapsed ICC has a poor prognosis, and current systemic nontargeted therapies are commonly extrapolated from those used in other gastrointestinal malignancies. We hypothesized that genomic profiling of clinical ICC samples would identify genomic alterations that are linked to targeted therapies and that could facilitate a personalized approach to therapy. METHODS DNA sequencing of hybridization-captured libraries was performed for 3,320 exons of 182 cancer-related genes and 36 introns of 14 genes frequently rearranged in cancer. Sample DNA was isolated from 40 μm of 28 formalin-fixed paraffin-embedded ICC specimens and sequenced to high coverage. RESULTS The most commonly observed alterations were within ARID1A (36%), IDH1/2 (36%), and TP53 (36%) as well as amplification of MCL1 (21%). Twenty cases (71%) harbored at least one potentially actionable alteration, including FGFR2 (14%), KRAS (11%), PTEN (11%), CDKN2A (7%), CDK6 (7%), ERBB3 (7%), MET (7%), NRAS (7%), BRCA1 (4%), BRCA2 (4%), NF1 (4%), PIK3CA (4%), PTCH1 (4%), and TSC1 (4%). Four (14%) of the ICC cases featured novel gene fusions involving the tyrosine kinases FGFR2 and NTRK1 (FGFR2-KIAA1598, FGFR2-BICC1, FGFR2-TACC3, and RABGAP1L-NTRK1). CONCLUSION Two thirds of patients in this study harbored genomic alterations that are associated with targeted therapies and that have the potential to personalize therapy selection for to individual patients.

Next-Generation Sequencing Reveals a Novel NSCLC ALK F1174V Mutation and Confirms ALK G1202R Mutation Confers High-Level Resistance to Alectinib (CH5424802/ RO5424802) in ALK -Rearranged NSCLC Patients Who Progressed on Crizotinib

Acquired secondary mutations in the anaplastic lymphoma kinase (ALK) gene have been identified in ALK-rearranged (ALK+) non-small-cell lung cancer (NSCLC) patients who developed disease progression while on crizotinib treatment. Here, we identified a novel secondary acquired NSCLC ALK F1174V mutation by comprehensive next-generation sequencing in one ALK+ NSCLC patient who progressed on crizotinib after a prolonged partial response to crizotinib. In a second case, we identified a secondary acquired ALK G1202R, which also confers resistance to alectinib (CH5424802/RO5424802), a second-generation ALK inhibitor that can inhibit ALK gatekeeper L1196M mutation in vitro. ALK G1202R is located at the solvent front of the ALK kinase domain and exhibits a high level of resistance to all other ALK inhibitors currently in clinical development in vitro. Comprehensive genomic profiling of resistant tumor is increasingly important in tailoring treatment decisions after disease progression on crizotinib in ALK+ NSCLC given the promise of second-generation ALK inhibitors and other therapeutic strategies.

Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

UNLABELLED Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (non-LCH), respectively. The discovery of BRAF(V600E) mutations in approximately 50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of patients with BRAF(V600E)-wild-type non-LCH are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole-exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in patients with BRAF(V600E)-wild-type non-LCH. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of patients with MAP2K1- and ARAF-mutated non-LCH using MEK and RAF inhibitors, respectively, resulted in clinical efficacy, demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders. SIGNIFICANCE We provide the first description of kinase fusions in systemic histiocytic neoplasms and activating ARAF and MAP2K1 mutations in non-Langerhans histiocytic neoplasms. Refractory patients with MAP2K1- and ARAF-mutant histiocytoses had clinical responses to MEK inhibition and sorafenib, respectively, highlighting the importance of comprehensive genomic analysis of these disorders.

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0–10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0–7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

Targeted Next Generation Sequencing Identifies Markers of Response to PD-1 Blockade

Mutational load, by whole exome sequencing, can correlate with immunotherapy responses. Assessing melanoma mutational load of a fraction of the genome, by hybrid capture-based NGS, provided an accurate surrogate for WES determinations, and predicted response to anti-PD-1. Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next-generation sequencing (NGS) panel available in the clinic was correlated with response to anti–PD-1 in melanoma. Using archival melanoma samples from anti–PD-1/PD-L1-treated patients, we performed hybrid capture–based NGS on 236–315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti–PD-1/PD-L1 had higher mutational loads in an initial cohort (median, 45.6 vs. 3.9 mutations/MB; P = 0.003) and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared with nonresponders. Response rate, progression-free survival, and overall survival were superior in the high, compared with intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median, 62.7 mutations/MB) and high response rates (74%), whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole-exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti–PD-1/PD-L1. Cancer Immunol Res; 4(11); 959–67. ©2016 AACR.

Fluorescence In Situ Hybridization, Immunohistochemistry, and Next-Generation Sequencing for Detection of EML4-ALK Rearrangement in Lung Cancer

BACKGROUND The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis. MATERIALS AND METHODS We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance. RESULTS Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC. CONCLUSION The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.

Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing

BACKGROUND To determine genomic alterations in head and neck squamous cell carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained through routine clinical practice, selected cancer-related genes were evaluated and compared with alterations seen in frozen tumors obtained through research studies. PATIENTS AND METHODS DNA samples obtained from 252 FFPE HNSCC were analyzed using next-generation sequencing-based (NGS) clinical assay to determine sequence and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV in situ hybridization (ISH) and p16 immunohistochemical (IHC) staining in a subset of tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and University of Chicago public datasets were analyzed for comparison. RESULTS Among 252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete concordance with NGS-derived HPV status. The most common genes with genomic alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were frequently altered. The HPV-positive oropharynx and HPV-positive nasal cavity/paranasal sinus carcinoma shared similar mutational profiles. CONCLUSION The genomic profile of FFPE HNSCC tumors obtained through routine clinical practice is comparable with frozen tumors studied in research setting, demonstrating the feasibility of comprehensive genomic profiling in a clinical setting. However, the clinical significance of these genomic alterations requires further investigation through application of these genomic profiles as integral biomarkers in clinical trials.BACKGROUND To determine genomic alterations in head and neck squamous cell carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained through routine clinical practice, selected cancer-related genes were evaluated and compared with alterations seen in frozen tumors obtained through research studies. PATIENTS AND METHODS DNA samples obtained from 252 FFPE HNSCC were analyzed using next-generation sequencing-based (NGS) clinical assay to determine sequence and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV in situ hybridization (ISH) and p16 immunohistochemical (IHC) staining in a subset of tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and University of Chicago public datasets were analyzed for comparison. RESULTS Among 252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete concordance with NGS-derived HPV status. The most common genes with genomic alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were frequently altered. The HPV-positive oropharynx and HPV-positive nasal cavity/paranasal sinus carcinoma shared similar mutational profiles. CONCLUSION The genomic profile of FFPE HNSCC tumors obtained through routine clinical practice is comparable with frozen tumors studied in research setting, demonstrating the feasibility of comprehensive genomic profiling in a clinical setting. However, the clinical significance of these genomic alterations requires further investigation through application of these genomic profiles as integral biomarkers in clinical trials.

Relapsed Classic E-Cadherin (CDH1)–Mutated Invasive Lobular Breast Cancer Shows a High Frequency of HER2 (ERBB2) Gene Mutations

Purpose: We queried whether comprehensive genomic profiling using a next-generation sequencing–based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC). Experimental Design: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC. Results: A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1–6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0–3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006). Conclusions: Comprehensive genomic profiling of relapsed CDH1-mutated ILC revealed actionable genomic alterations in 86% of cases, featured a high incidence of ERBB2 alterations, and can reveal actionable alterations that can inform treatment decisions for patients with ILC. Clin Cancer Res; 19(10); 2668–76. ©2013 AACR.