A phase Ib study of adavosertib, a selective Wee1 inhibitor, in patients with locally advanced or metastatic solid tumors.

Adavosertib selectively inhibits Wee1, which regulates intra-S and G2/M cell-cycle checkpoints. This study investigated dosing schedules for adavosertib monotherapy, determining the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) in patients with advanced solid tumors.Patients received oral adavosertib qd or bid on a 5/9 schedule (5 days on treatment, 9 days off) in 14-day cycles, or qd on one of two 5/2 schedules (weekly, or for 2 of 3 weeks) in 21-day cycles. Safety, efficacy, and pharmacokinetic analyses were performed.Sixty-two patients (female, 64.5%; median age, 61.5 years; most common primary tumors: lung [24.2%], ovary [21.0%]) received treatment (qd schedules, n = 50; bid schedules, n = 12) for 1.8 months (median). Median time to maximum adavosertib concentration was 2.2-4.1 h; mean half-life was 5-12 h. Adverse events (AEs) caused dose reductions, interruptions and discontinuations in 17 (27.4%), 25 (40.3%) and 4 (6.5%) patients, respectively. Most common grade ≥ 3 AEs were anemia, neutropenia (each n = 9, 14.5%) and diarrhea (n = 8, 12.9%). Seven (11.3%) patients experienced 10 treatment-related serious AEs (pneumonia n = 2 [3.2%], dehydration n = 2 [3.2%], anemia n = 1 [1.6%], febrile neutropenia n = 1 [1.6%], and thrombocytopenia n = 1 [1.6%]). Overall objective response rate was 3.4% (2/58); disease control rate was 48.4% (30/62); median progression-free survival was 2.7 months.MTDs were 125 mg (bid 5/9) and 300 mg (qd 5/9 and 5/2 for 2 of 3 weeks); RP2D was 300 mg (qd 5/2 for 2 of 3 weeks). The safety profile was manageable, acceptable, and generally concordant with the known safety profile.

An ErbB2 splice variant lacking exon 16 drives lung carcinoma.

Lung cancer causes more deaths annually than any other malignancy. A subset of non-small cell lung cancer (NSCLC) is driven by amplification and overexpression or activating mutation of the receptor tyrosine kinase (RTK) ERBB2 In some contexts, notably breast cancer, alternative splicing of ERBB2 causes skipping of exon 16, leading to the expression of an oncogenic ERBB2 isoform (ERBB2ΔEx16) that forms constitutively active homodimers. However, the broader implications of ERBB2 alternative splicing in human cancers have not been explored. Here, we have used genomic and transcriptomic analysis to identify elevated ERBB2ΔEx16 expression in a subset of NSCLC cases, as well as splicing site mutations facilitating exon 16 skipping and deletions of exon 16 in a subset of these lung tumors and in a number of other carcinomas. Supporting the potential of ERBB2ΔEx16 as a lung cancer driver, its expression transformed immortalized lung epithelial cells while a transgenic model featuring inducible ERBB2ΔEx16 specifically in the lung epithelium rapidly developed lung adenocarcinomas following transgene induction. Collectively, these observations indicate that ERBB2ΔEx16 is a lung cancer oncogene with potential clinical importance for a proportion of patients.

A FISH assay efficiently screens for BRAF gene rearrangements in pancreatic acinar-type neoplasms.

Approximately 1-2% of pancreatic neoplasms are acinar cell carcinomas. Recently, BRAF gene rearrangements were identified in over 20% of acinar-type neoplasms, which included both pure acinar cell carcinomas and mixed carcinomas with acinar differentiation, using next-generation sequencing-based platforms, providing a potential therapeutic target for patients with these neoplasms. Thus, it is clinically important to develop a rapid, cost- and material-efficient assay to screen for BRAF gene fusions in pancreatic acinar-type neoplasms. We developed a dual color, break-apart FISH assay to detect BRAF gene rearrangements in these neoplasms and evaluated its performance in comparison to next-generation sequencing-based studies. A blinded BRAF rearrangement FISH investigation was performed on 31 acinar-type neoplasms that had been studied previously by next-generation sequencing-based analysis as well as on 18 additional acinar-type neoplasms that were accrued over the past 2 years. In total, BRAF fusions were identified in 12/49 (24%) acinar-type neoplasms by FISH. BRAF fusion partners were uncovered by using targeted next-generation sequencing studies in 11 FISH-positive cases that had sufficient material for next-generation sequencing studies. SND1 was the most frequent fusion partner involved in BRAF-fusion acinar-type neoplasms (50%), followed by HERPUD1 (18%). No BRAF fusions were identified by next-generation sequencing in any of the FISH-negative cases investigated. FISH analysis showed that BRAF rearrangements were diffusely present across tumor-rich areas in BRAF-fusion acinar-type neoplasms, which is consistent with an oncogenic driver alteration pattern. Thus, we demonstrated that, in comparison to targeted next-generation sequencing-based technologies, the FISH assay is highly sensitive and specific as well as time- and cost-efficient in the detection of BRAF fusions in acinar-type neoplasms. The FISH assay can be easily implemented in diagnostic settings to identify acinar-type neoplasms patients potentially suitable for targeted therapy to inhibit MAPK pathway activity.

A Novel PRKAR1B-BRAF Fusion in Gastrointestinal Stromal Tumor Guides Adjuvant Treatment Decision-Making During Pregnancy.

Gastrointestinal stromal tumors (GISTs) are rare in pregnancy, with only 11 reported cases. Adjuvant imatinib therapy, which targets the most common driver mutations in GIST (KIT and PDGFRA), is recommended for patients with high-risk GIST, but it has known teratogenicity in the first trimester. A 34-year-old G3P2 woman underwent exploratory laparotomy at 16 weeks' gestation for a presumed adnexal mass. Surgical findings included normal adnexa and a 14-cm solid small bowel mass. The mass was resected en bloc with a segment of jejunum followed by a primary anastomosis. Histopathology and genomic analyses demonstrated a GIST with high-risk features but lack of KIT/PDGFRA mutations and identified the presence of a previously unreported, pathogenic PRKAR1B-BRAF gene fusion. Given her tumor profile, adjuvant therapy with imatinib was not recommended. GIST is rare in pregnancy, but can masquerade as an adnexal mass in women of childbearing age. Because neoadjuvant/adjuvant imatinib has risks of teratogenicity, tumor molecular profiling is critical as we identified a previously unreported gene fusion of PRKAR1B with BRAF that is predicted to be imatinib-resistant. In this case, testing provided the rationale for not offering adjuvant imatinib to avoid unnecessary toxicity to the patient and fetus.

The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.

Although the BRAF V600E base substitution is an approved target for the BRAF inhibitors in melanoma, BRAF gene fusions have not been investigated as anticancer drug targets. In our study, a wide variety of tumors underwent comprehensive genomic profiling for hundreds of known cancer genes using the FoundationOne™ or FoundationOne Heme™ comprehensive genomic profiling assays. BRAF fusions involving the intact in-frame BRAF kinase domain were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types, including 20 novel BRAF fusions. These comprised 29 unique 5' fusion partners, of which 31% (9) were known and 69% (20) were novel. BRAF fusions included 3% (14/531) of melanomas; 2% (15/701) of gliomas; 1.0% (3/294) of thyroid cancers; 0.3% (3/1,062) pancreatic carcinomas; 0.2% (8/4,013) nonsmall-cell lung cancers and 0.2% (4/2,154) of colorectal cancers, and were enriched in pilocytic (30%) vs. nonpilocytic gliomas (1%; p < 0.0001), Spitzoid (75%) vs. nonSpitzoid melanomas (1%; p = 0.0001), acinar (67%) vs. nonacinar pancreatic cancers (<1%; p < 0.0001) and papillary (3%) vs. nonpapillary thyroid cancers (0%; p < 0.03). Clinical responses to trametinib and sorafenib are presented. In conclusion, BRAF fusions are rare driver alterations in a wide variety of malignant neoplasms, but enriched in Spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers.

Nonamplification ERBB2 genomic alterations in 5605 cases of recurrent and metastatic breast cancer: An emerging opportunity for anti-HER2 targeted therapies.

BACKGROUND: Activating, nonamplification ERBB2 mutations (ERBB2mut) are not detected by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), but are detected by DNA sequencing and may predict clinical responses to human epidermal growth factor receptor (HER2)-targeted therapy. The authors queried 5605 advanced/metastatic breast cancers (mBC) to uncover the frequency of ERBB2mut genomic alterations. Clinical responses to anti-HER2 therapeutics were identified. METHODS: DNA was extracted from 40 µm of formalin-fixed paraffin-embedded (FFPE) sections. Comprehensive genomic profiling (CGP) was used to evaluate up to 315 genes (592× mean coverage depth). Results were analyzed for base substitutions, short indels, copy number changes, and selected rearrangements. RESULTS: Of 5605 cases, 698 (12.5%) featured ERBB2 alterations, including 596 (10.6%) ERBB2 amplifications (ERBB2amp) and 138 (2.4%) ERBB2mut; 38 cases (0.7%) had co-occurring ERBB2amp and ERBB2mut. ERBB2mut predominantly affected the kinase (124 cases; 90%) or extracellular (15 cases; 11%) domains. Both primary BC (52 cases; 38%) and metastatic site biopsies (86 cases; 62%) were found to harbor ERBB2mut, which were distributed across carcinoma not otherwise specified (NOS) (69 cases; 50%), invasive ductal carcinoma (IDC) (40 cases; 29%), invasive lobular carcinoma (ILC) (27 cases; 20%), and mucinous mBC (2 cases; 1%). Genes commonly coaltered with ERBB2 were tumor protein 53 (TP53) (49%); phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (42%); cadherin 1, type 1 (CDH1) (37%); MYC (17%); and cyclin D1 protein (CCND1) (16%). CDH1 mutations were enriched in ERBB2mut mBC (P<0.0006) and associated with recurrent mBC. Selected patients with ERBB2mut, without ERBB2amp, who responded to anti-HER2 targeted therapies are presented herein. CONCLUSIONS: Within this large series, 1.8% of cases harbored ERBB2mut, which are undetectable by standard-of-care IHC or FISH tests. Metastatic BC driven by ERBB2mut respond to anti-HER2 targeted therapies, and expanding clinical trials designed to detect ERBB2mut by CGP and optimize targeted treatments are warranted. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2654-2662. © 2016 American Cancer Society.

DNA sequencing of cancer: what have we learned?

DNA sequencing has taught us much about the structure of cancer genomes and enabled the discovery of novel genes that drive and maintain tumorigenesis. With the advent and application of next-generation massively parallel sequencing technologies, one can rapidly generate and analyze data from the cellular "-omes": genomes, exomes, and transcriptomes. This review highlights recent genomic discoveries in signal transduction, metabolism, epigenetic modifications, cell cycle and genome maintenance, RNA processing, and transcription. Additionally, genomic sequencing has revealed the complexity of the cancer genome and has enabled the discovery of functional rearrangements with therapeutic and diagnostic potentials.

Comprehensive Genomic Profiling Facilitates Implementation of the National Comprehensive Cancer Network Guidelines for Lung Cancer Biomarker Testing and Identifies Patients Who May Benefit From Enrollment in Mechanism-Driven Clinical Trials.

BACKGROUND: The National Comprehensive Cancer Network (NCCN) guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for EGFR, BRAF, ERBB2, and MET mutations; ALK, ROS1, and RET rearrangements; and MET amplification. We investigated the feasibility and utility of comprehensive genomic profiling (CGP), a hybrid capture-based next-generation sequencing (NGS) test, in clinical practice. METHODS: CGP was performed to a mean coverage depth of 576× on 6,832 consecutive cases of NSCLC (2012-2015). Genomic alterations (GAs) (point mutations, small indels, copy number changes, and rearrangements) involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, and KRAS were recorded. We also evaluated lung adenocarcinoma (AD) cases without GAs, involving these eight genes. RESULTS: The median age of the patients was 64 years (range: 13-88 years) and 53% were female. Among the patients studied, 4,876 (71%) harbored at least one GA involving EGFR (20%), ALK (4.1%), BRAF (5.7%), ERBB2 (6.0%), MET (5.6%), ROS1 (1.5%), RET (2.4%), or KRAS (32%). In the remaining cohort of lung AD without these known drivers, 273 cancer-related genes were altered in at least 0.1% of cases, including STK11 (21%), NF1 (13%), MYC (9.8%), RICTOR (6.4%), PIK3CA (5.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), NRAS (2.3%), BRCA1 (1.7%), MAP2K1 (1.2%), HRAS (0.7%), NTRK1 (0.7%), and NTRK3 (0.2%). CONCLUSION: CGP is practical and facilitates implementation of the NCCN guidelines for NSCLC by enabling simultaneous detection of GAs involving all seven driver oncogenes and KRAS. Furthermore, without additional tissue use or cost, CGP identifies patients with "pan-negative" lung AD who may benefit from enrollment in mechanism-driven clinical trials. IMPLICATIONS FOR PRACTICE: National Comprehensive Cancer Network guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for several genomic alterations (GAs). The feasibility and utility of comprehensive genomic profiling were studied in NSCLC and in lung adenocarcinoma (AD) without GAs. Of patients with NSCLC, 71% harbored at least one GA to a gene listed in the guidelines or KRAS; 273 cancer-related genes were altered in at least 0.1% of the AD cases. Although logistical and administrative hurdles limit the widespread use of next-generation sequencing, the data confirm the feasibility and potential utility of comprehensive genomic profiling in clinical practice.

Comprehensive Genomic Profiling of Advanced Penile Carcinoma Suggests a High Frequency of Clinically Relevant Genomic Alterations.

BACKGROUND: Advanced penile squamous cell carcinoma (PSCC) is associated with poor survival due to the aggressiveness of the disease and lack of effective systemic therapies. Comprehensive genomic profiling (CGP) was performed to identify clinically relevant genomic alterations (CRGAs). MATERIALS AND METHODS: DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections in patients with advanced PSCC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 692× for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes frequently rearranged in cancer. CRGAs were defined as genomic alterations (GAs) linked to targeted therapies on the market or under evaluation in mechanism-driven clinical trials. RESULTS: Twenty male patients with a median age of 60 years (range, 46-87 years) were assessed. Seventeen (85%) cases were stage IV and three cases (15%) were stage III. CGP revealed 109 GAs (5.45 per tumor), 44 of which were CRGAs (2.2 per tumor). At least one CRGA was detected in 19 (95%) cases, and the most common CRGAs were CDKN2A point mutations and homozygous deletion (40%), NOTCH1 point mutations and rearrangements (25%), PIK3CA point mutations and amplification (25%), EGFR amplification (20%), CCND1 amplification (20%), BRCA2 insertions/deletions (10%), RICTOR amplifications (10%), and FBXW7 point mutations (10%). CONCLUSION: CGP identified CRGAs in patients with advanced PSCC, including EGFR amplification and PIK3CA alterations, which can lead to the rational administration of targeted therapy and subsequent benefit for these patients. IMPLICATIONS FOR PRACTICE: Few treatment options exist for patients with advanced penile squamous cell carcinoma (PSCC). Outcomes are dismal with platinum-based chemotherapy, with median survival estimated at 1 year or less across multiple series. Biological studies of patients with PSCC to date have principally focused on human papillomavirus status, but few studies have elucidated molecular drivers of the disease. To this end, comprehensive genomic profiling was performed in a cohort of 20 patients with advanced PSCC. Findings of frequent mutations in CDKN2A, NOTCH1, PIK3CA, and EGFR (all in excess of 20%) point to potential therapeutic avenues. Trials of targeted therapies directed toward these mutations should be explored.

Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization.

INTRODUCTION: For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. MATERIALS AND METHODS: Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. RESULTS: A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. CONCLUSION: Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. IMPLICATIONS FOR PRACTICE: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.

FGFR1 and NTRK3 actionable alterations in "Wild-Type" gastrointestinal stromal tumors.

BACKGROUND: About 10-15% of adult, and most pediatric, gastrointestinal stromal tumors (GIST) lack mutations in KIT, PDGFRA, SDHx, or RAS pathway components (KRAS, BRAF, NF1). The identification of additional mutated genes in this rare subset of tumors can have important clinical benefit to identify altered biological pathways and select targeted therapies. METHODS: We performed comprehensive genomic profiling (CGP) for coding regions in more than 300 cancer-related genes of 186 GISTs to assess for their somatic alterations. RESULTS: We identified 24 GIST lacking alterations in the canonical KIT/PDGFRA/RAS pathways, including 12 without SDHx alterations. These 24 patients were mostly adults (96%). The tumors had a 46% rate of nodal metastases. These 24 GIST were more commonly mutated at 7 genes: ARID1B, ATR, FGFR1, LTK, SUFU, PARK2 and ZNF217. Two tumors harbored FGFR1 gene fusions (FGFR1-HOOK3, FGFR1-TACC1) and one harbored an ETV6-NTRK3 fusion that responded to TRK inhibition. In an independent sample set, we identified 5 GIST cases lacking alterations in the KIT/PDGFRA/SDHx/RAS pathways, including two additional cases with FGFR1-TACC1 and ETV6-NTRK3 fusions. CONCLUSIONS: Using patient demographics, tumor characteristics, and CGP, we show that GIST lacking alterations in canonical genes occur in younger patients, frequently metastasize to lymph nodes, and most contain deleterious genomic alterations, including gene fusions involving FGFR1 and NTRK3. If confirmed in larger series, routine testing for these translocations may be indicated for this subset of GIST. Moreover, these findings can be used to guide personalized treatments for patients with GIST. Trial registration NCT 02576431. Registered October 12, 2015.

Comprehensive Genomic Profiling of Clinically Advanced Medullary Thyroid Carcinoma.

OBJECTIVE: The aim of this study was to determine the genomic alterations of cancer-related genes in advanced medullary thyroid carcinoma during the course of clinical care. METHODS: Hybrid-capture-based comprehensive genomic profiling was performed on 34 consecutive medullary thyroid carcinoma cases to identify all four classes of genomic alterations, and outcome for an index patient was collected. RESULTS: RET was mutated in 88% (30/34) of cases, with RET M918T being responsible for 70% (21/30) of the RET alterations. The other RET alterations were RET E632_L633del, C634R, C620R, C618G/R/S, V804M, and RET amplification. Two of the four RET wild-type patients harbored mutations in KRAS or HRAS (1/34 each). The next most frequent genomic alterations were amplifications of CCND1, FGF3, and FGF19 and alterations in CDKN2A (3/34 each). One case with a RET M918T mutation developed acquired resistance to progressively dose-escalated vandetanib. When the mTOR inhibitor everolimus was added to continued vandetanib treatment, the patient achieved a second 25% reduction of tumor volume (RECIST 1.1) for 8 months. CONCLUSIONS: Comprehensive genomic profiling identified the full breadth of RET alterations in metastatic medullary thyroid carcinoma and possible cooperating oncogenic driver alterations. This approach may refine the use of targeted therapy for these patients.

Personalized Treatment for a Patient With a BRAF V600E Mutation Using Dabrafenib and a Tumor Treatment Fields Device in a High-Grade Glioma Arising From Ganglioglioma.

BACKGROUND: Gangliogliomas are slow-growing, low-grade central nervous system tumors affecting children and young adults. However, some patients will experience tumor recurrence and/or malignant progression. This article reports on the clinical history, molecular findings, and treatment response in a patient with BRAF V600-mutated high-grade glioma arising from ganglioglioma. METHODS: Hematoxylin-eosin staining and comprehensive genomic profiling via Foundation One were performed on the tumor sample from a male patient undergoing treatment at the Department of Neuro-Oncology at Baylor University Medical Center. RESULTS: The patient was eligible for participation in a clinical trial (ClinicalTrials.gov identifier: NCT00916409) of a tumor treatment fields (TTFields) device, NovoTTF-100A, with concurrent radiation and chemotherapy (CCRT). His disease relapsed 4 months after completion of his CCRT, with MRI showing areas of enhancement. Temozolomide was discontinued and he was offered dabrafenib, an oral selective inhibitor of BRAF V600E, with continued use of NovoTTF. At the time of this report, after 2 years of treatment with dabrafenib and TTFields, the patient shows a durable complete response in all areas with no active lesions or new areas of enhancement. CONCLUSIONS: This report suggests that TTFields delivered in combination with targeted therapy dabrafenib yielded a remarkable clinical and radiologic response in this recurrent high-grade glioma. Targeted therapy matched to genomic alterations combined with TTFields treatment could provide clinical benefit and should be prospectively explored in the near future.

Near universal detection of alterations in CTNNB1 and Wnt pathway regulators in desmoid-type fibromatosis by whole-exome sequencing and genomic analysis.

CTNNB1 mutations or APC abnormalities have been observed in ∼85% of desmoids examined by Sanger sequencing and are associated with Wnt/ß-catenin activation. We sought to identify molecular aberrations in "wild-type" tumors (those without CTNNB1 or APC alteration) and to determine their prognostic relevance. CTNNB1 was examined by Sanger sequencing in 117 desmoids; a mutation was observed in 101 (86%) and 16 were wild type. Wild-type status did not associate with tumor recurrence. Moreover, in unsupervised clustering based on U133A-derived gene expression profiles, wild-type and mutated tumors clustered together. Whole-exome sequencing of eight of the wild-type desmoids revealed that three had a CTNNB1 mutation that had been undetected by Sanger sequencing. The mutation was found in a mean 16% of reads (vs. 37% for mutations identified by Sanger). Of the other five wild-type tumors sequenced, two had APC loss, two had chromosome 6 loss, and one had mutation of BMI1. The finding of low-frequency CTNNB1 mutation or APC loss in wild-type desmoids was validated in the remaining eight wild-type desmoids; directed miSeq identified low-frequency CTNNB1 mutation in four and comparative genomic hybridization identified APC loss in one. These results demonstrate that mutations affecting CTNNB1 or APC occur more frequently in desmoids than previously recognized (111 of 117; 95%), and designation of wild-type genotype is largely determined by sensitivity of detection methods. Even true CTNNB1 wild-type tumors (determined by next-generation sequencing) may have genomic alterations associated with Wnt activation (chromosome 6 loss/BMI1 mutation), supporting Wnt/ß-catenin activation as the common pathway governing desmoid initiation.

Comprehensive Genomic Profiling of Advanced Esophageal Squamous Cell Carcinomas and Esophageal Adenocarcinomas Reveals Similarities and Differences.

BACKGROUND: Esophageal squamous cell carcinomas (ESCCs) and esophageal adenocarcinomas (EACs) account for >95% of esophageal malignancies and represent a major global health burden. ESCC is the dominant histology globally but represents a minority of U.S. cases, with EAC accounting for the majority of U.S. CASES: The patient outcomes for advanced ESCC and EAC are poor, and new therapeutic options are needed. Using a sensitive sequencing assay, we compared the genomic profiles of ESCC and EAC with attention to identification of therapeutically relevant genomic alterations. METHODS: Next-generation sequencing-based comprehensive genomic profiling was performed on hybridization-captured, adaptor ligation-based libraries to a median coverage depth of >650× for all coding exons of 315 cancer-related genes plus selected introns from 28 genes frequently rearranged in cancer. Results from a single sample were evaluated for all classes of genomic alterations (GAs) including point mutations, short insertions and deletions, gene amplifications, homozygous deletions, and fusions/rearrangements. Clinically relevant genomic alterations (CRGAs) were defined as alterations linked to approved drugs and those under evaluation in mechanism-driven clinical trials. RESULTS: There were no significant differences by sex for either tumor type, and the median age for all patients was 63 years. All ESCCs and EACs were at an advanced stage at the time of sequencing. All 71 ESCCs and 231 EACs featured GAs on profiling, with 522 GAs in ESCC (7.4 per sample) and 1,303 GAs in EAC (5.6 per sample). The frequency of clinically relevant GAs in ESCC was 94% (2.6 per sample) and 93% in EAC (2.7 per sample). CRGAs occurring more frequently in EAC included KRAS (23% EAC vs. 6% ESCC) and ERBB2 (23% EAC vs. 3% ESCC). ESCC samples were enriched for CRGA in PIK3CA (24% ESCC vs. 10% EAC), PTEN (11% ESCC vs. 4% EAC), and NOTCH1 (17% ESCC vs. 3% EAC). Other GAs that differed significantly between histologic tumor types included SMAD4 (14% EAC vs. 1% ESCC), RB1 (14% ESCC vs. 2% EAC), SOX2 (18% ESCC vs. 1% EAC), and NFE2L2 (24% ESCC vs. 1% EAC). CONCLUSION: ESCC and EAC share similarly high frequencies of overall and clinically relevant genomic alterations; however, the profiles of genomic alterations in the two diseases differ widely, with KRAS and ERBB2 far more frequently altered in EAC compared with ESCC and with mammalian target of rapamycin (MTOR) pathway genes (PIK3CA and PTEN) and NOTCH1 more frequently altered in ESCC compared with EAC. Comprehensive genomic profiling highlights the promise of identifying clinically relevant genomic alterations in both ESCC and EAC and suggests new avenues for molecularly directed therapies in esophageal cancer.

Oncogenic alterations in ERBB2/HER2 represent potential therapeutic targets across tumors from diverse anatomic sites of origin.

BACKGROUND: Targeted ERBB2/HER2 inhibitors are approved by the U.S. Food and Drug Administration for the treatment of breast, gastric, and esophageal cancers that overexpress or amplify HER2/ERBB2, as measured by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Activating mutations in ERBB2 have also been reported and are predicted to confer sensitivity to these targeted agents. Testing for these mutations is not performed routinely, and FISH and IHC are not applied outside of these approved indications. MATERIALS AND METHODS: We explored the spectrum of activating ERBB2 alterations across a collection of ∼ 7,300 solid tumor specimens that underwent comprehensive genomic profiling using next-generation sequencing. Results were analyzed for base substitutions, insertions and deletions, select rearrangements, and copy number changes. RESULTS: Known oncogenic ERBB2 alterations were identified in tumors derived from 27 tissues, and ERBB2 amplification in breast, gastric, and gastroesophageal cancers accounted for only 30% of these alterations. Activating mutations in ERBB2 were identified in 131 samples (32.5%); amplification was observed in 246 samples (61%). Two samples (0.5%) harbored an ERBB2 rearrangement. Ten samples (2.5%) harbored multiple ERBB2 mutations, yet mutations and amplifications were mutually exclusive in 91% of mutated cases. CONCLUSION: Standard slide-based tests for overexpression or amplification of ERBB2 would fail to detect the majority of activating mutations that occur overwhelmingly in the absence of copy number changes. Compared with current clinical standards, comprehensive genomic profiling of a more diverse set of tumor types may identify ∼ 3.5 times the number of patients who may benefit from ERBB2-targeted therapy.

Prospective comprehensive genomic profiling of advanced gastric carcinoma cases reveals frequent clinically relevant genomic alterations and new routes for targeted therapies.

BACKGROUND: Gastric cancer (GC) is a major global cancer burden and the second most common cause of global cancer-related deaths. The addition of anti-ERBB2 (HER2) targeted therapy to chemotherapy improves survival for ERBB2-amplified advanced GC patients; however, the majority of GC patients do not harbor this alteration and thus cannot benefit from targeted therapy under current practice paradigms. MATERIALS AND METHODS: Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic (90.0%) gastric cancer cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies approved by the U.S. Food and Drug Administration or targeted therapy-based clinical trials. RESULTS: Overall, 78% of GC cases harbored one clinically relevant GA or more, with the most frequent alterations being found in TP53 (50%), ARID1A (24%), KRAS (16%), CDH1 (15%), CDKN2A (14%), CCND1 (9.5%), ERBB2 (8.5%), PIK3CA (8.6%), MLL2 (6.9%), FGFR2 (6.0%), and MET (6.0%). Receptor tyrosine kinase genomic alterations were detected in 20.6% of cases, primarily ERBB2, FGFR2, and MET amplification, with ERBB2 alterations evenly split between amplifications and base substitutions. Rare BRAF mutations (2.6%) were also observed. One MET-amplified GC patient responded for 5 months to crizotinib, a multitargeted ALK/ROS1/MET inhibitor. CONCLUSION: Comprehensive genomic profiling of GC identifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.

Comprehensive genomic profiling of inflammatory breast cancer cases reveals a high frequency of clinically relevant genomic alterations.

Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for standard biomarkers (ER/PR/HER2). The objective of this study was to identify opportunities for benefit from targeted therapy, which are not currently identifiable in the standard workup for advanced breast cancer. Comprehensive genomic profiling on 53 IBC formalin-fixed paraffin-embedded specimens (mean, 800× + coverage) using the hybrid capture-based FoundationOne assay. Academic and community oncology clinics. From a series of 2208 clinical cases of advanced/refractory invasive breast cancers, 53 cases with IBC were identified. The presence of clinically relevant genomic alterations (CRGA) in IBC and responses to targeted therapies. CRGA were defined as genomic alterations (GA) associated with on label targeted therapies and targeted therapies in mechanism-driven clinical trials. For the 44 IBCs with available biomarker data, 19 (39 %) were ER-/PR-/HER2- (triple-negative breast cancer, TNBC). For patients in which the clinical HER2 status was known, 11 (25 %) were HER2+ with complete (100 %) concordance with ERBB2 (HER2) amplification detected by the CGP assay. The 53 sequenced IBC cases harbored a total of 266 GA with an average of 5.0 GA/tumor (range 1-15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 51/53 (96 %) of cases with an average of 2.6 CRGA/case. The most frequently altered genes were TP53 (62 %), MYC (32 %), PIK3CA (28 %), ERBB2 (26 %), FGFR1 (17 %), BRCA2 (15 %), and PTEN (15 %). In the TNBC subset of IBC, 8/19 (42 %) showed MYC amplification (median copy number 8X, range 7-20) as compared to 9/32 (28 %) in non-TNBC IBC (median copy number 7X, range 6-21). Comprehensive genomic profiling uncovered a high frequency of GA in IBC with 96 % of cases harboring at least 1 CRGA. The clinical benefit of selected targeted therapies in individual IBC cases suggests that a further study of CGP in IBC is warranted.

Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1.

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

Longitudinal Circulating Tumor DNA Modeling to Predict Disease Progression in First-Line Mutant Epidermal Growth Factor Receptor Non-Small Cell Lung Cancer.

This exploratory, post hoc analysis aimed to model circulating tumor DNA (ctDNA) dynamics and predict disease progression in patients with treatment-naïve locally advanced/metastatic epidermal growth factor receptor mutation (EGFRm)-positive non-small cell lung cancer, from the FLAURA trial (NCT02296125). Patients were randomized 1:1 and received osimertinib 80 mg once daily (q.d.) or comparator EGFR-TKIs (gefitinib 250 mg q.d. or erlotinib 150 mg q.d.). Plasma was collected at baseline and multiple timepoints until treatment discontinuation. Patients with Response Evaluation Criteria in Solid Tumors (RECIST) imaging data and detectable EGFR mutations (Ex19del/L858R) at baseline and ≥ 3 additional timepoints were evaluable. Joint modeling was conducted to characterize the relationship between longitudinal changes in ctDNA and probability of progression-free survival (PFS). A Bayesian joint model of ctDNA and PFS was developed solving differential equations with the ctDNA dynamics and the PFS time-to-event probability. Of 556 patients, 353 had detectable ctDNA at baseline. Evaluable patients (with available imaging and ≥ 3 additional timepoints, n = 320; ctDNA set) were divided into training (n = 259) and validation (n = 61) sets. In the validation set, the model predicted a median PFS of 17.7 months (95% confidence interval (CI): 11.9-28.3) for osimertinib (n = 23) and 9.1 months (95% CI: 6.3-14.8) for comparator (n = 38), consistent with observed RECIST PFS (16.4 months and 9.7, respectively). The model demonstrates that EGFRm ctDNA dynamics can predict the risk of disease progression in this patient population and could be used to predict RECIST-defined disease progression.