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Purpose: This study assessed the efficacy, safety, and pharmacokinetics of adavosertib in combination with four chemotherapy agents commonly used in patients with primary platinum-resistant ovarian cancer. Patients and Methods: Women with histologically or cytologically confirmed epithelial ovarian, fallopian tube, or peritoneal cancer with measurable disease were enrolled between January 2015 and January 2018 in this open-label, four-arm, multicenter, phase II study. Patients received adavosertib (oral capsules, 2 days on/5 days off or 3 days on/4 days off) in six cohorts from 175 mg once daily to 225 mg twice daily combined with gemcitabine, paclitaxel, carboplatin, or pegylated liposomal doxorubicin. The primary outcome measurement was overall response rate. Results: Three percent of patients (3/94) had confirmed complete response and 29% (27/94) had confirmed partial response. The response rate was highest with carboplatin plus weekly adavosertib, at 66.7%, with 100% disease control rate, and median progression-free survival of 12.0 months. The longest median duration of response was in the paclitaxel cohort (12.0 months). The most common grade ≥3 adverse events across all cohorts were neutropenia [45/94 (47.9%) patients], anemia [31/94 (33.0%)], thrombocytopenia [30/94 (31.9%)], and diarrhea and vomiting [10/94 (10.6%) each]. Conclusions: Adavosertib showed preliminary efficacy when combined with chemotherapy. The most promising treatment combination was adavosertib 225 mg twice daily on days 1–3, 8–10, and 15–17 plus carboplatin every 21 days. However, hematologic toxicity was more frequent than would be expected for carboplatin monotherapy, and the combination requires further study to optimize the dose, schedule, and supportive medications.

PURPOSE In this phase I study (BLOOM), osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was evaluated in patients with leptomeningeal metastases (LMs) from EGFR-mutated (EGFRm) advanced non–small-cell lung cancer (NSCLC) whose disease had progressed on previous EGFR-TKI therapy. PATIENTS AND METHODS Patients with cytologically confirmed LM received osimertinib 160 mg once daily. Objectives were to assess confirmed objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), pharmacokinetics (PK), and safety. Additional efficacy evaluations included changes from baseline in CSF cytology and neurologic examination. Measurable lesions were assessed by investigator according to RECIST version 1.1. LMs were assessed by neuroradiologic blinded central independent review (BICR) according to Response Assessment in Neuro-Oncology LM radiologic criteria and by investigator. RESULTS Forty-one patients were enrolled. LM ORR and DoR by neuroradiologic BICR were 62% (95% CI, 45% to 78%) and 15.2 months (95% CI, 7.5 to 17.5 months), respectively. Overall, ORR by investigator was 41% (95% CI, 26% to 58%), and median DoR was 8.3 months (95% CI, 5.6 to 16.5 months). Median investigator-assessed PFS was 8.6 months (95% CI, 5.4 to 13.7 months) with 78% maturity; median OS was 11.0 months (95% CI, 8.0 to 18.0 months) with 68% maturity. CSF tumor cell clearance was confirmed in 11 (28%; 95% CI, 15% to 44%) of 40 patients. Neurologic function was improved in 12 (57%) of 21 patients with an abnormal assessment at baseline. The adverse event and PK profiles were consistent with previous reports for osimertinib. CONCLUSION Osimertinib showed meaningful therapeutic efficacy in the CNS and a manageable safety profile at 160 mg once daily in patients with EGFRm NSCLC and LM.

Advanced non-small-cell lung cancer (NSCLC) patients with EGFR T790M-positive tumours benefit from osimertinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). Here we show that the size of the EGFR T790M-positive clone impacts response to osimertinib. T790M subclonality, as assessed by a retrospective NGS analysis of 289 baseline plasma ctDNA samples from T790M‐positive advanced NSCLC patients from the AURA3 phase III trial, is associated with shorter progression-free survival (PFS), both in the osimertinib and the chemotherapy-treated patients. Both baseline and longitudinal ctDNA profiling indicate that the T790M subclonal tumours are enriched for PIK3CA alterations, which we demonstrate to confer resistance to osimertinib in vitro that can be partially reversed by PI3K pathway inhibitors. Overall, our results elucidate the impact of tumour heterogeneity on response to osimertinib in advanced stage NSCLC patients and could help define appropriate combination therapies in these patients., A recent clinical trial shows that a proportion of lung cancer patients carrying the EGFR T790M mutation develop resistance to osimertinib. In this study, the authors show that T790M subclonality is associated with worse clinical outcome likely through co-occurring PIK3CA alterations, which can be targeted by PI3K pathway inhibitors.

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.

Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

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.

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.

Genomic profiling is widely predicted to become a standard of care in clinical oncology, but more effective data sharing to accelerate progress in precision medicine will be required. Here, we describe cancer-associated genomic profiles from 18,004 unique adult cancers. The dataset was composed of 162 tumor subtypes including multiple rare and uncommon tumors. Comparison of alteration frequencies to The Cancer Genome Atlas identified some differences and suggested an enrichment of treatment-refractory samples in breast and lung cancer cohorts. To illustrate novelty within the dataset, we surveyed the genomic landscape of rare diseases and identified an increased frequency of NOTCH1 alterations in adenoid cystic carcinomas compared with previous studies. Analysis of tumor suppressor gene patterns revealed disease specificity for certain genes but broad inactivation of others. We identified multiple potentially druggable, novel and known kinase fusions in diseases beyond those in which they are currently recognized. Analysis of variants of unknown significance identified an enrichment of SMAD4 alterations in colon cancer and other rare alterations predicted to have functional impact. Analysis of established, clinically relevant alterations highlighted the spectrum of molecular changes for which testing is currently recommended, as well as opportunities for expansion of indications for use of approved targeted therapies. Overall, this dataset presents a new resource with which to investigate rare alterations and diseases, validate clinical relevance, and identify novel therapeutic targets. Cancer Res; 77(9); 2464–75. ©2017 AACR.

BACKGROUND: Metastasis is the major cause of mortality in breast cancer (BrCa) patients. Our understanding of brain metastasis (BrM) is limited, reflected by a lack of effective treatments. We aimed to (1) determine BrCa gene signature differences between primary tumors and matched BrM and (2) uncover BrM-specific alterations that may be clinically actionable. MATERIALS and METHODS: NanoString expression profiling of 127 genes from 5 major prognostic tests (MammaPrint, EndoPredict, PAM50, OncotypeDX, MGI) was performed on 20 patient-matched primary (10 ER-neg, 10 ER-pos) and metastatic brain tumors. Subtype classification was performed using genefu. Protein changes in ER and HER2 (ERBB2) were confirmed by IHC. BrM-specific ERBB2 gains were corroborated in a publicly available dataset of 18 additional patient-matched cases (dbGAP phs000730.v1.p1). To test whether ERBB2 amplification and base pair mutation is metastasis-site specific, we further analyzed an expanded cohort of 7,884 breast tumors enriched for metastatic samples (52%) including liver (16.7%), lung (4.3%), bone (3.6%), and brain (2.0%) using comprehensive hybrid-capture sequencing of ERBB2. RESULTS: 17/20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17/20 BrM harbored expression changes (< or > 2-fold) in clinically actionable genes including gains of FGFR4 (30%), FLT1 (20%), AURKA (10%) and loss of ESR1 expression (45%). The most recurrently upregulated gene was ERBB2, showing a >2-fold expression increase in 35% of BrM. 3 of 13 (23.3%) cases originally HER2-negative, and thus HER2-therapy naive, in the primary BrCa were IHC-positive (3+) in the paired BrM with an observed metastasis-specific amplification of the ERBB2 locus. In an independent dataset, 2 of 9 (22.2%) HER2-negative BrCa switched to HER2-positive with one BrM acquiring ERBB2 amplification and the other showing metastastic enrichment of the activating V777L ERBB2 mutation. Analysis of a large cohort of breast tumors (n=7,884) showed that across all organs ERBB2 amplification and/or base pair mutation was similar (p=0.18) between primary (13%) and metastatic disease (12%), however, a strong and significant enrichment was seen for BrM (primary 13% vs BrM 24%, p CONCLUSIONS: Taken together, these results demonstrate that the majority (85%) of patient-matched BrM retain the intrinsic subtype of the primary cancer. However, despite this transcriptional similarity, alterations in clinically actionable genes are common, with BrM acquiring ERBB2 amplifications and/or base pair mutations at a frequency of ∼20%, even in HER2-therapy naive tumors. In a large cohort of primary and metastatic breast cancers, there is also a unique enrichment for ERBB2 alterations in BrM. This study provides a strong rationale to molecularly profile metastatic lesions to both better understand biological mechanisms of metastases and to perhaps refine therapeutic decision-making in advanced cancers. Citation Format: Priedigkeit N, Hartmaier RJ, Chen Y, Vareslija D, Basudan A, Thomas R, Leone JP, Lucas PC, Bhargava R, Hamilton RL, Chmielecki J, Davidson NE, Oesterreich S, Brufsky AM, Young L, Lee AV. Breast cancer brain metastases show limited intrinsic subtype switching, yet exhibit acquired ERBB2 amplifications and activating mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD1-05.

Purpose: The genomic landscape of gliomas has been characterized and now contributes to disease classification, yet the relationship between molecular profile and disease progression and treatment response remain poorly understood. Experimental Design: We integrated prospective clinical sequencing of 1,004 primary and recurrent tumors from 923 glioma patients with clinical and treatment phenotypes. Results: Thirteen percent of glioma patients harbored a pathogenic germline variant, including a subset associated with heritable genetic syndromes and variants mediating DNA repair dysfunctions (29% of the total) that were associated with somatic biallelic inactivation and mechanism-specific somatic phenotypes. In astrocytomas, genomic alterations in effectors of cell-cycle progression correlated with aggressive disease independent of IDH mutation status, arose preferentially in enhancing tumors (44% vs. 8%, P < 0.001), were associated with rapid disease progression following tumor recurrence (HR = 2.6, P = 0.02), and likely preceded the acquisition of alkylating therapy-associated somatic hypermutation. Thirty-two percent of patients harbored a potentially therapeutically actionable lesion, of whom 11% received targeted therapies. In BRAF-mutant gliomas, response to agents targeting the RAF/MEK/ERK signaling axis was influenced by the type of mutation, its clonality, and its cellular and genomic context. Conclusions: These data reveal genomic correlates of disease progression and treatment response in diverse types of glioma and highlight the potential utility of incorporating genomic information into the clinical decision-making for patients with glioma.

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.

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 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.

Background: Non-amplification ERBB2 alterations (ERBB2 mut) in advanced/metastatic breast cancer (mBC) are not detected by IHC or FISH, but when detected by DNA sequencing assays can lead to clinical responses to anti-HER2 targeted therapy. We queried a database of more than 43,000 clinical cases to uncover the frequency, type and associated genomic alterations (GA) in mBC driven by ERBB2 mut and highlight clinical responses to small molecule drug and antibody-based anti-HER2 therapeutics. Methods: DNA was extracted from 40 microns of FFPE sections from 5,605 mBC. Comprehensive genomic profiling (CGP) was performed using a hybrid-capture, adaptor ligation based next generation sequencing assay of up to 315 genes to a mean coverage depth of >600X. The results were analyzed for base substitutions, short insertions and deletions, selected rearrangements, and copy number changes. Results: 698 (12.5%) of 5,605 mBC featured ERBB2 alterations. 596 (10.6%) featured ERBB2 amplifications and 137 (2.4%) featured ERBB2mut. 35 (0.6%) of total mBC had both ERBB2amp and ERBB2mut, which accounted for 5.0% of all ERBB2 altered mBC. The 137 ERBB2mut mBC cases had a median age of 61 years (range 29 to 93 years) and were sequenced to a mean depth of 600X. Samples utilized for CGP included 52 (38%) from the patient's primary BC and 85 (62%) from metastatic sites including bone/soft tissue/skin (12%), liver (20%), LN (14%), serous cavities (6%), lung (4%) and miscellaneous sites (6%). 71 (52%) mBC were submitted as carcinoma NOS, 44 (32%) as IDC, 22 (16%) as ILC and 1 (1%) as mucinous mBC. Of the 137 ERBB2mut cases, 8 featured more than 1 ERBB2 mut. There were 124 (85%) ERBB2 kinase domain mutations and 15 (10%) extra-cellular domain ERBB2mut. The most common genes co-altered in ERBB2mut mBC were TP53 (49%), PIK3CA (42%), CDH1 (37%), MYC (17%), and CCND1 (16%). The enrichment of ERBB2mut in CDH1 mut mBR was significant (p=0.0006) and associated with relapsed lobular mBC. Multiple case examples of kinase domain and extra-cellular domain ERBB2mut mBC responding to a variety of anti-HER2 targeted therapies will be presented. Conclusions: In this large series of 5,605 mBC, 20% of the total ERBB2 alterations were non-amplification ERBB2mut not detectable by standard of care IHC and FISH slide-based HER2 tests. Given the demonstration of ERBB2mut driven mBC responsive to anti-HER2 targeted therapies in this study, expansion of clinical trials designed to detect these ERBB2mut cases with CGP and optimize the targeted therapies for these patients is strongly recommended. Citation Format: Ross JS, Wang K, Ali SM, Chumsri S, Elvin JA, Vergilio J-A, Suh J, Yelensky R, Lipson D, Chmielecki J, Miller VA, Stephens PJ. Non-amplification ERBB2 genomic alterations in 5,605 cases of refractory and metastatic breast cancer: An emerging opportunity for anti-HER2 targeted therapies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-05.

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.

The subset of metastatic colorectal adenocarcinomas that harbor BRAF V600E mutations are aggressive tumors with significantly shortened survival and limited treatment options. Here we present a colorectal cancer patient whose disease progressed through standard chemotherapy and who developed liver metastasis. Comprehensive genomic profiling (FoundationOne(®)) identified a BRAF V600E mutation in the liver lesion, as well as other genomic alterations consistent with colorectal cancers. Combination therapy of dabrafenib and trametinib with standard cytotoxic chemotherapy resulted in a durable major ongoing response for the patient. This report illustrates the utility of comprehensive genomic profiling with personalized targeted therapy for aggressive metastatic colorectal adenocarcinomas.

Circulating tumor DNA is released into the bloodstream from tumors and can reflect both intra-tumor heterogeneity and clonal evolution.1 As ctDNA levels are thought to reflect tumor burden, a decrease in ctDNA while on therapy may suggest treatment efficacy.2 We assessed whether longitudinal changes in ctDNA could supplement or improve RECIST-based measures for decision making during drug development. Plasma samples collected at regular intervals were analyzed to predict the risk of disease progression in patients with EGFRm (Ex19del or L858R) advanced NSCLC following initiation of first-line EGFR tyrosine kinase inhibitor (TKI) treatment in the FLAURA study (NCT02296125). Of 556 patients who received treatment (osimertinib 80 mg daily [QD] or comparator EGFR-TKI [gefitinib 250 mg QD or erlotinib 150 mg QD]), 353 (63%) had an available sample with detectable levels of ctDNA at baseline. Patients with both imaging and ctDNA samples at baseline and 3 additional timepoints (n=320, the analysis set) were divided (as implemented in caret R package)3 into training (n=259) and validation (n=61) cohorts. Clinical data from these patients (data cut-off: June 12 2017) were used to fit joint models of either ctDNA dynamics or sum of the longest tumor diameters (SLD) and progression-free survival (PFS). Longitudinal ctDNA levels were measured using droplet digital PCR (ddPCR; Biodesix®) for EGFR-TKI sensitizing mutations (Ex19del or L858R) in all available samples. Model covariates were selected based on their statistical significance for the longitudinal and hazard sub-models and were analyzed using JMbayes package as implemented in R software (3.5.1). Our analysis indicates that the joint model can independently characterize changes in ctDNA and SLD and can predict PFS in the validation cohort, using only ctDNA changes occurring within 6 weeks from the initiation of therapy. Similarly, in the training cohort, joint modelling indicates that the relationship between SLD and PFS using RECIST is consistent with the longitudinal ctDNA analysis. In the analysis set, our model estimated a PFS of 16.6 months (95% confidence interval [CI] 12.1, NC) for osimertinib (n=153) and 9.4 months (95% CI 8.0, 13.5) for comparator EGFR-TKI (n=167). Model-predicted PFS probability using either ctDNA or SLD data was similar, suggesting ctDNA dynamics may provide similar information to RECIST for clinical decision making. Based on this analysis, ctDNA dynamics could be utilized to predict RECIST-defined progression for decision making during drug development of treatments for patients with NSCLC.References: 1. Stewart CM, et al. J Pathol 2018;244(5):616-27 2. Herbreteau G, et al. J Thorac Dis 2019;11(Suppl 1):S113-26 3. R Core Team (2019). https://www.R-project.org/. Accessed Jan 27, 2020 Citation Format: Karthick Vishwanathan, James Dunyak, Philip Overend, Ryan Hartmaier, Aleksandra Markovets, Juliann Chmielecki, Ganesh Mugundu, Carl Barrett, Helen Tomkinson, Martin Johnson. Utility of longitudinal circulating tumor DNA (ctDNA) modeling to predict RECIST-defined progression in first-line patients with epidermal growth factor receptor mutation-positive (EGFRm) advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT024.

9553 Background: Genotyping is required to identify cancer patients (pts) eligible for targeted therapy; however, many do not receive biomarker testing, in part due to limitations associated with tissue-only genotyping practices and the growing list of biomarkers recommended to be tested. Liquid biopsy overcomes many of these limitations but is not yet fully adopted. We report here the clinical performance of a comprehensive liquid biopsy test based on next generation sequencing (NGS) of circulating tumor DNA (ctDNA) for the identification of NSCLC patients with EGFR exon 19 deletions (ex19del) or L858R mutations ( EGFRm) or EGFR T790M, eligible for treatment with osimertinib. Methods: 441 (79%) of 556 pts randomized in FLAURA (NCT02296125; first-line osimertinib vs comparator EGFR TKI in EGFRm NSCLC) and 300 (72%) of 419 pts from AURA3 (NCT012151981; osimertinib vs chemotherapy in NSCLC pts with T790M at progression on EGFR TKI) were retrospectively tested with Guardant360 (G360), a 74-gene ctDNA NGS assay assessing single nucleotide variants, insertion-deletions, amplifications, and fusions in genes relevant to targeted therapy selection as well as microsatellite instability. Progression-free survival (PFS) of pts with EGFRm or T790M detected by G360 was compared to pts detected by the cobas EGFR Mutation Test (cobas) using tissue or plasma with an unadjusted cox model. Results: Treatment with osimertinib was associated with a significant PFS benefit relative to control therapy in NSCLC pts with EGFRm (FLAURA) and T790M (AURA3) detected using G360 (Table). Observed clinical benefit for pts with EGFRm or T790M detected by G360 was similar to that for pts with EGFRm or T790M identified by cobas using tissue or plasma specimens. Conclusions: This analysis demonstrates that G360 accurately identifies pts for osimertinib therapy while simultaneously providing comprehensive genotyping for other therapeutic molecular targets. The application of NGS liquid biopsy has the potential to increase rates of pts genotyped and access to precision medicine. [Table: see text]

Background Tumor cells recapitulate cell-lineage transcriptional programs that are characteristic of normal tissues from which they arise. It is unclear why such lineage programs are fatefully maintained in tumors and if they contribute to cell proliferation and viability. Methods Here, we used the most common brain tumor, meningioma, which is strongly associated with female sex and high body mass index (BMI), as a model system to address these questions. We screened expression profiling data to identify the transcription factor (TF) genes which are highly enriched in meningioma, and characterized the expression pattern of those TFs and downstream genes in clinical meningioma samples as well as normal brain tissues. Meningioma patient-derived cell lines (PDCLs) were used for further validation and characterization. Results We identified 8 TFs highly enriched in meningioma. Expression of these TFs, which included sine oculis homeobox 1 (SIX1), readily distinguished meningiomas from other primary brain tumors and was maintained in PDCLs and even in pulmonary meningothelial nodules. In meningioma PDCLs, SIX1 and its coactivator eyes absent 2 (EYA2) supported the expression of the leptin receptor (LEPR), the cell-surface receptor for leptin (LEP), the adipose-specific hormone that is high in women and in individuals with high BMI. Notably, these transcriptional regulatory factors, LEPR and LEP, both contributed to support meningioma PDCLs proliferation and survival, elucidating a survival dependency on both a core transcriptional program and a metabolic cell-surface receptor. Conclusions These findings provide one rationale for why lineage TF expression is maintained in meningioma and for the epidemiological association of female sex and obesity with meningioma risk.

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.

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, What's new? New results may help target a rare genetic alteration that promotes cancer. Activation of the BRAF gene is already known to spur tumor growth, and usually that activation results from a single amino acid substitution. BRAF‐inhibiting treatments, then, target that mutation. However, in some cases, BRAF gets revved up by a gene fusion. In our study, the authors tested 20,000 tumors and identified 55 BRAF gene fusions in 12 different tumor types. They found the gene fusions occurred more frequently in certain histologic subtypes, information which will help guide treatment strategies for patients with these tumor subtypes.

Background: Targeted ERBB2/HER2 inhibitors are FDA-approved for the treatment of breast and gastric cancers. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to test for overexpression and amplification of ERBB2/HER2, respectively, are performed as part of routine clinical care. Recently, activating mutations in ERBB2 have been reported and may confer sensitivity to targeted agents. Testing for these mutations is not routine, and testing for amplifications is not done outside of approved indications. We explored the complete spectrum of activating ERBB2 mutations and amplifications across a collection of ∼7,300 solid tumor specimens, including a large number of breast cancer specimens, to determine a)how many breast cancer patients could benefit from HER2-targeted therapy due to activating mutations in ERBB2 and b)how widespread ERBB2 alterations are across the solid tumor spectrum. Methods: Extracted DNA from clinical tumor samples underwent comprehensive genomic profiling using FoundationOne . 3769 exons of 236 cancer-related genes and 47 introns from 19 genes that are frequently rearranged in cancer were fully sequenced to high, uniform coverage and results were analyzed for base substitutions, insertions and deletions, amplifications/deletions, and rearrangements. Results: Known oncogenic ERBB2 alterations were identified in approximately 6% of all solid tumors across 27 different histologies. Of all the ERBB2 alterations, activating mutations in ERBB2 were identified in 131 samples and amplifications were observed in 246 samples. Two samples harbored an ERBB2 rearrangement. Ten samples harbored multiple ERBB2 mutations, yet mutations and amplifications were mutually exclusive in 91% of mutated cases. ERBB2 amplification in breast and gastric cancers accounted for only 30% of these alterations. Breast cancer accounted for 37% of all the ERBB2 alterations detected, and included primarily amplifications. 25% of the alterations found in breast cancers were activating base substitutions. Standard tests for overexpression or amplification of ERBB2 would fail to detect these potentially treatable mutations. Non-amplification ERBB2 alterations were enriched in cases of relapsed lobular breast cancer. Multiple breast cancer patients with non-amplification ERBB2 alterations have responded to combinations of anti-HER2 targeted therapies. Conclusions: Comprehensive genomic profiling through FoundationOne identifies 25% more breast cancers that may be susceptible to HER2 targeted therapy due to the presence of activating mutations in ERBB2. Also, many more solid tumor cases could potentially benefit as well from ERBB2 testing. The current policy of testing only breast and gastric tumors for only HER2 amplifications is greatly limiting the potential value of the ERBB2/HER2 inhibitors. Citation Format: Gary A Palmer, Jeffrey S Ross, Kai Wang, Garrett M Frampton, Siraj M Ali, Norma Palma, Deborah Morosini, Vincent A Miller, Roman Yelensky, Doron Lipson, Philip J Stephens, Juliann Chmielecki. Activating mutations in ERBB2/HER2 as found by FoundationOneTM represent potential therapeutic targets in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-03.

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm associated with asbestos exposure. Although previous studies based on candidate gene approaches have identified important common somatic mutations in MPM, these studies have focused on small sets of genes and have provided a limited view of the genetic alterations underlying this disease. Here, we performed whole-exome sequencing on DNA from 22 MPMs and matched blood samples, and identified 517 somatic mutations across 490 mutated genes. Integrative analysis of mutations and somatic copy-number alterations revealed frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1. Our study presents the first unbiased view of the genomic basis of MPM. Cancer Res; 75(2); 264–9. ©2014 AACR.

Pancreatic acinar cell carcinomas (PACC) account for approximately 1% (∼500 cases) of pancreatic cancer diagnoses annually in the United States. Oncogenic therapuetic targets have proven elusive in this disease, and chemotherapy and radiotherapy have demonstrated limited efficacy against these tumors. Comprehensive genomic profiling of a large series of PACCs (n = 44) identified recurrent rearrangements involving BRAF and RAF1 (CRAF) in approximately 23% of tumors. The most prevalent fusion, SND1–BRAF, resulted in activation of the MAPK pathway, which was abrogated with MEK inhibition. SND1–BRAF-transformed cells were sensitive to treatment with the MEK inhibitor trametinib. PACCs lacking RAF rearrangements were significantly enriched for genomic alterations, causing inactivation of DNA repair genes (45%); these genomic alterations have been associated with sensitivity to platinum-based therapies and PARP inhibitors. Collectively, these results identify potentially actionable genomic alterations in the majority of PACCs and provide a rationale for using personalized therapies in this disease. Significance: PACC is genomically distinct from other pancreatic cancers. Fusions in RAF genes and mutually exclusive inactivation of DNA repair genes represent novel potential therapeutic targets that are altered in over two thirds of these tumors. Cancer Discov; 4(12); 1398–1405. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1355

Background: Endocrine therapy (ET) and mTOR inhibition are important treatment strategies in ER-positive breast cancer, but no specific genomic alterations reliably predict benefit. Because tumors are tested pretreatment, we hypothesized that this may not capture tumor interaction with therapy. Methods: We studied tumors from protocol NCT00570921 using fulvestrant and everolimus for metastatic ER-positive breast cancer after aromatase inhibitor (AI) failure. DNA from FFPE tumor tissue was subjected to next-generation genomic profiling using the FoundationOne® assay and alterations were then compared between available paired samples: primary/metastatic, before/after everolimus, and upon progression on everolimus. Results: The most common alterations encountered were in the PI3K/AKT/mTOR pathway with increased frequency of PIK3CA in endocrine-sensitive disease but no specific association with everolimus benefit (Massarweh et al, ASCO 2015). One patient with lobular carcinoma relapse on ET and a new contralateral primary, had PIK3CA, CDH1, and MAP3K1 in both tumors with no new alterations detected. Her disease was everolimus sensitive. Another patient with PIK3CA at baseline acquired a CTNNA1 mutation upon relapse with no everolimus benefit. Interestingly, one patient with liver metastasis and complete response to everolimus lasting 3 years had no known alterations reported in the primary tumor but had a PIK3CA mutation in one of two simultaneous biopsies of separate liver lesions. Another patient with liver metastasis and a GATA3 mutation at baseline had response to everolimus lasting 18 months, then developed a PIK3R2_c.1936A>T mutation on progression reported as a variant of unknown significance (VUS). Another patient with metastatic lobular carcinoma to skin and bone had PIK3CA, CDH1, and ERBB2 mutations at baseline, and acquired KRAS and MCL1 amplification on two sequential skin biopsies in the first month on everolimus. She remained on therapy for 1 year. One patient with locally advanced disease and de novo bone metastasis had TP53 and GATA3 mutations at baseline with resistance to multiple chemotherapy and endocrine treatments. Upon progression on AI, her tumor acquired PDGFRA and SMAD4, detected on day 1 biopsy of everolimus treatment. Repeat biopsy on day 28 revealed loss of PDGFRA and SMAD4 with emergence of PIK3CA and MLL2 mutations and loss of STK11. After 1 year on everolimus her tumor progressed and repeat breast biopsy revealed loss of the PIK3CA, STK11, and MLL2 events, with appearance of AKT1 and NF1 mutations. Interestingly, her tumor also acquired ESR1_c.1607T>G, MTOR_c.6104C>T, and NSD1_c.5938G>A mutations, all classified as VUS but were not previously encountered in her course. Further analysis and biologic relevance of these changes will be presented. Conclusion: This small study suggests that ER-positive breast cancer is a dynamic disease with genomic evolution on endocrine therapy and mTOR inhibition. In some patients, this change occurs early on therapy, possibly through clonal selection, but may also be related to tumor heterogeneity. The significance of this change is not fully understood, but study of early on-treatment biopsies may help us better understand tumor response to therapy. Citation Format: Massarweh S, Romond E, Stewart R, Sun J, Chmielecki J, Mehdi M, Black EP. Evolution of genomic alterations on endocrine therapy and mTOR inhibition in estrogen receptor (ER)-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-06.

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

Enhancements in clinical-grade next-generation sequencing (NGS) have fueled the advancement of precision medicine in the clinical oncology field. Here, we survey the molecular profiles of 1,113 patients with diverse malignancies who successfully underwent clinical-grade NGS (236–404 genes) in an academic tertiary cancer center. Among the individual tumors examined, the majority showed at least one detectable alteration (97.2%). Among 2,045 molecular aberrations was the involvement of 302 distinct genes. The most commonly altered genes were TP53 (47.0%), CDKN2A (18.0%), TERT (17.0%), and KRAS (16.0%), and the majority of patients had tumors that harbored multiple alterations. Tumors displayed a median of four alterations (range, 0–29). Most individuals had at least one potentially actionable alteration (94.7%), with the median number of potentially actionable alterations per patient being 2 (range, 0–13). A total of 1,048 (94.2%) patients exhibited a unique molecular profile, with either genes altered or loci within the gene(s) altered being distinct. Approximately 13% of patients displayed a genomic profile identical to at least one other patient; although genes altered were the same, the affected loci may have differed. Overall, our results underscore the complex heterogeneity of malignancies and argue that customized combination therapies will be essential to optimize cancer treatment regimens. Cancer Res; 77(22); 6313–20. ©2017 AACR.

Background High tumor mutational burden (TMB) is an emerging biomarker of sensitivity to immune checkpoint inhibitors and has been shown to be more significantly associated with response to PD-1 and PD-L1 blockade immunotherapy than PD-1 or PD-L1 expression, as measured by immunohistochemistry (IHC). The distribution of TMB and the subset of patients with high TMB has not been well characterized in the majority of cancer types. Methods In this study, we compare TMB measured by a targeted comprehensive genomic profiling (CGP) assay to TMB measured by exome sequencing and simulate the expected variance in TMB when sequencing less than the whole exome. We then describe the distribution of TMB across a diverse cohort of 100,000 cancer cases and test for association between somatic alterations and TMB in over 100 tumor types. Results We demonstrate that measurements of TMB from comprehensive genomic profiling are strongly reflective of measurements from whole exome sequencing and model that below 0.5 Mb the variance in measurement increases significantly. We find that a subset of patients exhibits high TMB across almost all types of cancer, including many rare tumor types, and characterize the relationship between high TMB and microsatellite instability status. We find that TMB increases significantly with age, showing a 2.4-fold difference between age 10 and age 90 years. Finally, we investigate the molecular basis of TMB and identify genes and mutations associated with TMB level. We identify a cluster of somatic mutations in the promoter of the gene PMS2, which occur in 10% of skin cancers and are highly associated with increased TMB. Conclusions These results show that a CGP assay targeting ~1.1 Mb of coding genome can accurately assess TMB compared with sequencing the whole exome. Using this method, we find that many disease types have a substantial portion of patients with high TMB who might benefit from immunotherapy. Finally, we identify novel, recurrent promoter mutations in PMS2, which may be another example of regulatory mutations contributing to tumorigenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0424-2) contains supplementary material, which is available to authorized users.

Pediatric cancers are generally characterized by low mutational burden and few recurrently mutated genes. Recent studies suggest that genomic alterations may help guide treatment decisions and clinical trial selection. Here, we describe genomic profiles from 1,215 pediatric tumors representing sarcomas, extracranial embryonal tumors, brain tumors, hematologic malignancies, carcinomas, and gonadal tumors. Comparable published datasets identified similar frequencies of clinically relevant alterations, validating this dataset as biologically relevant. We identified novel ALK fusions in a neuroblastoma (BEND5–ALK) and an astrocytoma (PPP1CB–ALK), novel BRAF fusions in an astrocytoma (BCAS1–BRAF) and a ganglioglioma (TMEM106B–BRAF), and a novel PAX3–GLI2 fusion in a rhabdomyosarcoma. Previously characterized ALK, NTRK1, and PAX3 fusions were observed in unexpected malignancies, challenging the "disease-specific" alterations paradigm. Finally, we identified recurrent variants of unknown significance in MLL3 and PRSS1 predicted to have functional impact. Data from these 1,215 tumors are publicly available for discovery and validation. Cancer Res; 77(2); 509–19. ©2017 AACR.

Metastatic triple-negative breast cancer comprises 12%–17% of breast cancers and carries a poor prognosis relative to other breast cancer subtypes. Treatment options in this disease are largely limited to systemic chemotherapy. A majority of clinical studies assessing efficacy of targeted therapeutics (e.g., the mammalian target of rapamycin [mTOR] inhibitor everolimus) in advanced breast cancer patients have not utilized predictive genomic biomarker-based selection and have reported only modest improvement in the clinical outcome relative to standard of care. However, recent reports have highlighted significant clinical responses of breast malignancies harboring alterations in genes involved in the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway to mTOR-inhibitor-involving regimens, underscoring the potential clinical benefit of treating subsets of breast cancer patients with molecularly matched targeted therapies. As the paradigm of cancer treatment shifts from chemotherapeutic regimens to more personalized approaches, the identification of additional reliable biomarkers is essential for identifying patients likely to derive maximum benefit from targeted therapies. Herein, we report a near-complete and ongoing 14-mo response to everolimus therapy of a heavily pretreated patient with biphenotypic, metastatic breast cancer. Genomic profiling of the metastatic triple-negative liver specimen identified a single reportable point mutation, STK11 F354L, that appears to have undergone loss of heterozygosity. No other alterations within the PI3K/mTOR pathway were observed. Published functional biochemical data on this variant are conflicting, and germline data, albeit with unclear zygosity status, are suggestive of a benign polymorphism role. Together with the preclinical data, this case suggests further investigation of this variant is warranted to better understand its role as a potential biomarker for mTOR inhibitor sensitivity in the appropriate clinical context.

AimsSmall cell lung cancer (SCLC) carries a poor prognosis, and the systemic therapies currently used as treatments are only modestly effective, as demonstrated by a low 5-year survival at only ∼5%. In this retrospective collected from March 2013 to study, we performed comprehensive genomic profiling of 98 small cell undifferentiated lung cancer (SCLC) samples to identify potential targets of therapy not currently searched for in routine clinical practice.MethodsDNA from 98 SCLC was sequenced to high, uniform coverage (Illumina HiSeq 2500) and analysed for all classes of genomic alterations.ResultsA total of 386 alterations were identified for an average of 3.9 alterations per tumour (range 1–10). Fifty-two (53%) of cases harboured at least 1 actionable alteration with the potential to personalise therapy including base substitutions, amplifications or homozygous deletions in RICTOR (10%), KIT (7%), PIK3CA (6%), EGFR (5%), PTEN (5%), KRAS (5%), MCL1 (4%), FGFR1 (4%), BRCA2, (4%), TSC1 (3%), NF1 (3%), EPHA3 (3%) and CCND1. The most common non-actionable genomic alterations were alterations in TP53 (86% of SCLC cases), RB1 (54%) and MLL2 (17%).ConclusionsGreater than 50% of the SCLC cases harboured at least one actionable alteration. Given the limited treatment options and poor prognosis of patients with SCLC, comprehensive genomic profiling has the potential to identify new treatment paradigms and meet an unmet clinical need for this disease.

Importance Patients with breast cancer (BrCa) brain metastases (BrM) have limited therapeutic options. A better understanding of molecular alterations acquired in BrM could identify clinically actionable metastatic dependencies. Objective To determine whether there are intrinsic subtype differences between primary tumors and matched BrM and to uncover BrM-acquired alterations that are clinically actionable. Design, Setting, and Participants In total, 20 cases of primary breast cancer tissue and resected BrM (10 estrogen receptor [ER]-negative and 10 ER-positive) from 2 academic institutions were included. Eligible cases in the discovery cohort harbored patient-matched primary breast cancer tissue and resected BrM. Given the rarity of patient-matched samples, no exclusion criteria were enacted. Two validation sequencing cohorts were used—a published data set of 17 patient-matched cases of BrM and a cohort of 7884 BrCa tumors enriched for metastatic samples. Main Outcomes and Measures Brain metastases expression changes in 127 genes within BrCa signatures, PAM50 assignments, and ERBB2 / HER2 DNA-level gains. Results Overall, 17 of 20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17 of 20 BrM harbored expression changes ( 2-fold) in clinically actionable genes including gains of FGFR4 (n = 6 [30%]), FLT1 (n = 4 [20%]), AURKA (n = 2 [10%]) and loss of ESR1 expression (n = 9 [45%]). The most recurrent expression gain was ERBB2 / HER2, which showed a greater than 2-fold expression increase in 7 of 20 BrM (35%). Three of these 7 cases were ERBB2/HER2-negative out of 13 ERBB2/HER2-negative in the primary BrCa cohort and became immunohistochemical positive (3+) in the paired BrM with metastasis-specific amplification of the ERBB2 / HER2 locus. In an independent data set, 2 of 9 (22.2%) ERBB2/HER2-negative BrCa switched to ERBB2/HER2-positive with 1 BrM acquiring ERBB2 / HER2 amplification and the other showing metastatic enrichment of the activating V777L ERBB2 / HER2 mutation. An expanded cohort revealed that ERBB2 / HER2 amplification and/or mutation frequency was unchanged between local disease and metastases across all sites; however, a significant enrichment was appreciated for BrM (13% local vs 24% BrM; P Conclusions and Relevance Breast cancer BrM commonly acquire alterations in clinically actionable genes, with metastasis-acquired ERBB2 / HER2 alterations in approximately 20% of ERBB2/HER2-negative cases. These observations have immediate clinical implications for patients with ERBB2/HER2–negative breast cancer and support comprehensive profiling of metastases to inform clinical care.

Background: Adavosertib is a highly selective inhibitor of Wee1, a crucial regulator of intra-S and G2/M cell cycle checkpoints. This Phase Ib study (NCT02610075) investigated dosing and schedule for adavosertib monotherapy and determined the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) in patients (pts) with advanced solid tumors. Methods: Pts received adavosertib orally once (QD) or twice daily (BID) on a 5/9 schedule (5 days on treatment followed by 9 days off) in 14-day cycles, or one of two 5/2 dosing schedules (5 days on followed by 2 days off; weekly or 2 out of 3 weeks) in 21-day cycles. MTD was determined using a 3+3 dose-escalation cohort design. DLTs were defined as toxicities related to adavosertib, occurring during the first cycle. PK and biomarker analyses were performed on blood samples. Pts’ response to treatment was assessed using RECIST v1.1. Results: 62 pts (female, 64.5%; median age, 61.5 years; most common primary tumor sites: lung [24.2%] and ovary [21.0%]) received treatment (QD schedule, n=50; BID schedule, n=12) (Table 1). Median treatment duration was 1.77 months. Adavosertib was steadily absorbed (median time to max concentration: 2.0-4.15 h) and slowly eliminated (mean half-life: 5-12 h). AEs leading to dose reductions, interruptions and discontinuations were reported by 17 (27.4%), 25 (40.3%) and 4 (6.5%) pts, respectively. The most common grade ≥3 AEs were anemia, neutropenia (both n=9, 14.5%) and diarrhea (n=8, 12.9%). The overall response rate was 3.4% (n=2 [thymoma; anal]), disease control rate 48.4% (n=30) and median progression-free survival 2.7 months. Circulating-tumor-derived DNA pharmacodynamic analyses will be presented. Conclusions: The MTD was determined to be 125 mg (BID 5/9) and 300 mg (QD 5/2 and 5/9) for 2/3 weeks, with 300 mg (QD 5/2) being the RP2D. The safety/tolerability profile was considered acceptable. Adavosertib monotherapy showed preliminary evidence of antitumor activity in a heavily pretreated patient population. Table 1CohortAdavosertib dose (schedule)N evaluableAny AE Grade ≥3, n (%)DLTs, n (%)DLTs:* AE preferred term/CTCAE gradeBID 1125 mg (5/9)64 (66.7)0–BID 2150 mg (5/9)65 (83.3)2 (33.3)Diarrhea/3 and nausea/2 Dehydration/3QD 1.1200 mg (5/9)51 (20.0)0–QD 1.2200 mg (5/2)53 (50.0)0–QD 2.1250 mg (5/9)42 (50.0)0–QD 2.2250 mg (5/2)33 (100.0)0–QD 2.3250 mg (5/2 weekly)95 (50.0)2 (22.2)Thrombocytopenia/3 and neutropenia/3 Thrombocytopenia/2QD 3.1300 mg (5/9)43 (75.0)0–QD 3.2300 mg (5/2)1512 (75.0)2 (13.3)Nausea/2 and weight decreased/1 Pneumonia/3QD 3.3300 mg (5/2 weekly)21 (50.0)2 (100.0)Thrombocytopenia/4 Nausea/2 and vomiting/2Total5939 (62.9)8125/2: doses given on days 1–5 and 8–12 per 21-day cycle; 5/2 weekly schedule: doses given on days 1–5, 8–12 and 15–19 per 21-day cycle; 5/9: doses given on days 1–5 per 14-day cycle. *Some patients experienced >1 DLT. CTCAE, Common Terminology Criteria for Adverse Events; DLT, dose-limiting toxicity Citation Format: Gerald S. Falchook, Jasgit Sachdev, Esteban Rodrigo Imedio, Sanjeev Kumar, Ganesh Mugundu, Juliann Chmielecki, Suzanne Jones, David R. Spigel, Melissa Johnson. A Phase Ib study of Wee1 inhibitor adavosertib in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT022.

Background: Preclinical data suggest that adavosertib (AZD1775), a highly selective Wee1 inhibitor, enhances the antitumor effect of PARP inhibitors such as olaparib. The dose-escalation part of this Phase Ib study (NCT02511795) investigated the safety and tolerability of adavosertib plus olaparib in patients (pts) with refractory solid tumors to determine a maximum tolerated dose (MTD) and recommended Phase II dose (RP2D). Methods: Pts received adavosertib (QD or BID) for 3 consecutive days with 4 days off treatment (3/4), or 5 consecutive days with 2 days off (5/2), plus olaparib (BID) for 14 or 21 days of a 21-day cycle (Table). The MTD was the highest dose at which 7 days, gr 3 thrombocytopenia with gr ≥2 bleeding, non-hematologic gr ≥3 AEs (excluding nausea, vomiting or diarrhea that responds to supportive care), liver function gr ≥3 AEs lasting >48 hours or changes consistent with Hy’s Law, or any other toxicity that disrupted dosing for >7 days. Results: 119 pts were treated (84 female; median age 59; most common primary tumor sites: ovary [21%], breast [16%], lung [12.6%]) (Table). The most common gr ≥3 AEs were anemia (n=28, 23.5%), neutropenia (n=26, 21.8%), and thrombocytopenia (n=20, 16.8%) (all grouped terms). The most common DLTs were thrombocytopenia (n=4) and neutropenia (n=4); two pts experienced both. There were 4 SAEs with an outcome of death, 1 was treatment related. ORR for the total population, cohort 4.2 and cohort 7.4 was 11.1%, 30.8% and 0%, respectively. DCR was 55.7%, 76.9% and 53.8%, respectively. PK and biomarker data will be presented. Conclusions: Treatment with adavosertib plus olaparib showed antitumor activity, mostly at the MTD/RP2D for the BID schedule, which was determined to be adavosertib 175 mg (3/4) for 2/3 weeks plus olaparib 200 mg BID. The RP2D for QD schedule was adavosertib 200 mg (3/4) for 2/3 weeks plus olaparib 200 mg BID. Summary of study cohortsCohortAdavosertib doseOlaparib doseAdavosertib schedule, daysOlaparib schedule, daysPatients, n (evaluable,* n)Patients with a DLT, n (%)Grade ≥3 AEs,† n (%)ORR, n (%)DCR, n (%)1125 mg BID (3/4)100 mg BID1–3/8–101–143 (2)01 (33.3)03 (100)2150 mg BID (3/4)100 mg BID1–3/8–101–144 (4)02 (50)1 (25)2 (50)3.1175 mg BID (3/4)100 mg BID1–3/8–101–144 (2)03 (75)03 (75)3.2150 mg BID (3/4)200 mg BID1–3/8–101–147 (5)04 (57.1)1 (14.3)5 (71.4)4.1175 mg BID (3/4)200 mg BID1–3/8–101–147 (7)04 (57.1)1 (14.3)4 (57.1)4.2175 mg BID (3/4)200 mg BID1–3/8–101–2114 (11)1 (9.1)9 (64.3)4 (30.8)10 (76.9)4.3175 mg BID (3/4)200 mg BID1–3/8–10/15–171–2114 (11)2 (18.2)13 (92.9)1 (7.7)7 (50)5175 mg BID (3/4)300 mg BID1–3/8–101–145 (5)1 (20.0)3 (60.0)2 (50)4 (100)6.1250 mg QD (5/2)200 mg BID1–5/8–121–217 (4)2 (50.0)6 (85.7)1 (20)1 (16.7)6.2200 mg QD (5/2)200 mg BID1–5/8–121–217 (5)2 (40.0)4 (57.1)01 (14.3)7.1250 mg QD (3/4)200 mg BID1–3/8–101–2116 (14)2 (14.3)12 (75)010 (62.5)7.2250 mg QD (3/4)200 mg BID1–3/8–10/15–171–214 (4)1 (25.0)3 (75)1 (25)3 (75)7.3300 mg QD (3/4)200 mg BID1–3/8–101–213 (3)1 (33.3)2 (66.7)007.4200 mg QD (3/4)200 mg BID1–3/8–101–2113 (12)1 (8.3)3 (23.1)07 (53.8)8.1200 mg QD (3/4)300 mg BID1–3/8–101–2111 (9)1 (11.1)4 (36.4)04 (40)*Evaluable patients received >75% of the planned dose of adavosertib and olaparib; †Common Terminology Criteria for Adverse Events. DCR, disease control rate; ORR, objective response rate Citation Format: Erika Hamilton, Gerald S. Falchook, Judy S. Wang, Siqing Fu, Amit Oza, So Karen, Esteban Rodrigo Imedio, Sanjeev Kumar, Lone Ottesen, Ganesh M. Mugundu, Juliann Chmielecki, Suzanne Jones, David R. Spigel, Bob T. Li. Phase Ib study of adavosertib in combination with olaparib in patients with refractory solid tumors: Dose escalation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT025.

Background Adavosertib (AZD1775) is a first-in-class, selective, small-molecule inhibitor of Wee1. Previously we reported safety and efficacy of adavosertib monotherapy in patients (pts) with advanced solid tumors (NCT02482311; Bauer et al Cancer Res 2016;76[14 Suppl]); here we report safety and efficacy data from expansion cohorts based on tumor type. Methods A total of 80 pts grouped into 6 biomarker-matched cohorts (Table) received adavosertib (175 mg PO bid; days 1–3 and 8–10 per 21-day cycle). Eligible pts had: confirmed diagnosis of ovarian cancer (OC), small-cell lung cancer (SCLC) or triple-negative breast cancer (TNBC); prior treatment (Tx) for metastatic/recurrent disease (≥3 prior Tx for pts with BRCAwt OC; progression following PARPi Tx for BRCA1/2m OC pts; ≤1 chemotherapy-based Tx for SCLC and ≥1 chemotherapy-based Tx for TNBC); measurable disease. Primary objective assessments: ORR; DCR; PFS (RECIST v1.1); safety. Tumor assessments were performed every 6 weeks in year 1 and every 12 weeks thereafter until disease progression or intolerable toxicity. Blood and tumor samples were collected for correlative biomarker and pharmacokinetic (PK) analyses. Results Median total Tx duration was 2.4 months. Most frequently reported adverse events (AEs) were diarrhea (61%), nausea (50%) and fatigue (43%). Most commonly reported grade ≥3 AEs were diarrhea (7.5%), nausea, fatigue and small intestine obstruction (6%). AEs leading to dose interruptions (22.5%), reductions (11.3%) or discontinuations (16.3%) were reported. The study showed preliminary antitumor activity, particularly in BRCAwt as well as PARPi-failure BRCAm OC pts (Table). PK and biomarker analyses will be presented. Conclusions Adavosertib was generally well tolerated and showed preliminary antitumor activity. DCR was modest across all patient cohorts. Patient cohortParameterOC, BRCAwtOC, BRCAm, PARPi failureTNBC, CCNE1/MYC/MYCL1/ MYCN non-amplifiedTNBC, CCNE1/MYC/MYCL1/ MYCN amplifiedSCLC, CCNE1/MYC/MYCL1/ MYCN non-amplifiedSCLC, CCNE1/MYC/MYCL1/MYCN amplifiedTotalPatients who received treatment, n163013612380Median age, years (range)62.5 (47–83)59.5 (44–73)58.0 (35–81)54.0 (43–78)64.5 (54–74)63.0 (56–69)60.0(35–83)Prior regimens, n (%) 1 2 3 4 5 6 >6 Median0 0 1 (6) 3 (19) 2 (13) 1 (6) 9 (56) 70 0 2 (7) 3 (10) 7 (23) 8 (27) 10 (33) 60 3 (23) 2 (15) 1 (8) 2 (15) 3 (23) 2 (15) 50 0 1 (17) 2 (33) 2 (33) 1 (17) 0 4.53 (25) 5 (42) 3 (25) 0 1 (8) 0 0 20 1 (33) 2 (67) 0 0 0 0 33 (4) 9 (11) 11 (14) 9 (11) 14 (18) 13 (16) 21 (26) 5ECOG PS, n (%) 0 18 (50) 8 (50)10 (33) 20 (67)4 (31) 9 (69)1 (17) 5 (83)2 (17) 10 (83)1 (33) 2 (67)26 (33)54 (68)ORR, n (%) CR* PR* SD ≥5 weeks PD NE1 (6) 0 1 (6) 10 (63) 4 (25) 1 (6)1 (3) 0 1 (3) 22 (73) 5 (17) 2 (7)0 0 0 9 (69) 3 (23) 1 (8)0 0 0 3 (50) 3 (50) 01 (8) 0 1 (8) 3 (25) 7 (58) 1 (8)0 0 0 1 (33) 2 (67) 03 (4) 0 3 (4) 48 (60) 24 (30) 5 (6)DCR, n (%)†11 (69)23 (77)9 (69)3 (50)4 (33)1 (33)51 (64)Median PFS, months4.53.93.12.01.31.23.0*Confirmed CR/PR; †DCR determined by CR + PR + SD. BRCA1/2m, BRCA1 or BRCA2 mutated; BRCAwt, BRCA1 and BRCA2 wild type; CR, complete response; DCR, disease control rate; ECOG PS, Eastern Cooperative Oncology Group performance status; NE, not evaluable; ORR, objective response rate; PARPi, PARP inhibitor; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease Citation Format: Todd M. Bauer, Kathleen Moore, Janet S. Rader, Fiona Simpkins, Alain Mita, J Thaddeus Beck, Lowell Hart, Quincy Chu, Amit Oza, Anna V. Tinker, Karen So, Esteban Rodrigo Imedio, Sanjeev Kumar, Ganesh M. Mugundu, Suzanne Jenkins, Juliann Chmielecki, Suzanne Jones, David R. Spigel, Siqing Fu. Open-label, multicenter, Phase Ib study to assess safety, tolerability and efficacy of adavosertib monotherapy in patients with advanced solid tumors: Expansion cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT012.

5513 Background: Adavosertib (AZD1775; A), a highly selective WEE1 inhibitor, demonstrated activity and tolerability in combination with carboplatin (C) in primary PROC. This study (NCT02272790) assessed the objective response rate (ORR) and safety of A in PROC. Methods: Pts with recurrent RECIST v1.1 measurable PROC received A with C, gemcitabine (G), weekly paclitaxel (P), or pegylated liposomal doxorubicin (PLD) in 3- (C) or 4-week (G, P, PLD) cycles (Table). Tumor assessments were performed every 2 cycles until disease progression. Primary objective: ORR; other objectives: disease control rate (DCR), progression-free survival (PFS) and safety. Results: In the 94 pts treated (median treatment duration 3 months; range 0–16 months), outcomes were greatest with A (weeks [W]1–3) + C (Table), with ORR of 67% and median PFS (mPFS) of 10.1 months for this cohort. Most common grade ≥3 treatment-emergent adverse events (TEAEs) are shown in the Table, with hematologic toxicity most notable with A (W1–3) + C. TEAEs led to A dose interruptions, reductions and discontinuations in 63%, 30% and 13% of the whole cohort, respectively. A possible positive relationship between CCNE1 amplification and response warrants further investigation. Conclusions: A shows preliminary efficacy when combined with CT. Pts receiving A (W1–3) + C showed greatest benefit. The increased but not unexpected hematologic toxicity is a challenge and could be further studied to optimize the dose schedule and supportive medications. Clinical trial information: NCT02272790. [Table: see text]

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.

Background Squamous cell cancers of the anal canal (ASCC) are increasing in frequency and lack effective therapies for advanced disease. Although an association with human papillomavirus (HPV) has been established, little is known about the molecular characterization of ASCC. A comprehensive genomic analysis of ASCC was undertaken to identify novel genomic alterations (GAs) that will inform therapeutic choices for patients with advanced disease. Patients and methods Hybrid-capture-based next-generation sequencing of exons from 236 cancer-related genes and intronic regions from 19 genes commonly rearranged in cancer was performed on 70 patients with ASCC. HPV status was assessed by aligning tumor sequencing reads to HPV viral genomes. GAs were identified using an established algorithm and correlated with HPV status. Results Sixty-one samples (87%) were HPV-positive. A mean of 3.5 GAs per sample was identified. Recurrent alterations in phosphoinositol-3-kinase pathway (PI3K/AKT/mTOR) genes including amplifications and homozygous deletions were present in 63% of cases. Clinically relevant GAs in genes involved in DNA repair, chromatin remodeling, or receptor tyrosine kinase signaling were observed in 30% of cases. Loss-of-function mutations in TP53 and CDKN2A were significantly enhanced in HPV-negative cases (P Conclusions This is the first comprehensive genomic analysis of ASCC, and the results suggest new therapeutic approaches. Differing genomic profiles between HPV-associated and HPV-negative ASCC warrants further investigation and may require novel therapeutic and preventive strategies.

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.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.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.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.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.

Background: Approximately 70% of newly diagnosed breast cancers express estrogen receptor alpha (ERα), and are treated with agents that block ER signaling. Acquired mutations in ESR1, the gene that encodes ERα, have been associated with resistance to aromatase inhibitor therapy in patients with ER positive metastatic breast cancer (ER+ mBC). The most frequently occurring ESR1 mutations are clustered between amino acids 536 to 538 within the ligand binding domain (LBD), although limited data exists characterizing the full mutation profile in a large number of breast cancer samples. Methods: We surveyed the Foundation Medicine dataset of 7590 primary and metastatic breast cancer tumor samples for ESR1 short variants and copy number alterations. Hormone receptor status was unavailable, therefore two assumptions were made to provide an estimate of prevalence in the ER+ HER2- population: 70% of the tumor samples are from ER+ HER2- patients, and all ESR1 mutations from non-HER2 amplified metastatic sites are from ER+ HER2- patients. In a separate cohort of 48 ER+ mBC patients, circulating tumor DNA (ctDNA) was analyzed for ESR1 mutations using the BEAMing method by Sysmex and with Foundation Medicine's sequencing assay, FoundationACT (Assay for Circulating Tumor DNA). Results: The prevalence of mutations in ER+ HER2- breast cancer was estimated to be 22% in samples from metastatic sites but less than 3% in samples from primary sites. ESR1 amplification was rare in samples from both primary and metastatic disease sites at 1.3% and 2.0% respectively. A total of 153 unique short variants of known and unknown status were identified. In addition to hotspot mutations at 537 and 538, previously undescribed rare mutations were identified throughout the entire length of the LBD, although 10 alterations at amino acids 380, 463, 536, 537, and 538 account for 86% of all ESR1 mutations in the ER+ HER2- metastatic sites. We also characterized the overlap of ESR1 alterations with commonly altered and clinically relevant genes in breast cancer, including PIK3CA mutations and HER2 amplification, and we report here a landscape of co-occurring alterations. In the cohort of patient samples where ctDNA was analyzed, BEAMing and FoundationAct assays both detected ESR1 mutations in 19 out of 48 samples, and overall concordance of mutation status (wild-type vs mutant) was 100%. A total of 51 individual mutations were detected with the BEAMing assay, 42 of which were detected with the FoundationACT assay. Seven mutations that were undetected by FoundationACT had mutant allele frequencies less than 0.1%. Ten ESR1 mutations were detected only by FoundationACT, 9 of which are not covered with the BEAMing assay. Alterations in PIK3CA, CDH1, TP53, ERBB2, and other breast cancer relevant genes were also detected with FoundationACT. Conclusions: Understanding the mutational landscape of ESR1 and co-occurring alterations is important for diagnostic development in conjunction with the clinical development of novel anti-endocrine therapies. Our data demonstrate a large spectrum of mutations in the LBD in addition to known hotspot mutations. In addition, the FoundationACT assay offers a robust NGS-based method to screen for mutations in ctDNA that is highly concordant with digital PCR methods. Citation Format: Spoerke JM, Schleifman E, Clark TA, Young G, Nahas M, Kennedy M, Young L, Chmielecki J, Otto GA, Lipson D, Wilson TR, Gendreau S, Lackner MR. The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-08.

Gene fusions involving the catalytic domain of tyrosine kinases (TKs) are found in a variety of hematological and solid tumor malignancies. Clinically, TK fusions have emerged as prime targets for therapy with small molecule kinase inhibitors. Unfortunately, identification of TK fusions has been hampered by experimental limitations. Here, we developed version 2.0 of a genomically based systematic kinase fusion screen and used it to detect a novel imatinib-sensitive C6orf204-PDGFRB fusion in a patient with precursor T lymphoblastic lymphoma (T-ALL) and an associated myeloproliferative neoplasm with eosinophilia. These data validate the ability of this targeted capture-sequencing approach to detect TK fusion events in small amounts of DNA extracted directly from patient samples.

Purpose: Patients with epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI) after a median of 10 to 16 months. In half of these cases, a second EGFR mutation, T790M, underlies acquired resistance. We undertook this study to examine the clinical course of patients harboring the T790M mutation following progression on TKI. Experimental Design: EGFR-mutant lung cancer patients with acquired resistance to EGFR TKIs were identified as part of a prospective rebiopsy protocol in which postprogression tumor specimens were collected for molecular analysis. Postprogression survival and characteristics of disease progression were compared in patients with and without T790M. Results: We identified T790M in the initial rebiopsy specimens from 58 of 93 patients (62%, 95% CI: 52–72). T790M was more common in biopsies of lung/pleura tissue and lymph nodes than in more distant sites (P = 0.014). Median postprogression survival was 16 months (interquartile range = 9–29 months); patients with T790M had a significantly longer postprogression survival (P = 0.036). Patients without T790M more often progressed in a previously uninvolved organ system (P = 0.014) and exhibited a poorer performance status at time of progression (P = 0.007). Conclusions: Among patients with acquired resistance to EGFR TKIs, the presence of T790M defines a clinical subset with a relatively favorable prognosis and more indolent progression. Knowledge of T790M status is therefore important both for the clinical care of these patients and for the optimal design and interpretation of clinical trials in this setting. Clin Cancer Res; 17(6); 1616–22. ©2010 AACR.

Changes in ctDNA can be used as a surrogate molecular marker of response to determine clinical efficacy and often precede imaging changes. This analysis is valuable to early clinical trials for dose optimization as response data may not be available for patients because of the short period of time on drug. Unlike other ctDNA applications where tumor-specific single gene mutations are followed over time, inclusion of “all comers” populations in phase I trials necessitates a comprehensive assay design optimized to detect multiple ctDNA changes from diverse tumors. Therefore, we developed a highly sensitive bespoke ctDNA panel (Resolution Bioscience) suitable for monitoring applications in “all comers” solid tumor populations. The panel uses next-generation sequencing technology to interrogate mutational hotspots from 8 oncogenes as well as the complete coding sequences of 3 tumor suppressor genes. Validation studies established the mutant allele frequency (MAF) limit of detection for short variants and small insertions/deletions as 0.1%; additionally, mutant alleles detected at or above this frequency can be followed below this threshold in serial samples. The assay requires 2mL plasma and results are reported within 14 days. In silico analysis predicted detection of at least one monitorable somatic alteration from ~63% of solid tumors that shed ctDNA. To demonstrate applicability, we used this assay to analyze serial ctDNA samples from patients enrolled in dose-escalation cohorts for the Wee1 inhibitor, AZD1775 (NCT02610075). Dosing schedules were compared for the degree of ctDNA clearance after completion of the first cycle, and correlated with subsequent RECIST response data, where available. To date, 15 patients were evaluable, with more anticipated as additional cohorts accrue. Greater ctDNA decreases were observed with higher drug dose, and longer drug exposure. Together with safety and exposure data, our studies demonstrate that ctDNA dynamics can be used to inform decisions around optimal dosing schedule. This enables rapid decision making before imaging studies are available, and complements traditional measurements of target engagement. Citation Format: Juliann Chmielecki, Zhongwu Lai, Kristy Potts, Kavita Garg, Mark Li, J Carl Barrett. Use of circulating tumor DNA (ctDNA) to predict superiority of dosing schedules in early clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5582.

Purpose: We sought to identify genomic alterations (GA) in salivary gland adenocarcinomas, not otherwise specified (NOS), salivary duct carcinomas (SDC), carcinoma ex pleomorphic adenoma (ca ex PA), and salivary carcinoma, NOS. Experimental Design: DNA was extracted from 149 tumors. Comprehensive genomic profiling (CGP) was performed on hybridization-captured adaptor ligation-based libraries of 182 or 315 cancer-related genes plus introns from 14 or 28 genes frequently rearranged for cancer and evaluated for all classes of GAs. Results: A total of 590 GAs were found in 157 unique genes (mean 3.9/tumor). GAs in the PI3K/AKT/mTOR pathway were more common in SDC (53.6%) than other histologies (P = 0.019) Cyclin-dependent kinase GAs varied among all histotypes: adenocarcinoma, NOS (34.6%); SDC (12.2%); ca ex PA (16.7%); carcinoma, NOS (31.2%; P = 0.043). RAS GAs were observed: adenocarcinoma, NOS (17.3%); SDC (26.8%); ca ex PA (4.2%); and carcinoma, NOS (9.4%; P = 0.054). ERBB2 GAs, including amplifications and mutations, were common: adenocarcinoma, NOS (13.5%); SDC (26.8%); ca ex PA (29.2%); carcinoma, NOS (18.8; P = 0.249). Other notable GAs include TP53 in >45% of each histotype; NOTCH1: adenocarcinoma, NOS (7.7%), ca ex PA (8.3%), carcinoma, NOS (21.6%); NF1: adenocarcinoma, NOS (9.6%), SDC (17.1%), carcinoma, NOS (18.8%). RET fusions were identified in one adenocarcinoma, NOS (CCDC6-RET) and two SDCs (NCOA4-RET). Clinical responses were observed in patients treated with anti-HER2 and anti-RET–targeted therapies. Conclusions: CGP of salivary adenocarcinoma, NOS, SDCs, ca ex PA, and carcinoma, NOS revealed diverse GAs that may lead to novel treatment options. Clin Cancer Res; 22(24); 6061–8. ©2016 AACR.

Purpose: Reliable detection of drug-sensitive activating EGFR mutations is critical in the care of advanced non–small cell lung cancer (NSCLC), but such testing is commonly performed using a wide variety of platforms, many of which lack rigorous analytic validation. Experimental Design: A large pool of NSCLC cases was assayed with well-validated, hybrid capture–based comprehensive genomic profiling (CGP) at the request of the individual treating physicians in the course of clinical care for the purpose of making therapy decisions. From these, 400 cases harboring EGFR exon 19 deletions (Δex19) were identified, and available clinical history was reviewed. Results: Pathology reports were available for 250 consecutive cases with classical EGFR Δex19 (amino acids 743–754) and were reviewed to assess previous non-hybrid capture–based EGFR testing. Twelve of 71 (17%) cases with EGFR testing results available were negative by previous testing, including 8 of 46 (17%) cases for which the same biopsy was analyzed. Independently, five of six (83%) cases harboring C-helical EGFR Δex19 were previously negative. In a subset of these patients with available clinical outcome information, robust benefit from treatment with EGFR inhibitors was observed. Conclusions: CGP identifies drug-sensitive EGFR Δex19 in NSCLC cases that have undergone prior EGFR testing and returned negative results. Given the proven benefit in progression-free survival conferred by EGFR tyrosine kinase inhibitors in patients with these alterations, CGP should be considered in the initial presentation of advanced NSCLC and when previous testing for EGFR mutations or other driver alterations is negative. Clin Cancer Res; 22(13); 3281–5. ©2016 AACR.

BACKGROUND: Primary CNS tumors, which include both low-grade and high-grade gliomas, are the most common pediatric solid malignancies. We queried whether CGP could uncover a significant frequency of CRGA that could inform treatment decisions and improve clinical outcome for these aggressive tumors. METHODS: DNA was extracted from 75 pediatric glioma formalin-fixed paraffin-embedded clinical specimens. Hybridization captured libraries of 236 (FoundationOne, n = 43) or 405 (FoundationOne Heme, n = 32) genes, plus select introns frequently rearranged in cancer were sequenced to high (>500x), uniform coverage. All classes of genomic alterations including base substitutions, small insertions and deletions, rearrangements, and copy number alterations, were evaluated and reported. CRGA were defined as GA associated with FDA approved therapies or targeted therapies in mechanism-driven clinical trials. RESULTS: The median age of the patients was 9 years (range 0 to 18 years). There were 41 male (57%) and 34 female (43%) patients. The study included glioblastomas (n = 30, 42%), astrocytomas not otherwise specified (NOS) (n = 17, 24%), gliomas NOS (n = 10, 14%), pilocytic astrocytomas and anaplastic astrocytomas (9 each, 13%). 89% of cases harbored at least one CRGA. Pediatric gliomas frequently harbored short variants or copy number alterations in TP53 (39%); BRAF (15%) for which 82% were the V600E base substitution; CDKN2A/B (15%), NF1 (14%), PIK3CA (14%), ATRX (13%) and EGFR (11%). Ten cases (14%) harbored a BRAF fusion, including 44% of pilocytic astrocytomas. BRAF fusions included the known KIAA1549-BRAF and the novel CCDC6-BRAF and BCAS1-BRAF. ATG7-RAF1, ALK-PPCB1 and QK1-RAF1 rearrangements were also identified. Outcomes from cases where targeted therapy was utilized will be presented. CONCLUSIONS: CRGA are frequently identified in pediatric glial tumors of both low-grade and high-grade histology and can be identified by CGP in the course of clinical care. CGP has the potential to identify opportunities for targeted therapies or enrollment in clinical trials for these patients.

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. Implications for Practice: Both esophageal squamous cell carcinoma and esophageal adenocarcinoma are aggressive cancers with poor patient response to conventional chemotherapy and radiation treatment. In this study, comprehensive genomic profiling was performed for 302 advanced esophageal cancers, and it was found that the frequently altered genes and biological pathways differed between the two subtypes. Also, a high frequency of clinically relevant genomic alterations was noted for both types of esophageal cancer as a means of finding a potential targeted therapy to be used in addition to or as an alternative to conventional treatment.

Recurrent, metastatic mesenchymal myxoid tumors of the gynecologic tract present a management challenge as there is minimal evidence to guide systemic therapy. Such tumors also present a diagnostic dilemma, as myxoid features are observed in leiomyosarcomas, inflammatory myofibroblastic tumors (IMT), and mesenchymal myxoid tumors. Comprehensive genomic profiling was performed in the course of clinical care on a case of a recurrent, metastatic myxoid uterine malignancy (initially diagnosed as smooth muscle tumor of uncertain malignant potential (STUMP)), to guide identify targeted therapeutic options. To our knowledge, this case represents the first report of clinical response to targeted therapy in a tumor harboring a DCTN1-ALK fusion protein. Hybridization capture of 315 cancer-related genes plus introns from 28 genes often rearranged or altered in cancer was applied to >50 ng of DNA extracted from this sample and sequenced to high, uniform coverage. Therapy was given in the context of a phase I clinical trial ClinicalTrials.gov Identifier: ( NCT01548144 ). Immunostains showed diffuse positivity for ALK1 expression and comprehensive genomic profiling identified an in frame DCTN1-ALK gene fusion. The diagnosis of STUMP was revised to that of an IMT with myxoid features. The patient was enrolled in a clinical trial and treated with an anaplastic lymphoma kinase (ALK) inhibitor (crizotinib/Xalkori®) and a multikinase VEGF inhibitor (pazopanib/Votrient®). The patient experienced an ongoing partial response (6+ months) by response evaluation criteria in solid tumors (RECIST) 1.1 criteria. For myxoid tumors of the gynecologic tract, comprehensive genomic profiling can identify clinical relevant genomic alterations that both direct treatment targeted therapy and help discriminate between similar diagnostic entities.

Background Collecting duct carcinoma (CDC) is a rare type of renal cell carcinoma (RCC) originating from the renal medulla. Clinical outcomes are poor, and there are no consensus guidelines to guide therapy. Objective To determine genomic alterations (GAs) in a series of patients with locally advanced or metastatic CDC for whom genomic profiling was performed during the course of clinical care. Design, setting, and participants Formalin-fixed, paraffin-embedded blocks or slides were obtained for 17 patients with CDC. DNA was extracted and comprehensive genomic profiling was performed in a laboratory certified under the Clinical Laboratory Improvement Amendments. Outcome measurements and statistical analysis Bayesian algorithms and local alignment algorithms were used to detect substitutions and insertions/deletions, respectively. A comparison to normal control samples was used to detect copy number alterations. Clinically relevant GAs (CRGAs) were defined as those linked to approved or investigational targeted therapies. Results and limitations The median age in the cohort was 53 yr (range 26–73), and 14 primary tumors and three metastatic sites assessed. A total of 36 GAs were detected in this series of patients, with an average of 2.1 GAs per case. The most common GAs were in NF2 (5/17, 29%), SETD2 (4/17, 24%), SMARCB1 (3/17, 18%), and CDKN2A (2/17, 12%). Of nine cases assessed for FH GAs, two patients had FH homozygous loss. A limitation is that targeted interrogation of genes known to be implicated in other cancers was performed, so mutations outside of these cannot be excluded. Conclusions Recurrent CRGAs were detected in this series of CDC cases and suggest a possible benefit from targeted therapy. In particular, mTOR inhibitors may be of interest in patients with NF2 alterations. Alterations in FH and SMARCB1 also occurred in a mutually exclusive manner to NF2 alterations. Patient summary This report provides important genomic insights into collecting duct carcinoma, a rare type of renal cell carcinoma with a very aggressive course. These insights could further rationalize the use of targeted therapies for rare tumors according to the individual genomic alterations harbored.