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Blood-based assays using various technologies and biomarkers are in commercial development for the purpose of detecting multiple cancer types concurrently at an early stage of disease. These multicancer early detection (MCED) assays have the potential to improve the detection of cancers, particularly those for which no current screening modality exists. However, the unknown clinical benefits and harms of using MCED assays for cancer screening necessitate the development and implementation of a randomized controlled trial (RCT) to ascertain their clinical effectiveness. This was the consensus of experts at a National Cancer Institute–hosted workshop to discuss initial design concepts for such a trial. Using these assays to screen simultaneously for multiple cancers poses novel uncertainties for patient care compared with conventional screening tests for single cancers, such as establishing the diagnostic workup to confirm the presence of cancer at any organ site; clarifying appropriate follow-up for a positive assay for which there is no definitive diagnosis; identifying potential harms such as overdiagnosis of indolent disease; determining clinically effective and efficient strategies for disseminating MCED screening in real-world practice; and understanding the ethical implications, such as potentially alleviating or exacerbating existing health disparities. These assays present new and complex challenges for designing an RCT. Issues that emerged from the meeting centered around the need for a flexibly designed, clinical utility RCT to rigorously capture the evidence required to fully understand the net benefit of this promising technology. Specific topic areas were endpoints, screening protocols, recruitment, diagnostic pathway, pilot phase, data elements, specimen collection, and ethical considerations.

Supplementary Figure A6-1 from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates

Supplementary Table 1 from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates

Gene expression analysis has identified biologically relevant subclasses of breast cancer. However, most classification schemes do not robustly cluster all HER2+ breast cancers, in part due to limitations and bias of clustering techniques used. In this article, we propose an alternative approach that first separates the HER2+ tumors using a gene amplification signal for Her2/neu amplicon genes and then applies consensus ensemble clustering separately to the HER2+ and HER2− clusters to look for further substructure. We applied this procedure to a microarray data set of 286 early-stage breast cancers treated only with surgery and radiation and identified two basal and four luminal subtypes in the HER2− tumors, as well as two novel and robust HER2+ subtypes. HER2+ subtypes had median distant metastasis-free survival of 99 months [95% confidence interval (95% CI), 83–118 months] and 33 months (95% CI, 11–54 months), respectively, and recurrence rates of 11% and 58%, respectively. The low recurrence subtype had a strong relative overexpression of lymphocyte-associated genes and was also associated with a prominent lymphocytic infiltration on histologic analysis. These data suggest that early-stage HER2+ cancers associated with lymphocytic infiltration are a biologically distinct subtype with an improved natural history. [Cancer Res 2007;67(22):10669–76]

Supplementary Table 3a from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates

Supplementary Appendix 1 from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates

Supplementary Table 2 from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates

Supplementary Figure A6-2 from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates

Purpose:Preclinical and clinical data suggest that downstream inhibition with an MEK inhibitor, such as binimetinib, might be efficacious for NRAS-mutated cancers.Patients and Methods:Patients enrolled in the NCI-MATCH trial master protocol underwent tumor biopsy and molecular profiling by targeted next-generation sequencing. Patients with NRAS-mutated tumors, except melanoma, were enrolled in subprotocol Z1A, a single-arm study evaluating binimetinib 45 mg twice daily. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS) and overall survival (OS). A post hoc analysis examined the association of NRAS mutation type with outcome.Results:In total, 47 eligible patients with a refractory solid tumor harboring a codon 12, 13, or 61 NRAS mutation were treated. Observed toxicity was moderate, and 30% of patients discontinued treatment because of binimetinib-associated toxicity. The ORR was 2.1% (1/47 patients). A patient with malignant ameloblastoma harboring a codon 61 NRAS mutation achieved a durable partial response (PR). A patient with NRAS codon 61–mutated colorectal cancer had an unconfirmed PR, and two other patients with NRAS codon 61–mutated colorectal had stable disease for at least 12 months. In an exploratory analysis, patients with colorectal cancer bearing a NRAS codon 61 mutation (n = 8) had a significantly longer OS (P = 0.03) and PFS (P = 0.007) than those with codon 12 or 13 mutations (n = 16).Conclusions:Single-agent binimetinib did not show promising efficacy in NRAS-mutated cancers. The observation of increased OS and PFS in patients with codon 61 NRAS-mutated colorectal cancer merits further investigation.

PURPOSE CALGB 40601 assessed whether dual versus single human epidermal growth factor receptor 2 (HER2) –targeting drugs added to neoadjuvant chemotherapy increased pathologic complete response (pCR). Here, we report relapse-free survival (RFS), overall survival (OS), and gene expression signatures that predict pCR and survival. PATIENTS AND METHODS Three hundred five women with untreated stage II and III HER2-positive breast cancer were randomly assigned to receive weekly paclitaxel combined with trastuzumab plus lapatinib (THL), trastuzumab (TH), or lapatinib (TL). The primary end point was pCR, and secondary end points included RFS, OS, and gene expression analyses. mRNA sequencing was performed on 264 pretreatment samples. RESULTS One hundred eighteen patients were randomly allocated to THL, 120 to TH, and 67 to TL. At more than 7 years of follow-up, THL had significantly better RFS and OS than did TH (RFS hazard ratio, 0.32; 95% CI, 0.14 to 0.71; P = .005; OS hazard ratio, 0.34; 95% CI, 0.12 to 0.94; P = .037), with no difference between TH and TL. Of 688 previously described gene expression signatures, significant associations were found in 215 with pCR, 45 with RFS, and only 22 with both pCR and RFS (3.2%). Specifically, eight immune signatures were significantly correlated with a higher pCR rate and better RFS. Among patients with residual disease, the immunoglobulin G signature was an independent, good prognostic factor, whereas the HER2-enriched signature, which was associated with a higher pCR rate, showed a significantly shorter RFS. CONCLUSION In CALGB 40601, dual HER2-targeting resulted in significant RFS and OS benefits. Integration of intrinsic subtype and immune signatures allowed for the prediction of pCR and RFS, both overall and within the residual disease group. These approaches may provide means for rational escalation and de-escalation treatment strategies in HER2-positive breast cancer.

PURPOSE BRAFV600 mutations are commonly found in melanoma and thyroid cancers and to a lesser degree in other tumor types. Subprotocol H (EAY131-H) of the NCI-MATCH platform trial sought to investigate the selective BRAF inhibitor dabrafenib and the MEK1/2 inhibitor trametinib in patients with solid tumors, lymphomas, or multiple myeloma whose tumors harbored a BRAFV600 mutation. PATIENTS AND METHODS EAY131-H is an open-label, single-arm study. Patients with melanoma, thyroid, or colorectal cancer were excluded; patients with non–small-cell lung cancer were later excluded in an amendment. Patients received dabrafenib 150 mg twice per day and trametinib 2 mg per day continuously until disease progression or intolerable toxicity. The primary end point was centrally assessed objective response rate (ORR); secondary end points included progression-free survival (PFS), 6-month PFS, and overall survival. RESULTS Thirty-five patients were enrolled, and 29 were included in the primary efficacy analysis as prespecified in the protocol. Median age was 59 years, and 45% of the patients had received ≥ 3 lines of therapy. The confirmed ORR was 38% (90% CI, 22.9% to 54.9%) with P < .0001 against a null rate of 5%, and PFS was 11.4 months (90% CI, 8.4 to 16.3 months); responses were seen in 7 distinct tumor types. Seven patients had a duration of response of > 12 months, including 4 patients with a duration of response of > 24 months. An additional 8 patients had a PFS > 6 months. The median overall survival was 28.6 months. Reported adverse events were comparable to those noted in previously reported profiles of dabrafenib and trametinib. CONCLUSION This study met its primary end point, with an ORR of 38% ( P < .0001) in this mixed histology, pretreated cohort. This promising activity warrants additional investigations in BRAFV600-mutated tumors outside of currently approved indications.

PURPOSE Therapeutically actionable molecular alterations are widely distributed across cancer types. The National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial was designed to evaluate targeted therapy antitumor activity in underexplored cancer types. Tumor biopsy specimens were analyzed centrally with next-generation sequencing (NGS) in a master screening protocol. Patients with a tumor molecular alteration addressed by a targeted treatment lacking established efficacy in that tumor type were assigned to 1 of 30 treatments in parallel, single-arm, phase II subprotocols. PATIENTS AND METHODS Tumor biopsy specimens from 5,954 patients with refractory malignancies at 1,117 accrual sites were analyzed centrally with NGS and selected immunohistochemistry in a master screening protocol. The treatment-assignment rate to treatment arms was assessed. Molecular alterations in seven tumors profiled in both NCI-MATCH trial and The Cancer Genome Atlas (TCGA) of primary tumors were compared. RESULTS Molecular profiling was successful in 93.0% of specimens. An actionable alteration was found in 37.6%. After applying clinical and molecular exclusion criteria, 17.8% were assigned (26.4% could have been assigned if all subprotocols were available simultaneously). Eleven subprotocols reached their accrual goal as of this report. Actionability rates differed among histologies (eg, > 35% for urothelial cancers and < 6% for pancreatic and small-cell lung cancer). Multiple actionable or resistance-conferring tumor mutations were seen in 11.9% and 71.3% of specimens, respectively. Known resistance mutations to targeted therapies were numerically more frequent in NCI-MATCH than TCGA tumors, but not markedly so. CONCLUSION We demonstrated feasibility of screening large numbers of patients at numerous accruing sites in a complex trial to test investigational therapies for moderately frequent molecular targets. Co-occurring resistance mutations were common and endorse investigation of combination targeted-therapy regimens.

PURPOSE NCI-MATCH is a nationwide, histology-agnostic, signal-finding, molecular profile–driven trial for patients with refractory cancers, lymphomas, or myelomas. Patients with tumors harboring actionable aberration(s) in fibroblast growth factor receptor ( FGFR) 1-3 were treated with AZD4547, an oral FGFR1-3 inhibitor. METHODS Patients’ tumors were screened by next-generation sequencing for predefined FGFR amplification, activating mutations, or fusions. Patients were treated with AZD4547, 80 mg orally twice daily until progression of disease or drug intolerance. A response rate of 16% was considered promising. RESULTS Between July 2016 and June 2017, 70 patients were assigned and 48 received protocol therapy and are eligible for analysis. Patients’ tumors harbored FGFR1 or FGFR2 amplification (n = 20), FGFR2 or FGFR3 single-nucleotide variants (n = 19), or FGFR1 or FGFR3 fusions (n = 9). The most common primary tumors were breast (33.3%), urothelial (12.5%), and cervical cancer (10.4%).Grade 3 adverse events were consistent with those described in previous clinical trials. Confirmed partial responses were seen in 8% (90% CI, 3% to 18%) and were observed only in patients whose tumors harbored FGFR1-3 point mutations or fusions. Stable disease was observed in 37.5% (90% CI, 25.8% to 50.4%). The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90% CI, 8% to 31%). For patients with tumors harboring FGFR fusions, the response rate was 22% (90% CI, 4.1% to 55%), and 6-month PFS rate was 56% (90% CI, 31% to 100%). CONCLUSION Preliminary signals of activity appeared to be limited to cancers harboring FGFR activating mutations and fusions, although AZD4547 did not meet the primary end point. Different FGFR somatic alterations may confer different levels of signaling potency and/or oncogene dependence.

Background Tumor molecular profiling from patients experiencing exceptional responses to systemic therapy may provide insights into cancer biology and improve treatment tailoring. This pilot study evaluates the feasibility of identifying exceptional responders retrospectively, obtaining pre-exceptional response treatment tumor tissues, and analyzing them with state-of-the-art molecular analysis tools to identify potential molecular explanations for responses. Methods Exceptional response was defined as partial (PR) or complete (CR) response to a systemic treatment with population PR or CR rate less than 10% or an unusually long response (eg, duration >3 times published median). Cases proposed by patients’ clinicians were reviewed by clinical and translational experts. Tumor and normal tissue (if possible) were profiled with whole exome sequencing and, if possible, targeted deep sequencing, RNA sequencing, methylation arrays, and immunohistochemistry. Potential germline mutations were tracked for relevance to disease. Results Cases reflected a variety of tumors and standard and investigational treatments. Of 520 cases, 476 (91.5%) were accepted for further review, and 222 of 476 (46.6%) proposed cases met requirements as exceptional responders. Clinical data were obtained from 168 of 222 cases (75.7%). Tumor was provided from 130 of 168 cases (77.4%). Of 117 of the 130 (90.0%) cases with sufficient nucleic acids, 109 (93.2%) were successfully analyzed; 6 patients had potentially actionable germline mutations. Conclusion Exceptional responses occur with standard and investigational treatment. Retrospective identification of exceptional responders, accessioning, and sequencing of pretreatment archived tissue is feasible. Data from molecular analyses of tumors, particularly when combining results from patients who received similar treatments, may elucidate molecular bases for exceptional responses.

Purpose: Substantial preclinical evidence and case reports suggest that MEK inhibition is an active approach in tumors with BRAF mutations outside the V600 locus, and in BRAF fusions. Thus, Subprotocol R of the NCI-MATCH study tested the MEK inhibitor trametinib in this population. Patients and Methods: The NCI-MATCH study performed genomic profiling on tumor samples from patients with solid tumors and lymphomas progressing on standard therapies or with no standard treatments. Patients with prespecified fusions and non-V600 mutations in BRAF were assigned to Subprotocol R using the NCI-MATCHBOX algorithm. The primary endpoint was objective response rate (ORR). Results: Among 50 patients assigned, 32 were eligible and received therapy with trametinib. Of these, 1 had a BRAF fusion and 31 had BRAF mutations (13 and 19 with class 2 and 3 mutations, respectively). There were no complete responses; 1 patient (3%) had a confirmed partial response (patient with breast ductal adenocarcinoma with BRAF G469E mutation) and 10 patients had stable disease as best response (clinical benefit rate 34%). Median progression-free survival (PFS) was 1.8 months, and median overall survival was 5.7 months. Exploratory subgroup analyses showed that patients with colorectal adenocarcinoma (n = 8) had particularly poor PFS. No new toxicity signals were identified. Conclusions: Trametinib did not show promising clinical activity in patients with tumors harboring non-V600 BRAF mutations, and the subprotocol did not meet its primary endpoint.

PURPOSE The National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial, the largest national precision oncology study to date (> 1,100 sites) of patients with relapsed or refractory malignancies, assigned patients to targeted therapy in parallel phase II studies based on tumor molecular alterations. The anti–programmed death receptor 1 inhibitor nivolumab previously showed activity in mismatch repair (MMR)–deficient colon cancer. We hypothesized that nivolumab would have activity in patients with MMR-deficient, noncolorectal tumors. PATIENTS AND METHODS Eligible patients with relapsed or refractory tumors, good end-organ function, and Eastern Cooperative Oncology Group performance status of ≤ 1 underwent tumor biopsy for centralized screening of molecular alterations. MMR deficiency was defined by complete loss of nuclear expression of MLH1 or MSH2 MMR gene products by immunohistochemistry (IHC). Patients with MMR-deficient colorectal cancer were excluded. Nivolumab, 3 mg/kg every 2 weeks (28-day cycles) and 480 mg every 4 weeks after cycle 4, was administered intravenously. Disease reassessment was performed every 2 cycles. The primary end point was RECIST 1.1 objective response rate (ORR). RESULTS Two percent of 4,902 screened patients had an MMR-deficient cancer by IHC. Forty-two evaluable patients were enrolled, with a median age of 60 years and a median of 3 prior therapies. The most common histologies were endometrioid endometrial adenocarcinoma (n = 13), prostate adenocarcinoma (n = 5), and uterine carcinosarcoma (n = 4). ORR was 36% (15 of 42 patients). An additional 21% of patients had stable disease. The estimated 6-, 12-, and 18-month progression-free survival rates were 51.3% (90% CI, 38.2% to 64.5%), 46.2% (90% CI, 33.1% to 59.3%), and 31.4% (90% CI, 18.7% to 44.2%), respectively. Median overall survival was 17.3 months. Toxicity was predominantly low grade. CONCLUSION A variety of refractory cancers (2.0% of those screened) had MMR deficiency as defined in NCI-MATCH. Nivolumab has promising activity in MMR-deficient noncolorectal cancers of a wide variety of histopathologic types.

Summary: Monoclonal antibodies (mAbs) targeting the oncogenic receptor tyrosine kinase ERBB2/HER2, such as Trastuzumab, are the standard of care therapy for breast cancers driven by ERBB2 overexpression and activation. However, a substantial proportion of patients exhibit de novo resistance. Here, by comparing matched Trastuzumab-naive and post-treatment patient samples from a neoadjuvant trial, we link resistance with elevation of H3K27me3, a repressive histone modification catalyzed by polycomb repressor complex 2 (PRC2). In ErbB2+ breast cancer models, PRC2 silences endogenous retroviruses (ERVs) to suppress anti-tumor type-I interferon (IFN) responses. In patients, elevated H3K27me3 in tumor cells following Trastuzumab treatment correlates with suppression of interferon-driven viral defense gene expression signatures and poor response. Using an immunocompetent model, we provide evidence that EZH2 inhibitors promote interferon-driven immune responses that enhance the efficacy of anti-ErbB2 mAbs, suggesting the potential clinical benefit of epigenomic reprogramming by H3K27me3 depletion in Trastuzumab-resistant disease. : Hirukawa et al. link Trastuzumab resistance in ErbB2+ breast cancers with activity of the methyltransferase EZH2, a key epigenetic regulator. By silencing retrotransposons, EZH2 suppresses type-I interferon signaling to limit immune surveillance. Retrotransposon de-repression following EZH2 inhibition triggers interferon responses and sensitizes immunocompetent in vivo models to ErbB2 antibody therapy. Keywords: ErbB2/HER2, breast cancer, Polycomb Repressor Complex 2, PRC2, epigenetics, Trastuzumab resistance, endogenous retroviruses, immune surveillance, type I interferon signaling, transcriptional silencing

With advances in genetic testing and its common usage, the field of precision medicine has exploded in the field of oncology. The National Cancer Institute is uniquely positioned to lead in this area of research through its wide network of investigators, partnerships with pharmaceutical companies in drug development, and laboratory capabilities. It has developed a portfolio of trials as part of a Precision Medicine Initiative, that uses various basket/umbrella designs to increase the understanding of treatment of cancer through genetic selection and targeted therapies. This article describes these trials, ALCHEMIST, LungMAP, NCI/NRG ALK Trial, MPACT, NCI-MATCH, and pediatric MATCH, and their contributions to the area of precision medicine.

Academic, industry, regulatory leaders and patient advocates in cancer clinical research met in November 2018 at the Innovation and Biomarkers in Cancer Drug Development meeting in Brussels to address the existing dichotomy between increasing calls for personalised oncology approaches based on individual molecular profiles and the need to make resource and regulatory decisions at the societal level in differing health-care delivery systems around the globe. Novel clinical trial designs, the utility and limitations of real-world evidence (RWE) and emerging technologies for profiling patient tumours and tumour-derived DNA in plasma were discussed. While randomised clinical trials remain the gold standard approach to defining clinical utility of local and systemic therapeutic interventions, the broader adoption of comprehensive tumour profiling and novel trial designs coupled with RWE may allow patient and physician autonomy to be appropriately balanced with broader assessments of safety and overall societal benefit. (C) 2019 Published by Elsevier Ltd.

Background: In CALGB 40601 (Alliance, NCT00770809), a neoadjuvant phase III trial of paclitaxel and trastuzumab with or without lapatinib for 12 weeks for patients with HER2-positive breast cancer, 33% of pretreatment tumors were Luminal A subtype, however, 69% of post-treatment samples with residual disease were Luminal A subtype. In addition, 71% of Luminal B (12/17) and 67% of HER2-Enriched (6/9) tumors changed into Luminal A, while 80% of Luminal A (20/24) remained Luminal A (Carey et al. J Clin Oncol. 2016). It is not known whether this shift to Luminal A was transient or permanent. Methods: We selected matched pairs of pre- and post-treatment 40601 samples with tumor purity >10% based upon DNA analyses to ensure all samples contained tumor. PAM50 intrinsic subtyping was applied to the 40601 samples gene expression data using a two-step normalization process based on The Genome Cancer Atlas, and PAM50 training set. In addition, a HER2-enriched expression subtype patient-derived xenograft (PDX) tumor called WHIM35, was studied and was either untreated (n=10), or treated with lapatinib at 220 mg/kg for 1 week (wk) (n=5), for 2 wks (n=8), or for 3 wks (n=4). We also treated WHIM35 tumors with lapatinib for 2 wks (on) and then removed laptinib for 1 wk (off) (n=6), or for 2 wks on and 2-4 wk off (n=6), and finally for 3 wks on, and 1 wk off (n=3). PAM50 intrinsic subtyping was applied to the PDX gene expression data and subtype assessed as well as a genomic-based proliferation score. ANOVA p-values were calculated by comparing median values across all gene signature or correlation scores. Results: We found 10 pairs of 40601 samples that kept their tumor purity values, however, their subtype changed to Luminal A after treatment (i.e., in the residual disease), and in these cases no minor tumor subclone became a dominant clone in the post treatment sample. Pretreatment subtypes were 6 Luminal B, 3 Luminal A, and 1 HER2-enriched. The tumor purity values did not change after the treatments, but correlation to Luminal A was significantly higher (p=0.01), while correlation to HER2-enriched (p=0.004) and proliferation signature scores (p=0.003) were significantly lower in the post-treatment samples. Among the WHIM35 PDX tumors, one sample changed its subtype from HER2-enriched to Luminal A after the lapatinib treatment and the rest remained HER2-enriched, suggesting environmental differences between patient samples and the PDX model. However, correlation to Luminal A was significantly higher in all lapatinib treated WHIM35 samples (p=8.3e-12), and notably went back to the initial low levels just one week after removing lapatinib. Likewise, correlation to HER2-enriched (p=1.2e-10) and proliferation signature scores (p=6.2e-12) also got lower while treated with lapatinib, but went back to the initial levels after cessation of treatment. Conclusions: Our findings suggest that the apparent subtype change during HER2-targeting therapy is not permanent, but is more likely a transient state change from a HER2-enriched subtype into a more Luminal A-like state. When we plan additional treatment strategies using residual disease phenotypes, it may not be clear what is the true subtype of the sample due to this inherent plasticity. Citation Format: Tanioka M, Parker JS, Henry LN, Tolaney S, Dang C, Krop IE, Harris L, Polley M, Berry DA, Winer EP, Carey LA, Perou CM. Transient state change, but not permanent subtype change, after HER2-targeted therapy for HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-04.

2616 Background: On arm Z1D of the NCI-MATCH trial, the PD-1 inhibitor nivolumab was found to have activity among patients with mismatch repair-deficient (MMRd) tumors as defined by complete loss of MLH1 or MSH2 nuclear expression determined by immunohistochemistry, with 6-month progression free survival of 51%. We aimed to identify molecular predictors of response in this population. Methods: Among patients treated on NCI-MATCH Z1D, we evaluated genomic and tissue predictors of clinical benefit (CB), defined as patients with RECIST v1.1 complete or partial response or stable disease for ≥ 6months. WES files were processed and filtered using GATK best practices preceding TMB and MSI calculations according to MSI sensor score, a WES-based MSI rating system. Cutoffs were set to define TMB (TMB-Low: ≤10 mutation/Mb; TMB-High: >10) and MSI (MSS: ≤10% unstable loci; MSI-Low: 10 > x ≤ 20; MSI-High: >20). Multiplex immunofluorescence (mIF) used formalin-fixed paraffin-embedded slides stained using a BOND RX automated stainer. Expression analyses followed normalization in DEseq2's median of ratios method. Gene set enrichment analysis was conducted by “empirical phenotype-based permutation test.” Additional RNA, WES, and mIF comparisons used the Wilcoxon rank-sum test. Results: Among 36 patients accrued to NCI-MATCH Z1D with pretreatment correlative samples available, 7 were unevaluable for response, and 1 was misclassified as having an MMRd tumor. Of the remaining 28, 15 had CB (2 CR, 10 PR, 3 SD ≤ 6 months) and correlative data were available for 26 (WES), 27 (RNAseq), and between 10-20 for mIF based on the marker assessed. According to MSI-sensor score, 11 were MSI-high, 8 were MSI-low, and 7 were MSS. MSI-sensor status, but not TMB was associated with CB (p=0.037 and p=0.185, respectively). Similar results were seen when using CR+PR vs SD+PD evaluation. Using RNAseq gene set enrichment analyses, CB patients had increased expression of interferon alpha (p=0.01), interferon gamma (p=0.03), PI3K-AKT-mTOR (p=0.02), cytotoxicity (p=0.05) and antigen processing (p=0.01) gene sets, while hedgehog signaling genes were increased in non-CB patients (p=0.04). The ESTIMATE immune index and infiltration of CD4+/PD1+/Ki67+ cell populations as determined by mIF were nominally higher in patients with CB (p=0.051 and p=0.075). Conclusions: Among patients with MMRd tumors treated with PD-1 checkpoint blockade, correlative analyses demonstrate associations between CB and MSI-sensor score as well as biomarkers indicative of immune infiltration and antigen presentation. This suggests that these measures may help differentiate patient response in MSI tumors. Clinical trial information: NCT02465060.

3010 Background: NCI-MATCH (EAY131) is a platform trial enrolling patients (pts) with solid tumors, lymphomas, or multiple myeloma to targeted therapies based on matching genomic alterations (NCT02465060). Subprotocol Arm T evaluated vismodegib (GDC0449), a hedgehog signaling pathway inhibitor with anti-tumor activity in pts with tumors harboring PTCH1 and SMO mutations. Methods: Pts whose tumors had SMO or PTCH1 mutations were eligible; results were confirmed by NCI-MATCH central labs if possible. Pts received oral vismodegib (150 mg daily) for 4-week cycles until progression/toxicity. Tumor response was assessed every 2 cycles. Primary endpoint was ORR; secondary endpoints included PFS, 6-month PFS, OS, and predictive biomarkers. Cutaneous basal cell carcinomas were excluded. Results: Of 34 pts enrolled (6/20/16 – 9/22/20); 2 were ineligible and 1 did not start therapy. The 31 analyzable pts’ demographics were primary tumor sites/histology [gastrointestinal (n = 9), skin/soft tissue (n = 7), gynecologic (n = 5), lung (n = 4), unknown primary (n = 4), ductal breast (n = 1), meningioma (n = 1)]; median age 64 (range 19-81); 48.4% women; 61.3% (19/31) > 3 lines of prior therapy; 74% (23/31) > 1 co-occurring mutation [median 2 co-alterations (range 1-20)]. 8/31 > 4 co-occurring alterations. 9 pts had SMO mutant tumors (all SNVs); 5/9 had > 1 co-occurring gene alterations. 22 pts had PTCH1 alterations (7 SNVs and 15 indels); 18/22 pts had > 1 additional gene alteration. Of 31 analyzable pts, 22 were MATCH-confirmed (i.e. had central confirmation of tumor PTCH1/SMO mutations). MATCH-confirmed pts had ORR 9.1% (2/22) while all analyzable pts had ORR 6.5% (2/31). 2 PRs were seen in pts with a skin/soft tissue sarcoma ( PTCH) and a meningioma ( SMO) with a median duration of response 14 months. The 6-month PFS rate was similar in MATCH-confirmed and analyzable pts (22.4% and 23.2% respectively) and median PFS was identical at 1.8 months. Median OS was 9.1 months in MATCH-confirmed and 7.3 months in analyzable pts. Within analyzable SMO variants: 1 PR, 3 SD, 4 PD, and 1 unevaluable responses were documented. Within analyzable PTCH1 variants: 1 PR, 7 SD, 10 PD, and 4 unevaluable responses were seen. 4 pts (12.9%) discontinued therapy due to AE. Among 33 pts starting therapy, 18 (54.5%) had grade 1-2 toxicity, while 2 (6.1%) had grade 3 treatment-related toxicity. Most common toxicities: grade 1-2 fatigue (n = 11), anorexia (n = 8), weight loss (n = 7), alopecia (n = 7), and dysgeusia (n = 6). There were 4 on-study deaths, but none were treatment related. Conclusions: Although the primary endpoint was not reached, vismodegib was well-tolerated with mostly grade 1-2 toxicities and substantial responses were seen in patients with SMOPro641Ala and PTCHGlu947Ter alterations. Further study of the impact of concomitant molecular alterations may yield additional insights into vismodegib mechanisms of response. Clinical trial information: NCT02465060.

Purpose: Response to a complex trastuzumab-based regimen is affected by multiple features of the tumor and its microenvironment. Developing a predictive algorithm is key to optimizing HER2-targeting therapy. Experimental Design: We analyzed 137 pretreatment tumors with mRNA-seq and DNA exome sequencing from CALGB 40601, a neoadjuvant phase III trial of paclitaxel plus trastuzumab with or without lapatinib in stage II to III HER2-positive breast cancer. We adopted an Elastic Net regularized regression approach that controls for covarying features within high-dimensional data. First, we applied 517 known gene expression signatures to develop an Elastic Net model to predict pCR, which we validated on 143 samples from four independent trials. Next, we performed integrative analyses incorporating clinicopathologic information with somatic mutation status, DNA copy number alterations (CNA), and gene signatures. Results: The Elastic Net model using only gene signatures predicted pCR in the validation sets (AUC = 0.76). Integrative analyses showed that models containing gene signatures, clinical features, and DNA information were better pCR predictors than models containing a single data type. Frequently selected variables from the multiplatform models included amplifications of chromosome 6p, TP53 mutation, HER2-enriched subtype, and immune signatures. Variables predicting resistance included Luminal/ER+ features. Conclusions: Models using RNA only, as well as integrated RNA and DNA models, can predict pCR with improved accuracy over clinical variables. Somatic DNA alterations (mutation, CNAs), tumor molecular subtype (HER2E, Luminal), and the microenvironment (immune cells) were independent predictors of response to trastuzumab and paclitaxel-based regimens. This highlights the complexity of predicting response in HER2-positive breast cancer. Clin Cancer Res; 24(21); 5292–304. ©2018 AACR.

Purpose: Immunoprofiling to identify biomarkers and integration with clinical trial outcomes are critical to improving immunotherapy approaches for patients with cancer. However, the translational potential of individual studies is often limited by small sample size of trials and the complexity of immuno-oncology biomarkers. Variability in assay performance further limits comparison and interpretation of data across studies and laboratories. Experimental Design: To enable a systematic approach to biomarker identification and correlation with clinical outcome across trials, the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) Network was established through support of the Cancer MoonshotSM Initiative of the National Cancer Institute (NCI) and the Partnership for Accelerating Cancer Therapies (PACT) with industry partners via the Foundation for the NIH. Results: The CIMAC-CIDC Network is composed of four academic centers with multidisciplinary expertise in cancer immunotherapy that perform validated and harmonized assays for immunoprofiling and conduct correlative analyses. A data coordinating center (CIDC) provides the computational expertise and informatics platforms for the storage, integration, and analysis of biomarker and clinical data. Conclusions: This overview highlights strategies for assay harmonization to enable cross-trial and cross-site data analysis and describes key elements for establishing a network to enhance immuno-oncology biomarker development. These include an operational infrastructure, validation and harmonization of core immunoprofiling assays, platforms for data ingestion and integration, and access to specimens from clinical trials. Published in the same volume are reports of harmonization for core analyses: whole-exome sequencing, RNA sequencing, cytometry by time of flight, and IHC/immunofluorescence.

Importance In the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial, agents targeting genetic tumor abnormalities are administered to patients. In the NCI-MATCH subprotocol EAY131-Y trial, patients with anAKT1 E17K-mutated metastatic tumor received the pan-AKTinhibitor capivasertib. Objective To assess the objective response rate (ORR) of capivasertib in patients with anAKT1 E17K-mutated tumor. Design, Setting, and Participants Between July 13, 2016, and August 10, 2017, patients in the NCI-MATCH trial were enrolled and assigned to the subprotocol EAY131-Y nonrandomized trial. Patients included adults with anAKT1 E17K-mutated metastatic tumor that had progressed with standard treatment, and these patients were assigned to receive capivasertib. Tumor assessments were repeated every 2 cycles. Data analysis of this evaluable population was performed from November 8, 2019, to March 12, 2020. Interventions The study treatment was capivasertib, 480 mg, orally twice daily for 4 days on and 3 days off weekly in 28-day cycles until disease progression or unacceptable toxic effect. If patients continued hormone therapy for metastatic breast cancer, the capivasertib dose was 400 mg. Main Outcomes and Measures The primary end point was the ORR (ie, complete response [CR] and partial response) according to the Response Evaluation Criteria in Solid Tumors criteria, version 1.1. Secondary end points included progression-free survival (PFS), 6-month PFS, overall survival, and safety. Results In total, 35 evaluable and analyzable patients were included, of whom 30 were women (86%), and the median (range) age was 61 (32-73) years. The most prevalent cancers were breast (18 [51%]), including 15 patients with hormone receptor (HR)–positive/ERBB2-negative and 3 with triple-negative disease, and gynecologic (11 [31%]) cancers. The ORR rate was 28.6% (95% CI, 15%-46%). One patient with endometrioid endometrial adenocarcinoma achieved a CR and remained on therapy at 35.6 months. Patients with confirmed partial response had the following tumor types: 7 had HR-positive/ERBB2-negative breast cancer, 1 had uterine leiomyosarcoma, and 1 had oncocytic parotid gland carcinoma and continued receiving treatment at 28.8 months. Sixteen patients (46%) had stable disease as the best response, 2 (6%) had progressive disease, and 7 (20%) were not evaluable. With a median follow-up of 28.4 months, the overall 6-month PFS rate was 50% (95% CI, 35%-71%). Capivasertib was discontinued because of adverse events in 11 of 35 patients (31%). Grade 3 treatment-related adverse events included hyperglycemia (8 [23%]) and rash (4 [11%]). One grade 4 hyperglycemic adverse event was reported. Conclusions and Relevance This nonrandomized trial found that, in patients with anAKT1 E17K-mutated tumor treated with capivasertib, a clinically significant ORR was achieved, including 1 CR. Clinically meaningful activity with single-agent capivasertib was demonstrated in refractory malignant neoplasms, including rare cancers. Trial Registration ClinicalTrials.gov Identifier:NCT00700882

Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse., eLife digest Cells in the body remain healthy by tightly preventing and repairing random changes, or mutations, in their genetic material. In cancer cells, however, these mechanisms can break down. When these cells grow and multiply, they can then go on to accumulate many mutations. As a result, cancer cells in the same tumor can each contain a unique combination of genetic changes. This genetic heterogeneity has the potential to affect how cancer responds to treatment, and is increasingly becoming appreciated clinically. For example, if a drug only works against cancer cells carrying a specific mutation, any cells lacking this genetic change will keep growing and cause a relapse. However, it is still difficult to quantify and understand genetic heterogeneity in cancer. Copy number alterations (or CNAs) are a class of mutation where large and small sections of genetic material are gained or lost. This can result in cells that have an abnormal number of copies of the genes in these sections. Here, Baslan et al. set out to explore how CNAs might vary between individual cancer cells within the same tumor. To do so, thousands of individual cancer cells were isolated from human breast tumors, and a technique called single-cell genome sequencing used to screen the genetic information of each of them. These experiments confirmed that CNAs did differ – sometimes dramatically – between patients and among cells taken from the same tumor. For example, many of the cells carried extra copies of well-known cancer genes important for treatment, but the exact number of copies varied between cells. This heterogeneity existed for individual genes as well as larger stretches of DNA: this was the case, for instance, for an entire section of chromosome 8, a region often affected in breast and other tumors. The work by Baslan et al. captures the sheer extent of genetic heterogeneity in cancer and in doing so, highlights the power of single-cell genome sequencing. In the future, a finer understanding of the genetic changes present at the level of an individual cancer cell may help clinicians to manage the disease more effectively.

This abstract was not presented at the symposium.

Background: In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML. The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted "myeloid panel" NGS assay platform (s-NGS). Methods: DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) >5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used. Results: Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these "edge case" variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B). Conclusions: NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. Figure 1 Figure 1. Disclosures Zhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

3087 Background: The NCI-MATCH trial assigns patients (pts) with solid tumors, lymphomas, or multiple myeloma to targeted therapies based on genetic alterations identified in tumor biopsies. Neurofibromatosis 2 (NF2)-inactivated tumors demonstrate increased sensitivity to FAK inhibition in preclinical models. Arm U evaluated the FAK inhibitor defactinib in pts with NF2 altered tumors. Methods: Patients found to harbor an inactivating NF2 mutation on NGS were assigned to the ARM U substudy MATCH. Defactinib 400 mg was given by mouth twice daily until progression or intolerable toxicity. The primary endpoint was objective response rate (ORR). Secondary endpoints included toxicity, progression-free survival (PFS), and 6-month PFS. Results: Of 5,548 cases with sufficient tissue for genomic analysis, 51 pts were found to have NF2 alterations (< 1% of the total analyzed). While NF2 alterations are known to occur more commonly in meningiomas and mesotheliomas, alterations were also detected in an array of other tumor types, including renal cell carcinomas and ovarian cancers. Thirty-five pts were ultimately enrolled; 33 patients were started on therapy, with 2 of those determined to be ineligible for outcome analysis. All pts had received at least one prior therapy, with 52% (16/31) having received 3 or more prior lines of therapy. Median follow-up was 35.9 months. ORR [90% CI] was 3% (1/31, [0.16, 14.86]), with the one partial response in a pt with choroid meningioma. Of the twelve pts whose best response was stable disease (39%, 12/31), 8 demonstrated some degree of tumor shrinkage (Table) with a disease control rate of 42% (13/31). Median PFS was 1.9 months for the 31 eligible pts who received study treatment, with median PFS of 9.3 months for the 9 patients who had a best response of stable disease or better. Six pts achieved a PFS of greater than 5.5 months. Among all treated pts (n=33), the most common treatment-related toxicities were fatigue (36%), nausea (33%), and hyperbilirubinemia (27%). There were no grade 4 or 5 toxicities; 27% of pts had grade 3 toxicities. No correlation could be made between clinical outcomes and tumor histology or specific NF2 genotype. Conclusions: Defactinib monotherapy had limited clinical activity in this cohort of previously treated patients with solid tumors exhibiting NF2 loss. Clinical trial information: NCT04439331. [Table: see text]

6565 Background: Patients (pts) with cancer are at increased risk of SARS-CoV-2 infection and severe COVID-19 disease. Longitudinal follow-up is needed to characterize the severity, sequelae and outcomes in pts with cancer who develop COVID-19. Methods: NCCAPS is a prospective, longitudinal study (NCT04387656) aiming to accrue 2,000 pts with cancer undergoing active treatment or prior stem cell transplant for hematologic or solid tumor malignancy. Adult patients are eligible to enroll within 14 days of their first positive SARS-CoV-2 test; pediatric patients may also enroll retrospectively. Clinical data, patient-reported outcomes, blood specimens, and imaging are collected for up to 2 years. This abstract provides initial baseline and 2-month follow-up data. Results: As of Jan 22, 2021, 585 pts (552 adults and 33 pediatric pts) had complete baseline data and of these pts, 215 adults had 2 months of complete follow-up data. 23.4% of adults and 42.4% of pediatric pts were of non-White race and/or Hispanic/Latinx ethnicity. The most common cancer diagnoses were breast (19.6%), lung (9.9%) and multiple myeloma (8.9%) in adults and acute leukemia (AML/ALL; 63.6%) in children. The most recent treatment was chemotherapy in 38.2%, immunotherapy in 9.6%, and radiation in 5.4%. Median time from positive SARS-CoV-2 test to study enrollment was 10.5 days in adults and 18 days in pediatric pts. Preliminary analysis of plasma cytokines will be presented. At enrollment, 84.6% of adults had COVID-19 symptoms. 55.9% reported symptoms 2 weeks after their positive SARS-CoV-2 test; this fell to 39.0% at 1 month and 28.8% at 2 months (see Table). Of the 215 adults with complete data at 2 months, sequelae included pulmonary (n=22, 10%), cardiovascular (n=12, 6%) thromboembolic (n=9, 4%), bleeding (n=9, 4%) and gastrointestinal (n=11, 5%). 144 (67%) reported at least one cancer treatment disruption in the first 2 months, most commonly delayed therapy (n=98; 46%).Of the 348 adults with baseline data and SARS-CoV-2 test date prior to Nov 23, 2020, 6.3% had died (median time from SARS-CoV-2 test to death: 27 days), and 22.1% reported at least one hospitalization for COVID-19. No deaths were reported in the pediatric population. Conclusion: Cancer pts with COVID-19 report ongoing symptoms after acute infection and a substantial number develop sequelae. Cancer treatment disruptions are common in the initial months following SARS-CoV-2 infection. Longer follow-up will inform whether these treatment disruptions are associated with adverse outcomes. Clinical trial information: NCT04387656. [Table: see text]

HLA-DRB1*07:01 allele carriage was characterised as a risk biomarker for lapatinib-induced liver injury in a large global study evaluating lapatinib, alone and in combination with trastuzumab and taxanes, as adjuvant therapy for advanced breast cancer (adjuvant lapatinib and/or trastuzumab treatment optimisation). HLA-DRB1*07:01 carriage was associated with serum alanine aminotransferase (ALT) elevations in lapatinib-treated patients (odds ratio 6.5, P=3 × 10-26, n=4482) and the risk and severity of ALT elevation for lapatinib-treated patients was higher in homozygous than heterozygous HLA-DRB1*07:01 genotype carriers. A higher ALT case incidence plus weaker HLA association observed during concurrent administration of lapatinib and taxane suggested a subset of liver injury in this combination group that was HLA-DRB1*07:01 independent. Furthermore, the incidence of ALT elevation demonstrated an expected correlation with geographic HLA-DRB1*07:01 carriage frequency. Robust ALT elevation risk estimates for HLA-DRB1*07:01 may support causality discrimination and safety risk management during the use of lapatinib combination therapy for the treatment of metastatic breast cancer.

Background: HER2+ breast cancers are heterogeneous at both clinical and molecular levels. We and others have determined that the HER2-Enriched subtype exhibits the highest rate of pathologic complete response (pCR) to neoadjuvant chemotherapy and trastuzumab (T), while the HER2-Basal subtype is resistant to anti-HER2 therapy (Carey et al, JCO 2015;Varadan et al, CCR 2016). Additionally, we reported that signatures of immune cell infiltration and immune cell subsets evaluated after one dose of T can predict pCR to preoperative T and chemotherapy (Varadan et al, CCR 2016). Given recent evidence for improved immune response with increasing mutational load, we chose to characterize the association of somatic mutations and copy-number alterations with subtypes of HER2+ breast cancer and immune modulation after one dose of T. Methods: Fresh tumor core biopsies were taken at baseline and 2 weeks after one dose of either T or nab-paclitaxel (N) from 60 patients with stage II-III HER2+ cancers enrolled on a multicenter trial (BrUOG 211B). All patients then received 18 weeks of T+N+carboplatin. PAM50 subtyping was performed using gene expression data from patient tumor biopsies and tumors were classified into HER2-Enriched, HER2-Luminal and HER2-Basal subtypes. Whole-exome sequencing (WES) was performed on a total of 86 samples (49 baseline, 37 brief-exposure), sequenced at an average depth of 90X. Somatic mutations were detected by applying multiple mutation-detection algorithms on the WES data, followed by stringent quality control using public and in-house variant databases, and mutation data curated from 11,000 tumors sequenced by the TCGA. Somatic copy-number alterations were estimated using a published algorithm, ENVE (Varadan et al, Genome Med 2015) that robustly detects somatic copy-number alterations in WES tumor profiles. We employed previously defined gene-expression signatures (Varadan et al, CCR 2016) of total immune infiltration and immune cell subsets, to assess for association with genomic aberrations. Results: HER2-Basal tumors exhibited lower average copy number for HER2 and were less likely to have high-level amplifications of co-amplicons (e.g. 11q13, 20q13) with the exception of the MYC amplicon (8q24). They also exhibited a non-significant (P=0.33) trend towards higher mutational burden (Avg=85) compared to HER2-Luminals (Avg=79). A majority of somatic mutations (62%, 2282/3666) persisted after a single-dose of either T or N, while 17% (624/3666) were not detectable after brief-exposure. There was no association between immune infiltration and mutational burden in any HER2 subtype. Tumors harboring FGFR1 (8p11) amplifications exhibited higher gene-signature levels for macrophages (P=0.0073) and T-cells (P=0.0493) but not B-cells (P=0.213). Conclusions: The HER2-Basal subtype is less likely to respond to trastuzumab-based neoadjuvant therapy and exhibits lower numbers of common amplicons. The disappearance of mutations after brief-exposure to therapy may be due to either tumor heterogeneity/sampling or clonal selection. The association of 8p11 amplifications with increased T-cell infiltration suggests that this amplicon may play an immunogenic role in HER2+ breast cancer. These results warrant further investigation in larger cohorts. Citation Format: Singh S, Gilmore H, Somlo G, Abu-Khalaf M, Sikov W, Harris L, Varadan V. Association of co-amplicons with immune infiltration in subtypes of HER2-Positive breast cancer [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 P1-05-09.

Background The National Cancer Institute—Molecular Analysis for Therapy Choice (NCI-MATCH) is a national precision medicine study incorporating centralized genomic testing to direct refractory cancer patients to molecularly targeted treatment subprotocols. This treatment subprotocol was designed to screen for potential signals of efficacy of ado-trastuzumab emtansine (T-DM1) in HER2-amplified histologies other than breast and gastroesophageal tumors. Methods Eligible patients had HER2 amplification at a copy number (CN) >7 based on targeted next-generation sequencing (NGS) with a custom Oncomine AmpliSeq™ (ThermoFisher Scientific) panel. Patients with prior trastuzumab, pertuzumab or T-DM1 treatment were excluded. Patients received T-DM1 at 3.6 mg/kg i.v. every 3 weeks until toxicity or disease progression. Tumor assessments occurred every three cycles. The primary end point was centrally assessed objective response rate (ORR). Exploratory end points included correlating response with HER2 CN by NGS. The impact of co-occurring genomic alterations and PTEN loss by immunohistochemistry were also assessed. Results Thirty-eight patients were enrolled and 36 included in efficacy analysis. Median prior therapies in the metastatic setting was 3 (range 0–9; unknown in one patient). Median HER2 CN was 17 (range 7–139). Partial responses were observed in two (5.6%) patients: one mucoepidermoid carcinoma of parotid gland and one parotid gland squamous cell cancer. Seventeen patients (47%) had stable disease including 8/10 (80%) with ovarian and uterine carcinomas, with median duration of 4.6 months. The 6-month progression-free survival rate was 23.6% [90% confidence interval 14.2% to 39.2%]. Common toxicities included fatigue, anemia, fever and thrombocytopenia with no new safety signals. There was a trend for tumor shrinkage with higher levels of gene CN as determined by the NGS assay. Conclusion T-DM1 was well tolerated. While this subprotocol did not meet the primary end point for ORR in this heavily pre-treated diverse patient population, clinical activity was seen in salivary gland tumors warranting further study in this tumor type in dedicated trials.

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis., Polycomb Repressor Complex 2 (PRC2) is frequently up-regulated in cancers. Here, the authors show that the tyrosine kinase c-Src stimulates mitochondrial function to signal energy sufficiency to mTORC1, increasing translation of the PRC2 subunits EZH2 and SUZ12 to support ErbB2-dependent tumours.

A small fraction of cancer patients with advanced disease survive significantly longer than patients with clinically comparable tumors. Molecular mechanisms for exceptional responses to therapy have been identified by genomic analysis of tumor biopsies from individual patients. Here, we analyzed tumor biopsies from an unbiased cohort of 111 exceptional responder patients using multiple platforms to profile genetic and epigenetic aberrations as well as the tumor microenvironment. Integrative analysis uncovered plausible mechanisms for the therapeutic response in nearly a quarter of the patients. The mechanisms were assigned to four broad categories���DNA damage response, intracellular signaling, immune engagement, and genetic alterations characteristic of favorable prognosis���with many tumors falling into multiple categories. These analyses revealed synthetic lethal relationships that may be exploited therapeutically and rare genetic lesions that favor therapeutic success, while also providing a wealth of testable hypotheses regarding oncogenic mechanisms that may influence the response to cancer therapy. Profiling multi-platform genomics of 110 cancer patients with an exceptional therapeutic response, Wheeler et al. identify putative molecular mechanisms explaining this survival phenotype in ���23% of cases. Therapeutic success is related to rare molecular features of responding tumors, exploiting synthetic lethality and oncogene addiction.

// Callie R. Merry 1, 2 , Sarah McMahon 1 , Megan E. Forrest 1 , Cynthia F. Bartels 1 , Alina Saiakhova 1 , Courtney A. Bartel 3 , Peter C. Scacheri 1, 3 , Cheryl L. Thompson 3, 5 , Mark W. Jackson 3 , Lyndsay N. Harris 3, 4 , Ahmad M. Khalil 1, 2, 3 1 Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, USA 2 Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA 3 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA 4 Department of Medicine and Case Western Reserve University, Cleveland, OH 44106, USA 5 Department of Nutrition, Case Western Reserve University, Cleveland, OH 44106, USA Correspondence to: Ahmad M. Khalil, email: Dr.Ahmad.Khalil@gmail.com Keywords: breast cancer, HER2, drug resistance, trastuzumab-resistance, cancer therapy Received: March 11, 2016 Accepted: July 09, 2016 Published: July 16, 2016 ABSTRACT Approximately, 25–30% of early-stage breast tumors are classified at the molecular level as HER2-positive, which is an aggressive subtype of breast cancer. Amplification of the HER2 gene in these tumors results in a substantial increase in HER2 mRNA levels, and consequently, HER2 protein levels. HER2, a transmembrane receptor tyrosine kinase (RTK), is targeted therapeutically by a monoclonal antibody, trastuzumab (Tz), which has dramatically improved the prognosis of HER2-driven breast cancers. However, ~30% of patients develop resistance to trastuzumab and recur; and nearly all patients with advanced disease develop resistance over time and succumb to the disease. Mechanisms of trastuzumab resistance (TzR) are not well understood, although some studies suggest that growth factor signaling through other receptors may be responsible. However, these studies were based on cell culture models of the disease, and thus, it is not known which pathways are driving the resistance in vivo . Using an integrative transcriptomic approach of RNA isolated from trastuzumab-sensitive and trastuzumab-resistant HER2+ tumors, and isogenic cell culture models, we identified a small set of mRNAs and lincRNAs that are associated with trastuzumab-resistance (TzR). Functional analysis of a top candidate gene, S100P, demonstrated that inhibition of S100P results in reversing TzR. Mechanistically, S100P activates the RAS/MEK/MAPK pathway to compensate for HER2 inhibition by trastuzumab. Finally, we demonstrated that the upregulation of S100P appears to be driven by epigenomic changes at the enhancer level. Our current findings should pave the path toward new therapies for breast cancer patients.

Purpose: Recent data suggest that intrinsic subtype and immune cell infiltration may predict response to trastuzumab-based therapy. We studied the interaction between these factors, changes in immune signatures following brief exposure to trastuzumab, and achievement of pathologic complete response (pCR) to subsequent preoperative trastuzumab and chemotherapy in HER2-positive breast cancer. Experimental Design: In patients enrolled on two multicenter trials (03-311 and 211B), tumor core biopsies were obtained at baseline and after brief exposure to single-agent trastuzumab or nab-paclitaxel. Gene expression profiles were assessed to assign PAM50 subtypes, measure immune cell activation, and were correlated with response. Results: The pCR rate was significantly higher in HER2-enriched tumors in the Discovery, 03-311 (36%, P = 0.043) dataset, as compared with other subtypes, which validated in 211B (50%, P = 0.048). Significant increases in a signature of immune cell admixture (Immune Index) were observed only following brief exposure to trastuzumab in HER2-enriched tumors (Discovery/03-311, P = 0.05; Validation/211B, P = 0.02). Increased Immune Index was predictive of response after brief exposure (03-311, P = 0.03; 211B, P = 0.04), but not at baseline, in addition to increased expression of a CD4+ follicular helper T-cell signature (03-311, P = 0.05; 211B, P = 0.04). Brief exposure to trastuzumab significantly increased gene expression of the T-cell marker PD-1 in HER2-enriched tumors (Discovery/03-311, P = 0.045) and PD-1 positivity by IHC (Validation/211B, P = 0.035). Conclusions: Correlations between pCR rates, increases in Immune Index and markers of T-cell activity following brief exposure to trastuzumab in HER2-enriched tumors provide novel insights into the interaction between tumor biology, antitumor immunity, and response to treatment, and suggest potential clinically useful biomarkers in HER2+ breast cancers. Clin Cancer Res; 22(13); 3249–59. ©2016 AACR.

Purpose To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer. Methods A literature search and prospectively defined study selection sought systematic reviews, meta-analyses, randomized controlled trials, prospective-retrospective studies, and prospective comparative observational studies published from 2006 through 2014. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert panel members used informal consensus to develop evidence-based guideline recommendations. Results The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that met guideline criteria for clinical utility were found to guide choice of specific treatments or regimens. Recommendations In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2, the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX, EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens. Treatment decisions should also consider disease stage, comorbidities, and patient preferences.

Using transgenic mouse models, cell line-based functional studies, and clinical specimens, we show that cyclin D1/CDK4 mediate resistance to targeted therapy for HER2-positive breast cancer. This is overcome using CDK4/6 inhibitors. Inhibition of CDK4/6 not only suppresses Rb phosphorylation, but also reduces TSC2 phosphorylation and thus partially attenuates mTORC1 activity. This relieves feedback inhibition of upstream EGFR family kinases, resensitizing tumors to EGFR/HER2 blockade. Consequently, dual inhibition of EGFR/HER2 and CDK4/6 invokes a more potent suppression of TSC2 phosphorylation and hence mTORC1/S6K/S6RP activity. The suppression of both Rb and S6RP enhances G1 arrest and a phenotype resembling cellular senescence. In vivo, CDK4/6 inhibitors sensitize patient-derived xenograft tumors to HER2-targeted therapies and delay tumor recurrence in a transgenic model of HER2-positive breast cancer.

Background: RNA profiling and mutational analyses in CALGB 40601 (NCT00770809) found significant impact on pathologic complete response (pCR) rates from tumor (intrinsic subtype, p53 mutation) and microenvironmental (immune cell) features. Integrated analysis across platforms is needed to better understand the roles of these different factors with respect to response to HER2-targeted therapies. Methods: We performed a comprehensive genomic analyses on pCR, defined as no invasive tumor in the breast, by integrating clinicopathological information with somatic mutation status, 422 segment-level DNA Copy Number Alterations (CNAs), and 510 gene expression signatures using mRNAseq and DNA exome sequencing from 213 pre-treatment tumors. Excluding 48 samples in the TL arm that was closed early due to futility, and 4 Normal-like tumors, the dataset consisted of 161 patients from TH and THL arms including 47 HER2-enriched (HER2E), 8 Basal-like, 54 Luminal A, and 52 Luminal B, all of whom received H. The main analysis was performed using the Elastic Net on multivariate logistic regression models for predicting pCR. The samples were divided into a training and a test set, then models were built to predict pCR by 10-fold cross-validation in the training set, then applying the best model onto the test set to construct ROC curves and evaluate prediction accuracy by calculating area under ROC (AUC). We also used the DawnRank, a network-based bioinformatics tool that integrates DNA and RNA data to identify driver genes, to find predictors of resistance to H-containing therapies. Results: Among clinicopathological factors, clinical estrogen/progesterone receptor (ER/PgR) status and intrinsic subtype by PAM50 were statistically associated with pCR, but treatment arm (TH vs THL) and stage were not. In the Elastic Net analysis, the models incorporating either gene signatures (AUC: 0.724) or CNAs (AUC: 0.777) were more predictive of response than mutation status model (AUC: 0.635). Gene signatures and CNAs were further combined with either mutation status (AUC: 0.773), clinical ER/PgR status (AUC: 0.787) or ER/PgR status plus intrinsic subtype (AUC: 0.784). The combination with the highest AUC comprised gene signatures, CNAs, and ER/PgR status, and demonstrated that CNAs at Chromosome (Chr.) 6p, 10q22, or 11q23, the signature of Correlation to HER2E, and a T-cell signature, positively predicted pCR and that Luminal and PgR gene signatures were negative predictors. The CN gain of Chr.6p, which contains the HLA genes, predicted for pCR and was associated with higher expression of HLA genes and B cell / IgG signatures. The CN loss of Chr.11q23 including CD3D, CD3E, and CD3G was also identified by DawnRank as a region associated with resistance. Conclusions: Tumor genetics (CNAs), tumor RNA subtype (HER2E, Luminal), and the microenvironment (immune cells) were independently predictive of response to H-containing therapies and biologically and clinically important for HER2-positive breast cancer, supporting integrated RNA- and DNA-based tumor assessments to clarify response to HER2-targeting. Support: U10CA031946/033601/180821/180882/180888. Citation Format: Tanioka M, Fan C, Carey LA, Hyslop T, Pitcher BN, Parker JA, Hoadley KA, Henry NL, Tolaney S, Dang C, Krop IE, Harris L, Berry DA, Mardis E, Perou CM, Winer EP, Hudis CA. Integrated analysis of multidimensional genomic data on CALGB 40601 (Alliance), a randomized neoadjuvant phase III trial of weekly paclitaxel (T) and trastuzumab (H) with or without lapatinib (L) for HER2-positive breast cancer [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 S3-05.

Background: NRAS-mutations are established oncologic drivers in many malignancies with no effective targeted therapy options. Preclinical and clinical data have suggested that downstream inhibition with a MEK inhibitor, such as binimetinib, might be efficacious for NRAS-mutated cancers. Methods: Patients who enrolled in the multicenter NCI-MATCH trial master protocol underwent tumor biopsy and molecular profiling by targeted next generation sequencing with a custom Oncomine AmpliSeq™ panel. Patients with refractory solid tumors harboring codon 12, 13, or 61 NRAS-mutations were enrolled in subprotocol Z1A, a single arm study of binimetinib 45 mg twice daily. Patients with melanoma were excluded. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS) and overall survival (OS). A post-hoc analysis examined association of NRAS-mutation allele with outcome and histology. Results: 47 eligible patients with refractory solid tumors harboring codon 12, 13, or 61 NRAS-mutations were treated on this trial. The most common cancer types enrolled on this subprotocol were colorectal adenocarcinoma (24/47, 51%), cholangiocarcinoma (7/47, 15%), low-grade papillary serous carcinoma of the ovary (3/47, 6%), and endometrioid endometrial adenocarcinoma patients (3/47, 6%). Observed toxicity was moderate, similar to previous reports, and 27.7% (13 of 47) of eligible patients discontinued binimetinib because of adverse events. The ORR was 2.1% (1 of 47 patients), and the median PFS was 3.5 months. The sole confirmed partial response (PR) was observed in a codon 61 NRAS-mutated indolent malignant ameloblastoma. One patient with a colorectal cancer harboring a NRAS codon 61 mutation had an unconfirmed PR, and two others with NRAS codon 61 mutated colorectal cancer had stable disease for at least 12 months. In a post-hoc analysis, patients with cancers bearing a codon 61 NRAS-mutation (n=22) had a significantly longer OS (p=0.04) and PFS (p=0.006) than those with tumors harboring codon 12 or 13 NRAS-mutations (n=25). Similarly, colorectal cancer patients with NRAS codon 61 mutations treated with binimetinib (n=8) had a significantly longer OS (p=0.03) and PFS (p=0.007) than those with NRAS codon 12 or 13 mutated (n=16) tumors. Conclusions: Single-agent binimetinib did not demonstrate promising efficacy in NRAS-mutated solid tumors. Further studies are needed to clarify whether the increased OS and PFS observed in codon 61 NRAS-mutated cancers reflects a more favorable prognosis for this subtype. Citation Format: James M. Cleary, Victoria Wang, Rebecca Heist, Scott Kopetz, Edith P. Mitchell, James Zwiebel, Helen X. Chen, Shuli Li, Robert Gray, Lisa McShane, Larry Rubinstein, David Patton, Funda Meric-Bernstam, Melissa Dillmon, Mickey Williams, Stanley Hamilton, Barbara Conley, Peter O'Dwyer, Lyndsay Harris, Carlos Arteaga, Alice Chen, Keith Flaherty. Binimetinib in patients with tumors with NRAS mutations: NCI-MATCH ECOG-ACRIN Cancer Research Group subprotocol EAY131-Z1A [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 CT061.

3506 Background: The NCI-MATCH (EAY131) is a platform trial that enrolls patients (pts) with solid tumors, lymphomas, or multiple myeloma to targeted therapies based on matching genomic alterations of interest (NCT02465060). Arm Z1F evaluated copanlisib, a highly selective, pan-Class 1 PI3K inhibitor with predominant activity against both the δ and α isoforms in pts with PIK3CA mutations. Methods: Pts received copanlisib (60 mg IV) on days 1, 8, and 15 in 28-day cycles until progression/toxicity. Tumor assessment was every 2 cycles. The primary endpoint was objective response rate (ORR); secondary endpoints were PFS, 6-month PFS, and predictive biomarkers. Pts with KRAS mutations, HER2+ve breast cancers, lymphomas were excluded. Results: 35 pts were enrolled (from 8/2/18 to 12/27/18), of which, 28 pts were available for analysis (7 patients, not eligible or did not start therapy). Multiple histologies were enrolled with gynecologic (n = 7), gastrointestinal (n = 6), and genitourinary (n = 5) the most common tumors. Median age 61 (range 42-78). 75% of pts had ≥ 3 lines of prior therapy. 54% of PIK3CA mutations were located in the helical domain, 32% in kinase domain and 14% in other domains. Twenty-six pts had co-occurring gene alterations (median 3; range 1-9), with 9 patients having 4 or more gene alterations. The ORR was 11% (3/28, 90% CI: 3%-25%). Partial responses were seen in uterine cancer, clear cell carcinoma of anterior abdominal wall, and liposarcoma. 6 pts had > 6 months of stable disease and clinical benefit rate was 32% (9/28). Two pts are still on treatment. The most common reason for protocol discontinuation was disease progression (n = 18, 69%). Thirty pts were included for toxicity analysis. Ten pts (33%) had grade 1 or 2 toxicities, 16 pts (53%) had grade 3 toxicities, and one patient (3%) had grade 4 toxicity (CTCAE v5.0). Most common toxicities include hyperglycemia (n = 19), fatigue (n = 11), hypertension (n = 10), diarrhea (n = 10), and nausea (n = 9). Total of 5 deaths were reported, none related to treatment. Conclusions: Copanlisib showed meaningful clinical activity across various tumors with PIK3CA mutation in the late-line refractory setting. Further study either alone or in combinations in select tumors is warranted. G3/4 toxicities observed were consistent with reported toxicities for PI3K pathway inhibition. Clinical trial information: NCT02465060 .