NSABP FB-10: a phase Ib/II trial evaluating ado-trastuzumab emtansine (T-DM1) with neratinib in women with metastatic HER2-positive breast cancer.

BACKGROUND: We previously reported our phase Ib trial, testing the safety, tolerability, and efficacy of T-DM1 + neratinib in HER2-positive metastatic breast cancer patients. Patients with ERBB2 amplification in ctDNA had deeper and more durable responses. This study extends these observations with in-depth analysis of molecular markers and mechanisms of resistance in additional patients. METHODS: Forty-nine HER2-positive patients (determined locally) who progressed on-treatment with trastuzumab + pertuzumab were enrolled in this phase Ib/II study. Mutations and HER2 amplifications were assessed in ctDNA before (C1D1) and on-treatment (C2D1) with the Guardant360 assay. Archived tissue (TP0) and study entry biopsies (TP1) were assayed for whole transcriptome, HER2 copy number, and mutations, with Ampli-Seq, and centrally for HER2 with CLIA assays. Patient responses were assessed with RECIST v1.1, and Molecular Response with the Guardant360 Response algorithm. RESULTS: The ORR in phase II was 7/22 (32%), which included all patients who had at least one dose of study therapy. In phase I, the ORR was 12/19 (63%), which included only patients who were considered evaluable, having received their first scan at 6 weeks. Central confirmation of HER2-positivity was found in 83% (30/36) of the TP0 samples. HER2-amplified ctDNA was found at C1D1 in 48% (20/42) of samples. Patients with ctHER2-amp versus non-amplified HER2 ctDNA determined in C1D1 ctDNA had a longer median progression-free survival (PFS): 480 days versus 60 days (P = 0.015). Molecular Response scores were significantly associated with both PFS (HR 0.28, 0.09-0.90, P = 0.033) and best response (P = 0.037). All five of the patients with ctHER2-amp at C1D1 who had undetectable ctDNA after study therapy had an objective response. Patients whose ctHER2-amp decreased on-treatment had better outcomes than patients whose ctHER2-amp remained unchanged. HER2 RNA levels show a correlation to HER2 CLIA IHC status and were significantly higher in patients with clinically documented responses compared to patients with progressive disease (P = 0.03). CONCLUSIONS: The following biomarkers were associated with better outcomes for patients treated with T-DM1 + neratinib: (1) ctHER2-amp (C1D1) or in TP1; (2) Molecular Response scores; (3) loss of detectable ctDNA; (4) RNA levels of HER2; and (5) on-treatment loss of detectable ctHER2-amp. HER2 transcriptional and IHC/FISH status identify HER2-low cases (IHC 1+ or IHC 2+ and FISH negative) in these heavily anti-HER2 treated patients. Due to the small number of patients and samples in this study, the associations we have shown are for hypothesis generation only and remain to be validated in future studies. Clinical Trials registration NCT02236000.

First-line pembrolizumab and trastuzumab in HER2-positive oesophageal, gastric, or gastro-oesophageal junction cancer: an open-label, single-arm, phase 2 trial.

BACKGROUND: Addition of trastuzumab to first-line chemotherapy improves overall survival in patients with HER2-positive metastatic gastric cancer. We assessed the safety and activity of pembrolizumab in combination with trastuzumab and chemotherapy in first-line HER2-positive metastatic oesophagogastric (gastric, oesophageal, or gastroesophageal junction) cancer. METHODS: This study was an investigator-initiated, open-label, non-randomised, single-arm, single centre, phase 2 trial in patients aged 18 years or older with HER2-positive metastatic oesophagogastric cancer. Eligible patients had measurable or evaluable non-measurable disease, Eastern Cooperative Oncology Group performance status of 0, 1, or 2, and left ventricular ejection fraction of at least 53%. Patients were eligible to receive an initial induction cycle of 200 mg flat dose of intravenous pembrolizumab and 8 mg/kg loading dose of intravenous trastuzumab. For subsequent cycles, patients received 130 mg/m2 of intravenous oxaliplatin or 80 mg/m2 of cisplatin on day 1, 850 mg/m2 of oral capecitabine twice a day for 2 weeks followed by 1 week off (or intravenous 5-fluorouracil, 800 mg/m2 per day on days 1-5), and a 200 mg flat dose of intravenous pembrolizumab, and 6 mg/kg of trastuzumab, administered on day 1 of each 3-week cycle. The primary endpoint was 6-month progression-free survival, defined as the proportion of patients alive and free of progression at 6 months, assessed in patients who received at least one dose of trastuzumab and pembrolizumab. The regimen would be considered worthy of further investigation if 26 or more of 37 patients were progression-free at 6 months. This trial is registered with ClinicalTrials.gov, NCT02954536, and is ongoing, but closed to enrolment. FINDINGS: Between Nov 11, 2016, and Jan 23, 2019, 37 patients were enrolled. At the time of data cutoff on Aug 6, 2019, median follow-up among survivors was 13·0 months (IQR 11·7-23·5). The primary endpoint was achieved; 26 (70%; 95% CI 54-83) of 37 patients were progression-free at 6 months. The most common treatment-related adverse event of any grade was neuropathy, which was reported in 36 (97%) of 37 patients. The most common grade 3 or 4 adverse events were lymphocytopenia (seven [19%] patients with grade 3 and two [5%] with grade 4), grade 3 decreased electrolytes (six [16%] patients), and grade 3 anaemia (four [11%] patients). Serious adverse events occurred in two patients patients (both grade 3 nephritis leading to treatment discontinuation). Four patients discontinued pembrolizumab because of immune-related adverse events. There were no treatment-related deaths. INTERPRETATION: Pembrolizumab can be safely combined with trastuzumab and chemotherapy and has promising activity in HER2-positive metastatic oesophagogastric cancer. A randomised phase 3 clinical trial assessing the efficacy and safety of pembrolizumab versus placebo in combination with trastuzumab and chemotherapy in first-line HER2-positive metastatic oesophagogastric cancer is underway. FUNDING: Merck & Co.

Comprehensive Analysis of AR Alterations in Circulating Tumor DNA from Patients with Advanced Prostate Cancer.

BACKGROUND: Somatic alterations in circulating tumor DNA (ctDNA) may be associated with treatment response or prognosis in prostate cancer (PCa). The goal was to characterize androgen receptor gene (AR) amplifications and mutations detected in ctDNA from patients with PCa and to further understand the somatic genetic heterogeneity of advanced prostate cancer. PATIENTS AND METHODS: This study included a heterogeneous group of 892 patients with advanced PCa (predominantly castrate-resistant prostate cancer) with AR alterations detected in ctDNA that underwent next-generation sequencing of 54 to 73 genes via Guardant360 testing (Guardant Health, Inc., Redwood City, CA). Distribution and summary of AR alterations detected, the association of AR alterations with other genes, and a pathway analysis are reported. RESULTS: The median absolute plasma copy number of AR amplifications was 3.3 (range, 1.2-165.2). Many patients had multiple AR mutations; a total of 112 unique mutations were identified in AR, including L702H (25%), T878A (14%), H875Y (11%), W742C (8%), W742L (4%), F877L (2%), and T878S (2%). Other ctDNA gene alterations in the Guardant assays included TP53 (50%), MYC (34%), BRAF (32%), PIK3CA (29%), MET (25%), CDK6 (26%), EGFR (24%), FGFR1 (21%), and APC (12%). Many of these non-AR alterations are not tissue verified in other studies. AR amplification cosegregated with alterations in MYC (p < .001), BRAF (p < .001), PIK3CA (p < .001), MET (p < .001), CDK6 (p < .001), EGFR (p < .001), FGFR1 (p = .391), and more. Alterations in APC were significantly associated with mutations in AR (p < .001). CONCLUSION: Several AR alterations and concomitant non-AR alterations that associate with drug resistance were detected. These findings provide additional insights into the heterogeneity of advanced prostate cancer. IMPLICATIONS FOR PRACTICE: The goal was to characterize androgen receptor gene (AR) amplifications and mutations detected in circulating tumor DNA (ctDNA) from patients with prostate cancer in relation to non-AR gene alterations detected in the ctDNA landscape. The study included 892 patients with prostate cancer with AR alterations in ctDNA. AR alterations were significantly associated with other gene alterations detected in ctDNA. The common AR mutations found are linked to resistance to abiraterone, enzalutamide, or bicalutamide. Characterization of the circulating AR landscape and gene alterations provides potential additional insight into the somatic genetic heterogeneity of advanced prostate cancer.

Circulating tumor DNA alterations in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: Because cell-free DNA (cfDNA) analysis facilitates the noninvasive genomic profiling of metastatic castration-resistant prostate cancer (mCRPC), the authors evaluated the association between cfDNA alterations and outcomes and evolution with therapy. METHODS: Patients with mCRPC underwent cfDNA genomic profiling using Guardant360, which examines major cancer-associated genes. Clinical factors, therapy information, failure-free survival, and overall survival (OS) were obtained for select patients. The association between genomic alterations and outcomes was investigated. RESULTS: Of 514 men with mCRPC, 482 (94%) had ≥1 circulating tumor DNA (ctDNA) alteration. The most common recurrent somatic mutations were in TP53 (36%), androgen receptor (AR) (22%), adenomatous polyposis coli (APC) (10%), neurofibromin 1 (NF1) (9%), epidermal growth factor receptor (EGFR), catenin beta-1 (CTNNB1), and AT-rich interactive domain-containing protein 1A (ARID1A) (6% each); and BRCA1, BRCA2, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (5% each) The most common genes with increased copy numbers were AR (30%), MYC (20%), and BRAF (18%). Clinical outcomes were available for 163 patients, 46 of whom (28.8%) were untreated for mCRPC. A higher number of ctDNA alterations, AR alterations, and amplifications of MYC and BRAF were associated with worse failure-free survival and/or OS. On multivariable analysis, MYC amplification remained significantly associated with OS. Prior therapy and serial profiling demonstrated the evolution of alterations in AR and other genes. CONCLUSIONS: ctDNA frequently was detected in this large cohort of "real-world" patients with mCRPC, and the alterations appeared to be similar to previously reported tumor tissue alterations. A higher number of alterations, and AR and MYC alterations, appear to compromise clinical outcomes, suggesting a role for immune checkpoint inhibitors and novel AR and BET inhibitors in selected patients.

Characterization of metastatic urothelial carcinoma via comprehensive genomic profiling of circulating tumor DNA.

BACKGROUND: Biomarker-guided clinical trials are increasingly common in metastatic urothelial carcinoma (mUC), yet patients for whom contemporary tumor tissue is not available are not eligible. Technological advancements in sequencing have made cell-free circulating DNA (cfDNA) next-generation sequencing (NGS) readily available in the clinic. The objective of the current study was to determine whether the genomic profile of mUC detected by NGS of cfDNA is similar to historical tumor tissue NGS studies. A secondary objective was to determine whether the frequency of genomic alterations (GAs) differed between lower tract mUC (mLTUC) and upper tract mUC (mUTUC). METHODS: Patients from 13 academic medical centers in the United States who had a diagnosis of mUC between 2014 and 2017 and for whom cfDNA NGS results were available were included. cfDNA profiling was performed using a commercially available platform (Guardant360) targeting 73 genes. RESULTS: Of 369 patients with mUC, 294 were diagnosed with mLTUC and 75 with mUTUC. A total of 2130 GAs were identified in the overall mUC cohort: 1610 and 520, respectively, in the mLTUC and mUTUC cohorts. In the mLTUC cohort, frequently observed GAs were similar between cfDNA NGS and historical tumor tissue studies, including tumor protein p53 (TP53) (P = 1.000 and .115, respectively), AT-rich interaction domain 1A (ARID1A) (P = .058 and .058, respectively), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (P = .058 and .067, respectively), erb-b2 receptor tyrosine kinase 2 (ERBB2) (P = .565 and .074, respectively), and fibroblast growth factor receptor 3 (FGFR3) (P = .164 and .014, respectively). No significant difference was observed with regard to the frequency of GAs between patients with mLTUC and mUTUC. CONCLUSIONS: Among patients with mUC for whom no tumor tissue was available, cfDNA NGS was able to identify a similar profile of GAs for biomarker-driven clinical trials compared with tumor tissue. Despite the more aggressive clinical course, cases of mUTUC demonstrated a circulating tumor DNA genomic landscape that was similar to that of mLTUC. Cancer 2018;124:2115-24. © 2018 American Cancer Society.

Determinants of Survival with Combined HER2 and PD-1 Blockade in Metastatic Esophagogastric Cancer.

PURPOSE: We report updated clinical outcomes from a phase II study of pembrolizumab, trastuzumab, and chemotherapy (PTC) in metastatic esophagogastric cancer in conjunction with outcomes from an independent Memorial Sloan Kettering (MSK) cohort. PATIENTS AND METHODS: The significance of pretreatment 89Zr-trastuzumab PET, plasma circulating tumor DNA (ctDNA) dynamics, and tumor HER2 expression and whole exome sequencing was evaluated to identify prognostic biomarkers and mechanisms of resistance in patients treated on-protocol with PTC. Additional prognostic features were evaluated using a multivariable Cox regression model of trastuzumab-treated MSK patients (n = 226). Single-cell RNA sequencing (scRNA-seq) data from MSK and Samsung were evaluated for mechanisms of therapy resistance. RESULTS: 89Zr-trastuzumab PET, scRNA-seq, and serial ctDNA with CT imaging identified how pre-treatment intrapatient genomic heterogeneity contributes to inferior progression-free survival (PFS). We demonstrated that the presence of intensely avid lesions by 89Zr-trastuzumab PET declines in tumor-matched ctDNA by 3 weeks, and clearance of tumor-matched ctDNA by 9 weeks were minimally invasive biomarkers of durable PFS. Paired pre- and on-treatment scRNA-seq identified rapid clearance of HER2-expressing tumor clones with expansion of clones expressing a transcriptional resistance program, which was associated with MT1H, MT1E, MT2A, and MSMB expression. Among trastuzumab-treated patients at MSK, ERBB2 amplification was associated with improved PFS, while alterations in MYC and CDKN2A/B were associated with inferior PFS. CONCLUSIONS: These findings highlight the clinical relevance of identifying baseline intrapatient heterogeneity and serial ctDNA monitoring of HER2-positive esophagogastric cancer patients to identify early evidence of treatment resistance, which could guide proactive therapy escalation or deescalation.

Biomarker Discovery and Outcomes for Comprehensive Cell-Free Circulating Tumor DNA Versus Standard-of-Care Tissue Testing in Advanced Non-Small-Cell Lung Cancer.

PURPOSE: Treatment guidelines for advanced non-small-cell lung cancer (aNSCLC) recommend broad molecular profiling for targeted therapy selection. This study prospectively assessed comprehensive next-generation sequencing (NGS) of cell-free circulating tumor DNA (cfDNA) compared with standard-of-care (SOC) tissue-based testing to identify guideline-recommended alterations in aNSCLC. PATIENTS AND METHODS: Patients with treatment-naïve aNSCLC were tested using a well-validated NGS cfDNA panel, and results were compared with SOC tissue testing. The primary objective was noninferiority of cfDNA vs. tissue analysis for the detection of two guideline-recommended biomarkers (EGFR and ALK) and an additional six actionable biomarkers. Secondary analyses included tissue versus cfDNA biomarker discovery, overall response rate (ORR), progression-free survival (PFS) to targeted therapy, and positive predictive value (PPV) of cfDNA. RESULTS: The primary objective was met with cfDNA identifying actionable mutations in 46 patients versus 48 by tissue (P < .05). In total, 0/186 patients were genotyped for all eight biomarkers with tissue, compared with 90.8% using cfDNA. Targetable alterations or KRAS were identified in 80.7% when cfDNA was used first versus 57.1% when tissue was used first. PPV for cfDNA-detected EGFR was 100.0% (25/25). ORR and PFS in patients receiving targeted therapy based on tissue or cfDNA were similar to those previously reported. CONCLUSION: This prospective study confirms a previous report that comprehensive cfDNA testing is noninferior to SOC tissue testing in detecting aNSCLC-recommended biomarkers. Furthermore, cfDNA-based first-line therapy produced outcomes similar to tissue-based testing, demonstrating the clinical utility of comprehensive cfDNA genotyping as the initial genotyping modality in patients with treatment-naïve aNSCLC when tissue is insufficient or when all actionable biomarkers cannot be rapidly assessed.

Serial Monitoring of Circulating Tumor DNA by Next-Generation Gene Sequencing as a Biomarker of Response and Survival in Patients With Advanced NSCLC Receiving Pembrolizumab-Based Therapy.

Although the majority of patients with metastatic non-small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging. MATERIALS AND METHODS: Patients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded. RESULTS: Among 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P < .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P < .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION: Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies.

Circulating Tumor DNA Alterations in Advanced Urothelial Carcinoma and Association with Clinical Outcomes: A Pilot Study.

Cell-free circulating DNA (cfDNA) can be used for noninvasive profiling of tumor genomic aberrations. We hypothesized that molecular alterations may inform prognostication in advanced urothelial carcinoma (aUC). We evaluated 124 aUC patients who underwent cfDNA analysis using a 73-gene sequencing panel (Guardant360). The association of molecular alterations and clinical factors with overall survival (OS) and failure-free-survival (FFS) was evaluated using the Kaplan-Meier method and Cox proportional-hazards regression. The median age was 72yr, and 65 patients (52.4%) received prior therapy with platinum, 21 (17.1%) with a taxane, and ten (8.1%) with a PD-1/PD-L1 inhibitor. At least one genomic alteration was detected in 112 patients (90.3%). The median number of alterations per sample was four (range 0-80). Commonly altered genes included TP53 (54.8%), PIK3CA (24.2%), ARID1A (22.6%), ERBB2 (19.4%), EGFR (16.1%), NF1 (13.7%), RB1 (12.9%), FGFR3 (11.3%), BRAF (10.5%), BRCA1 (10.5%), and RAF1 (8.9%). BRCA1 and RAF1 alterations were associated with worse OS (hazard ratio [HR] 2.48; p=0.07; HR 4.87; p=0.007) and FFS (HR 2.35; p=0.016; HR 2.40; p=0.047). Poor Eastern Cooperative Oncology Group performance status and the presence of visceral metastasis were associated with shorter OS; genomic evolution was observed. In conclusion, cfDNA molecular alterations were detected in most aUC patients. BRCA1 and RAF1 alterations were negatively prognostic, supporting further evaluation of DNA damage response and RAF kinase inhibitors. PATIENT SUMMARY: Noninvasive testing of cell-free circulating DNA in advanced urothelial carcinoma identifies clinically relevant molecular aberrations. Alterations in BRCA1 and RAF1 genes appear to be negatively associated with clinical outcomes, supporting further study of DNA damage response and RAF kinase inhibitors in selected patients.

Alterations in PTEN and ESR1 promote clinical resistance to alpelisib plus aromatase inhibitors.

Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial's primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

MET Alterations Are a Recurring and Actionable Resistance Mechanism in ALK-Positive Lung Cancer.

PURPOSE: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. EXPERIMENTAL DESIGN: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance. RESULTS: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. CONCLUSIONS: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Molecular Landscape of BRAF-Mutant NSCLC Reveals an Association Between Clonality and Driver Mutations and Identifies Targetable Non-V600 Driver Mutations.

INTRODUCTION: Approximately 4% of NSCLC harbor BRAF mutations, and approximately 50% of these are non-V600 mutations. Treatment of tumors harboring non-V600 mutations is challenging because of functional heterogeneity and lack of knowledge regarding their clinical significance and response to targeted agents. METHODS: We conducted an integrative analysis of BRAF non-V600 mutations using genomic profiles of BRAF-mutant NSCLC from the Guardant360 database. BRAF mutations were categorized by clonality and class (1 and 2: RAS-independent; 3: RAS-dependent). Cell viability assays were performed in Ba/F3 models. Drug screens were performed in NSCLC cell lines. RESULTS: A total of 305 unique BRAF mutations were identified. Missense mutations were most common (276, 90%), and 45% were variants of unknown significance. F468S and N581Y were identified as novel activating mutations. Class 1 to 3 mutations had higher clonality than mutations of unknown class (p < 0.01). Three patients were treated with MEK with or without BRAF inhibitors. Patients harboring G469V and D594G mutations did not respond, whereas a patient with the L597R mutation had a durable response. Trametinib with or without dabrafenib, LXH254, and lifirafenib had more potent inhibition of BRAF non-V600-mutant NSCLC cell lines than other MEK, BRAF, and ERK inhibitors, comparable with the inhibition of BRAF V600E cell line. CONCLUSIONS: In BRAF-mutant NSCLC, clonality is higher in known functional mutations and may allow identification of variants of unknown significance that are more likely to be oncogenic drivers. Our data indicate that certain non-V600 mutations are responsive to MEK and BRAF inhibitors. This integration of genomic profiling and drug sensitivity may guide the treatment for BRAF-mutant NSCLC.

Baseline Plasma Tumor Mutation Burden Predicts Response to Pembrolizumab-based Therapy in Patients with Metastatic Non-Small Cell Lung Cancer.

PURPOSE: The role of plasma-based tumor mutation burden (pTMB) in predicting response to pembrolizumab-based first-line standard-of-care therapy for metastatic non-small cell lung cancer (mNSCLC) has not been explored. EXPERIMENTAL DESIGN: A 500-gene next-generation sequencing panel was used to assess pTMB. Sixty-six patients with newly diagnosed mNSCLC starting first-line pembrolizumab-based therapy, either alone or in combination with chemotherapy, were enrolled (Clinicaltrial.gov identifier: NCT03047616). Response was assessed using RECIST 1.1. Associations were made for patient characteristics, 6-month durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS). RESULTS: Of 66 patients, 52 (78.8%) were pTMB-evaluable. Median pTMB was 16.8 mutations per megabase (mut/Mb; range, 1.9-52.5) and was significantly higher for patients achieving DCB compared with no durable benefit (21.3 mut/Mb vs. 12.4 mut/Mb, P = 0.003). For patients with pTMB ≥ 16 mut/Mb, median PFS was 14.1 versus 4.7 months for patients with pTMB < 16 mut/Mb [HR, 0.30 (0.16-0.60); P < 0.001]. Median OS for patients with pTMB ≥ 16 was not reached versus 8.8 months for patients with pTMB < 16 mut/Mb [HR, 0.48 (0.22-1.03); P = 0.061]. Mutations in ERBB2 exon 20, STK11, KEAP1, or PTEN were more common in patients with no DCB. A combination of pTMB ≥ 16 and absence of negative predictor mutations was associated with PFS [HR, 0.24 (0.11-0.49); P < 0.001] and OS [HR, 0.31 (0.13-0.74); P = 0.009]. CONCLUSIONS: pTMB ≥ 16 mut/Mb is associated with improved PFS after first-line standard-of-care pembrolizumab-based therapy in mNSCLC. STK11/KEAP1/PTEN and ERBB2 mutations may help identify pTMB-high patients unlikely to respond. These results should be validated in larger prospective studies.

Efficacy and Determinants of Response to HER Kinase Inhibition in HER2-Mutant Metastatic Breast Cancer.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib. SIGNIFICANCE: HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer.This article is highlighted in the In This Issue feature, p. 161.

BRAF Mutations Classes I, II, and III in NSCLC Patients Included in the SLLIP Trial: The Need for a New Pre-Clinical Treatment Rationale.

BRAF V600 mutations have been found in 1-2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50-80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decisions.

Identification of osimertinib-resistant EGFR L792 mutations by cfDNA sequencing: oncogenic activity assessment and prevalence in large cfDNA cohort.

Cell-free DNA (cfDNA) next-generation sequencing has the potential to capture tumor heterogeneity and genomic evolution under treatment pressure in a non-invasive manner. Here, we report the detection of EGFR L792 mutations, a non-covalent mechanism of osimertinib resistance, using Guardant360 cfDNA testing in a patient with metastatic EGFR-mutant non-small cell lung cancer (NSCLC) whose disease progressed on osimertinib. We subsequently analyzed a large cohort of over 1800 additional patient samples harboring an EGFR T790M mutation and identified a concomitant L792 mutation in a total of 22 (1.2%) cases. In vitro functional assays demonstrated that the EGFR L858R/T790M/L792F/H mutations conferred intermediate-level resistance to osimertinib. Further understanding of potential acquired resistance mechanisms to targeted therapy may help inform treatment strategy in EGFR-mutant NSCLC.

Therapeutic outcomes in non-small cell lung cancer with BRAF mutations: a single institution, retrospective cohort study.

BACKGROUND: Data describing therapeutic outcomes in patients with non-small cell lung cancers (NSCLC) with BRAF mutations remains limited. METHODS: We conducted a retrospective cohort study of 31 patients with metastatic NSCLC treated at Duke University Hospital who had been identified by next-generation sequencing methods to bear a BRAF mutation in their tumor in order to evaluate clinical response to immunotherapy and chemotherapy. RESULTS: Sixty-five percent of patients identified in this cohort were current or former smokers. Fourteen (45.2%) of patients had a BRAF V600E mutation and 17 (54.8%) had a non-V600E mutation. Median progression-free survival (PFS) in the 23 patients who received first-line chemotherapy was 6.4 months [95% confidence interval (CI), 2.3 to 13.0]. Overall survival (OS) in patients who received first-line chemotherapy showed a median survival of 18 months (95% CI, 7.4 to 28.6). OS comparing patients who had never received immunotherapy at any point was 18.4 months (95% CI, 4.1 to NE) compared to 19.0 months (95% CI, 9.9 to 28.6) in those who had received immunotherapy. We did not find a statistically significant difference in OS in patients with BRAF V600E, BRAF amplification, or non-V600E mutations. There was also no difference in OS in patients treated with targeted BRAF inhibitors compared to those who were not treated with targeted BRAF inhibitors. CONCLUSIONS: We describe therapeutic outcomes for patients with metastatic NSCLC with BRAF mutations treated with either cytotoxic chemotherapy or immunotherapy. Although the sample size is small, the survival curves do not suggest improved clinical activity in this population when treated with immunotherapy.

Genomic Profiling for KRAS, NRAS, BRAF, Microsatellite Instability, and Mismatch Repair Deficiency Among Patients With Metastatic Colon Cancer.

PURPOSE: Genomic testing is recognized in national guidelines as essential to guide appropriate therapy selection in metastatic colorectal cancer. Previous studies report adherence to testing guidelines is suboptimal, but current testing rates have not been assessed. This study reports testing rates in metastatic colon cancer (mCC) for guideline-recommended biomarkers in a US-based population. MATERIALS AND METHODS: A retrospective review of data extracted from electronic medical records was performed to identify patients with pathologically confirmed mCC and describe patterns of guideline-aligned biomarker testing. Data were extracted from the electronic health records of 1,497 patients treated at 23 practices across the United States. Both community and academic centers were represented. RESULTS: A total of 1,497 patients with mCC diagnosed between January 1, 2013 and December 31, 2017 were identified. Guideline-aligned biomarker testing rates for RAS, BRAF, and microsatellite instability/mismatch repair deficiency over this study period were 41%, 43%, and 51%, respectively. Patients were more likely to have guideline-aligned testing for RAS and BRAF if they were treated at an academic center, were diagnosed with de novo metastatic disease, and were female. In addition, patients < 65 years of age were more likely to have guideline-aligned RAS testing. Of the 177 patients (12% of cohort) who received anti-epidermal growth factor receptor therapy, only 50 (28%) had complete guideline-aligned biomarker testing. CONCLUSION: Despite guideline recommendations and significant therapeutic implications, overall biomarker testing rates in mCC remain suboptimal. Adherence to guideline-recommended biomarker testing would potentially reduce exposure to expensive and ineffective therapies, resulting in improved patient outcomes.

Plasma HER2 (ERBB2) Copy Number Predicts Response to HER2-targeted Therapy in Metastatic Colorectal Cancer.

PURPOSE: ERBB2 (HER2) amplification is an emerging biomarker in colon cancer, conferring sensitivity to combination anti-HER2 therapy. Measurement of HER2 copy number is typically performed using surgical specimens, but cell-free circulating tumor DNA (ctDNA) analysis may be a noninvasive alternative. We determined the sensitivity of plasma copy number (pCN) for detecting ERBB2 amplifications and whether pCN correlated with tissue-detected copy number. We also assessed response to HER2-targeted therapy based on pCN and suggest a pCN threshold predictive of response. EXPERIMENTAL DESIGN: Forty-eight pretreatment and progression plasma samples from 29 HER2-positive patients in the HERACLES A clinical trial were tested using the Guardant360 cfDNA assay. We correlated ERRB2 pCN with progression-free survival (PFS) and best objective response (BOR) and applied an adjustment method based on tumor DNA shedding using the maximum mutant allele fraction as a surrogate for tumor content to accurately determine the pCN threshold predictive of response. RESULTS: Forty-seven of 48 samples had detectable ctDNA, and 46 of 47 samples were ERBB2-amplified on the basis of cfDNA [2.55-122 copies; 97.9% sensitivity (95% confidence interval, 87.2%-99.8%)]. An adjusted ERBB2 pCN of ≥25.82 copies correlated with BOR and PFS (P = 0.0347). CONCLUSIONS: cfDNA is a viable alternative to tissue-based genotyping in the metastatic setting. The cfDNA platform utilized correctly identified 28 of 29 (96.6%) of pretreatment samples as ERBB2-amplified and predicted benefit from HER2-targeted therapy. In this study, an observed pCN of 2.4 and an adjusted pCN of 25.82 copies of ERBB2 are proposed to select patients who will benefit from HER2-targeted therapy.