Targeted mutation detection in breast cancer using MammaSeq™.

BACKGROUND: Breast cancer is the most common invasive cancer among women worldwide. Next-generation sequencing (NGS) has revolutionized the study of cancer across research labs around the globe; however, genomic testing in clinical settings remains limited. Advances in sequencing reliability, pipeline analysis, accumulation of relevant data, and the reduction of costs are rapidly increasing the feasibility of NGS-based clinical decision making. METHODS: We report the development of MammaSeq, a breast cancer-specific NGS panel, targeting 79 genes and 1369 mutations, optimized for use in primary and metastatic breast cancer. To validate the panel, 46 solid tumors and 14 plasma circulating tumor DNA (ctDNA) samples were sequenced to a mean depth of 2311× and 1820×, respectively. Variants were called using Ion Torrent Suite 4.0 and annotated with cravat CHASM. CNVKit was used to call copy number variants in the solid tumor cohort. The oncoKB Precision Oncology Database was used to identify clinically actionable variants. Droplet digital PCR was used to validate select ctDNA mutations. RESULTS: In cohorts of 46 solid tumors and 14 ctDNA samples from patients with advanced breast cancer, we identified 592 and 43 protein-coding mutations. Mutations per sample in the solid tumor cohort ranged from 1 to 128 (median 3), and the ctDNA cohort ranged from 0 to 26 (median 2.5). Copy number analysis in the solid tumor cohort identified 46 amplifications and 35 deletions. We identified 26 clinically actionable variants (levels 1-3) annotated by OncoKB, distributed across 20 out of 46 cases (40%), in the solid tumor cohort. Allele frequencies of ESR1 and FOXA1 mutations correlated with CA.27.29 levels in patient-matched blood draws. CONCLUSIONS: In solid tumor biopsies and ctDNA, MammaSeq detects clinically actionable mutations (OncoKB levels 1-3) in 22/46 (48%) solid tumors and in 4/14 (29%) of ctDNA samples. MammaSeq is a targeted panel suitable for clinically actionable mutation detection in breast cancer.

The molecular landscape of premenopausal breast cancer.

INTRODUCTION: Breast cancer in premenopausal women (preM) is frequently associated with worse prognosis compared to that in postmenopausal women (postM), and there is evidence that preM estrogen receptor-positive (ER+) tumors may respond poorly to endocrine therapy. There is, however, a paucity of studies characterizing molecular alterations in premenopausal tumors, a potential avenue for personalizing therapy for this group of women. METHODS: Using TCGA and METABRIC databases, we analyzed gene expression, copy number, methylation, somatic mutation, and reverse-phase protein array data in breast cancers from >2,500 preM and postM women. RESULTS: PreM tumors showed unique gene expression compared to postM tumors, however, this difference was limited to ER+ tumors. ER+ preM tumors showed unique DNA methylation, copy number and somatic mutations. Integrative pathway analysis revealed that preM tumors had elevated integrin/laminin and EGFR signaling, with enrichment for upstream TGFß-regulation. Finally, preM tumors showed three different gene expression clusters with significantly different outcomes. CONCLUSION: Together these data suggest that ER+ preM tumors have distinct molecular characteristics compared to ER+ postM tumors, particularly with respect to integrin/laminin and EGFR signaling, which may represent therapeutic targets in this subgroup of breast cancers.

Candidate mechanisms of acquired resistance to first-line osimertinib in EGFR-mutated advanced non-small cell lung cancer.

Osimertinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), potently and selectively inhibits EGFR-TKI-sensitizing and EGFR T790M resistance mutations. In the Phase III FLAURA study (NCT02296125), first-line osimertinib improved outcomes vs comparator EGFR-TKIs in EGFRm advanced non-small cell lung cancer. This analysis identifies acquired resistance mechanisms to first-line osimertinib. Next-generation sequencing assesses circulating-tumor DNA from paired plasma samples (baseline and disease progression/treatment discontinuation) in patients with baseline EGFRm. No EGFR T790M-mediated acquired resistance are observed; most frequent resistance mechanisms are MET amplification (n = 17; 16%) and EGFR C797S mutations (n = 7; 6%). Future research investigating non-genetic acquired resistance mechanisms is warranted.

Analysis of acquired resistance mechanisms to osimertinib in patients with EGFR-mutated advanced non-small cell lung cancer from the AURA3 trial.

Osimertinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), potently and selectively inhibits EGFR-TKI-sensitizing and EGFR T790M resistance mutations. This analysis evaluates acquired resistance mechanisms to second-line osimertinib (n = 78) in patients with EGFR T790M advanced non-small cell lung cancer (NSCLC) from AURA3 (NCT02151981), a randomized phase 3 study comparing osimertinib with chemotherapy. Plasma samples collected at baseline and disease progression/treatment discontinuation are analyzed using next-generation sequencing. Half (50%) of patients have undetectable plasma EGFR T790M at disease progression and/or treatment discontinuation. Fifteen patients (19%) have >1 resistance-related genomic alteration; MET amplification (14/78, 18%) and EGFR C797X mutation (14/78, 18%).

Early Clearance of Plasma Epidermal Growth Factor Receptor Mutations as a Predictor of Outcome on Osimertinib in Advanced Non-Small Cell Lung Cancer; Exploratory Analysis from AURA3 and FLAURA.

PURPOSE: Plasma circulating tumor DNA (ctDNA) analysis is used for genotyping advanced non-small cell lung cancer (NSCLC); monitoring dynamic ctDNA changes may be used to predict outcomes. PATIENTS AND METHODS: This was a retrospective, exploratory analysis of two phase III trials [AURA3 (NCT02151981), FLAURA (NCT02296125)]. All patients had EGFR mutation-positive (EGFRm; ex19del or L858R) advanced NSCLC; AURA3 also included T790M-positive NSCLC. Osimertinib (FLAURA, AURA3), or comparator EGFR-tyrosine kinase inhibitor (EGFR-TKI; gefitinib/erlotinib; FLAURA), or platinum-based doublet chemotherapy (AURA3) was given. Plasma EGFRm was analyzed at baseline and Weeks 3/6 by droplet digital PCR. Outcomes were assessed by detectable/non-detectable baseline plasma EGFRm and plasma EGFRm clearance (non-detection) at Weeks 3/6. RESULTS: In AURA3 (n = 291), non-detectable versus detectable baseline plasma EGFRm had longer median progression-free survival [mPFS; HR, 0.48; 95% confidence interval (CI), 0.33-0.68; P < 0.0001]. In patients with Week 3 clearance versus non-clearance (n = 184), respectively, mPFS (months; 95% CI) was 10.9 (8.3-12.6) versus 5.7 (4.1-9.7) with osimertinib and 6.2 (4.0-9.7) versus 4.2 (4.0-5.1) with platinum-pemetrexed. In FLAURA (n = 499), mPFS was longer with non-detectable versus detectable baseline plasma EGFRm (HR, 0.54; 95% CI, 0.41-0.70; P < 0.0001). For Week 3 clearance versus non-clearance (n = 334), respectively, mPFS was 19.8 (15.1 to not calculable) versus 11.3 (9.5-16.5) with osimertinib and 10.8 (9.7-11.1) versus 7.0 (5.6-8.3) with comparator EGFR-TKI. Similar outcomes were observed by Week 6 clearance/non-clearance. CONCLUSIONS: Plasma EGFRm analysis as early as 3 weeks on-treatment has the potential to predict outcomes in EGFRm advanced NSCLC.

Osimertinib + Savolitinib to Overcome Acquired MET-Mediated Resistance in Epidermal Growth Factor Receptor-Mutated, MET-Amplified Non-Small Cell Lung Cancer: TATTON.

MET-inhibitor and EGFR tyrosine kinase inhibitor (EGFR-TKI) combination therapy could overcome acquired MET-mediated osimertinib resistance. We present the final phase Ib TATTON (NCT02143466) analysis (Part B, n = 138/Part D, n = 42) assessing oral savolitinib 600 mg/300 mg once daily (q.d.) + osimertinib 80 mg q.d. in patients with MET-amplified, EGFR-mutated (EGFRm) advanced non-small cell lung cancer (NSCLC) and progression on prior EGFR-TKI. An acceptable safety profile was observed. In Parts B and D, respectively, objective response rates were 33% to 67% and 62%, and median progression-free survival (PFS) was 5.5 to 11.1 months and 9.0 months. Increased antitumor activity may occur with MET copy number ≥10. EGFRm circulating tumor DNA clearance on treatment predicted longer PFS in patients with detectable baseline ctDNA, while acquired resistance mechanisms to osimertinib + savolitinib were mediated by MET, EGFR, or KRAS alterations. SIGNIFICANCE: The savolitinib + osimertinib combination represents a promising therapy in patients with MET-amplified/overexpressed, EGFRm advanced NSCLC with disease progression on a prior EGFR-TKI. Acquired resistance mechanisms to this combination include those via MET, EGFR, and KRAS. On-treatment ctDNA dynamics can predict clinical outcomes and may provide an opportunity to inform earlier decision-making. This article is highlighted in the In This Issue feature, p. 1.

Improving ChIP-seq peak-calling for functional co-regulator binding by integrating multiple sources of biological information.

BACKGROUND: Chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) is increasingly being applied to study genome-wide binding sites of transcription factors. There is an increasing interest in understanding the mechanism of action of co-regulator proteins, which do not bind DNA directly, but exert their effects by binding to transcription factors such as the estrogen receptor (ER). However, due to the nature of detecting indirect protein-DNA interaction, ChIP-seq signals from co-regulators can be relatively weak and thus biologically meaningful interactions remain difficult to identify. RESULTS: In this study, we investigated and compared different statistical and machine learning approaches including unsupervised, supervised, and semi-supervised classification (self-training) approaches to integrate multiple types of genomic and transcriptomic information derived from our experiments and public database to overcome difficulty of identifying functional DNA binding sites of the co-regulator SRC-1 in the context of estrogen response. Our results indicate that supervised learning with naïve Bayes algorithm significantly enhances peak calling of weak ChIP-seq signals and outperforms other machine learning algorithms. Our integrative approach revealed many potential ERα/SRC-1 DNA binding sites that would otherwise be missed by conventional peak calling algorithms with default settings. CONCLUSIONS: Our results indicate that a supervised classification approach enables one to utilize limited amounts of prior knowledge together with multiple types of biological data to enhance the sensitivity and specificity of the identification of DNA binding sites from co-regulator proteins.

Who's driving anyway? Herculean efforts to identify the drivers of breast cancer.

The continuing advancement of sequencing technologies has made the systematic identification of all driving somatic events in cancer a possibility. In the June 2012 issue of Nature, five papers show some significant headway in this endeavor, each a herculean effort of genome sequencing, and transcriptome and copy number analysis resulting in data on thousands of breast cancers. Integrating these massive datasets, the authors were able to further subdivide breast cancer and identify a number of novel driver genes. While the studies represent a leap forward in describing the genomics of breast cancer, and clearly highlight the tremendous diversity between tumors, the studies only scrape the surface of molecular changes in breast tumors, with more granularity to come from the study of epigenomics, single cell sequencing, and so on. The immediate importance of the data to clinical care is currently unknown, and will depend upon detailed identification and functional analysis of driver mutations.

Osimertinib plus Selumetinib in EGFR-Mutated Non-Small Cell Lung Cancer After Progression on EGFR-TKIs: A Phase Ib, Open-Label, Multicenter Trial (TATTON Part B).

BACKGROUND: MEK/ERK inhibition can overcome acquired resistance to osimertinib in preclinical models. Osimertinib [EGFR-tyrosine kinase inhibitor (TKI)] plus selumetinib (MEK1/2 inhibitor) was assessed in the global TATTON study. METHODS: This multicenter, open-label, phase Ib study expansion cohort enrolled patients (aged ≥18 years) with MET-negative, EGFRm advanced NSCLC who had progressed on EGFR-TKIs. Patients were assigned to one of two cohorts by prior first- or second-generation or T790M-directed EGFR-TKI and received osimertinib 80 mg every day and intermittent selumetinib 75 mg twice a day orally. Safety and tolerability (primary objective) and antitumor activity determined by objective response rate (ORR), and progression-free survival (PFS) using RECIST v1.1 were assessed. Data cutoff: March 4, 2020. RESULTS: Forty-seven patients received treatment (prior first- or second-generation EGFR-TKI, n = 12; prior T790M-directed EGFR-TKI, n = 35). Forty-four (94%) patients were Asian; 30 (64%) had baseline exon 19 deletion. Most common AEs were diarrhea (89%), decreased appetite (40%), and stomatitis (32%); 11/47 patients (23%) had an AE Grade ≥3 possibly causally selumetinib-related. ORR was 66.7% [95% confidence interval (CI), 34.9-90.1] in the prior first- or second-generation EGFR-TKI group, 22.9% (95% CI, 10.4-40.1) in the prior T790M-directed EGFR-TKI group, and 34.0% (95% CI, 20.9-49.3) overall; median PFS was 15.0 (95% CI, 2.7-33.0), 2.8 (95% CI, 1.6-5.5), and 4.2 months (95% CI, 2.7-7.2), respectively. CONCLUSIONS: In this small study, AEs and tolerability of osimertinib plus selumetinib were as expected, on the basis of previous studies. The combination demonstrated antitumor activity supportive of further investigation in patients with MET-negative, EGFRm advanced NSCLC who had progressed on a previous EGFR-TKI.

A Novel Mouse Model for SNP in Steroid Receptor Co-Activator-1 Reveals Role in Bone Density and Breast Cancer Metastasis.

The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.

The genomic landscape of metastatic breast cancer: Insights from 11,000 tumors.

BACKGROUND: Metastatic breast cancer is the leading cause of cancer death in women, but the genomics of metastasis in breast cancer are poorly studied. METHODS: We explored a set of 11,616 breast tumors, including 5,034 metastases, which had undergone targeted sequencing during standard clinical care. RESULTS: Besides the known hotspot mutations in ESR1, we observed a metastatic enrichment of previously unreported, lower-prevalence mutations in the ligand-binding domain, implying that these mutations may also be functional. Furthermore, individual ESR1 hotspots are significantly enriched in specific metastatic tissues and histologies, suggesting functional differences between these mutations. Other alterations enriched across all metastases include loss of function of the CDK4 regulator CDKN1B, and mutations in the transcription factor CTCF. Mutations enriched at specific metastatic sites generally reflect biology of the target tissue and may be adaptations to growth in the local environment. These include PTEN and ASXL1 alterations in brain metastases and NOTCH1 alterations in skin. We observed an enrichment of KRAS, KEAP1, STK11 and EGFR mutations in lung metastases. However, the patterns of other mutations in these tumors indicate that these are misdiagnosed lung primaries rather than breast metastases. CONCLUSIONS: An order-of-magnitude increase in samples relative to previous studies allowed us to detect novel genomic characteristics of metastatic cancer and to expand and clarify previous findings.

Nuclear receptor coregulator SNP discovery and impact on breast cancer risk.

BACKGROUND: Coregulator proteins are "master regulators", directing transcriptional and posttranscriptional regulation of many target genes, and are critical in many normal physiological processes, but also in hormone driven diseases, such as breast cancer. Little is known on how genetic changes in these genes impact disease development and progression. Thus, we set out to identify novel single nucleotide polymorphisms (SNPs) within SRC-1 (NCoA1), SRC-3 (NCoA3, AIB1), NCoR (NCoR1), and SMRT (NCoR2), and test the most promising SNPs for associations with breast cancer risk. METHODS: The identification of novel SNPs was accomplished by sequencing the coding regions of these genes in 96 apparently normal individuals (48 Caucasian Americans, 48 African Americans). To assess their association with breast cancer risk, five SNPs were genotyped in 1218 familial BRCA1/2-mutation negative breast cancer cases and 1509 controls (rs1804645, rs6094752, rs2230782, rs2076546, rs2229840). RESULTS: Through our resequencing effort, we identified 74 novel SNPs (30 in NCoR, 32 in SMRT, 10 in SRC-3, and 2 in SRC-1). Of these, 8 were found with minor allele frequency (MAF) >5% illustrating the large amount of genetic diversity yet to be discovered. The previously shown protective effect of rs2230782 in SRC-3 was strengthened (OR = 0.45 [0.21-0.98], p = 0.04). No significant associations were found with the other SNPs genotyped. CONCLUSIONS: This data illustrates the importance of coregulators, especially SRC-3, in breast cancer development and suggests that more focused studies, including functional analyses, should be conducted.

ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer.

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.

A Novel Next-Generation Sequencing Approach to Detecting Microsatellite Instability and Pan-Tumor Characterization of 1000 Microsatellite Instability-High Cases in 67,000 Patient Samples.

Microsatellite instability (MSI) is an important biomarker for predicting response to immune checkpoint inhibitor therapy, as emphasized by the recent checkpoint inhibitor approval for MSI-high (MSI-H) solid tumors. Herein, we describe and validate a novel method for determining MSI status from a next-generation sequencing comprehensive genomic profiling assay using formalin-fixed, paraffin-embedded samples. This method is 97% (65/67) concordant with current standards, PCR and immunohistochemistry. We further apply this method to >67,000 patient tumor samples to identify genes and pathways that are enriched in MSI-stable or MSI-H tumor groups. Data show that although rare in tumors other than colorectal and endometrial carcinomas, MSI-H samples are present in many tumor types. Furthermore, the large sample set revealed that MSI-H tumors selectively share alterations in genes across multiple common pathways, including WNT, phosphatidylinositol 3-kinase, and NOTCH. Last, MSI is sufficient, but not necessary, for a tumor to have elevated tumor mutation burden. Therefore, MSI can be determined from comprehensive genomic profiling with high accuracy, allowing for efficient MSI-H detection across all tumor types, especially those in which routine use of immunohistochemistry or PCR-based assays would be impractical because of a rare incidence of MSI. MSI-H tumors are enriched in alterations in specific signaling pathways, providing a rationale for investigating directed immune checkpoint inhibitor therapies in combination with pathway-targeted therapies.

Frequent ESR1 and CDK Pathway Copy-Number Alterations in Metastatic Breast Cancer.

DNA sequencing has identified a limited number of driver mutations in metastatic breast cancer beyond single base-pair mutations in the estrogen receptor (ESR1). However, our previous studies and others have observed that structural variants, such as ESR1 fusions, may also play a role. Therefore, we expanded upon these observations by performing a comprehensive and highly sensitive characterization of copy-number (CN) alterations in a large clinical cohort of metastatic specimens. NanoString DNA hybridization was utilized to measure CN gains, amplifications, and deletions of 67 genes in 108 breast cancer metastases, and in 26 cases, the patient-matched primary tumor. For ESR1, a copyshift algorithm was applied to identify CN imbalances at exon-specific resolution and queried large data sets (>15,000 tumors) that had previously undergone next-generation sequencing (NGS). Interestingly, a subset of ER+ tumors showed increased ESR1 CN (11/82, 13%); three had CN amplifications (4%) and eight had gains (10%). Increased ESR1 CN was enriched in metastatic specimens versus primary tumors, and this was orthogonally confirmed in a large NGS data set. ESR1-amplified tumors showed a site-specific enrichment for bone metastases and worse outcomes than nonamplified tumors. No ESR1 CN amplifications and only one gain was identified in ER- tumors. ESR1 copyshift was present in 5 of the 11 ESR1-amplified tumors. Other frequent amplifications included ERBB2, GRB7, and cell-cycle pathway members CCND1 and CDK4/6, which showed mutually exclusivity with deletions of CDKN2A, CDKN2B, and CDKN1B. IMPLICATIONS: Copy-number alterations of ESR1 and key CDK pathway genes are frequent in metastatic breast cancers, and their clinical relevance should be tested further.

FGFR4 overexpression and hotspot mutations in metastatic ER+ breast cancer are enriched in the lobular subtype.

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.

BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations.

PURPOSE: Dabrafenib and trametinib are approved for the management of advanced non-small-cell lung cancers (NSCLCs) that harbor BRAF V600E mutations. Small series and pan-cancer analyses have identified non-V600 alterations as therapeutic targets. We sought to examine a large genomic data set to comprehensively characterize non-V600 BRAF alterations in lung cancer. PATIENTS AND METHODS: A total of 23,396 patients with lung cancer provided data to assay with comprehensive genomic profiling. Data were reviewed for predicted pathogenic BRAF base substitutions, short insertions and deletions, copy number changes, and rearrangements. RESULTS: Adenocarcinomas represented 65% of the occurrences; NSCLC not otherwise specified (NOS), 15%; squamous cell carcinoma, 12%; and small-cell lung carcinoma, 5%. BRAF was altered in 4.5% (1,048 of 23,396) of all tumors; 37.4% (n = 397) were BRAF V600E, 38% were BRAF non-V600E activating mutations, and 18% were BRAF inactivating. Rearrangements were observed at a frequency of 4.3% and consisted of N-terminal deletions (NTDs; 0.75%), kinase domain duplications (KDDs; 0.75%), and BRAF fusions (2.8%). The fusions involved three recurrent fusion partners: ARMC10, DOCK4, and TRIM24. BRAF V600E was associated with co-occurrence of SETD2 alterations, but other BRAF alterations were not and were instead associated with CDKN2A, TP53, and STK11 alterations (P < .05). Potential mechanisms of acquired resistance to BRAF V600E inhibition are demonstrated. CONCLUSION: This series characterized the frequent occurrence (4.4%) of BRAF alterations in lung cancers. Recurrent BRAF alterations in NSCLC adenocarcinoma are comparable to the frequency of other NSCLC oncogenic drivers, such as ALK, and exceed that of ROS1 or RET. This work supports a broad profiling approach in lung cancers and suggests that non-V600E BRAF alterations represent a subgroup of lung cancers in which targeted therapy should be considered.

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations.

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma.

KRAS is the most common oncogenic driver in lung adenocarcinoma (LUAC). We previously reported that STK11/LKB1 (KL) or TP53 (KP) comutations define distinct subgroups of KRAS-mutant LUAC. Here, we examine the efficacy of PD-1 inhibitors in these subgroups. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%), and K-only (28.6%) subgroups (P < 0.001) in the Stand Up To Cancer (SU2C) cohort (174 patients) with KRAS-mutant LUAC and in patients treated with nivolumab in the CheckMate-057 phase III trial (0% vs. 57.1% vs. 18.2%; P = 0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P < 0.001) and overall (P = 0.0015) survival compared with KRASMUT;STK11/LKB1WT LUAC. Among 924 LUACs, STK11/LKB1 alterations were the only marker significantly associated with PD-L1 negativity in TMBIntermediate/High LUAC. The impact of STK11/LKB1 alterations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1-positive non-small cell lung cancer. In Kras-mutant murine LUAC models, Stk11/Lkb1 loss promoted PD-1/PD-L1 inhibitor resistance, suggesting a causal role. Our results identify STK11/LKB1 alterations as a major driver of primary resistance to PD-1 blockade in KRAS-mutant LUAC.Significance: This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets. Cancer Discov; 8(7); 822-35. ©2018 AACR.See related commentary by Etxeberria et al., p. 794This article is highlighted in the In This Issue feature, p. 781.