Memorial Sloan Kettering-Integrated Mutation Profiling Of Actionable Cancer Targets Research Articles

Associations Between Cancer Predisposition Mutations and Clonal Hematopoiesis in Patients With Solid Tumors.

Clonal hematopoiesis (CH), the expansion of clones in the hematopoietic system, has been linked to different internal and external features such as aging, genetic ancestry, smoking, and oncologic treatment. However, the interplay between mutations in known cancer predisposition genes and CH has not been thoroughly examined in patients with solid tumors. We used prospective tumor-blood paired sequencing data from 46,906 patients who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing to interrogate the associations between CH and rare pathogenic or likely pathogenic (P/LP) germline variants. We observed an enrichment of CH-positive patients among those carrying P/LP germline mutations and identified a significant association between P/LP germline variants in ATM and CH. Germline and CH comutation patterns in ATM, TP53, and CHEK2 suggested biallelic inactivation as a potential mediator of clonal expansion. Moreover, we observed that CH-PPM1D mutations, similar to somatic tumor-associated PPM1D mutations, were depleted in patients with P/LP germline mutations in the DNA damage response (DDR) genes ATM, CHEK2, and TP53. Patients with solid tumors and harboring P/LP germline mutations, CH mutations, and mosaicism chromosomal alterations might be at an increased risk of developing secondary leukemia while germline variants in TP53 were identified as an independent risk factor (hazard ratio, 36; P < .001) for secondary leukemias. Our results suggest a close relationship between inherited variants and CH mutations within the DDR genes in patients with solid tumors. Associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities in patients with cancer harboring these germline mutations.

Comprehensive analysis of germline drivers in endometrial cancer.

We sought to determine the prevalence of germline pathogenic variants (gPVs) in unselected patients with endometrial cancer (EC), define biallelic gPVs within tumors, and describe their associations with clinicopathologic features. Germline assessment of at least 76 cancer predisposition genes was performed in patients with EC undergoing clinical tumor-normal Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing from January 1, 2015, to June 30, 2021. In patients with gPVs, biallelic alterations in ECs were identified through analysis of loss of heterozygosity and somatic PVs. Clinicopathologic variables were compared using nonparametric tests. Of 1625 patients with EC, 216 (13%) had gPVs, and 15 patients had 2 gPVs. There were 231 gPVs in 35 genes (75 [32%] high penetrance; 39 [17%] moderate penetrance; and 117 [51%] low, recessive, or uncertain penetrance). Compared with those without gPVs, patients with gPVs were younger (P = .002), more often White (P = .009), and less obese (P = .025) and had differences in distribution of tumor histology (P = .017) and molecular subtype (P < .001). Among 231 gPVs, 74 (32%) exhibited biallelic inactivation within tumors. For high-penetrance gPVs, 63% (47 of 75) of ECs had biallelic alterations, primarily affecting mismatch repair (MMR) and homologous recombination related genes, including BRCA1,BRCA2, RAD51D, and PALB2. Biallelic inactivation varied across molecular subtypes with highest rates in microsatellite instability-high (MSI-H) or copy-number (CN)-high subtypes (3 of 12 [25%] POLE, 30 of 77 [39%] MSI-H, 27 of 60 [45%] CN-high, 9 of 57 [16%] CN-low; P < .001). Of unselected patients with EC, 13% had gPVs, with 63% of gPVs in high-penetrance genes (MMR and homologous recombination) exhibiting biallelic inactivation, potentially driving cancer development. This supports germline assessment in EC given implications for treatment and cancer prevention.

Comprehensive Molecular Characterization of Gallbladder Carcinoma and Potential Targets for Intervention.

Gallbladder carcinoma (GBC) is an uncommon and aggressive disease, which remains poorly defined at a molecular level. Here, we aimed to characterize the molecular landscape of GBC and identify markers with potential prognostic and therapeutic implications. GBC samples were analyzed using the MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) platform (targeted NGS assay that analyzes 505 cancer-associated genes). Variants with therapeutic implications were identified using OncoKB database. The associations between recurrent genetic alterations and clinicopathologic characteristics (Fisher exact tests) or overall survival (univariate Cox regression) were evaluated. P values were adjusted for multiple testing. Overall, 244 samples (57% primary tumors and 43% metastases) from 233 patients were studied (85% adenocarcinomas, 10% carcinomas with squamous differentiation, and 5% neuroendocrine carcinomas). The most common oncogenic molecular alterations appeared in the cell cycle (TP53 63% and CDKN2A 21%) and RTK_RAS pathways (ERBB2 15% and KRAS 11%). No recurrent structural variants were identified. There were no differences in the molecular landscape of primary and metastasis samples. Variants in SMAD4 and STK11 independently associated with reduced survival in patients with metastatic disease. Alterations considered clinically actionable in GBC or other solid tumor types (e.g., NTRK1 fusions or oncogenic variants in ERBB2, PIK3CA, or BRCA1/2) were identified in 35% of patients; 18% of patients with metastatic disease were treated off-label or enrolled in a clinical trial based on molecular findings. GBC is a genetically diverse malignancy. This large-scale genomic analysis revealed alterations with potential prognostic and therapeutic implications and provides guidance for the development of targeted therapies.

Methylation Analyses Reveal Promoter Hypermethylation as a Rare Cause of "Second Hit" in Germline BRCA1-Associated Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5-6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a "second hit" mechanism in patients with gBRCA1-PDAC. We evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed. Of 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38-84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation. In patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

B2M and JAK1/2-mutated MSI-H Colorectal Carcinomas Can Benefit From Anti-PD-1 Therapy.

β2-microglobulin (B2M) and Janus kinases 1 and 2 (JAK1/2) mutations have been suggested as genetic mechanisms of immune evasion for anti–programmed cell death protein 1 (PD-1) therapy. Whether B2M and JAK1/2 lose-of-function mutation can cause primary resistance to anti-PD-1 therapy in colorectal carcinoma (CRC) patients remains controversial. Here, we sought to compare the efficacy of anti-PD-1 therapy in DNA mismatch repair deficient/microsatellite instability–high CRC patients with or without B2M or JAK1/2 mutations. Thirty-Five CRC patients who received anti-PD-1 therapy were enrolled in this study. All tumor samples underwent next-generation sequencing. The clinical and molecular data from 110 CRC patients sequenced with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay and accessed through cBioportal were also analyzed in this study. Of the 35 CRC patients from our center, 10 (28.6%) had a B2M loss-of-function mutation, and 8 (22.9%) had a JAK1/2 loss-of-function mutation. Compared with B2M wild-type CRCs, B2M-mutated CRCs did not show a higher frequency of resistance to anti-PD-1 therapy (P=0.71). There was even better response to anti-PD-1 therapy in patients with JAK1/2 mutation than in those without (P=0.015). Of the 110 CRC patients in the MSK-IMPACT datasets, 13 (11.8%) had a B2M mutation, and 15 (13.6%) had a JAK1/2 mutation. After analyzing the response to anti-PD-1 therapy in these 110 patients, we found similar results (P=0.438 and 0.071, respectively). Moreover, patients with B2M or JAK1/2 mutation had a lower tumor mutational burden score compared with those without. B2M and JAK1/2 loss-of-function mutations occur frequently in microsatellite instability–high CRC. Our study demonstrated that patients with CRC harboring B2M or JAK1/2 mutations should not be excluded from anti-PD-1 therapy.

TP53 Mutations Identify High-Risk Peripheral T-Cell Lymphoma Patients Treated with CHOP-Based Chemotherapy

TP53 Mutations Identify High-Risk Peripheral T-Cell Lymphoma Patients Treated with CHOP-Based Chemotherapy

Contextualizing Genes by Using Text-Mined Co-Occurrence Features for Cancer Gene Panel Discovery.

Developing a biomedical-explainable and validatable text mining pipeline can help in cancer gene panel discovery. We create a pipeline that can contextualize genes by using text-mined co-occurrence features. We apply Biomedical Natural Language Processing (BioNLP) techniques for literature mining in the cancer gene panel. A literature-derived 4,679 × 4,630 gene term-feature matrix was built. The EGFR L858R and T790M, and BRAF V600E genetic variants are important mutation term features in text mining and are frequently mutated in cancer. We validate the cancer gene panel by the mutational landscape of different cancer types. The cosine similarity of gene frequency between text mining and a statistical result from clinical sequencing data is 80.8%. In different machine learning models, the best accuracy for the prediction of two different gene panels, including MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets), and Oncomine cancer gene panel, is 0.959, and 0.989, respectively. The receiver operating characteristic (ROC) curve analysis confirmed that the neural net model has a better prediction performance (Area under the ROC curve (AUC) = 0.992). The use of text-mined co-occurrence features can contextualize each gene. We believe the approach is to evaluate several existing gene panels, and show that we can use part of the gene panel set to predict the remaining genes for cancer discovery.

Regulations, Open Data and Healthcare Innovation: A Case of MSK-IMPACT and Its Implications for Better Cancer Care.

Simple SummaryThe advancement in both science and technology has contributed to the development of novel diagnostic technologies; such technologies enable medical practitioners to diagnose diseases that could not be previously detected. However, in order to translate new technologies into practical applications, various types of challenges need to be overcome. To address these challenges, including those in clinical management and regulatory science, healthcare policies have been constantly implemented to promote the practical application of outcomes generated by healthcare innovation. This study conducted comparative analyses of three tumor profiling tests approved by the U.S. Food and Drug Administration (FDA) in 2017, hypothesizing that the FDA’s regulatory reforms, early application of new technologies to both research and clinical settings, and open data accumulated as a result of large-scale research programs have promoted new drug development in oncology. The study then discussed the implications potentially suggested by the outcomes and challenges of the three tests.This study investigated a case of Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a tumor profiling test approved by the U.S. Food and Drug Administration (FDA) in 2017, to examine what factors would contribute to healthcare innovation. First, we set the following three parameters to observe cases: (i) the FDA regulatory reforms, (ii) early application of new technologies, such as next-generation sequencing (NGS), to both research and clinical settings, and (iii) accumulation of open data. Then, we performed a comparative analysis of MSK-IMPACT with FoundationOne CDx and Oncomine Dx Target Test, both of which were FDA-approved tumor profiling tests launched in 2017. As a result, we found that MSK-IMPACT secures neutrality as a non-profit organization, achieves the active incorporation of basic research results, and performs superiorly in clinical operations, such as patient enrollment. On the contrary, we confirmed that FoundationOne CDx was the most prominent case in terms of the number of new drugs and expanded indications approved in which the FDA’s expedited approval programs were considerably utilized. Consequently, to uncover the full potential of MSK-IMPACT, it is suggested that more intersectoral collaborative activities between various healthcare stakeholders, in particular, pharmaceutical companies, for driving clinical development must be carried out based on an organizational framework that facilitates collaboration.

Abstract B20: Characterization of tumor mutational landscape in smokers and nonsmokers with urothelial carcinoma

Abstract Introduction and Objectives: Smoking is a major risk factor for development of bladder cancer. It has been previously shown that smoking-related and non-smoking-related cancers have characteristic mutational signatures. Hypothesizing that somatic alterations differ between smokers and nonsmokers with urothelial carcinoma, we used next-generation sequencing to evaluate for differences in genomic alterations between these two groups of patients. Methods: We identified in our prospectively maintained institutional database all patients with urothelial carcinoma whose tumors underwent next-generation sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay. A panel of genes previously reported by The Cancer Genome Atlas (TCGA) as being commonly mutated in bladder cancer was chosen for analysis. Results: A total of 1,044 samples from 968 patients were included in the analysis. Seven hundred eighty-five (85%) of the samples represented bladder tumors and 259 (25%) of the samples represented tumors of the upper tract. Patients with smoking history comprised 66% of the cohort. The most frequently altered genes in active or former smokers and never smokers were TERT promoter (55% and 60%), TP53 (43% and 41%), KDM6A (30% and 26%), and FGFR3 (27% and 26%). Conclusions: Mutational profiling of urothelial tumors in this large cohort of patients showed high incidence of genomic alterations consistent with prior studies. The frequency of alterations in these genes was similar between smokers and nonsmokers. To further understand smoking-related mutational process, we are examining mutational signatures and differences in genomic alterations as a function of smoking intensity exposure. Citation Format: Aleksandra Walasek, Nima Almassi, Min Yuen Teo, Samuel Funt, Nikolaus Schultz, Guido Dalbagni, Wenhu Hu, Hikmat Al-Ahmadie, Dean F Bajorin, Jonathan E Rosenberg, Gopakumar Iyer, Bernard H. Bochner, David B. Solit, Eugene J. Pietzak. Characterization of tumor mutational landscape in smokers and nonsmokers with urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B20.

Molecular profiling and analysis of genetic aberrations aimed at identifying potential therapeutic targets in fibrolamellar carcinoma of the liver.

Fibrolamellar carcinoma (FLC) is a rare primary liver cancer of young adults. A functional chimeric transcript resulting from the in-frame fusion of the DNAJ homolog, subfamily B, member 1 (DNAJB1), and the catalytic subunit of protein kinase A (PRKACA) genes on chromosome 19 is believed to be unique in FLC, with a possible role in pathogenesis, yet with no established therapeutic value. The objective of the current study was to understand the molecular landscape of FLC and to identify potential novel therapeutic targets. Archival fresh, formalin-fixed, paraffin-embedded samples from patients with FLC who prospectively consented to an institutional review board-approved protocol were analyzed using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a next-generation sequencing assay encompassing up to 468 key cancer genes. Custom targeted RNA-Seq was performed in selected patients. Demographics, treatment, and outcome data were collected prospectively. Survival outcomes were estimated and correlated with mutation and/or copy number alterations. A total of 33 tumor samples from 31 patients with FLC were analyzed. The median age of the patients at the time of diagnosis was 18years and approximately 53% were women. The DNAJB1-PRKACA fusion transcript was detected in 100% of patients. In 10 of 31 patients in which MSK-IMPACT did not detect the fusion, its presence was confirmed by targeted RNA-Seq. TERT promoter mutation was the second most common, and was detected in 7 patients. The median follow up was 30months (range, 6-153months). The 3-year overall survival rate was 84% (95% CI, 61%-93%). The DNAJB1-PRKACA fusion transcript is nonspecific and nonsensitive to FLC. Its potential therapeutic value currently is under evaluation. Opportunities currently are under development for therapy that may be driven or related to the DNAJB1-PRKACA fusion transcript or any therapeutic target identified from next-generation sequencing in patients with FLC.

Alterations in driver genes are predictive of survival in patients with resected pancreatic ductal adenocarcinoma.

KRAS, TP53, CDKN2A, and SMAD4 are established driver genes in pancreatic ductal adenocarcinoma (PDAC). This study was aimed at determining whether the mutational status of driver genes and those involved in DNA repair pathways are associated with clinical outcomes for individuals who undergo resection. Eligible individuals were those who underwent resection of PDAC and consented to targeted sequencing of their primary tumor via Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT). Genomic alterations were determined on the basis of MSK-IMPACT results from formalin-fixed, paraffin-embedded samples. Associations between genomic alterations and clinical outcomes were assessed. Targeted sequencing was performed on 283 primary tumors resected between 2004 and 2017. The median follow-up was 23 months among survivors. Alterations in KRAS and TP53 were associated with worse overall survival (OS) in comparison to wild type (median for KRAS, 38.8 months [95% CI, 33.0-45.5 months] vs 91.0 months [95% CI, 34.8 months to not available (NA)]; P=.043; median for TP53, 37.4 months [95% CI, 32.1-42.8 months] vs 65.0 months [95% CI, 33.0 months to NA]; P=.035). KRAS G12D mutations were associated with worse OS (median, 31.6 months [95% CI, 25.3-45.5 months] vs 39.2 months [95% CI, 37.4-75.2 months]; P=.012). TP53 truncating mutations (median, 39.6 months [95% CI, 32.4-75.2 months] vs 33.9 months [95% CI, 24.0-39.0 months]; P=.020) and those associated with loss of heterozygosity (median, 26.6 months [95% CI, 21.6-44.2 months] vs 39.2 months [95% CI, 34.5-49.1 months]; P=.048) had decreased OS. TP53 alterations were independently associated with OS in a multivariate analysis (hazard ratio, 1.54; 95% CI, 1.01-2.33; P=.042). Individuals with germline alterations in homologous recombination deficiency (HRD) genes had improved OS in comparison with those without them (median, not reached vs 37.0 months; 95% CI, 33.0-49.8 months; P=.035). In patients with resected PDAC, genomic alterations in KRAS and TP53 are associated with worse outcomes, whereas alterations in HRD genes are associated with a favorable prognosis. Further studies are needed to better define these alterations as biomarkers in resected PDAC.

Enrichment of kinase fusions in ESR1 wild-type, metastatic breast cancer revealed by a systematic analysis of 4854 patients

Kinase fusions are rare and poorly characterized in breast cancer (BC). We aimed to characterize kinase fusions within a large cohort of advanced BC. A total of 4854 patients with BC were analyzed by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) targeted DNAseq and MSK-Fusion targeted RNAseq during the study time period. Twenty-seven of 4854 (0.6%) patients harbored fusions: 11 FGFR (five FGFR2, three FGFR3, three FGFR1), five BRAF, four NTRK1, two RET, two ROS1, one ALK, one ERBB2, and one MET. A history of endocrine therapy was present in 15 (56%) of fusion-positive BC; eight of the 15 cases had available pre-treatment samples, of which six were fusion-negative. None of the fusion-positive BC samples harbored ESR1 hotspot mutations. Two patients with acquired LMNA-NTRK1 fusions and metastatic disease received larotrectinib and demonstrated clinical benefit. Kinase fusions in BC are extremely rare, and appear to be enriched in hormone-resistant, metastatic carcinomas and mutually exclusive with ESR1 mutations. The present study expands the spectrum of genetic alterations activating mitogen-activated protein kinase (MAPK) signaling that can substitute for ESR1 mutations in this setting. Molecular testing at progression after endocrine therapy should include fusion testing, particularly in the absence of ESR1 hotspot alterations, in an effort to identify additional therapeutic options which may provide substantial clinical benefit.

Tumor mutation burden and depression in lung cancer: Association with inflammation.

e23159 Background: Depression and inflammation are both associated with lung cancer. Tumor Mutation Burden (TMB) can be used to predict immunotherapy response which relies on tumor immunogenicity, a form of inflammation. This study evaluated whether higher TMB was associated with increased depression and various forms of inflammation. Methods: Patients with metastatic Non-small Cell Lung Cancer (NSCLC) and Small Cell Lung Cancer (SCLC) receiving anticancer treatments were evaluated for depression severity using the Hospital Anxiety and Depression Scale (HADS). TMB was measured using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT). Inflammation was evaluated using C - reactive protein (CRP) and the Neutrophil to Lymphocyte Ratio (NLR). Results: Ninety-six out of 140 participants with adequate TMB testing were evaluated (68.6%). The average number of mutations was 10.8 (SD 10.8). Twenty-four percent met screening criteria for depression. TMB correlated with depression severity (r = .34, p = .001) and NLR (r = .37, p = .002) and absolute neutrophil count (r = .3, p = .006) but not CRP (r = .193, p = .06). TMB was higher in patients receiving chemotherapy (12.0) and immunotherapy (14.4) over targeted therapy (4.8). A multivariate model found that TMB (β = .25, p = .032) and CRP (β = .36, p = .003) predicted for depression severity. Conclusions: TMB is an independent predictor of depression. Inflammatory indices suggest a possible relationship but the exact mechanism should be further evaluated and the results warrant replication. Ultimately, these findings may help identify lung cancer patients at risk for depression. [Table: see text]

Acquired MET Exon 14 Alteration Drives Secondary Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in EGFR-Mutated Lung Cancer.

Novel resistance mechanisms to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer continue to be defined.1 With the recent approval of the third-generation EGFR TKI osimertinib as first-line treatment, and expanded use of large-panel next-generation sequencing (NGS)–based testing, many novel resistance mechanisms are emerging. Resistance to osimertinib can result from on-target mechanisms, such as the acquisition of second-site EGFR mutations, or from the activation of off-target mechanisms or bypass pathways, including acquired oncogenic fusions of RET, ALK, BRAF, and FGFR1,2 and amplification or mutation of HER2, BRAF, MEK, KRAS, PIK3CA, and MET.3 MET amplification is reported in 5% to 22% of EGFR TKI resistance.1,4 Preclinical studies have shown that acquired resistance to EGFR TKIs resulting from MET amplification can be reversed by combined therapy with EGFR and MET inhibitors.4 MET exon 14 skipping alteration (METex14) is present in 3% to 4% of lung adenocarcinomas,5 and the MET receptor lacking exon 14 shows decreased protein turnover because of loss of the ubiquitination site encoded by exon 14, resulting in aberrant MET activation and oncogenesis.6 In preclinical and clinical studies, responses to MET inhibitors, such as crizotinib, have been reported in patients with lung cancer with METex14 as a primary driver.6-11 However, it has not been previously implicated in acquired resistance to EGFR-TKIs. In this study, we used targeted NGS with Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT),12 immunohistochemistry, cell-free DNA testing, and fluorescence in situ hybridization to evaluate acquired resistance mediated by METex14. Furthermore, we used in vitro functional studies to establish METex14 as a novel mechanism of acquired resistance to EGFR TKIs. We used MSK-IMPACT, a large-panel NGS assay, to detect mutations, copy-number alterations, and select gene fusions involving up to 468 cancer-associated genes.12 METex14 was introduced into PC9 and H1975 cells as follows. Briefly, full-length METex14 was polymerase chain reaction amplified and subcloned into pLenti-CMV-blast lentiviral vector (plasmid 17451; Addgene, Cambridge, MA). The lentiviral plasmids were cotransfected with packaging plasmids into HEK 293 T cells using FuGENE HD (Promega, Madison, WI), and lentiviruses were generated. Cells were infected with lentivirus-expressing METex14 cDNA, followed by selection with blasticidin (20 µg/mL) for 8 days. The Data Supplement provides more detailed methods.

Abstract PD7-05: Correlation of CDH1 alterations and aberrant E-cadherin expression in lobular carcinomas

Abstract Background: E-cadherin (ECAD), encoded by the CDH1 gene, is expressed in the majority of invasive ductal carcinomas (IDC). In contrast, ECAD expression is lost in ˜90% of invasive lobular carcinomas (ILC) due to genomic alterations of CDH1. Immunohistochemical (IHC) staining for ECAD has thus increasingly been utilized to differentiate between ductal and lobular lesions. This study examines the correlation between CDH1 mutation profile, morphology and ECAD IHC status in invasive breast carcinomas (BC). Methods: The Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platform is a hybrid capture next-generation sequencing assay to detect somatic alterations in exons and selected introns of 468 cancer-related genes. Invasive BC tested from 1/2014 – 5/2018 which yielded a CDH1 alteration were identified and only cases with ECAD IHC results were included. Histologic features were extracted from pathology reports. All available H&amp;E/ECAD slides were reviewed by two pathologists to confirm the histology and immunophenotype. Results: CDH1 alterations were identified in 455 out of 4103 BC analyzed (11%) and ECAD IHC were available for 209 (46%) tumors. Most of the tumors were reported as ILC (70%, 147/209). The majority (77%, 161/209) were ECAD-negative but 23% (48/209) showed some ECAD staining. Table 1 summarizes the histologic subtypes and IHC results. Upon review of H&amp;E/ECAD slides in cases with ECAD staining, the histologic subtype was re-classified in 52% (25/48) of cases (Table 2). For ECAD-negative BC, at least 61% (98/161) of CDH1 alterations were truncating mutations (frameshift deletion 47/161, nonsense 51/161) and 39% (63/161) were variants of unknown significance (VUS). In cases with ECAD staining, 48% (23/48) of CDH1 alterations were truncating mutations (frameshift deletion 12/48, nonsense 11/48) however 52% (25/48) were VUS. Conclusion: In this study, 88% (141/161) of cases with negative ECAD staining and CDH1 alteration were classified as ILC, confirming the known observation that CDH1 alteration and loss of ECAD expression is relatively specific for a lobular phenotype. The correlation is, however, imperfect. A subset of BC (23%; 48/209) with CDH1 alteration still exhibits ECAD expression. Morphologically this subset consists of ILC in 60% (29/48), IMC in 25% (12/48) and IDC in 15% (7/48). Our preliminary findings suggest that aberrant ECAD expression can occur in BC with morphologic features of ILC due to certain CDH1 alterations that most likely result in non-functional ECAD complex. BC with histologic features of ILC should therefore not be re-classified as IDC/IMC based solely on the status of ECAD expression. Given that ILC is clinically distinct from IDC, correct classification based on morphological and molecular features is prudent. Table 1:Histologic subtypes and ECAD IHC ILC (n = 147)IDC (n = 13)IMC (n = 49)ECAD staining Negative141020Strong, diffuse4135Heterogenous0020Reduced/weak204IMC: invasive mammary with mixed ductal/lobular features Table 2:Morphologic review of cases with ECAD stainingECAD stainingOriginal DX (n)Morphology Review DX ILCIDCIMCStrong, diffuseILC (4)301 IDC (13)670 IMC (5)500HeterogenousIMC (20)10010Reduced/weakILC (2)200 IMC (4)301 Citation Format: Grabenstetter A, Mohanty AS, DeLair DF, Tan LK, Ross DS. Correlation of CDH1 alterations and aberrant E-cadherin expression in lobular carcinomas [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 PD7-05.

Ampullary cancer: Evaluation of somatic and germline genetic alterations and association with clinical outcomes.

Ampullary carcinoma (AC) is a rare gastrointestinal cancer. Pathogenic germline alterations (PGAs) in BRCA2 and potentially targetable somatic alterations (SAs) in ERBB2 and ELF3 have been previously described in AC. Memorial Sloan Kettering Cancer Center has implemented an opt-in strategy for germline testing (GT) and somatic testing (ST) for patients with AC to further evaluate the spectrum of PGAs and SAs. Forty-five patients with pathologically confirmed AC prospectively consented with the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) test (410-468 genes). A subset of the cohort (23 of the 45 patients) also consented to GT with MSK-IMPACT (76-88 genes). Germline data for 21 of the remaining 22 patients who had not consented to GT were obtained in a de-identified fashion without clinical correlation. Clinicopathologic features, treatment histories, and survival data for consenting patients were collected and analyzed. Pancreaticobiliary, intestinal, and mixed features of the 2 types were the primary pathologic subtypes of AC identified in this cohort. No difference in median overall survival was found between pathologic subtypes. Eight of 44 patients (18%) were identified as harboring pathogenic mutations in BRCA2, ATM, RAD50, and MUTYH. In addition, this study found a wide spectrum of SAs in genes such as KRAS, MDM2, ERBB2, ELF3, and PIK3CA. Two patients in the cohort underwent SA-targeted therapy, and 1 had a partial radiographic response. Mutations in multiple somatic and germline genes were identified in this cohort. Significantly, actionable targets were identified in the tumors, and broader testing for PGAs and SAs should be considered for all patients with AC.

Immunohistochemical analysis of estrogen receptor in breast cancer with ESR1 mutations detected by hybrid capture-based next-generation sequencing

Estrogen receptor-α (ER-α), encoded by ESR1, is detected by immunohistochemistry in approximately 70% of invasive breast cancers and serves as a strong predictive biomarker. ESR1-activating mutations in the ligand-binding domain have been reported in up to 35–40% of ER-positive metastatic breast cancers and are associated with endocrine therapy resistance and disease progression. At present, it is unclear whether ESR1 mutations alter the immunohistochemical detection of ER performed in routine clinical practice. In this study, ESR1 mutations in breast cancer were identified utilizing Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a Food and Drug Administration-approved hybridization capture-based next-generation sequencing assay. Five hundred and eighty-six breast cancers from patients with locally advanced or metastatic disease were analyzed using MSK-IMPACT in the study period. ESR1 somatic alterations were identified in 67 breast cancer samples from 66 patients. Immunohistochemical analysis of ER, progesterone receptor, and human epidermal growth factor receptor 2 was performed on the primary and treated breast cancers from these patients at the time of diagnosis. Twenty unique ESR1 mutations were identified involving the ligand-binding domain, all in breast cancer samples from patients previously treated with endocrine therapy. The most frequent mutations were D538G (n = 22), Y537S (n = 7), and E380Q (n = 7). All breast cancer samples with an ESR1 mutation were ER-positive by immunohistochemistry. Review of the ER immunohistochemistry in the paired untreated primary tumor and treated tumor from 34 patients showed no detectable change in the ER-positive immunohistochemical status (median percentage of invasive tumor cells with nuclear staining: untreated primary tumor 90%, treated tumor 95%). We conclude that ESR1 mutations do not appreciably diminish ER-positive staining by immunohistochemistry. In addition to standard biomarker testing by immunohistochemistry, the assessment of ESR1 mutations by molecular testing can help guide the clinical management of patients with ER-positive breast cancer in the setting of endocrine resistance and progression of disease.

Multicenter experience with large panel next-generation sequencing in patients with advanced solid cancers in Japan.

Application of next-generation DNA sequencing (NGS) has recently become increasingly common in the field of clinical oncology in several countries around the world. In Japan also, a system for applying NGS to routine clinical practice is gradually being established. During this process, we introduced in Japan the tumor-profiling MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) assay. We present here our initial experience with the use of MSK-IMPACT in 68 patients selected from two institutions in Japan between June 2016 and October 2017. MSK-IMPACT sequencing was successful and yielded results in specimens obtained from 64 of the 68 patients, representing an overall assay success rate of 94.1%. The top three cancer types tested were endometrial cancer (17.2%), pancreatic cancer (15.6%) and colorectal cancer (12.5%). Evaluation of the clinical actionability of the genetic alterations revealed that 25.0% of patients (n = 16) harbored at least one actionable alteration. However, enrolling the patients in a genomically matched clinical trial was difficult, mainly because most clinical trials are limited to tumors arising from a specific organ/site. One patient with microsatellite instability-high status, as determined by MSK-IMPACT, was treated with pembrolizumab and showed partial response. Although tumor profiling by NGS and administration of genomically matched therapy is a promising strategy, because of its high cost, we need to consider how we can fit it into the Japanese medical system. Towards this end, we believe that it is important to share our initial experience for furthering precision medicine in Japan.

Commentary on “DNA damage response and repair gene alterations are associated with improved survival in patients with platinum-treated advanced urothelial carcinoma.”

PurposePlatinum-based chemotherapy remains the standard treatment for advanced urothelial carcinoma by inducing DNA damage. We hypothesize that somatic alterations in DNA damage response and repair (DDR) genes are associated with improved sensitivity to platinum-based chemotherapy. Experimental designPatients with diagnosis of locally advanced and metastatic urothelial carcinoma treated with platinum-based chemotherapy who had exon sequencing with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay were identified. Patients were dichotomized based on the presence/absence of alterations in a panel of 34 DDR genes. DDR alteration status was correlated with clinical outcomes and disease features. ResultsOne hundred patients were identified, of which 47 harbored alterations in DDR genes. Patients with DDR alterations had improved progression-free survival (9.3 vs. 6.0 months, log-rank P = 0.007) and overall survival (23.7 vs. 13.0 months, log-rank P = 0.006). DDR alterations were also associated with higher number mutations and copy-number alterations. A trend toward positive correlation between DDR status and nodal metastases and inverse correlation with visceral metastases were observed. Different DDR pathways also suggested variable effect on clinical outcomes. ConclusionsSomatic DDR alteration is associated with improved clinical outcomes in platinum-treated patients with advanced urothelial carcinoma. Once validated, it can improve patient selection for clinical practice and future study enrollment.

Real-Time Genomic Characterization of Metastatic Pancreatic Neuroendocrine Tumors Has Prognostic Implications and Identifies Potential Germline Actionability.

We assessed the usefulness of real-time molecular profiling through next-generation sequencing (NGS) in predicting the tumor biology of advanced pancreatic neuroendocrine tumors (panNETs) and in characterizing genomic evolution. Patients with metastatic panNETs were recruited in the routine clinical practice setting (between May 2014 and March 2017) for prospective NGS of their tumors as well as for germline analysis using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing platform. When possible, NGS was performed at multiple time points. NGS was performed in 96 tumor samples from 80 patients. Somatic alterations were identified in 76 of 80 patients (95%). The most commonly altered genes were MEN1 (56%), DAXX (40%), ATRX (25%), and TSC2 (25%). Alterations could be defined in pathways that included chromatin remodeling factors, histone methyltransferases, and mammalian target of rapamycin pathway genes. Somatic loss of heterozygosity was particularly prevalent (50 of 96 samples [52%]), and the presence of loss of heterozygosity resulted in inferior overall survival (P < .01). Sequencing of pre- and post-treatment samples revealed tumor-grade progression; clonal evolution patterns were also seen (molecular resistance mechanisms and chemotherapy-associated mutagenesis). Germline genetic analysis identified clinically actionable pathogenic or likely pathogenic variants in 14 of 88 patients (16%), including mutations in high-penetrance cancer susceptibility genes (MEN1, TSC2, and VHL). A clinical NGS platform reveals pertubations of biologic pathways in metastatic panNETs that may inform prognosis and direct therapies. Repeat sequencing at disease progression reveals increasing tumor grade and genetic evolution, demonstrating that panNETs adopt a more aggressive behavior through time and therapies. In addition to frequent somatic mutations in MEN1 and TSC2, germline mutations in these same genes underlie susceptibility to panNETs and highlight the need to re-evaluate whether germline genetic analysis should be performed for all patients with panNETs.