Search

Purpose: EGFR exon 20 insertions (ex20ins) are an uncommon genotype in non-small cell lung cancer (NSCLC) for which targeted therapies are under development. We sought to describe treatment outcomes and genomic and immunophenotypic characteristics of these tumors., Experimental Design: We identified sequential patients with NSCLC with EGFR ex20ins and compared their clinical outcomes and pathologic features with other patients with NSCLC., Results: Among 6,290 patients with NSCLC, 106 (2%) had EGFR ex20ins. Patients with EGFR ex20ins were more likely to be Black (14% vs. 6%; P < 0.001) or Asian (22% vs. 10%; P < 0.001) compared with all other patients with NSCLC. Median tumor mutational burden (TMB; 3.5 vs. 5.9; P < 0.001) and proportion of tumors with PD-L1 expression ≥1% (22% vs. 60%; P < 0.001) were lower in EGFR ex20ins compared with other NSCLCs (TMB, n = 5,851 and PD-L1 expression, n = 282) and EGFR del 19/L858R (median TMB, 3.5; P = 0.001 and 39% PD-L1 ≥ 1%; P = 0.02). Compared with a 2:1 cohort of patients with metastatic NSCLC without targetable alterations ( n = 192), EGFR ex20ins patients had longer overall survival (median 20 vs. 12 months; HR, 0.56; P = 0.007) and longer time to treatment discontinuation (TTD) for platinum chemotherapy (median, 7 vs. 4 months; HR, 0.6; P = 0.02) and no improvement in TTD for immune checkpoint inhibitors (ICI; HR, 1.75; P = 0.05)., Conclusions: With better outcomes on platinum chemotherapy, patients with EGFR ex20ins NSCLC have improved prognosis, lower PD-L1 expression and TMB, and derive less benefit from ICIs compared with patients with NSCLC without targetable oncogenes. Improving molecularly targeted therapies could provide greater benefit for patients with EGFR ex20ins., (©2021 American Association for Cancer Research.)

Purpose: KRAS mutations are identified in approximately 30% of patients with non-small cell lung cancer (NSCLC). Novel direct inhibitors of KRAS G12C have shown activity in early-phase clinical trials. We hypothesized that patients with KRAS G12C mutations may have distinct clinical characteristics and responses to therapies., Experimental Design: Through routine next-generation sequencing, we identified patients with KRAS -mutant NSCLC treated at Memorial Sloan Kettering Cancer Center (New York, NY) from 2014 to 2018 and reviewed tumor characteristics, overall survival, and treatment outcomes., Results: We identified 1,194 patients with KRAS -mutant NSCLC, including 770 with recurrent or metastatic disease. KRAS G12C mutations were present in 46% and KRAS non-G12C mutations in 54%. Patients with KRAS G12C had a higher tumor mutation burden (median, 8.8 vs. 7 mut/Mb; P = 0.006) and higher median PD-L1 expression (5% vs. 1%). The comutation patterns of STK11 (28% vs. 29%) and KEAP1 (23% vs. 24%) were similar. The median overall survivals from diagnosis were similar for KRAS G12C (13.4 months) and KRAS non-G12C mutations (13.1 months; P = 0.96). In patients with PD-L1 ≥50%, there was not a significant difference in response rate with single-agent immune checkpoint inhibitor for patients with KRAS G12C mutations (40% vs. 58%; P = 0.07)., Conclusions: We provide outcome data for a large series of patients with KRAS G12C-mutant NSCLC with available therapies, demonstrating that responses and duration of benefit with available therapies are similar to those seen in patients with KRAS non-G12C mutations. Strategies to incorporate new targeted therapies into the current treatment paradigm will need to consider outcomes specific to patients harboring KRAS G12C mutations., (©2021 American Association for Cancer Research.)

Breast implant associated anaplastic large cell lymphoma (BIA-ALCL) is a distinct type of ALCL, and a new provisional entity by the 2016 revision of the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues. In contrast to systemic and primary cutaneous ALCLs, BIA-ALCLs have been genetically characterized by the absence of fusions and frequent activation of the JAK-STAT3 pathway through mutations in JAK1 and STAT3. In this study, we report the results of the genetic profiling of 9 BIA-ALCL cases supporting the role of the JAK-STAT pathway activation in this entity, including the identification of an activating STAT3-JAK2 fusion similar to those recently reported in T-cell lymphoproliferative disorders of the gastrointestinal tract. To our knowledge, this is the first fusion reported in BIA-ALCL, providing further insight into the overall genetic landscape of this rare entity as well as uncovering potential options for targeted therapy in cases with advanced disease., (© 2021 Wiley Periodicals LLC.)

Purpose: Analysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions., Design: Retrospective cross-sectional study of the association of plasma ctDNA from retinoblastoma and simulating lesions with disease course., Participants: Fifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions., Methods: The ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease., Main Outcome Measures: Presence or absence of molecular aberrations in the RB1 gene and correlations with clinical features., Results: RB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature [PFV]). In 2 patients, we identified 2 independent RB1 mutations in plasma., Conclusions: Mutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future., (© 2021 by the American Academy of Ophthalmology.)

Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon-producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML., (© 2021 by The American Society of Hematology.)

Aims: The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the testing guideline in 2018 to address issues arising from uncommon human epidermal growth factor receptor 2 (HER2) fluorescence in-situ hybridisation (FISH) results according to the 2013 guideline. Next-generation sequencing (NGS) may be used to better classify patients. The aim of this study was to assess the ERBB2 amplification status of invasive breast carcinoma with equivocal HER2 immunohistochemistry (IHC) results by using NGS, focusing on Group 4 (HER2/CEP17 ratio of <2.0; average HER2 signals/cell of ≥4.0 and <6.0)., Methods and Results: We retrospectively reviewed HER2 FISH and NGS data of HER2 IHC-equivocal breast carcinomas at our centre between January 2009 and September 2019, wherein all three assays were performed on the same tissue block, and compared HER2 FISH results, according to the 2018 ASCO/CAP guideline, and the ERBB2 amplification status determined with NGS. A total of 52 HER2 FISH and NGS results from 51 patients with HER2 IHC-equivocal breast carcinomas were reviewed. The cohort included eight cases classified as 2018 ASCO/CAP in-situ hybridisation Group 1, three classified as Group 2, three classified as Group 3, 14 classified as Group 4, and 24 classified as Group 5. Thirteen of 14 (92.9%) Group 4 (HER2-negative) cases were classified as ERBB2-non-amplified by the use of NGS; the discordant case was later classified as Group 1 with alternative sample FISH testing. NGS revealed no significant difference in somatic mutations or copy number alterations between Groups 4 and 5., Conclusions: Our NGS findings support the reclassification of HER2 FISH-equivocal cases as HER2-negative under the 2018 ASCO/CAP guideline., (© 2020 John Wiley & Sons Ltd.)

Clonal hematopoiesis (CH) describes somatic mutations in hematopoietic stem and progenitor cells resulting in clonal expansion in individuals with no overt hematologic disease. Since CH increases in an age-related manner, understanding its role in hematopoietic cell transplantation (HCT) has become increasingly relevant to an aging transplant population. Multiple factors distinguish post-transplant hematopoiesis from unperturbed, steady-state hematopoiesis, including the influence of immunosuppressants, cytotoxic reagents, and marked proliferative stress, all of which may enhance or diminish the opportunity for clonal expansion. We reviewed the available clinical evidence on the consequences of CH at time of transplant in patients undergoing autologous HCT, and the impact of donor and recipient CH on allogeneic HCT outcomes. In the absence of evidence-based guidelines, we share our suggestions for managing donors and recipients found to have CH. Large-scale studies are needed to guide an evidence-based, uniform approach for the management of CH in the setting of HCT., (Copyright © 2020. Published by Elsevier Ltd.)

Double unit cord blood (dCB) transplantation (dCBT) is associated with high engraftment rates but delayed myeloid recovery. We investigated adding haplo-identical CD34+ cells to dCB grafts to facilitate early haplo-identical donor-derived neutrophil recovery (optimal bridging) prior to CB engraftment. Seventy-eight adults underwent myeloablation with cyclosporine-A/mycophenolate mofetil immunoprophylaxis (no antithymocyte globulin, ATG). CB units (median CD34+ dose 1.1 × 10 5 /kg/unit) had a median 5/8 unit-recipient human leukocyte antigen (HLA)-match. Haplo-identical grafts had a median CD34+ dose of 5.2 × 10 6 /kg. Of 77 evaluable patients, 75 had sustained CB engraftment that was mediated by a dominant unit and heralded by dominant unit-derived T cells. Optimal haplo-identical donor-derived myeloid bridging was observed in 34/77 (44%) patients (median recovery 12 days). Other engrafting patients had transient bridging with second nadir preceding CB engraftment (20/77 (26%), median first recovery 12 and second 26.5 days) or no bridge (21/77 (27%), median recovery 25 days). The 2 (3%) remaining patients had graft failure. Higher haplo-CD34+ dose and better dominant unit-haplo-CD34+ HLA-match significantly improved the likelihood of optimal bridging. Optimally bridged patients were discharged earlier (median 28 versus 36 days). ATG-free haplo-dCBT can speed neutrophil recovery but successful bridging is not guaranteed due to rapid haplo-identical graft rejection.

Mutations in the epidermal growth factor receptor (EGFR) are the most common targetable alterations in lung adenocarcinoma. To facilitate rapid testing, the Idylla EGFR assay was incorporated as a screening method before next-generation sequencing (NGS). Validation and experience using an in-house developed analysis pipeline, enhanced with a manual review algorithm is described. Results are compared with corresponding NGS results. In all, 1249 samples were studied. Validation demonstrated 98.57% (69/70) concordance with the reference methods. The limit of detection varied from 2% to 5% variant allele frequency for total EGFR quantitation cycle between 20 and 23. Of the 1179 clinical cases, 23.41% were EGFR-positive by Idylla. Concurrent NGS was successfully performed on 94.9% (799/842) requests. Concordance of Idylla with NGS was 98.62% (788/799) and 98.50% (787/799) using our in-house and Idylla analysis pipelines, respectively. Discordances involved missed mutations by both assays associated with low tumor/low input. Incorporating a manual review algorithm to supplement automated calls improved accuracy from 98.62% to 99.37% and sensitivity from 94.68% to 97.58%. Overall reporting time, from receipt of material to official clinical report, ranged from 1 to 3 days. Therefore, Idylla EGFR testing enables rapid and sensitive screening without compromising subsequent comprehensive NGS, when required. Automated calling, enhanced with a manual review algorithm, reduces false-negative calls associated with low tumor/low input samples., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

TERT gene promoter mutations are known in multiple cancer types. Other TERT alterations remain poorly characterized. Sequencing data from 30,773 tumors analyzed by a hybridization capture next-generation sequencing assay (Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets) were analyzed for the presence of TERT alterations. Promoter rearrangements (500 bases upstream of the transcriptional start site), hypermethylation (n = 57), and gene expression (n = 155) were evaluated for a subset of cases. Mutually exclusive and recurrent promoter mutations were identified at three hot spots upstream of the transcriptional start site in 11.3% of cases (-124: 74%; -146: 24%; and -138: <2%). Mutually exclusive amplification events were identified in another 2.3% of cases, whereas mutually exclusive rearrangements proximal to the TERT gene were seen in 24 cases. The highest incidence of TERT promoter mutations was seen in cutaneous melanoma (82%), whereas amplification events significantly outnumbered promoter mutations in well-differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%). Gene expression analysis suggests that the highest levels of gene expression are seen in cases with amplifications and rearrangements. Hypermethylation events upstream of the TERT coding sequence were not mutually exclusive with known pathogenic alterations. Studies aimed at defining the prevalence and prognostic impact of TERT alterations should incorporate other pathogenic TERT alterations as these may impact telomerase function., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Chromosome translocations involving the RUNX1 gene at 21q22 are recurring abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), that is, t(8;21) and t(3;21) and in B-cell acute lymphoblastic leukemia with t(12;21). These translocations result in the fusion of RUNX1 with RUNX1T1, MECOM, and ETV6, respectively, and are implicated in leukemogenesis. Here we describe 10 rare RUNX1 fusion gene partners, including six novel fusions, in myeloid neoplasia. Comprehensive molecular testing revealed the partner genes and features of these fusions in all the tested patients, and detected various recurring myeloid related gene mutations in eight patients. In two patients, RUNX1 mutations were identified. Most of these fusions were detected in patients with high-grade MDS and AML with a relatively short survival. Integration of conventional chromosome analysis, FISH testing and molecular genetic studies allow a comprehensive characterization of these rare RUNX1 fusions. Our study may help define myeloid neoplasms with rare and novel RUNX1 translocations and support appropriate patient management., (© 2020 Wiley Periodicals LLC.)

The 2016 International Myeloma Working Group consensus recommendations emphasize high-sensitivity methods for minimal residual disease (MRD) detection, treatment response assessment, and prognostication. Next-generation sequencing (NGS) of IGH gene rearrangements is highly specific and sensitive, but its description in routine clinical practice and performance comparison with high-sensitivity flow cytometry (hsFC) remain limited. In this large, single-institution study including 438 samples from 251 patients, the use of NGS targeting the IGH and IGK genes for clonal characterization and monitoring, with comparison to hsFC, is described. The index clone characterization success rate was 93.6% (235/251), which depended on plasma cell (PC) cellularity, reaching 98% when PC ≥10% and below 80% when PC <5%. A total of 85% of cases were successfully characterized using leader and FR1 primer sets, and most clones showed high somatic hypermutation rates (median, 8.1%). Among monitoring samples from 124 patients, 78.6% (147/187) had detectable disease by NGS. Concordance with hsFC was 92.9% (170/183). Discordant cases encompassed 8 of 124 hsFC MRD+/NGS MRD- patients (6.5%) and 4 of 124 hsFC MRD-/NGS MRD+ patients (3.2%), all with low-level disease near detection limits for both assays. Among concordant hsFC MRD-/NGS MRD- cases, only 5 of 24 patients (20.8%) showed subsequent overt relapse at 3-year follow-up. HsFC and NGS showed similar operational sensitivity, and the choice of test may depend on practical, rather than test performance, considerations., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: MET tyrosine kinase inhibitors (TKIs) can achieve modest clinical outcomes in MET exon 14-altered lung cancers, likely secondary to primary resistance. Mechanisms of primary resistance remain poorly characterized and comprehensive proteomic analyses have not previously been performed., Experimental Design: We performed hybrid capture-based DNA sequencing, targeted RNA sequencing, cell-free DNA sequencing, selected reaction monitoring mass spectrometry (SRM-MS), and immunohistochemistry on patient samples of MET exon 14-altered lung cancers treated with a MET TKI. Associations between overall response rate (ORR), progression-free survival (PFS), and putative genomic alterations and MET protein expression were evaluated., Results: Seventy-five of 168 MET exon 14-altered lung cancers received a MET TKI. Previously undescribed (zygosity, clonality, whole-genome duplication) and known (copy-number focality, tumor mutational burden, mutation region/type) genomic factors were not associated with ORR/PFS ( P > 0.05). In contrast, MET expression was associated with MET TKI benefit. Only cases with detectable MET expression by SRM-MS ( N = 15) or immunochemistry ( N = 22) responded to MET TKI therapy, and cancers with H-score ≥ 200 had a higher PFS than cancers below this cutoff (10.4 vs. 5.5 months, respectively; HR, 3.87; P = 0.02)., Conclusions: In MET exon 14-altered cancers treated with a MET TKI, a comprehensive analysis of previously unknown and known genomic factors did not identify a genomic mechanism of primary resistance. Instead, MET expression correlated with benefit, suggesting the potential role of interrogating the proteome in addition to the genome in confirmatory prospective trials., (©2020 American Association for Cancer Research.)

Inhibition of the MEK/ERK pathway is critical for Bcl-2-like protein 11 (BIM)-mediated epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-induced apoptosis, and dysregulation of this pathway may be a mechanism of acquired resistance. Therefore, MEK inhibition with trametinib and an EGFR TKI may resensitize tumors with acquired resistance. Limited targeted therapies are available after progression on EGFR TKIs, and it is in this setting that we completed a phase I/II study of erlotinib and trametinib., Methods: Patients with metastatic EGFR -mutant lung adenocarcinoma and acquired resistance to an EGFR TKI received combination erlotinib 75 mg and trametinib 1.5 mg daily until progression or unacceptable side effects. The primary objective was objective response rate determined using RECIST version 1.1., Results: Twenty-three patients were accrued; patients had received a median of two lines of prior TKI therapy (61% prior osimertinib), and 48% had acquired EGFR T790M. We confirmed one partial response (1/23, 4%, 95% CI, 0 to 22). The median progression-free survival was 1.8 months, and the median overall survival was 21 months. Diarrhea (87%), acneiform rash (87%), and fatigue (52%) were the most common treatment-related adverse events. Two patients who had tumor shrinkage both harbored a BRAF fusion., Conclusion: Addition of trametinib to erlotinib in the acquired resistance setting in an unselected population is not efficacious. Future studies should focus on targeted therapies in molecularly selected populations. Acquired BRAF fusions in patients with EGFR -sensitizing mutations may be a molecular subset where EGFR and MEK combination therapy could be studied further., (© 2021 by American Society of Clinical Oncology.)

IDH1/2 hotspot mutations occur in glioma, cholangiocarcinoma, chondrosarcoma, sinonasal carcinoma, and T-cell lymphoma and have diagnostic, prognostic, and/or therapeutic value. Availability of immunohistochemistry (IHC) protocols for specific IDH2 mutation detection is limited. A targeted exome sequencing assay MSK-IMPACT cohort comprising >38,000 cancer cases was explored for the presence of IDH1/2 mutations in solid malignancies and select T-cell lymphomas. Seventy-four formalin-fixed paraffin-embedded IDH1/2-mutated (n = 62) and wild-type (n = 12) samples were used for testing and optimization of anti-IDH2 monoclonal antibodies (mAbs) 14H7, 3C11, and MMab1 targeting R172K, R172G, and R172M mutant proteins, respectively. IDH1/2 mutations were common in glioma (26.8% and 1.6%), intrahepatic cholangiocarcinoma (23.1% and 5.7%), chondrosarcoma (19.4% and 10.7%), sinonasal undifferentiated/large-cell neuroendocrine carcinoma (0% and 84.2%), angioimmunoblastic T-cell lymphoma (0% and 22%), and peripheral T-cell lymphoma (0 and 5.1%). In other cancers, IDH2 mutations were rare. IDH2 R172 variants included R172K (39%), R172S (29%), R172W (12%), R172G (10%), R172M (5%), and R172T (4%). 14H7, 3C11, and MMab1 detected all IDH2 R172K, R172G, and R172M, respectively, and produced a crisp, granular cytoplasmic staining pattern. 3C11 was also positive in 5 of 6 IDH1 R132G mutants showing a homogeneous, smooth cytoplasmic staining. All 3 mAbs were negative in other IDH1/2 mutant or wild-type cases. IHC using mAbs 14H7, 3C11, and MMab1 can facilitate molecular diagnosis as a reliable, fast, and inexpensive alternative for specific IDH2 variant detection. Given the distinct distribution of IDH2 R172 mutations in cancers, these mAbs could also serve as useful pathologic diagnostic markers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A subset of glioblastomas (GBMs) harbors potentially druggable oncogenic FGFR3-TACC3 (F3T3) fusions. However, their associated molecular and clinical features are poorly understood. Here we analyze the frequency of F3T3-fusion positivity, its associated genetic and methylation profiles, and its impact on survival in 906 IDH-wildtype GBM patients. We establish an F3T3 prevalence of 4.1% and delineate its associations with cancer signaling pathway alterations. F3T3-positive GBMs had lower tumor mutational and copy-number alteration burdens than F3T3-wildtype GBMs. Although F3T3 fusions were predominantly mutually exclusive with other oncogenic RTK pathway alterations, they did rarely co-occur with EGFR amplification. They were less likely to harbor TP53 alterations. By methylation profiling, they were more likely to be assigned the mesenchymal or RTK II subclass. Despite being older at diagnosis and having similar frequencies of MGMT promoter hypermethylation, patients with F3T3-positive GBMs lived about 8 months longer than those with F3T3-wildtype tumors. While consistent with IDH-wildtype GBM, F3T3-positive GBMs exhibit distinct biological features, underscoring the importance of pursuing molecular studies prior to clinical trial enrollment and targeted treatment.

Acquired mutations are pervasive across normal tissues. However, understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMNs). We find that mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response genes (TP53, PPM1D, CHEK2). Sequential sampling provides definitive evidence that DNA damage response clones outcompete other clones when exposed to certain therapies. Among cases in which CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies.

Purpose: SMARCA4 mutations are among the most common recurrent alterations in non-small cell lung cancer (NSCLC), but the relationship to other genomic abnormalities and clinical impact has not been established., Experimental Design: To characterize SMARCA4 alterations in NSCLC, we analyzed the genomic, protein expression, and clinical outcome data of patients with SMARCA4 alterations treated at Memorial Sloan Kettering., Results: In 4,813 tumors from patients with NSCLC, we identified 8% ( n = 407) of patients with SMARCA4 -mutant lung cancer. We describe two categories of SMARCA4 mutations: class 1 mutations (truncating mutations, fusions, and homozygous deletion) and class 2 mutations (missense mutations). Protein expression loss was associated with class 1 mutation (81% vs. 0%, P < 0.001). Both classes of mutation co-occurred more frequently with KRAS, STK11 , and KEAP1 mutations compared with SMARCA4 wild-type tumors ( P < 0.001). In patients with metastatic NSCLC, SMARCA4 alterations were associated with shorter overall survival, with class 1 alterations associated with shortest survival times ( P < 0.001). Conversely, we found that treatment with immune checkpoint inhibitors (ICI) was associated with improved outcomes in patients with SMARCA4 -mutant tumors ( P = 0.01), with class 1 mutations having the best response to ICIs ( P = 0.027)., Conclusions: SMARCA4 alterations can be divided into two clinically relevant genomic classes associated with differential protein expression as well as distinct prognostic and treatment implications. Both classes co-occur with KEAP1, STK11 , and KRAS mutations, but individually represent independent predictors of poor prognosis. Despite association with poor outcomes, SMARCA4 -mutant lung cancers may be more sensitive to immunotherapy., (©2020 American Association for Cancer Research.)

Purpose: Although primary germ cell tumors (GCTs) have been extensively characterized, molecular analysis of metastatic sites has been limited. We performed whole-exome sequencing and targeted next-generation sequencing on paired primary and metastatic GCT samples in a patient cohort enriched for cisplatin-resistant disease., Patients and Methods: Tissue sequencing was performed on 100 tumor specimens from 50 patients with metastatic GCT, and sequencing of plasma cell-free DNA was performed for a subset of patients., Results: The mutational landscape of primary and metastatic pairs from GCT patients was highly discordant (68% of all somatic mutations were discordant). Whereas genome duplication was common and highly concordant between primary and metastatic samples, only 25% of primary-metastasis pairs had ≥ 50% concordance at the level of DNA copy number alterations (CNAs). Evolutionary-based analyses revealed that most mutations arose after CNAs at the respective loci in both primary and metastatic samples, with oncogenic mutations enriched in the set of early-occurring mutations versus variants of unknown significance (VUSs). TP53 pathway alterations were identified in nine cisplatin-resistant patients and had the highest degree of concordance in primary and metastatic specimens, consistent with their association with this treatment-resistant phenotype., Conclusion: Analysis of paired primary and metastatic GCT specimens revealed significant molecular heterogeneity for both CNAs and somatic mutations. Among loci demonstrating serial genetic evolution, most somatic mutations arose after CNAs, but oncogenic mutations were enriched in the set of early-occurring mutations as compared with VUSs. Alterations in TP53 were clonal when present and shared among primary-metastasis pairs., Competing Interests: Conflicts of Interest Statement:The authors have declared that no competing interests exist.Conflicts of Interest Statement:Authors’ disclosures of potential conflicts of interest and contributions are found at the end of this article.The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Michael L. ChengHonoraria: The Lynx Group (supported by Bristol Myers Squibb), WebMD (supported by AstraZeneca), PCME (supported by Merck and Lilly) Consulting or Advisory Role: AstraZeneca, Inivata Travel, Accommodations, Expenses: Daiichi Sankyo, Allergan, Sanofi, Natera, AstraZeneca, Guardant Health, WebMD, PCMEFrançois AudenetHonoraria: Nucleix Travel, Accommodations, Expenses: Ferring, IpsenEugene J. PietzakHonoraria: UpToDate Consulting or Advisory Role: Merck, Chugai PharmaSumit IsharwalOpen Payments Link: https://openpaymentsdata.cms.gov/physician/1232544Gopa IyerConsulting or Advisory Role: Bayer, Janssen, Mirati Therapeutics Research Funding: Mirati Therapeutics (Inst), Novartis (Inst), Debiopharm Group (Inst), Bayer (Inst)Samuel FuntStock and Other Ownership Interests: Kite Pharma, Urogen Pharma (I), Allogene Therapeutics, Neogene Therapeutics (I), Kronos Bio (I), Vida Ventures (I), Vaxigene Consulting or Advisory Role: AstraZeneca/MedImmune, Merck, Immunai Research Funding: Genentech (Inst), AstraZeneca (Inst), Decibel Therapeutics (Inst) Travel, Accommodations, Expenses: Bristol Myers Squibb, AstraZeneca/MedImmuneDean F. BajorinHonoraria: Merck Sharp & Dohme Consulting or Advisory Role: Merck, Fidia Farmaceutici, Hoffman-La Roche, Dragonfly Therapeutics Research Funding: Novartis (Inst), Genentech (Inst), Merck (Inst), Bristol Myers Squibb (Inst), AstraZeneca (Inst), Astellas Pharma (Inst), Seattle Genetics/Astellas (Inst) Travel, Accommodations, Expenses: Genentech, MerckVictor E. ReuterConsulting or Advisory Role: Cepheid Uncompensated Relationships: PaigeAIGabriella JosephStock and Other Ownership Interests: AmgenMaria E. ArcilaHonoraria: Invivoscribe, Biocartis Consulting or Advisory Role: AstraZeneca Travel, Accommodations, Expenses: AstraZeneca, Invivoscribe, Raindance TechnologiesDana W.Y. TsuiHonoraria: Cowen, BofA Merrill Lynch Patents, Royalties, Other Intellectual Property: I am a co-inventor on a provisional patent application filed by Memorial Sloan Kettering Cancer CenterAhmet ZehirHonoraria: IlluminaMichael F. BergerConsulting or Advisory Role: Roche Research Funding: Grail Patents, Royalties, Other Intellectual Property: Provisional patent pending for “Systems and Methods for Detecting Cancer via cfDNA Screening”Eliezer Van AllenStock and Other Ownership Interests: Syapse, Tango Therapeutics, Genome Medical, Microsoft, Ervaxx Consulting or Advisory Role: Syapse, Roche, Third Rock Ventures, Takeda, Novartis, Genome Medical, InVitae, Illumina, Tango Therapeutics, Ervaxx, Janssen Speakers' Bureau: Illumina Research Funding: Bristol Myers Squibb, Novartis Patents, Royalties, Other Intellectual Property: Patent on discovery of retained intron as source of cancer neoantigens (Inst); patent on discovery of chromatin regulators as biomarkers of response to cancer immunotherapy (Inst); patent on clinical interpretation algorithms using cancer molecular data (Inst) Travel, Accommodations, Expenses: GenentechBarry S. TaylorConsulting or Advisory Role: Boehringer Ingelheim, Loxo Oncology at Lilly Research Funding: GenentechHikmat Al-AhmadieConsulting or Advisory Role: Bristol Myers Squibb, EMD Serono, AstraZeneca/MedImmune, Janssen BiotechDavid B. SolitStock and Other Ownership Interests: Loxo Consulting or Advisory Role: Pfizer, Loxo, Illumina, Vivideon Therapeutics, Lilly Oncology, QED Therapeutics, BridgeBio PharmaDarren R. FeldmanResearch Funding: Novartis, Seattle Genetics, Decibel Therapeutics (Inst), Astellas Pharma Other Relationship: UpToDate No other potential conflicts of interest were reported., (© 2020 by American Society of Clinical Oncology.)

Objective: To report the mutational profile and clinical outcomes of a cohort of patients with KIT-mutant seminomas and nonseminomatous germ-cell tumors (SGCT/NSGCTs)., Patients and Methods: Retrospective cohort study of all patients with KIT-mutant GCTs sequenced at Memorial Sloan Kettering between March 2014 and March 2020. Tumors were assessed with MSK-IMPACT, a DNA next-generation sequencing assay for targeted sequencing of up to 468 key cancer genes., Results: Among 568 patients with GCTs, 8.1% had somatic KIT mutations, including 28 seminomas and 18 mixed/NSGCTs. Exons 17 (67.3%), 11 (22.4%), and 13 (6.1%) were most commonly affected. KIT-mutant cases were enriched for oncogenic RAS/MAPK pathway alterations compared to KIT-wildtype cases (34.8% vs 19.2%, P = .02). Among KIT-mutant cases, concurrent mutations were noted in KRAS (21.7%), RRAS2 (11.8%), CBL (6.5%), NRAS (4.3%), MAP2K1 (2.2%), and RAC1 (2.2%). Mutations in KRAS, RRAS2, and NRAS were mutually exclusive. In all, 73.9% of patients developed metastases and 95.7% received chemotherapy. No patients received KIT-directed tyrosine kinase inhibitors (TKIs). Classification as a NSGCT rather than a SGCT was associated with an increased risk of death (hazard ratio 9.1, 95% confidence interval 1.1-78.4, P = .04) while the presence of a concurrent RAS/MAPK pathway alteration was not (hazard ratio 0.8, 95% confidence interval 0.1-4.3, P = .76)., Conclusion: Mitogenic driver alterations can co-occur with activating KIT mutations, which may explain the lack of efficacy of KIT-directed TKIs in prior trials. Novel KIT-directed TKIs that target exon 17 mutations may benefit chemotherapy-refractory patients with KIT-mutant GCTs without RAS/MAPK alterations. Dual MEK/KIT inhibitor therapy in KIT-mutant GCTs with concurrent RAS/MAPK alterations could also be a plausible therapeutic strategy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: For patients with advanced NSCLC, cytologic samples may be the only diagnostic specimen available for molecular profiling. Although both rapid and comprehensive assessment are essential in this setting, an integrated multitest approach remains an important strategy in many laboratories, despite the risks and challenges when working with scant samples. In this study, we describe our experience and high success rate in using a multitest approach, focusing on the clinical validation and incorporation of ultrarapid EGFR testing using the Idylla system followed by comprehensive next-generation sequencing (NGS)., Methods: Cytology samples received for routine molecular testing were included in this study. The performance characteristics of the EGFR Idylla assay were assessed; tissue suitability parameters and interpretation criteria to supplement automated mutation calling were established. The assay performance was monitored for 1 year, comparing the results with those of concurrent NGS testing by MSK-IMPACT (primarily) or MSK-AmpliSeq and MSK-Fusion solid panel in a subset of cases., Results: Overall, 301 samples were studied; 83 samples were included in validation (60.2% [50 of 83] were positive for EGFR mutations). Concordance with the reference method was 96.4% (80 of 83) of the samples with excellent reproducibility. The limit of detection was variable depending on the total tissue input and the specific mutation tested. Unextracted tissue inputs that maintained total EGFR cycle of quantification at less than 23 allowed all mutations to be detected if present at greater than 5% variant allele frequency. Mutations could be detected at 1% variant allele frequency with total EGFR cycle of quantification of 18. During the clinical implementation phase, 218 NSCLC samples were tested by Idylla (24.3% [53 of 218] were EGFR mutation positive). Concurrent NGS testing was requested on 165 samples and successfully performed on 96.4% (159 of 165) of the samples. The Idylla automated results were concordant with those obtained by NGS in 96.2% (153 of 159) of cases and improved to 98.7% (157 of 159) after incorporation of manual review criteria to supplement automated calling, resulting in a diagnostic sensitivity of 95.6% (95% confidence interval: 84.9%-99.5%). In general, 9% (14 of 159) of the cases tested by NGS had EGFR mutations not covered by the Idylla assay, primarily insertions in exon 19 and 20 and minor mutations cooccurring with canonical sensitizing mutations., Conclusions: Comprehensive molecular testing is feasible and has a high success rate in NSCLC cytology samples when using a multitest approach. Testing with the Idylla system enables rapid and accurate determination of the EGFR status without compromising subsequent NGS testing., Competing Interests: Disclosure: Dr. Arcila has received honoraria from Biocartis and Invivoscribe; Dr. Nafa has received honoraria from Biocartis; Dr. Yang has received consulting fees from Invitae. The remaining authors declare no conflict of interest.

Purpose: Although MET exon 14 ( MET ex14)-altered lung cancers were first identified more than a decade and a half ago, the frequency of CNS metastatic disease remains poorly defined. Furthermore, the seminal trial of crizotinib in these patients (PROFILE 1001) did not report patterns of CNS response or progression., Patients and Methods: Patients with pathologically confirmed, advanced non-small-cell lung cancers (NSCLC) harboring a MET ex14 alteration by targeted DNA/RNA sequencing were studied. The incidence of brain metastases and the outcomes of MET inhibition with crizotinib were analyzed., Results: Eighty-three patients with MET ex14-altered metastatic NSCLC were identified. The incidence of CNS metastases at diagnosis was 17% (95% CI, 10% to 27%). The lifetime incidence was 36% (95% CI, 26% to 47%); 83% of patients had parenchymal disease, and 17% had leptomeningeal disease. The probability of having brain metastasis at 1, 2, and 3 years was 24%, 35%, and 38%, respectively. Fifty-four patients received crizotinib. The median time to radiologic CNS progression was 5.8 months (range, 3.7-20.0 months). Patterns of crizotinib progression were as follows: intracranial only in 10% of patients, intracranial and extracranial in 12%, and extracranial only in 78%. In patients with brain metastases before treatment, the median time on crizotinib was 7.5 months (range, 7.2-11.7 months)., Conclusion: CNS metastases, including leptomeningeal disease, occurred in more than a third of patients with MET ex14-altered lung cancers. In crizotinib-treated patients with or without CNS metastases, CNS failure was seen in less than a quarter of patients on progression., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Michael OffinHonoraria: OncLive Consulting or Advisory Role: PharmaMar, Novartis, Targeted Oncology Travel, Accommodations, Expenses: Bristol Myers Squibb, Merck Sharp & DohmeJia LuoHonoraria: Targeted OncologyJordan DienstagStock and Other Ownership Interests: AbbVie, Bristol Myers Squibb, Thermo Fisher Scientific, AllerganCharles M. RudinConsulting or Advisory Role: AbbVie, Harpoon Therapeutics, Genentech, AstraZeneca, Ascentage Pharma, Bicycle Therapeutics, Celgene, Daiichi Sankyo, Ipsen, Loxo, PharmaMar, Bridge Medicines, Amgen, Jansen, Syros Pharmaceuticals Research Funding: AbbVie/Stemcentrx (Inst), Viralytics (Inst), Merck (Inst), Daiichi Sankyo (Inst), Genentech (Inst)Gregory RielyResearch Funding: Novartis (Inst), Genentech (Inst), GlaxoSmithKline (Inst), Pfizer (Inst), Infinity Pharmaceuticals (Inst), Mirati Therapeutics (Inst), Merck (Inst), Takeda (Inst) Patents, Royalties, Other Intellectual Property: Patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases (Inst) Travel, Accommodations, Expenses: Merck Sharp & Dohme Other Relationship: Pfizer, Genentech, TakedaNatasha RekhtmanHonoraria: Physicians’ Education ResourceMaria E. ArcilaHonoraria: Invivoscribe, Biocartis Consulting or Advisory Role: AstraZeneca Travel, Accommodations, Expenses: AstraZeneca, Invivoscribe, Raindance TechnologiesGlenn HellerResearch Funding: Janssen DiagnosticsMarc LadanyiConsulting or Advisory Role: Bristol Myers Squibb, Bayer Research Funding: Loxo (Inst), Helsinn Therapeutics, Merus NV (Inst), Elevation Oncology (Inst)Bob T. LiConsulting or Advisory Role: Guardant Health, Hengrui Therapeutics, Eli Lilly (Inst), Genentech (Inst) Research Funding: AstraZeneca (Inst), GRAIL (Inst), Daiichi Sankyo (Inst), Hengrui Therapeutics (Inst), Guardant Health (Inst), Amgen (Inst), Eli Lilly (Inst), MORE Health (Inst) Patents, Royalties, Other Intellectual Property: US62/514,661 (Inst), US62/685,057 (Inst)Mark G. KrisConsulting or Advisory Role: AstraZeneca, Regeneron, Pfizer, Daiichi Sankyo Travel, Accommodations, Expenses: AstraZeneca, Pfizer Other Relationship: Memorial Sloan Kettering Cancer Center, Genentech Open Payments Link: https://openpaymentsdata.cms.gov/physician/markgkris/summaryPaul PaikHonoraria: Celgene, Takeda, Calithera Biosciences, Boehringer Ingelheim, EMD Serono, AstraZeneca Consulting or Advisory Role: Celgene, EMD Serono, Takeda, Calithera Biosciences, AstraZeneca, Boehringer Ingelheim Research Funding: Celgene, EMD Serono Travel, Accommodations, Expenses: EMD SeronoAlexander DrilonHonoraria: Medscape, OncLive, PeerVoice, Physicians Education Resources, Targeted Oncology, MORE Health, Research to Practice, Foundation Medicine, Peerview Consulting or Advisory Role: Ignyta, Loxo, TP Therapeutics, AstraZeneca, Pfizer, Blueprint Medicines, Genentech, Helsinn Therapeutics, BeiGene, Hengrui Therapeutics, Exelixis, Bayer, Tyra Biosciences, Verastem, Takeda/Ariad/Millennium, BerGenBio, MORE Health, Eli Lilly, Verastem, AbbVie, 14ner/Elevation Oncology, Remedica, Archer Biosciences, Monopteros Research Funding: Foundation Medicine Patents, Royalties, Other Intellectual Property: Wolters Kluwer (royalties for Pocket Oncology) Other Relationship: Merck, GlaxoSmithKline, Teva, Taiho Pharmaceutical, Pfizer, PharmaMar, Puma Biotechnology No other potential conflicts of interest were reported., (© 2020 by American Society of Clinical Oncology.)

Importance: The combination of erlotinib and bevacizumab as initial treatment of epidermal growth factor receptor (EGFR [OMIM 131550])-mutant lung cancers improves progression-free survival (PFS) compared with erlotinib alone. Because osimertinib prolongs PFS compared with erlotinib, this trial was designed to study the combination of osimertinib and bevacizumab as first-line treatment., Objectives: To determine the safety and tolerability of osimertinib and bevacizumab combination treatment and assess the 12-month PFS of the combination in patients with metastatic EGFR-mutant lung cancers., Design, Setting, and Particiants: From August 15, 2016, to May 15, 2018, 49 patients with metastatic EGFR-mutant lung cancers were enrolled in this interventional clinical trial, conducted at a single academic cancer center. In the phase 1 portion of the study, a standard 3 + 3 dose de-escalation design was used to determine the maximum tolerated dose of osimertinib and bevacizumab. In the phase 2 portion of the study, patients were treated at the maximum tolerated dose defined in the phase 1 portion. Statistical analysis was performed from August 1 to October 1, 2019., Interventions: All patients received osimertinib, 80 mg daily, and bevacizumab, 15 mg/kg once every 3 weeks., Main Outcomes and Measures: The primary objective of the phase 2 portion of the study was to determine the number of patients receiving the combination of osimertinib and bevacizumab who were progression free at 12 months. Secondary end points included overall response rate, median PFS, overall survival, and definition of the toxic effects of the combination treatment., Results: Among the 49 patients in the study (34 women; median age, 60 years [range, 36-83 years]), PFS at 12 months was 76% (95% CI, 65%-90%). The overall response rate was 80% (95% CI, 67%-91%), and median PFS was 19 months (95% CI, 15-24 months). Of the 6 patients with measurable central nervous system disease, all had a partial or complete central nervous system response. Persistent detection of EGFR-mutant circulating tumor (ct)DNA at 6 weeks was associated with shorter median PFS (clearance at 6 weeks, 16.2 months [95% CI, 13 months to not reached]; and no clearance at 6 weeks, 9.8 months [95% CI, 4 months to not reached]; P = .04) and median overall survival (clearance at 6 weeks, not reached; and no clearance at 6 weeks, 10.1 months [95% CI, 6 months to not reached]; P = .002). Identified mechanisms of resistance included squamous cell transformation (n = 2) pleomorphic transformation (n = 1), and acquired EGFR L718Q (n = 1) and C797S (n = 1) mutations., Conclusions and Relevance: The combination of osimertinib and bevacizumab met the study's prespecified effectiveness end point. Persistent EGFR-mutant circulating tumor DNA at 6 weeks was associated with early progression and shorter survival. A randomized phase 3 study comparing osimertinib and bevacizumab with osimertinib alone is planned., Trial Registration: ClinicalTrials.gov Identifier: NCT02803203.

Purpose: Patterns of resistance to first-line osimertinib are not well-established and have primarily been evaluated using plasma assays, which cannot detect histologic transformation and have differential sensitivity for copy number changes and chromosomal rearrangements., Experimental Design: To characterize mechanisms of resistance to osimertinib, patients with metastatic EGFR -mutant lung cancers who received osimertinib at Memorial Sloan Kettering Cancer Center and had next-generation sequencing performed on tumor tissue before osimertinib initiation and after progression were identified., Results: Among 62 patients who met eligibility criteria, histologic transformation, primarily squamous transformation, was identified in 15% of first-line osimertinib cases and 14% of later-line cases. Nineteen percent (5/27) of patients treated with first-line osimertinib had off-target genetic resistance (2 MET amplification, 1 KRAS mutation, 1 RET fusion, and 1 BRAF fusion) whereas 4% (1/27) had an acquired EGFR mutation ( EGFR G724S). Patients with squamous transformation exhibited considerable genomic complexity; acquired PIK3CA mutation, chromosome 3q amplification, and FGF amplification were all seen. Patients with transformation had shorter time on osimertinib and shorter survival compared with patients with on-target resistance. Initial EGFR sensitizing mutation, time on osimertinib treatment, and line of therapy also influenced resistance mechanism that emerged. The compound mutation EGFR S768 + V769L and the mutation MET H1094Y were identified and validated as resistance mechanisms with potential treatment options., Conclusions: Histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes. See related commentary by Piotrowska and Hata, p. 2441 ., (©2020 American Association for Cancer Research.)

TET2 and DNMT3A mutations are frequently identified in T-cell lymphomas of T follicular helper cell origin (TCL-TFH), clonal hematopoiesis (CH), and myeloid neoplasms (MNs). The relationships among these 3 entities, however, are not well understood. We performed comprehensive genomic studies on paired bone marrow and tissue samples as well as on flow cytometry-sorted bone marrow and peripheral blood subpopulations from a cohort of 22 patients with TCL-TFH to identify shared CH-type mutations in various hematopoietic cell compartments. Identical mutations were detected in the neoplastic T-cell and myeloid compartments of 15 out of 22 patients (68%), including TET2 (14/15) and DNMT3A (10/15). Four patients developed MNs, all of which shared CH-type mutations with their TCL-TFH; additional unique genetic alterations were also detected in each patient's TCL-TFH and MN. These data demonstrate that CH is prevalent in patients with TCL-TFH and that divergent evolution of a CH clone may give rise to both TCL-TFH and MNs.

Non-Hodgkin lymphoma encompasses a diverse group of B-cell and T-cell neoplasms. Current classification is based on clinical information, histologic assessment, immunophenotypic characteristics, and molecular alterations. A wide range of genetic alterations, including large chromosomal structural rearrangements, aneuploidies, point mutations, and copy number alterations, have been reported across all types of lymphomas. Many of these are now incorporated into the World Health Organization-defined criteria for the diagnostic evaluation of patients with lymphoid proliferations and, therefore, their accurate identification is paramount for diagnosis, subclassification, and selection of treatment. In addition to their value in the diagnostic setting, many alterations that are not routinely evaluated in standard clinical practice may still define specific disease entities as they have important implications in risk stratification, as well as roles in emerging alternate therapies and disease monitoring. Because of the complexity and range of alterations, their accurate and sensitive assessment requires a careful selection of technology. Here, we discuss the most commonly used molecular techniques in current clinical practice and highlight some of the benefits and pitfalls based on the type of alteration.

Amplification of and oncogenic mutations in ERBB2 , the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2 -amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFICANCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2 . This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy. See related commentary by Rolfo and Russo, p. 643 . This article is highlighted in the In This Issue feature, p. 627 ., (©2020 American Association for Cancer Research.)

TAFRO syndrome, a clinical subtype of idiopathic multicentric Castleman disease (iMCD), consists of a constellation of symptoms/signs including thrombocytopenia, anasarca, fever, reticulin fibrosis/renal dysfunction, and organomegaly. The etiology of iMCD-TAFRO and the basis for cytokine hypersecretion commonly seen in iMCD-TAFRO patients has not been elucidated. Here, we identified a somatic MEK2 P128L mutation and a germline RUNX1 G60C mutation in two patients with iMCD-TAFRO, respectively. The MEK2 P128L mutation, which has been identified previously in solid tumor and histiocytosis patients, caused hyperactivated MAP kinase signaling, conferred IL-3 hypersensitivity and sensitized the cells to various MEK inhibitors. The RUNX1 G60C mutation abolished the transcriptional activity of wild-type RUNX1 and functioned as a dominant negative form of RUNX1, resulting in enhanced self-renewal activity in hematopoietic stem/progenitor cells. Interestingly, ERK was heavily activated in both patients, highlighting a potential role for activation of MAPK signaling in iMCD-TAFRO pathogenesis and a rationale for exploring inhibition of the MAPK pathway as a therapy for iMCD-TAFRO. Moreover, these data suggest that iMCD-TAFRO might share pathogenetic features with clonal inflammatory disorders bearing MEK and RUNX1 mutations such as histiocytoses and myeloid neoplasms.

Clinical testing for MLH1 promoter hypermethylation status is important in the workup of patients with MLH1-deficient colorectal and uterine carcinomas when evaluating patients for Lynch syndrome. Current assays use single gene-based methods to assess promoter hypermethylation. Herein, we describe the development and report the performance of a clinical assay for MLH1 promoter hypermethylation using the Infinium methylationEPIC (850k) bead-array platform. Using four cytosine-guanine dinucleotide (CpG) sites within the MLH1 gene promoter, a qualitative MLH1 promoter hypermethylation assay was developed and validated using 63 gastrointestinal and uterine carcinoma samples of known hypermethylation status based on a pyrosequencing reference test. The array-based method achieves clinically robust and reproducible results at an analytical sensitivity level of 8%. Of importance, the 850k array contains probes targeting >850,000 additional CpG sites across the genome, covering sites in most known genes as well as important enhancer regions provided by the Encyclopedia of DNA Elements and Functional Annotation of The Mammalian Genome projects. Thus, the testing modality presented may also be applied to determine the methylation status of other clinically relevant genes or regulatory regions, potentially providing a single laboratory testing workflow for all clinical methylation assays. Furthermore, the concomitant acquisition of genome-wide methylation information provides a workflow that seamlessly enables wider translational epigenetic research., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

We have encountered pancreatic tumors with unique histologic features, which do not conform to any of the known tumors of the pancreas or other anatomical sites. We aimed to define their clinicopathologic features and whether they are characterized by recurrent molecular signatures. Eight cases were identified; studied histologically and by immunohistochemistry. Selected cases were also subjected to whole-exome sequencing (WES; n = 4), RNA-sequencing (n = 6), Archer FusionPlex assay (n = 5), methylation profiling using the Illumina MethylationEPIC (850k) array platform (n = 6), and TERT promoter sequencing (n = 5). Six neoplasms occurred in females. The mean age was 43 years (range: 26-75). Five occurred in the head/neck of the pancreas. All patients were treated surgically; none received neoadjuvant/adjuvant therapy. All patients are free of disease after 53 months of median follow-up (range: 8-94). The tumors were well-circumscribed, and the median size was 1.8 cm (range: 1.3-5.8). Microscopically, the unencapsulated tumors had a geographic pattern of epithelioid cell nests alternating with spindle cell fascicles. Some areas showed dense fibrosis, in which enmeshed tumor cells imparted a slit-like pattern. The predominant epithelioid cells had scant cytoplasm and round-oval nuclei with open chromatin. The spindle cells displayed irregular, hyperchromatic nuclei. Mitoses were rare. No lymph node metastases were identified. All tumors were positive for vimentin, CD99 and cytokeratin (patchy), while negative for markers of solid pseudopapillary neoplasm, neuroendocrine, acinar, myogenic/rhabdoid, vascular, melanocytic, or lymphoid differentiation, gastrointestinal stromal tumor as well as MUC4. Whole-exome sequencing revealed no recurrent somatic mutations or amplifications/homozygous deletions in any known oncogenes or tumor suppressor genes. RNA-sequencing and the Archer FusionPlex assay did not detect any recurrent likely pathogenic gene fusions. Single sample gene set enrichment analysis revealed that these tumors display a likely mesenchymal transcriptomic program. Unsupervised analysis (t-SNE) of their methylation profiles against a set of different mesenchymal neoplasms demonstrated a distinct methylation pattern. Here, we describe pancreatic neoplasms with unique morphologic/immunophenotypic features and a distinct methylation pattern, along with a lack of abnormalities in any of key genetic drivers, supporting that these neoplasms represent a novel entity with an indolent clinical course. Given their mesenchymal transcriptomic features, we propose the designation of "sclerosing epithelioid mesenchymal neoplasm" of the pancreas.

RAS-pathway mutations are recurrent events in myeloid malignancies. However, there is limited data on the significance of RAS-pathway mutations in patients with myelofibrosis (MF). We analyzed next-generation sequencing data of 16 genes, including RAS-pathway genes, from 723 patients with primary and secondary MF across three international centers and evaluated their significance. N/KRAS variants were present in 6% of patients and were typically sub-clonal (median VAF = 20%) relative to other genes variants. RAS variants were associated with advanced MF features including leukocytosis (p = 0.02), high somatic mutation burden (p < 0.01) and the presence of established "molecular high-risk" (MHR) mutations. MF patients with N/KRAS mutations had shorter 3-year overall survival (OS) (34% vs 58%, p < 0.001) and higher incidence of acute myeloid leukemia at 3 years (18% vs 11%, p = 0.03). In a multivariate Cox model, RAS mutations were associated with decreased OS (HR 1.93, p < 0.001). We created a novel score to predict OS incorporating RAS mutations, and it predicted OS across training and validation cohorts. Patients with intermediate risk/high-risk DIPSS with RAS mutations who received ruxolitinib had a nonsignificant longer 2-year OS relative to those who did not receive ruxolitinib. These data demonstrate the importance of identifying RAS mutations in MF patients.

Background: Mutations in human epidermal growth factor receptor 2 (HER2; also known as ERBB2) are found in approximately 2% of lung adenocarcinomas. The frequency and clinical course of brain metastases in this oncogenic subset are ill defined., Methods: Baseline and subsequent development of brain metastases was evaluated in consecutive patients with HER2-mutant (n = 98), epidermal growth factor receptor (EGFR)-mutant (n = 200), and KRAS-mutant lung cancers (n = 200)., Results: At metastatic diagnosis, the odds ratio (ORs) for brain metastases was similar for patients whose tumors harbored HER2 mutations (19%) in comparison with patients with KRAS mutations (24%; OR for HER2 vs KRAS, 0.7; P = .33) but lower compared to patients with EGFR mutations (31%; OR for HER2 vs EGFR, 0.5; P = .03). Patients with lung cancer and HER2 mutations developed more brain metastases on treatment than patients with KRAS mutations (28% vs 8%; hazard ratio [HR], 5.2; P < .001) and trended more than patients with EGFR mutations (28% vs 16%; HR, 1.7; P = .06). Patients with HER2 YVMA mutations also developed more brain metastases on treatment than patients with KRAS mutations (HR, 5.9; P < .001). The median overall survival (OS) was shorter for patients with HER2-mutant (1.6 years; P < .001) or KRAS-mutant lung cancers (1.1 years; P < .001) than patients with EGFR-mutant lung cancers (3.0 years). Brain metastases occurred in 47% of patients with HER2-mutant lung cancers, which imparted shorter OS (HR, 2.7; P < .001)., Conclusions: These data provide a framework for brain imaging surveillance in patients with HER2-mutant lung cancers and underpin the need to develop HER2-targeted agents with central nervous system activity., (© 2019 American Cancer Society.)

Clonal heterogeneity and evolution of mantle cell lymphoma (MCL) remain unclear despite the progress in our understanding of its biology. Here, we report a 71-yr-old male patient with an aggressive MCL and depict the clonal evolution from initial diagnosis of typical MCL to relapsed blastoid MCL. During the course of the disease, the patient was diagnosed with classic Hodgkin lymphoma (CHL) and received a CHL therapeutic regimen. Molecular analysis by next-generation sequencing of both MCL and CHL demonstrated clonally related CHL with characteristic immunophenotype and PDL1/2 gains. Moreover, our data illustrate the clonal heterogeneity and acquisition of additional genetic aberrations including a rare fusion of SEC22B-NOTCH2 in the process of clonal evolution. Evidence obtained from our comprehensive immunophenotypic and genetic studies indicates that MCL and CHL can originate from a common precursor by divergent clonal evolution, which may pose a therapeutic challenge., (© 2019 Tashkandi et al.; Published by Cold Spring Harbor Laboratory Press.)

Histiocytoses are clonal hematopoietic disorders frequently driven by mutations mapping to the BRAF and MEK1 and MEK2 kinases. Currently, however, the developmental origins of histiocytoses in patients are not well understood, and clinically meaningful therapeutic targets outside of BRAF and MEK are undefined. In this study, we uncovered activating mutations in CSF1R and rearrangements in RET and ALK that conferred dramatic responses to selective inhibition of RET (selpercatinib) and crizotinib, respectively, in patients with histiocytosis.

Minimal residual disease (MRD) tracking, by next generation sequencing of immunoglobulin sequences, is moving towards clinical implementation in multiple myeloma. However, there is only sparse information available to address whether clonal sequences remain stable for tracking over time, and to what extent light chain sequences are sufficiently unique for tracking. Here, we analyzed immunoglobulin repertoires from 905 plasma cell myeloma and healthy control samples, focusing on the third complementarity determining region (CDR3). Clonal heavy and/or light chain expression was identified in all patients at baseline, with one or more subclones related to the main clone in 3.2%. In 45 patients with 101 sequential samples, the dominant clonal CDR3 sequences remained identical over time, despite differential clonal evolution by whole exome sequencing in 49% of patients. The low frequency of subclonal CDR3 variants, and absence of evolution over time in active multiple myeloma, indicates that tumor cells at this stage are not under selective pressure to undergo antibody affinity maturation. Next, we establish somatic hypermutation and non-templated insertions as the most important determinants of light chain clonal uniqueness, identifying a potentially trackable sequence in the majority of patients. Taken together, we show that dominant clonal sequences identified at baseline are reliable biomarkers for long-term tracking of the malignant clone, including both IGH and the majority of light chain clones., (© 2019 Wiley Periodicals, Inc.)

Purpose: In patients with >1 non-small cell lung carcinoma (NSCLC), the distinction between separate primary lung carcinomas (SPLCs) and intrapulmonary metastases (IPMs) is a common diagnostic dilemma with critical staging implications. Here, we compared the performance of comprehensive next-generation sequencing (NGS) with standard histopathologic approaches for distinguishing NSCLC clonal relationships in clinical practice., Experimental Design: We queried 4,119 NSCLCs analyzed by 341-468 gene MSK-IMPACT NGS assay for patients with >1 surgically resected tumor profiled by NGS. Tumor relatedness predicted by prospective histopathologic assessment was contrasted with comparative genomic profiling by subsequent NGS., Results: Sixty patients with NGS performed on >1 NSCLCs were identified, yielding 76 tumor pairs. NGS classified tumor pairs into 51 definite SPLCs (median, 14; up to 72 unique somatic mutations per pair), and 25 IPMs (24 definite, one high probability; median, 5; up to 16 shared somatic mutations per pair). Prospective histologic prediction was discordant with NGS in 17 cases (22%), particularly in the prediction of IPMs (44% discordant). Retrospective review highlighted several histologic challenges, including morphologic progression in some IPMs. We subsampled MSK-IMPACT data to model the performance of less comprehensive assays, and identified several clinicopathologic differences between NGS-defined tumor pairs, including increased risk of subsequent recurrence for IPMs., Conclusions: Comprehensive NGS allows unambiguous delineation of clonal relationship among NSCLCs. In comparison, standard histopathologic approach is adequate in most cases, but has notable limitations in the recognition of IPMs. Our results support the adoption of broad panel NGS to supplement histology for robust discrimination of NSCLC clonal relationships in clinical practice., (©2019 American Association for Cancer Research.)

Disruption of intestinal microbial communities appears to underlie many human illnesses, but the mechanisms that promote this dysbiosis and its adverse consequences are poorly understood. In patients who received allogeneic hematopoietic cell transplantation (allo-HCT), we describe a high incidence of enterococcal expansion, which was associated with graft-versus-host disease (GVHD) and mortality. We found that Enterococcus also expands in the mouse gastrointestinal tract after allo-HCT and exacerbates disease severity in gnotobiotic models. Enterococcus growth is dependent on the disaccharide lactose, and dietary lactose depletion attenuates Enterococcus outgrowth and reduces the severity of GVHD in mice. Allo-HCT patients carrying lactose-nonabsorber genotypes showed compromised clearance of postantibiotic Enterococcus domination. We report lactose as a common nutrient that drives expansion of a commensal bacterium that exacerbates an intestinal and systemic inflammatory disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Plasma cfDNA evaluation at acquired resistance to targeted therapies in lung cancer is routine, however, reports of extended clinical application and pitfalls in laboratory practice are still limited. In this study we describe our experience with cfDNA testing using EGFR T790M as a prototype. Methods: Patients with metastatic EGFR -mutant NSCLC patients who underwent plasma EGFR T790M testing at acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) from January 2016 through August 2017 were identified. Molecular laboratory records were reviewed to assess performance of testing by digital PCR, concordance between plasma and tissue testing, turnaround time (TAT), plasma T790M variant allele frequency (VAF), and its correlations with metastatic sites and clinical outcomes. Results: 177 patients underwent T790M cfDNA testing during this period. Plasma T790M was positive in 32% of patients. The median TAT was shorter for plasma T790M compared to tissue PCR (9 vs. 15 days, p  < .0001), and led to osimertinib use in 84% of positive patients. In 52 patients with plasma and tissue T790M evaluation, the concordance was 77%. Plasma T790M VAF did not correlate with time to osimertinib discontinuation ( p  = .4). Plasma T790M status correlated with a higher number of metastatic sites (4 vs. 3, p  < .001) and bone metastases ( p  = .0002). Conclusion: Plasma EGFR T790M testing had shorter TAT compared to tissue testing, however, it was longer than anticipated. Test sensitivity is higher in patients with osseous metastases and with higher metastatic burden suggesting a more limited role for early detection. T790M VAF was not associated with clinical outcomes.

TFEB is overexpressed in TFEB-rearranged renal cell carcinomas as well as in renal tumors with amplifications of TFEB at 6p21.1. As recent literature suggests that renal tumors with 6p21.1 amplification behave more aggressively than those with rearrangements of TFEB, we compared relative TFEB gene expression in these tumors. This study included 37 TFEB-altered tumors: 15 6p21.1-amplified and 22 TFEB-rearranged (including 5 cases from The Cancer Genome Atlas data set). TFEB status was verified using a combination of fluorescent in situ hybridization (n=27) or comprehensive molecular profiling (n=13) and digital droplet polymerase chain reaction was used to quantify TFEB mRNA expression in 6p21.1-amplified (n=9) and TFEB-rearranged renal tumors (n=19). These results were correlated with TFEB immunohistochemistry. TFEB-altered tumors had higher TFEB expression when normalized to B2M (mean: 168.9%, n=28), compared with non-TFEB-altered controls (mean: 7%, n=18, P=0.005). Interestingly, TFEB expression in tumors with rearrangements (mean: 224.7%, n=19) was higher compared with 6p21.1-amplified tumors (mean: 51.2%, n=9; P=0.06). Of note, classic biphasic morphology was only seen in TFEB-rearranged tumors and when present correlated with 6.8-fold higher TFEB expression (P=0.00004). Our results suggest that 6p21.1 amplified renal tumors show increased TFEB gene expression but not as much as t(6;11) renal tumors. These findings correlate with the less consistent/diffuse expression of downstream markers of TFEB activation (cathepsin K, melan A, HMB45) seen in the amplified neoplasms. This suggests that the aggressive biological behavior of 6p21.1 amplified renal tumors might be secondary to other genes at the 6p21.1 locus that are co-amplified, such as VEGFA and CCND3, or other genetic alterations.

Introduction: EGFR-mutant lung cancers are clinically and genomically heterogeneous with concurrent RB transcriptional corepressor 1 (RB1)/tumor protein p53 (TP53) alterations identifying a subset at increased risk for small cell transformation. The genomic alterations that induce lineage plasticity are unknown., Methods: Patients with EGFR/RB1/TP53-mutant lung cancers, identified by next-generation sequencing from 2014 to 2018, were compared to patients with untreated, metastatic EGFR-mutant lung cancers without both RB1 and TP53 alterations. Time to EGFR-tyrosine kinase inhibitor discontinuation, overall survival, SCLC transformation rate, and genomic alterations were evaluated., Results: Patients with EGFR/RB1/TP53-mutant lung cancers represented 5% (43 of 863) of EGFR-mutant lung cancers but were uniquely at risk for transformation (7 of 39, 18%), with no transformations in EGFR-mutant lung cancers without baseline TP53 and RB1 alterations. Irrespective of transformation, patients with EGFR/TP53/RB1-mutant lung cancers had a shorter time to discontinuation than EGFR/TP53- and EGFR-mutant -only cancers (9.5 versus 12.3 versus 36.6 months, respectively, p = 2 × 10 -9 ). The triple-mutant population had a higher incidence of whole-genome doubling compared to NSCLC and SCLC at large (80% versus 34%, p < 5 × 10 -9 versus 51%, p < 0.002, respectively) and further enrichment in triple-mutant cancers with eventual small cell histology (seven of seven pre-transformed plus four of four baseline SCLC versus 23 of 32 never transformed, respectively, p = 0.05). Activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like mutation signature was also enriched in triple-mutant lung cancers that transformed (false discovery rate = 0.03)., Conclusions: EGFR/TP53/RB1-mutant lung cancers are at unique risk of histologic transformation, with 25% presenting with de novo SCLC or eventual small cell transformation. Triple-mutant lung cancers are enriched in whole-genome doubling and Activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like hypermutation which may represent early genomic determinants of lineage plasticity., (Copyright © 2019. Published by Elsevier Inc.)

Amplifications of JAK2, PD-L1, and PD-L2 at 9p24.1 lead to constitutive expression of PD-L1. This, coupled with JAK2-activation dependent upregulation of PD-L1 and adaptive/induced expression leads to higher tumor PD-L1 expression and immune evasion. Renal tumors were therefore evaluated for 9p24.1 amplifications. A combination of next generation sequencing-based copy number analysis, fluorescence in situ hybridization for JAK2/INSL6 and PD-L1/PD-L2 and immunohistochemistry for phospho-STAT3 (downstream target of JAK2), PD-L1, PD-L2, and PD-1 was performed. In this study we interrogated a "Discovery" cohort of 593 renal tumors, a "Validation" cohort of 398 high-grade renal tumors, The Cancer Genome Atlas (879 cases) and other public datasets (846 cases). 9p24.1 amplifications were significantly enriched in renal tumors with sarcomatoid transformation (5.95%, 15/252) when compared to all histologic subtypes in the combined "Discovery", "Validation" and public datasets (16/2636, 0.6%, p < 0.00001). Specifically, 9p24.1 amplifications amongst sarcomatoid tumors in public datasets, the "Discovery" and "Validation" cohorts were 7.7% (6/92), 15.1% (5/33), and 3.1% (4/127), respectively. Herein, we describe 13 cases and amplification status for these was characterized using next generation sequencing (n = 9) and/or fluorescence in situ hybridization (n = 10). Correlation with PD-L1 immunohistochemistry (n = 10) revealed constitutive expression (mean H-score: 222/300, n = 10). Analysis of outcomes based on PD-L1 expression in tumor cells performed on 282 cases ("Validation" cohort) did not reveal a significant prognostic effect and was likely reflective of advanced disease. A high incidence of constitutive PD-L1 expression in tumor cells in the "Validation" cohort (H-Score ≥250/300) was noted amongst 83 rhabdoid (6%) and 127 sarcomatoid renal tumors (7.1%). This suggests additional mechanisms of constitutive expression other than amplification events. Importantly, two patients with 9p24.1-amplified sarcomatoid renal tumors showed significant response to immunotherapy. In summary, a subset of renal tumors with sarcomatoid transformation exhibits constitutive PD-L1 overexpression and these patients should be evaluated for enhanced response to immunotherapy.

Purpose: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 ( MET ex14) alterations in DNA sequencing (DNAseq) driver-negative lung cancers., Experimental Design: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq., Results: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) MET ex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 MET ex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver-negative cases with low TMB [0-5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions ( P < 0.0001)., Conclusions: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver-negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq. See related commentary by Davies and Aisner, p. 4586 ., (©2019 American Association for Cancer Research.)

Background: Although EGFR mutant tumors exhibit low response rates to immune checkpoint blockade overall, some EGFR mutant tumors do respond to these therapies; however, there is a lack of understanding of the characteristics of EGFR mutant lung tumors responsive to immune checkpoint blockade., Patients and Methods: We retrospectively analyzed de-identified clinical and molecular data on 171 cases of EGFR mutant lung tumors treated with immune checkpoint inhibitors from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, University of California Los Angeles, and Dana Farber Cancer Institute. A separate cohort of 383 EGFR mutant lung cancer cases with sequencing data available from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, and The Cancer Genome Atlas was compiled to assess the relationship between tumor mutation burden and specific EGFR alterations., Results: Compared with 212 EGFR wild-type lung cancers, outcomes with programmed cell death 1 or programmed death-ligand 1 (PD-(L)1) blockade were worse in patients with lung tumors harboring alterations in exon 19 of EGFR (EGFRΔ19) but similar for EGFRL858R lung tumors. EGFRT790M status and PD-L1 expression did not impact response or survival outcomes to immune checkpoint blockade. PD-L1 expression was similar across EGFR alleles. Lung tumors with EGFRΔ19 alterations harbored a lower tumor mutation burden compared with EGFRL858R lung tumors despite similar smoking history., Conclusions: EGFR mutant tumors have generally low response to immune checkpoint inhibitors, but outcomes vary by allele. Understanding the heterogeneity of EGFR mutant tumors may be informative for establishing the benefits and uses of PD-(L)1 therapies for patients with this disease., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Background: Mutations in the promoter region of the TERT gene have been detected in a variety of cancers. These mutations can potentially lead to unlimited cell divisions and result in poor clinical prognosis., Objective: To determine the role and relevance of TERT promoter region mutations in both clear cell (ccRCC) and non-clear cell (nccRCC) renal cell carcinoma using ultra-deep and whole-genome sequencing methods on primary tumor samples., Design, Setting, and Participants: DNA from 281 kidney tumors (147 ccRCC and 134 nccRCC) was sequenced between 2013 and 2015, and clinical outcomes for these patients from a single institution were retrospectively analyzed., Outcome Measurements and Statistical Analysis: Differences in patient characteristics and mutational status were tested using Fisher's exact test for categorical variables and the Wilcoxon rank sum test for continuous variables. Survival times were estimated using the Kaplan-Meier method and differences were tested using the log-rank test., Results and Limitations: TERT mutations occurred in 12.2% of ccRCC and 10.4% of nccRCC cases. In >80% of the cases, mutations were located at C228T and were found to co-occur only rarely with other relevant RCC driver genes. The median follow-up among survivors overall was 2.5 yr (range 0.1-18.3). TERT promoter mutations were significantly associated with cancer-specific survival in ccRCC (hazard ratio 2.68, 95% confidence interval 1.19-6.01; p=0.013). In nccRCC, TERT mutations were significantly associated with larger tumors and metastatic development. Assessment of further relevant clinical associations was precluded in the nccRCC group by the heterogeneous and small sample size., Conclusions: Our data suggests that TERT mutational status reflects a distinct pathogenesis with an aggressive disease course in RCC. Stratifying patients with this unique tumorigenesis that leads to poor clinical outcomes could be a putative target for novel therapeutics., Patient Summary: We show a previously unrecognized frequency of TERT promoter mutations in both clear cell and non-clear cell renal cell carcinoma. TERT promoter mutations were associated with some worse outcomes in patients with clear cell renal cell carcinoma., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) is commercially available and increasingly adopted in clinical practice despite a paucity of prospective data to support its use., Methods: Patients with advanced lung cancers who had no known oncogenic driver or developed resistance to current targeted therapy (n = 210) underwent plasma NGS, targeting 21 genes. A subset of patients had concurrent tissue NGS testing using a 468-gene panel (n = 106). Oncogenic driver detection, test turnaround time (TAT), concordance, and treatment response guided by plasma NGS were measured. All statistical tests were two-sided., Results: Somatic mutations were detected in 64.3% (135/210) of patients. ctDNA detection was lower in patients who were on systemic therapy at the time of plasma collection compared with those who were not (30/70, 42.9% vs 105/140, 75.0%; OR = 0.26, 95% CI = 0.1 to 0.5, P < .001). The median TAT of plasma NGS was shorter than tissue NGS (9 vs 20 days; P < .001). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 56.6% (60/106, 95% CI = 46.6% to 66.2%). Among patients who tested plasma NGS positive, 89.6% (60/67; 95% CI = 79.7% to 95.7%) were also concordant on tissue NGS and 60.6% (60/99; 95% CI = 50.3% to 70.3%) vice versa. Patients who tested plasma NGS positive for oncogenic drivers had tissue NGS concordance of 96.1% (49/51, 95% CI = 86.5% to 99.5%), and directly led to matched targeted therapy in 21.9% (46/210) with clinical response., Conclusions: Plasma ctDNA NGS detected a variety of oncogenic drivers with a shorter TAT compared with tissue NGS and matched patients to targeted therapy with clinical response. Positive findings on plasma NGS were highly concordant with tissue NGS and can guide immediate therapy; however, a negative finding in plasma requires further testing. Our findings support the potential incorporation of plasma NGS into practice guidelines., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: Multiple genetic mechanisms have been identified in EGFR-mutant lung cancers as mediators of acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs), but many cases still lack a known mechanism., Methods: To identify novel mechanisms of AR, we performed targeted large panel sequencing of samples from 374 consecutive patients with metastatic EGFR-mutant lung cancer, including 174 post-TKI samples, of which 38 also had a matched pre-TKI sample. Alterations hypothesized to confer AR were introduced into drug-sensitive EGFR-mutant lung cancer cell lines (H1975, HCC827, and PC9) by using clustered regularly interspaced short palindromic repeats/Cas9 genome editing. MSK-LX138cl, a cell line with EGFR exon 19 deletion (ex19del) and praja ring finger ubiquitin ligase 2 gene (PJA2)/BRAF fusion, was generated from an EGFR TKI-resistant patient sample., Results: We identified four patients (2.3%) with a BRAF fusion (three with acylglycerol kinase gene (AGK)/BRAF and one with PJA2/BRAF) in samples obtained at AR to EGFR TKI therapy (two posterlotinib samples and two posterlotinib and postosimertinib samples). Pre-TKI samples were available for two of four patients and both were negative for BRAF fusion. Induction of AGK/BRAF fusion in H1975 (L858R + T790M), PC9 (ex19del) and HCC827 (ex19del) cells increased phosphorylation of BRAF, MEK1/2, ERK1/2, and signal transducer and activator of transcription 3 and conferred resistance to growth inhibition by osimertinib. MEK inhibition with trametinib synergized with osimertinib to block growth. Alternately, a pan-RAF inhibitor as a single agent blocked growth of all cell lines with mutant EGFR and BRAF fusion., Conclusion: BRAF fusion is a mechanism of AR to EGFR TKI therapy in approximately 2% of patients. Combined inhibition of EGFR and MEK (with osimertinib and trametinib) or BRAF (with a pan-RAF inhibitor) are potential therapeutic strategies that should be explored., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)