A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging.

The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.

Zurletrectinib is a next-generation TRK inhibitor with strong intracranial activity against NTRK fusion-positive tumours with on-target resistance to first-generation agents.

BACKGROUND: While NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority. METHODS: In silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations. RESULTS: In vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05). CONCLUSION: Our data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.

Comparison of Immunohistochemistry, Next-generation Sequencing and Fluorescence In Situ Hybridization for Detection of MTAP Loss in Pleural Mesothelioma.

9p21 deletions involving MTAP/CDKN2A genes are detected in diffuse pleural mesotheliomas (DPM) but are absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study were to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam) and 1813 (NBP2-75730, Novus Biologicals) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted next-generation sequencing. 9p21 Copy number status was assessed by Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) analysis and also by CDKN2A fluorescence in situ hybridization in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining, and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or nonspecific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/fluorescence in situ hybridization calls (κ = 0.85; 95% CI, 0.71-0.99; P < .001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific, and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 because mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.

Precision medicine in non-small cell lung cancer: Current applications and future directions.

Advances in biomarkers, targeted therapies, and immuno-oncology have transformed the clinical management of patients with advanced NSCLC. For oncogene-driven tumors, there are highly effective targeted therapies against EGFR, ALK, ROS1, BRAF, TRK, RET, and MET. In addition, investigational therapies for KRAS, NRG1, and HER2 have shown promising results and may become standard-of-care in the near future. In parallel, immune-checkpoint therapy has emerged as an indispensable treatment modality, especially for patients lacking actionable oncogenic drivers. While PD-L1 expression has shown modest predictive utility, biomarkers for immune-checkpoint inhibition in NSCLC have remained elusive and represent an area of active investigation. Given the growing importance of biomarkers, optimal utilization of small tissue biopsies and alternative genotyping methods using circulating cell-free DNA have become increasingly integrated into clinical practice. In this review, we will summarize the current landscape and emerging trends in precision medicine for patients with advanced NSCLC with a special focus on predictive biomarker testing.

Neurofibromatosis Type 2-Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif Dual Immunohistochemistry Is a Reliable Marker for the Detection of Neurofibromatosis Type 2 Alterations in Diffuse Pleural Mesothelioma.

Neurofibromatosis type 2 (NF2) loss occurs in approximately 30% to 50% of diffuse pleural mesothelioma (DPM) with accumulation of yes-associated protein (YAP) 1 and transcriptional coactivator with PDZ-binding motif (TAZ) in tumor nuclei. NF2 and YAP/TAZ represent potential therapeutic targets. We investigated the performance of NF2-YAP/TAZ dual immunohistochemistry (IHC) in identifying DPM that harbors NF2 alterations and in distinguishing DPM from benign mesothelial proliferations. NF2-YAP/TAZ IHC was subsequently performed in a Discovery cohort of DPMs with (n = 10) or without (n = 10) NF2 alterations detected by next-generation sequencing (NGS) and 9 benign cases. The cutoff values for loss of NF2 expression and YAP/TAZ overexpression using IHC were determined in the Discovery cohort. The performance characteristics of NF2-YAP/TAZ IHC were investigated in a Validation cohort (20 DPMs and 10 benign cases). In the Discovery cohort, all DPMs with NF2 alterations using NGS showed NF2 IHC scores of <2, whereas all NF2-wild-type DPMs showed scores of ≥2. NF2-altered DPMs had significantly higher YAP/TAZ H-scores (P < .001) than NF2-wild-type DPM and benign pleura (median H-scores: 237.5 [range, 185-275], 130.0 [range, 40-225], and 10.0 [range, 0-75], respectively). NF2-YAP/TAZ IHC demonstrated 95.2% sensitivity, 100% specificity, 100% positive predictive value, and 95% negative predictive value for detecting NF2 alterations in DPM (n = 40) with NGS as the gold standard and 87.5% sensitivity and 100% specificity for distinguishing DPM (n = 40) from benign mesothelial proliferations (n = 19). NF2-YAP/TAZ IHC has a high sensitivity and specificity for detecting NF2 alterations in DPM and a high specificity for malignancy, highlighting potential utility for guiding NF2-targeted therapies and distinguishing DPM from benign mimics.

Integrated genomic characterization of ERBB2/HER2 alterations in invasive breast carcinoma: a focus on unusual FISH groups.

In patients with invasive breast cancer, fluorescence in situ hybridization (FISH) testing for HER2 typically demonstrates the clear presence or lack of ERBB2 (HER2) amplification (i.e., groups 1 or 5). However, a small subset of patients can present with unusual HER2 FISH patterns (groups 2-4), resulting in diagnostic confusion. To provide clarity, the 2018 CAP/ASCO HER2 testing guideline recommends additional testing using HER2 immunohistochemistry (IHC) for determining the final HER2 status. Despite this effort, the genomic correlates of unusual HER2 FISH groups remain poorly understood. Here, we used droplet digital PCR (ddPCR) and targeted next-generation sequencing (NGS) to characterize the genomic features of both usual and unusual HER2 FISH groups. In this study, 51 clinical samples were selected to represent FISH groups 1-5. Furthermore, group 1 was subdivided into two groups, with groups 1A and 1B corresponding to cases with HER2 signals/cell ≥6.0 and 4-6, respectively. Overall, our findings revealed a wide range of copy number alterations in HER2 across the different FISH groups. As expected, groups 1A and 5 showed the clear presence and lack of HER2 copy number gain, respectively, as measured by ddPCR and NGS. In contrast, group 1B and other uncommon FISH groups (groups 2-4) were characterized by a broader range of HER2 copy levels with only a few select cases showing high-level gain. Notably, these cases with increased HER2 copy levels also showed HER2 overexpression by IHC, thus highlighting the correlation between HER2 copy number and HER2 protein expression. Given the concordance between the genomic and protein results, our findings suggest that HER2 IHC may inform HER2 copy number status in patients with unusual FISH patterns. Hence, our results support the current recommendation for using IHC to resolve HER2 status in FISH groups 2-4.

Will oncotype DX DCIS testing guide therapy? A single-institution correlation of oncotype DX DCIS results with histopathologic findings and clinical management decisions.

Given the increased detection rates of ductal carcinoma in situ (DCIS) and the limited overall survival benefit from adjuvant breast irradiation after breast-conserving surgery, there is interest in identifying subsets of patients who have low rates of ipsilateral breast tumor recurrence such that they might safely forgo radiation. The Oncotype DCIS score is a reverse transcription-PCR (RT-PCR)-based assay that was validated to predict which DCIS cases are most likely to recur. Clinically, these results may be used to assist in selecting which patients with DCIS might safely forgo radiation therapy after breast-conserving surgery; however, little is currently published on how this test is being used in practice. Our study examines traditional histopathologic features used in predicting DCIS risk with Oncotype DCIS results and how these results affect clinical decision-making at our academic institution. Histopathologic features and management decisions for 37 cases with Oncotype DCIS results over the past 4 years were collected. Necrosis, high nuclear grade, biopsy site change, estrogen receptor and progesterone receptor positivity <90% on immunohistochemistry, and Van Nuys Prognostic Index score of 8 or greater were significant predictors of an intermediate-high recurrence score on multivariate regression analysis (P<0.02). Low Oncotype DCIS scores and low nuclear grade were associated with lower rate of radiation therapy (P<0.008). There were seven cases (19%) with Oncotype DCIS results that we considered unexpected in relation to the histopathologic findings (ie, high nuclear grade with comedonecrosis and a low Oncotype score, or hormone receptor discrepancies). Overall, pathologic features correlate with Oncotype DCIS scores but unexpected results do occur, making individual recommendations sometimes challenging.

Diffuse Pleural Mesothelioma: Advances in Molecular Pathogenesis, Diagnosis, and Treatment.

Diffuse pleural mesothelioma (DPM) is a highly aggressive malignant neoplasm arising from the mesothelial cells lining the pleural surfaces. While DPM is a well-recognized disease linked to asbestos exposure, recent advances have expanded our understanding of molecular pathogenesis and transformed our clinical practice. This comprehensive review explores the current concepts and emerging trends in DPM, including risk factors, pathobiology, histologic subtyping, and therapeutic management, with an emphasis on a multidisciplinary approach to this complex disease.

Diffuse Pleural Mesotheliomas with Genomic Near-Haploidization: A Newly Recognized Subset with Distinct Clinical, Histologic, and Molecular Features.

PURPOSE: Diffuse pleural mesotheliomas (DPM) with genomic near-haploidization (GNH) represent a novel subtype first recognized by The Cancer Genome Atlas project; however, its clinicopathologic and molecular features remain poorly defined. EXPERIMENTAL DESIGN: We analyzed clinical genomic profiling data from 290 patients with DPM using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay. Allele-specific copy number analysis was performed using the Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) algorithm. RESULTS: A total of 210 patients were evaluable for loss of heterozygosity (LOH) analysis using FACETS from MSK-IMPACT tumor:normal sequencing data. In this cohort, GNH, defined as LOH across >80% of the genome, was detected in 10 cases (4.8%). Compared with non-GNH tumors, GNH DPMs were associated with younger age and less frequent self-reported history of occupational asbestos exposure. Histologically, GNH DPMs were enriched in biphasic subtype (80% vs. 14.5%) and showed abundant tumor-infiltrating lymphocytes (TILs). Genomic analysis revealed a higher frequency of TP53 alterations, whereas SETDB1 mutations were present in nearly all and only in this subset. The clinicopathologic and molecular findings were further validated in a separate cohort. Despite the younger age, patients with GNH DPMs had a shorter overall survival (10.9 vs. 25.4 months, P = 0.004); the poor prognostic impact of GNH remained significant after controlling for biphasic histology. Of three patients with GNH DPMs who received immune checkpoint blockade, two achieved a clinician-assessed partial response. CONCLUSIONS: GNH defines an aggressive subtype of mainly biphasic DPMs in younger patients with recurrent alterations in SETDB1 and TP53. The enrichment in biphasic histology and TILs, together with our preliminary immune checkpoint blockade response data and anecdotal clinical trial data, suggests that further evaluation of immunotherapy may be warranted in this subset.

Microsatellite Instability and Mismatch Repair Deficiency Define a Distinct Subset of Lung Cancers Characterized by Smoking Exposure, High Tumor Mutational Burden, and Recurrent Somatic MLH1 Inactivation.

INTRODUCTION: Microsatellite instability (MSI) and mismatch repair (MMR) deficiency represent a distinct oncogenic process and predict response to immune checkpoint inhibitors (ICIs). The clinicopathologic features of MSI-high (MSI-H) and MMR deficiency (MMR-D) in lung cancers remain poorly characterized. METHODS: MSI status from 5171 patients with NSCLC and 315 patients with SCLC was analyzed from targeted next-generation sequencing data using two validated bioinformatic pipelines. RESULTS: MSI-H and MMR-D were identified in 21 patients with NSCLC (0.41%) and six patients with SCLC (1.9%). Notably, all patients with NSCLC had a positive smoking history, including 11 adenocarcinomas. Compared with microsatellite stable cases, MSI-H was associated with exceptionally high tumor mutational burden (37.4 versus 8.5 muts/Mb, p < 0.0001), MMR mutational signatures (43% versus 0%, p < 0.0001), and somatic biallelic alterations in MLH1 (52% versus 0%, p < 0.0001). Loss of MLH1 and PMS2 expression by immunohistochemistry was found in MLH1 altered and wild-type cases. Similarly, the majority of patients with MSI-H SCLC had evidence of MLH1 inactivation, including two with MLH1 promoter hypermethylation. A single patient with NSCLC with a somatic MSH2 mutation had Lynch syndrome as confirmed by the presence of a germline MSH2 mutation. Among patients with advanced MSI-H lung cancers treated with ICIs, durable clinical benefit was observed in three of eight patients with NSCLC and two of two patients with SCLC. In NSCLC, STK11, KEAP1, and JAK1 were mutated in nonresponders but wild type in responders. CONCLUSIONS: We present a comprehensive clinicogenomic landscape of MSI-H lung cancers and reveal that MSI-H defines a rare subset of lung cancers associated with smoking, high tumor mutational burden, and MLH1 inactivation. Although durable clinical benefit to ICI was observed in some patients, the broad range of responses suggests that clinical activity may be modulated by co-mutational landscapes.

Tumor-agnostic genomic and clinical analysis of BRAF fusions identify actionable targets.

BACKGROUND: Even though BRAF fusions are increasingly detected in standard multigene next-generation sequencing panels, few reports have explored their structure and impact on clinical course. PATIENTS/METHODS: We collected data from patients with BRAF fusion-positive cancers identified through a genotyping protocol of 97,024 samples. Fusions were characterized and reviewed for oncogenic potential (in-frame status, non-BRAF partner gene, intact BRAF kinase domain). RESULTS: We found 241 BRAF fusion-positive tumors from 212 patients with 82 unique 5' fusion partners spanning 52 histologies. 39 fusion partners were not previously reported, and 61 were identified once. BRAF fusion incidence was enriched in pilocytic astrocytomas, gangliomas, low-grade neuroepithelial tumors, and acinar cell carcinoma of the pancreas. 24 patients spanning multiple histologies were treated with MAPK-directed therapies of which 20 were evaluable for RECIST. Best response was partial response (N=2), stable disease (N=11), and progressive disease (N=7). The median time on therapy was 1 month with MEK plus BRAF inhibitors ([N=11], range 0-18 months) and 8 months for MEK inhibitors ([N=14], range 1-26 months). 9 patients remained on treatment for longer than 6 months [pilocytic astrocytomas (N=6), Erdheim-Chester disease (N=1), extraventricular neurocytoma (N=1), melanoma (N=1)]. Fifteen patients had acquired BRAF fusions. CONCLUSIONS: BRAF fusions are found across histologies and represent an emerging actionable target. BRAF fusions have a diverse set of fusion partners. Durable responses to MAPK therapies were seen, particularly in pilocytic astrocytomas. Acquired BRAF fusions were identified after targeted therapy underscoring the importance of post-progression biopsies to optimize treatment at relapse in these patients.

The mutagenic forces shaping the genomic landscape of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) accounts for up to 25% of all lung cancers and has been associated with exposure to secondhand tobacco smoke and air pollution in observational studies. Here, we evaluate the mutagenic exposures in LCINS by examining deep whole-genome sequencing data from a large international cohort of 871 treatment-naïve LCINS recruited from 28 geographical locations within the Sherlock-Lung study. KRAS mutations were 3.8-fold more common in adenocarcinomas of never smokers from North America and Europe, while a 1.6-fold higher prevalence of EGFR and TP53 mutations was observed in adenocarcinomas from East Asia. Signature SBS40a, with unknown cause, was found in most samples and accounted for the largest proportion of single base substitutions in adenocarcinomas, being enriched in EGFR-mutated cases. Conversely, the aristolochic acid signature SBS22a was almost exclusively observed in patients from Taipei. Even though LCINS exposed to secondhand smoke had an 8.3% higher mutational burden and 5.4% shorter telomeres, passive smoking was not associated with driver mutations in cancer driver genes or the activities of individual mutational signatures. In contrast, patients from regions with high levels of air pollution were more likely to have TP53 mutations while exhibiting shorter telomeres and an increase in most types of somatic mutations, including a 3.9-fold elevation of signature SBS4 (q-value=3.1 × 10-5), previously linked mainly to tobacco smoking, and a 76% increase of clock-like signature SBS5 (q-value=5.0 × 10-5). A positive dose-response effect was observed with air pollution levels, which correlated with both a decrease in telomere length and an elevation in somatic mutations, notably attributed to signatures SBS4 and SBS5. Our results elucidate the diversity of mutational processes shaping the genomic landscape of lung cancer in never smokers.

Rare molecular subtypes of lung cancer.

Oncogenes that occur in ≤5% of non-small-cell lung cancers have been defined as 'rare'; nonetheless, this frequency can correspond to a substantial number of patients diagnosed annually. Within rare oncogenes, less commonly identified alterations (such as HRAS, NRAS, RIT1, ARAF, RAF1 and MAP2K1 mutations, or ERBB family, LTK and RASGRF1 fusions) can share certain structural or oncogenic features with more commonly recognized alterations (such as KRAS, BRAF, MET and ERBB family mutations, or ALK, RET and ROS1 fusions). Over the past 5 years, a surge in the identification of rare-oncogene-driven lung cancers has challenged the boundaries of traditional clinical grade diagnostic assays and profiling algorithms. In tandem, the number of approved targeted therapies for patients with rare molecular subtypes of lung cancer has risen dramatically. Rational drug design has iteratively improved the quality of small-molecule therapeutic agents and introduced a wave of antibody-based therapeutics, expanding the list of actionable de novo and resistance alterations in lung cancer. Getting additional molecularly tailored therapeutics approved for rare-oncogene-driven lung cancers in a larger range of countries will require ongoing stakeholder cooperation. Patient advocates, health-care agencies, investigators and companies with an interest in diagnostics, therapeutics and real-world evidence have already taken steps to surmount the challenges associated with research into low-frequency drivers.

RET-Altered Cancers-A Tumor-Agnostic Review of Biology, Diagnosis and Targeted Therapy Activity.

RET alterations, such as fusions or mutations, drive the growth of multiple tumor types. These alterations are found in canonical (lung and thyroid) and non-canonical (e.g., gastrointestinal, breast, gynecological, genitourinary, histiocytic) cancers. RET alterations are best identified via comprehensive next-generation sequencing, preferably with DNA and RNA interrogation for fusions. Targeted therapies for RET-dependent cancers have evolved from older multikinase inhibitors to selective inhibitors of RET such as selpercatinib and pralsetinib. Prospective basket trials and retrospective reports have demonstrated the activity of these drugs in a wide variety of RET-altered cancers, notably those with RET fusions. This paved the way for the first tumor-agnostic selective RET inhibitor US FDA approval in 2022. Acquired resistance to RET kinase inhibitors can take the form of acquired resistance mutations (e.g., RET G810X) or bypass alterations.

Clinical and Genomic Features of Response and Toxicity to Sotorasib in a Real-World Cohort of Patients With Advanced KRAS G12C-Mutant Non-Small Cell Lung Cancer.

PURPOSE: With the recent approval of the KRAS G12C inhibitor sotorasib for patients with advanced KRAS G12C-mutant non-small cell lung cancer (NSCLC), there is a new need to identify factors associated with activity and toxicity among patients treated in routine practice. MATERIALS AND METHODS: We conducted a multicenter retrospective study of patients treated with sotorasib outside of clinical trials to identify factors associated with real-world progression free survival (rwPFS), overall survival (OS), and toxicity. RESULTS: Among 105 patients with advanced KRAS G12C-mutant NSCLC treated with sotorasib, treatment led to a 5.3-month median rwPFS, 12.6-month median OS, and 28% real-world response rate. KEAP1 comutations were associated with shorter rwPFS and OS (rwPFS hazard ratio [HR], 3.19; P = .004; OS HR, 4.10; P = .003); no significant differences in rwPFS or OS were observed across TP53 (rwPFS HR, 1.10; P = .731; OS HR, 1.19; P = .631) or STK11 (rwPFS HR, 1.66; P = .098; OS HR, 1.73; P = .168) comutation status. Notably, almost all patients who developed grade 3 or higher treatment-related adverse events (G3+ TRAEs) had previously been treated with anti-PD-(L)1 therapy. Among these patients, anti-PD-(L)1 therapy exposure within 12 weeks of sotorasib was strongly associated with G3+ TRAEs (P < .001) and TRAE-related sotorasib discontinuation (P = .014). Twenty-eight percent of patients with recent anti-PD-(L)1 therapy exposure experienced G3+ TRAEs, most commonly hepatotoxicity. CONCLUSION: Among patients treated with sotorasib in routine practice, KEAP1 comutations were associated with resistance and recent anti-PD-(L)1 therapy exposure was associated with toxicity. These observations may help guide use of sotorasib in the clinic and may help inform the next generation of KRAS G12C-targeted clinical trials.

Molecular Biomarkers of Disease Outcomes and Mechanisms of Acquired Resistance to First-Line Osimertinib in Advanced EGFR-Mutant Lung Cancers.

INTRODUCTION: Preferred first-line treatment for patients with metastatic EGFR-mutant lung cancer is osimertinib, yet it is not known whether patient outcomes may be improved by identifying and intervening on molecular markers associated with therapeutic resistance. METHODS: All patients with metastatic EGFR-mutant lung cancer treated with first-line osimertinib at the Memorial Sloan Kettering Cancer Center (n = 327) were identified. Available pretreatment and postprogression tumor samples underwent targeted gene panel sequencing and mutational signature analysis using SigMA algorithm. Progression-free survival (PFS) and overall survival were estimated using the Kaplan-Meier method. RESULTS: Using multivariate analysis, baseline atypical EGFR (median PFS = 5.8 mo, p < 0.001) and concurrent TP53/RB1 alterations (median PFS = 10.5 mo, p = 0.015) were associated with shorter PFS on first-line osimertinib. Of 95 patients with postprogression biopsies, acquired resistance mechanisms were identified in 52% (off-target, n = 24; histologic transformation, n = 14; on-target, n = 12), with MET amplification (n = 9), small cell lung transformation (n = 7), and acquired EGFR amplification (n = 7), the most frequently identified mechanisms. Although there was no difference in postprogression survival on the basis of identified resistance (p = 0.07), patients with subsequent second-line therapy tailored to postprogression biopsy results had improved postprogression survival (hazard ratio = 0.09, p = 0.006). The paired postprogression tumors had higher tumor mutational burden (p = 0.008) and further dominant APOBEC mutational signatures (p = 0.07) compared with the pretreatment samples. CONCLUSIONS: Patients with EGFR-mutant lung cancer treated with first-line osimertinib have improved survival with treatment adaptation on the basis of identified mechanisms of resistance at time of progression using tissue-based genomic analysis. Further survival gains may be achieved using risk-based treatment adaptation of pretreatment genomic alterations.

Diagnosis of Hypersensitivity Pneumonitis: Review and Summary of American College of Chest Physicians Statement.

Assessment of lung biopsies for the diagnosis of hypersensitivity pneumonitis (HP) is one of the most difficult diagnostic problems for surgical pathologists. It is a form of interstitial lung disease resulting from an immune reaction provoked by an inhaled antigen in susceptible individuals. Although this definition sounds simple, in practice, the diagnosis of HP can be challenging. To address these issues, the American College of Chest Physicians (CHEST) has recently published a guideline for the diagnosis of HP. In this review, we will explore the multidisciplinary diagnostic evaluation of HP with a focus on the pathologic features as outlined in the CHEST guidelines. The histologic criteria are divided into 4 diagnostic categories: (1) Typical nonfibrotic HP or fibrotic HP; (2) Compatible with nonfibrotic HP or fibrotic HP; (3) Indeterminate for nonfibrotic or fibrotic HP; and (4) Alternative Diagnosis. It is important to emphasize that patterns 1 to 3 do not represent discrete histologic entities or pathologic diagnoses. Rather, these categories are meant to serve as a practical guide for organizing a complex set of overlapping histologic patterns into an integrated diagnostic framework for facilitating multidisciplinary discussion. High-resolution computed tomography features are also summarized, emphasizing how the correlation of lung biopsies with computed tomography findings can help to favor the diagnosis, particularly in cases where biopsies are not typical for HP. This review highlights details of the histologic spectrum of HP as well as the utility of different types of biopsies and bronchoalveolar lavage. We also emphasize the importance of multidisciplinary discussion and the complex differential diagnosis.

Clinical Utility and Performance of an Ultrarapid Multiplex RNA-Based Assay for Detection of ALK, ROS1, RET, and NTRK1/2/3 Rearrangements and MET Exon 14 Skipping Alterations.

Several kinase fusions are established targetable drivers in lung cancers. However, rapid and comprehensive detection remains challenging because of diverse partner genes and breakpoints. We assess the clinical utility and performance of a rapid microfluidic multiplex real-time PCR-based assay for simultaneous query of fusions involving ALK, ROS1, RET, and NTRK1/2/3, as well as MET exon 14 skipping, using a 3-hour automated process. Dual analytic strategies were utilized: fusion-specific amplification and 3' to 5' expression imbalance. One-hundred and forty-three independent, formalin-fixed, paraffin-embedded tumor samples (112 surgical specimens, 31 cytologic cell blocks) were analyzed: 133 with known kinase gene alterations and 10 negative samples based on clinically validated next-generation sequencing. Testing was successful in 142 (99%) cases. The assay demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27), and 100% (20/20) for ALK, RET, ROS1, and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens aged up to 5 years, with >10% tumor, and inputs of at least 9 mm2 (surgical specimens) and 9000 cells (cytologic cell blocks). The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared with immunohistochemistry and molecular methods, while also circumventing the infrastructure dependencies associated with next-generation sequencing and fluorescence in situ hybridization.

Molecular Characterization of Peritoneal Mesotheliomas.

INTRODUCTION: Malignant peritoneal mesothelioma (MPeM) is clinically distinct and less studied than malignant pleural mesothelioma. We report the genomic and immunophenotypic features of a prospectively collected MPeM cohort. METHODS: Next-generation sequencing (NGS) was performed on MPeM tumors. Genomic near-haploidization (GNH) was assessed. WT1, BAP1, mesothelin, VISTA, and programmed death-ligand 1 were evaluated by immunohistochemistry (IHC) when tissue was available. Overall survival was stratified by selected genomic and IHC features. RESULTS: A total of 50 consented patients with MPeM (45 epithelioid, 5 nonepithelioid) were studied exhibiting common alterations in BAP1 (60%; 30 of 50), NF2 (24%; 12 of 50) SETD2 (22%; 11 of 50), and TP53 (16%; 8 of 50). A total of 76% (38 of 50) of specimens were assessable for allele-specific copy number analysis; 8% (3 of 38) had GNH. IHC positivity rates were 93% (37 of 40) for mesothelin, 96% (46 of 48) for WT1, 50% (19 of 38) for programmed death-ligand 1, and 89% (34 of 38) for VISTA. BAP1 loss by IHC was observed in 76% (29 of 38), including five wild-type on NGS. Combining NGS and IHC for BAP1, overall survival was worse with alteration or loss compared with wild-type or retained in all patients (n = 37 versus 13, 43.8 versus 117.3 mo, p = 0.04) Three of 30 patients had a pathogenic germline variant: POT1 I78T, MUTYH R109Y, and BAP1 E402∗. CONCLUSIONS: MPeM has distinct biology and genomic composition. CDKN2A/B alterations were rare in MPeM, whereas BAP1, NF2, TP53, SETD2, and LATS2 were common. BAP1 alteration/loss was associated with shorter survival when all patients were included. A notable minority of specimens had GNH associated with NF2, TP53, and SETDB1 mutations. Pathogenic germline mutations were found in 3 of 30 patients.

Bioinformatically Expanded Next-Generation Sequencing Analysis Optimizes Identification of Therapeutically Relevant MET Copy Number Alterations in >50,000 Tumors.

PURPOSE: Clinical relevance thresholds and laboratory methods are poorly defined for MET amplification, a targetable biomarker across malignancies. EXPERIMENTAL DESIGN: The utility of next-generation sequencing (NGS) in assessing MET copy number alterations was determined in >50,000 solid tumors. Using fluorescence in situ hybridization as reference, we validated and optimized NGS analysis. RESULTS: Incorporating read-depth and focality analyses achieved 91% concordance, 97% sensitivity, and 89% specificity. Tumor heterogeneity, neoplastic cell proportions, and genomic focality affected MET amplification assessment. NGS methodology showed superiority in capturing overall amplification status in heterogeneous tumors and defining amplification focality among other genomic alterations. MET copy gains and amplifications were found in 408 samples across 23 malignancies. Total MET copy number inversely correlated with amplified segment size. High-level/focal amplification was enriched in certain genomic subgroups and associated with targeted therapy response. CONCLUSIONS: Leveraging our integrated bioinformatic approach, targeted therapy benefit was observed across diverse MET amplification contexts.