Cellular Cutaneous Epithelioid Hemangioma Harboring the Rare GATA6::FOXO1 Gene Fusion.

ABSTRACT: Epithelioid hemangioma (EH) is a benign vascular tumor displaying diverse histomorphologies. Among these, one EH subtype comprises cellular sheets of atypical epithelioid cells, posing potential challenges in distinguishing it from malignant vascular lesions. In this case report, we present a cutaneous cellular EH that carries the rare GATA6::FOXO1 gene fusion, a recent discovery. Our aim is to provide an updated insight into the evolving knowledge of EHs while delving into the histologic and molecular characteristics of the primary differential diagnoses.

Deep-Learning Model for Tumor-Type Prediction Using Targeted Clinical Genomic Sequencing Data.

Tumor type guides clinical treatment decisions in cancer, but histology-based diagnosis remains challenging. Genomic alterations are highly diagnostic of tumor type, and tumor-type classifiers trained on genomic features have been explored, but the most accurate methods are not clinically feasible, relying on features derived from whole-genome sequencing (WGS), or predicting across limited cancer types. We use genomic features from a data set of 39,787 solid tumors sequenced using a clinically targeted cancer gene panel to develop Genome-Derived-Diagnosis Ensemble (GDD-ENS): a hyperparameter ensemble for classifying tumor type using deep neural networks. GDD-ENS achieves 93% accuracy for high-confidence predictions across 38 cancer types, rivaling the performance of WGS-based methods. GDD-ENS can also guide diagnoses of rare type and cancers of unknown primary and incorporate patient-specific clinical information for improved predictions. Overall, integrating GDD-ENS into prospective clinical sequencing workflows could provide clinically relevant tumor-type predictions to guide treatment decisions in real time. SIGNIFICANCE: We describe a highly accurate tumor-type prediction model, designed specifically for clinical implementation. Our model relies only on widely used cancer gene panel sequencing data, predicts across 38 distinct cancer types, and supports integration of patient-specific nongenomic information for enhanced decision support in challenging diagnostic situations. See related commentary by Garg, p. 906. This article is featured in Selected Articles from This Issue, p. 897.

Pathologic Assessment and Staging of Multiple Non-Small Cell Lung Carcinomas: A Paradigm Shift with the Emerging Role of Molecular Methods.

Non-small cell lung carcinomas (NSCLCs) commonly present as 2 or more separate tumors. Biologically, this encompasses 2 distinct processes: separate primary lung carcinomas (SPLCs), representing independently arising tumors, and intrapulmonary metastases (IPMs), representing intrapulmonary spread of a single tumor. The advent of computed tomography imaging has substantially increased the detection of multifocal NSCLCs. The strategies and approaches for distinguishing between SPLCs and IPMs have evolved significantly over the years. Recently, genomic sequencing of somatic mutations has been widely adopted to identify targetable alterations in NSCLC. These molecular techniques have enabled pathologists to reliably discern clonal relationships among multiple NSCLCs in clinical practice. However, a standardized approach to evaluating and staging multiple NSCLCs using molecular methods is still lacking. Here, we reviewed the historical context and provided an update on the growing applications of genomic testing as a clinically relevant benchmark for determining clonal relationships in multiple NSCLCs, a practice we have designated "comparative molecular profiling." We examined the strengths and limitations of the morphology-based distinction of SPLCs vs IPMs and highlighted pivotal clinical and pathologic insights that have emerged from studying multiple NSCLCs using genomic approaches as a gold standard. Lastly, we suggest a practical approach for evaluating multiple NSCLCs in the clinical setting, considering the varying availability of molecular techniques.

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.

Alveolar Differentiation Drives Resistance to KRAS Inhibition in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD), commonly driven by KRAS mutations, is responsible for 7% of all cancer mortality. The first allele-specific KRAS inhibitors were recently approved in LUAD, but the clinical benefit is limited by intrinsic and acquired resistance. LUAD predominantly arises from alveolar type 2 (AT2) cells, which function as facultative alveolar stem cells by self-renewing and replacing alveolar type 1 (AT1) cells. Using genetically engineered mouse models, patient-derived xenografts, and patient samples, we found inhibition of KRAS promotes transition to a quiescent AT1-like cancer cell state in LUAD tumors. Similarly, suppressing Kras induced AT1 differentiation of wild-type AT2 cells upon lung injury. The AT1-like LUAD cells exhibited high growth and differentiation potential upon treatment cessation, whereas ablation of the AT1-like cells robustly improved treatment response to KRAS inhibitors. Our results uncover an unexpected role for KRAS in promoting intratumoral heterogeneity and suggest that targeting alveolar differentiation may augment KRAS-targeted therapies in LUAD. SIGNIFICANCE: Treatment resistance limits response to KRAS inhibitors in LUAD patients. We find LUAD residual disease following KRAS targeting is composed of AT1-like cancer cells with the capacity to reignite tumorigenesis. Targeting the AT1-like cells augments responses to KRAS inhibition, elucidating a therapeutic strategy to overcome resistance to KRAS-targeted therapy. This article is featured in Selected Articles from This Issue, p. 201.

A cutaneous epithelioid vascular tumor harboring a TPM3::ALK fusion.

Substantial progress has been made in understanding the molecular pathways associated with vascular tumors over the last two decades. In addition to mutations and copy number aberrations, fusions have emerged as significant contributors to the pathogenesis of a notable subset of vascular tumors. In this report, we present a case of an unusual intradermal vascular tumor with epithelioid cytomorphology. Immunohistochemistry revealed diffuse positivity for CD31, ERG and Factor VIII, supporting its endothelial lineage. RNA sequencing (ArcherFusion Plex) revealed the presence of an in-frame fusion between the genes TPM3 Exon 8 and ALK Exon 20. Immunohistochemistry confirmed ALK expression by the endothelial cells. To our knowledge, this is the first documented case of a vascular tumor harboring an ALK fusion. It may fall within the spectrum of epithelioid hemangiomas; nevertheless, we cannot definitively exclude the possibility of it being a distinct and potentially unique benign entity on its own.

Alveolar differentiation drives resistance to KRAS inhibition in lung adenocarcinoma.

Lung adenocarcinoma (LUAD), commonly driven by KRAS mutations, is responsible for 7% of all cancer mortality. The first allele-specific KRAS inhibitors were recently approved in LUAD, but clinical benefit is limited by intrinsic and acquired resistance. LUAD predominantly arises from alveolar type 2 (AT2) cells, which function as facultative alveolar stem cells by self-renewing and replacing alveolar type 1 (AT1) cells. Using genetically engineered mouse models, patient-derived xenografts, and patient samples we found inhibition of KRAS promotes transition to a quiescent AT1-like cancer cell state in LUAD tumors. Similarly, suppressing Kras induced AT1 differentiation of wild-type AT2 cells upon lung injury. The AT1-like LUAD cells exhibited high growth and differentiation potential upon treatment cessation, whereas ablation of the AT1-like cells robustly improved treatment response to KRAS inhibitors. Our results uncover an unexpected role for KRAS in promoting intra-tumoral heterogeneity and suggest targeting alveolar differentiation may augment KRAS-targeted therapies in LUAD. Significance: Treatment resistance limits response to KRAS inhibitors in LUAD patients. We find LUAD residual disease following KRAS targeting is composed of AT1-like cancer cells with the capacity to reignite tumorigenesis. Targeting the AT1-like cells augments responses to KRAS inhibition, elucidating a therapeutic strategy to overcome resistance to KRAS-targeted therapy.

Deep Learning Model for Tumor Type Prediction using Targeted Clinical Genomic Sequencing Data.

Tumor type guides clinical treatment decisions in cancer, but histology-based diagnosis remains challenging. Genomic alterations are highly diagnostic of tumor type, and tumor type classifiers trained on genomic features have been explored, but the most accurate methods are not clinically feasible, relying on features derived from whole genome sequencing (WGS), or predicting across limited cancer types. We use genomic features from a dataset of 39,787 solid tumors sequenced using a clinical targeted cancer gene panel to develop Genome-Derived-Diagnosis Ensemble (GDD-ENS): a hyperparameter ensemble for classifying tumor type using deep neural networks. GDD-ENS achieves 93% accuracy for high-confidence predictions across 38 cancer types, rivalling performance of WGS-based methods. GDD-ENS can also guide diagnoses on rare type and cancers of unknown primary, and incorporate patient-specific clinical information for improved predictions. Overall, integrating GDD-ENS into prospective clinical sequencing workflows has enabled clinically-relevant tumor type predictions to guide treatment decisions in real time.

Diagnosis of thoracic SMARCA4-deficient undifferentiated tumor in cytology.

INTRODUCTION: Although alterations in SMARCA4-deficient occur in non-small cell lung carcinoma (SD-NSCLC), thoracic SMARCA4-deficient undifferentiated tumor (TSDUT) is recognized as a distinct entity in the 2021 World Health Organization Classification of Thoracic Tumors because of unique morphologic, immunophenotypic and molecular features, and worse survival compared with SD-NSCLC. Cytologic diagnosis of TSDUT is clinically important because of its aggressive behavior and because it is often diagnosed by fine-needle aspiration because TSDUTs are usually unresectable at presentation. Here, we identify cytologic features that can be used for recognition of TSDUT and distinction from SD-NSCLC. MATERIALS AND METHODS: Cytomorphologic features were investigated in cytology specimens from patients with TSDUT (n = 11) and compared with a control group of patients with SD-NSCLC (n = 20). RESULTS: The presence of classic rhabdoid morphology, at least focally, was entirely specific for TSDUT (n = 6, 55%) compared with SD-NSCLC (n = 0) in this study. TSDUT more frequently showed tumor necrosis (n = 11, 100% vs. n = 8, 40%; p = .001), dominant single-cell pattern on aspirate smears or touch preparation slides (n = 8 [of 9], 80% vs. n = 3, 15%; p = .010), nuclear molding (n = 5, 45% vs. n = 1, 5%; p = .013), and indistinct cell borders (n = 11, 100% vs. n = 5, 25%; P < .001) compared with SD-NSCLC, respectively. CONCLUSIONS: Cytomorphologic features occurring more frequently in TSDUT include tumor necrosis, dominant single-cell pattern, nuclear molding indistinct cell borders, and focal rhabdoid cells. Presence of these features in a cytology specimen of an undifferentiated tumor, particularly in a patient with a thoracic mass, should raise suspicion for TSDUT and prompt appropriate ancillary workup.

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.

Non-small cell lung carcinomas with diffuse coexpression of TTF1 and p40: clinicopathological and genomic features of 14 rare biphenotypic tumours.

Thyroid transcription factor 1 (TTF1) and p40 are widely-utilized diagnostic markers of lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), respectively. Diffuse coexpression of TTF1 and p40 has been described in only rare case reports. In a multi-institutional study, we collected the largest cohort of these unusual tumours to-date (n = 14), with the goal of elucidating their clinicopathological and genomic characteristics. Lung tumours with diffuse coexpression (labelling 50-100% tumour cells) of TTF1 clone 8G7G3/1 and p40 clone BC28 were identified. Detailed clinicopathological and immunohistochemical parameters were analyzed. Eight tumours were analyzed by next-generation sequencing (NGS) and the results were compared to those in > 9 K LUAD and > 1 K LUSC. All tumours with diffuse TTF1/p40 coexpression were poorly differentiated non-small cell lung carcinomas (NSCLC), 42% of which had basaloid features. Some tumours exhibited focal keratinization (14%), napsin A and/or mucicarmine labelling (46%) or both squamous and glandular features (7%). NGS revealed a uniquely high rate of FGFR1 amplifications (70%) compared to either LUAD (0.7%, P < 0.0001) or LUSC (11%, P = 0.001). LUAD-type targetable driver alterations were identified in 38% of cases (one EGFR, two KRAS G12C). The tumours were clinically aggressive, exhibiting metastatic disease in most patients. Lung carcinomas with diffuse TTF1/p40 coexpression represent poorly differentiated NSCLCs with frequent basaloid features, but some show evidence of focal squamous, glandular or dual differentiation with a distinctly high rate of FGFR1 amplifications. The presence of targetable LUAD-type alterations (EGFR, KRAS G12C) emphasizes the importance of molecular testing in these tumours.

Reversion mutations in germline BRCA1/2-mutant tumors reveal a BRCA-mediated phenotype in non-canonical histologies.

The association between loss of BRCA1/2 and a homologous recombination deficiency phenotype is lineage dependent. In BRCA-associated cancers such as breast, ovarian, pancreas and prostate, this phenotype confers sensitivity to PARP inhibitors and platinum-therapies. Somatic reversion mutations restoring BRCA1/2 function mediate resistance, and have exclusively been reported in BRCA-associated tumors. In this study, we analyze matched tumor and normal sequencing from 31,927 patients and identify 846 (2.7%) patients with germline BRCA1/2 variants across 43 different cancer types, including 11 with somatic reversion mutations. While nine are in BRCA-associated tumors, we find two reversion mutations in non-BRCA-associated histologies, namely lung and esophagogastric adenocarcinomas. Both were detected following platinum therapy. Whole exome sequencing confirms the homologous recombination deficiency phenotype of these tumors. While reversion mutations arise in all BRCA-associated cancer types, here we show that reversion mutations arising post-platinum in non-BRCA associated histologies, while rare, may indicate BRCA1/2 mediated tumorigenesis.

Rb Tumor Suppressor in Small Cell Lung Cancer: Combined Genomic and IHC Analysis with a Description of a Distinct Rb-Proficient Subset.

PURPOSE: RB1 mutations and loss of retinoblastoma (Rb) expression represent consistent but not entirely invariable hallmarks of small cell lung cancer (SCLC). The prevalence and characteristics of SCLC retaining wild-type Rb are not well-established. Furthermore, the performance of targeted next-generation sequencing (NGS) versus immunohistochemistry for Rb assessment is not well-defined. EXPERIMENTAL DESIGN: A total of 208 clinical SCLC samples were analyzed by comprehensive targeted NGS, covering all exons of RB1, and Rb IHC. On the basis of established coordination of Rb/p16/cyclinD1 expression, p16-high/cyclinD1-low profile was used as a marker of constitutive Rb deficiency. RESULTS: Fourteen of 208 (6%) SCLC expressed wild-type Rb, accompanied by a unique p16-low/cyclinD1-high profile supporting Rb proficiency. Rb-proficient SCLC was associated with neuroendocrine-low phenotype, combined SCLC with non-SCLC (NSCLC) histology and aggressive behavior. These tumors exclusively harbored CCND1 amplification (29%), and were markedly enriched in CDKN2A mutations (50%) and NSCLC-type alterations (KEAP1, STK11, FGFR1). The remaining 194 of 208 SCLC were Rb-deficient (p16-high/cyclinD1-low), including 184 cases with Rb loss (of which 29% lacked detectable RB1 alterations by clinical NGS pipeline), and 10 cases with mutated but expressed Rb. CONCLUSIONS: This is the largest study to date to concurrently analyze Rb by NGS and IHC in SCLC, identifying a 6% rate of Rb proficiency. Pathologic-genomic data implicate NSCLC-related progenitors as a putative source of Rb-proficient SCLC. Consistent upstream Rb inactivation via CDKN2A/p16↓ and CCND1/cyclinD1↑ suggests the potential utility of CDK4/6 inhibitors in this aggressive SCLC subset. The study also clarifies technical aspects of Rb status determination in clinical practice, highlighting the limitations of exon-only sequencing for RB1 interrogation. See related commentary by Mahadevan and Sholl, p. 4603.

POU2F3 in SCLC: Clinicopathologic and Genomic Analysis With a Focus on Its Diagnostic Utility in Neuroendocrine-Low SCLC.

INTRODUCTION: POU2F3 is a recent marker of a small cell lung carcinoma (SCLC) subtype related to chemosensory tuft cells (SCLC-P). The characteristics of SCLC-P have not been fully defined, and the data on POU2F3 expression in other lung tumors are scarce. METHODS: We screened 254 SCLC for POU2F3 expression and comprehensively analyzed histopathologic, genomic, and clinical characteristics of POU2F3-positive tumors. We also explored POU2F3 expression in other major lung cancer types (n = 433) and a targeted set of potential diagnostic mimics of SCLC (n = 123). RESULTS: POU2F3 was expressed in 30 of 254 (12%) SCLC and was strongly associated with low expression of standard neuroendocrine markers (synaptophysin, chromogranin A, CD56, INSM1). Notably, POU2F3 was expressed in 75% of SCLC with entirely negative or minimal neuroendocrine marker expression (15/20) and was helpful in supporting the diagnosis of SCLC in such cases. Broad targeted next-generation sequencing revealed that SCLC-P (n = 12) exhibited enrichment in several alterations, including PTEN inactivation, MYC amplifications, and 20q13 amplifications, but similar rates of RB1 and TP53 alterations as other SCLC (n = 155). Beyond SCLC, POU2F3 expression was exclusively limited to large cell neuroendocrine carcinoma (12%) and basaloid squamous cell carcinoma (22%). CONCLUSIONS: This is the largest cohort of SCLC-P clinical samples to date, where we describe the diagnostic utility of POU2F3 in a challenging subset of SCLC with low or absent expression of standard neuroendocrine markers. The distinct genomic alterations in SCLC-P may offer a novel avenue for therapeutic targeting. The role of POU2F3 in a narrow subset of other lung cancer types warrants further study.

First Report of Thoracic Carcinoma With DEK::AFF2 Rearrangement: A Case Report.

DEK::AFF2 carcinomas of the head and neck region have been recently described and reported to have aggressive clinical behavior but exceptional sensitivity to immunotherapy. We report a case of a 26-year-old female, never smoker, with a 5.2-cm left lower lobe central lung mass, with morphologic features identical to those reported for DEK::AFF2 head and neck carcinomas, including mixed papillary exophytic and invasive components, squamous/basaloid features, and monomorphic cytomorphology. DEK (exon 7)::AFF2 (exon 9) fusion was identified by whole-transcriptome RNA sequencing. This is the first report of thoracic DEK::AFF2 carcinoma, indicating that these tumors are not confined to the head and neck region but can involve both upper and lower respiratory tracts. This entity should be considered in the differential diagnosis of squamous cell carcinomas in never smokers lacking other known oncogenic mutations.

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.

FGFR2::TACC2 fusion as a novel KIT-independent mechanism of targeted therapy failure in a multidrug-resistant gastrointestinal stromal tumor.

Genetic alterations in FGF/FGFR pathway are infrequent in gastrointestinal stromal tumors (GIST), with rare cases of quadruple wildtype GISTs harboring FGFR1 gene fusions and mutations. Additionally, FGF/FGFR overexpression was shown to promote drug resistance to kinase inhibitors in GISTs. However, FGFR gene fusions have not been directly implicated as a mechanism of drug resistance in GISTs. Herein, we report a patient presenting with a primary small bowel spindle cell GIST and concurrent peritoneal and liver metastases displaying an imatinib-sensitive KIT exon 11 in-frame deletion. After an initial 9-month benefit to imatinib, the patient experienced intraabdominal peritoneal recurrence owing to secondary KIT exon 13 missense mutation and FGFR4 amplification. Despite several additional rounds of tyrosine kinase inhibitors (TKI), the patient's disease progressed after 2 years and presented with multiple peritoneal and liver metastases, including one pericolonic mass harboring secondary KIT exon 18 missense mutation, and a concurrent transverse colonic mass with a FGFR2::TACC2 fusion and AKT2 amplification. All tumors, including primary and recurrent masses, harbored an MGA c.7272 T > G (p.Y2424*) nonsense mutation and CDKN2A/CDKN2B/MTAP deletions. The transcolonic mass showed elevated mitotic count (18/10 HPF), as well as significant decrease in CD117 and DOG1 expression, in contrast to all the other resistant nodules that displayed diffuse and strong CD117 and DOG1 immunostaining. The FGFR2::TACC2 fusion resulted from a 742 kb intrachromosomal inversion at the chr10q26.3 locus, leading to a fusion between exons 1-17 of FGFR2 and exons 7-17 TACC2, which preserves the extracellular and protein tyrosine kinase domains of FGFR2. We present the first report of a multidrug-resistant GIST patient who developed an FGFR2 gene fusion as a secondary genetic event to the selective pressure of various TKIs. This case also highlights the heterogeneous escape mechanisms to targeted therapy across various tumor nodules, spanning from both KIT-dependent and KIT-independent off-target activation pathways.

Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation.

BACKGROUND: Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies. METHODS: We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment. RESULTS: Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib. CONCLUSIONS: Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.

Spectrum of BRAF Mutations and Gene Rearrangements in Ovarian Serous Carcinoma.

Low-grade serous carcinoma (LGSC) is a rare type of ovarian cancer, which commonly arises from serous borderline tumor (SBT) and is characterized by frequent activating mutations in the mitogen-activated protein kinase pathway, including BRAF. The BRAF V600E mutation is associated with improved prognosis in SBT and LGSC, and responses to BRAF inhibitor therapy have been reported. We sought to characterize the clinicopathologic and molecular features of BRAF-driven tubo-ovarian and primary peritoneal serous tumors. METHODS: Retrospective analysis of our institutional cohort of SBTs (n = 22), LGSCs (n = 119) and high-grade serous carcinomas (HGSCs, n = 1,290) subjected to targeted massively parallel sequencing was performed to identify cases with BRAF genetic alterations. Putative BRAF rearrangements were confirmed using targeted RNA sequencing and/or fluorescence in situ hybridization (FISH). BRAFV600E oncoprotein expression was assessed by immunohistochemistry on selected cases. RESULTS: BRAF somatic genetic alterations were identified in 29 of 1,431 (2%) serous tumors and included mutations (n = 24), gene rearrangements (n = 3), and amplification (n = 2). BRAF mutations were more frequent in SBTs (7 of 22; 32%) compared with LGSCs (11 of 119; 9%, P = .009) and HGSCs (6 of 1,290; 0.5%; P < .0001, SBT/LGSC v HGSC). The BRAF V600E hotspot mutation was most common (n = 16); however, other BRAF driver mutations were also detected (n = 8). BRAF mutations were often clonal or truncal in SBTs and LGSCs, but subclonal in most HGSCs. Pathogenic BRAF gene fusions were identified in LGSCs (n = 2) and HGSC (n = 1) and involved distinct fusion partners (AGK, MKRN1, and AGAP3). Three patients with BRAF-mutant LGSC were treated with targeted mitogen-activated protein kinase inhibitors, one of whom was maintained on therapy for over 3 years with clinical benefit. CONCLUSION: Recognition of BRAF alterations beyond V600E mutation in LGSC may have clinical implications for appropriate targeted therapy selection.

Multiomic Analysis of Lung Tumors Defines Pathways Activated in Neuroendocrine Transformation.

Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUAD) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre/posttransformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre/posttransformation samples, supports that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in the PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacologic inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer. SIGNIFICANCE: The difficulty in collection of transformation samples has precluded the performance of molecular analyses, and thus little is known about the lineage plasticity mechanisms leading to LUAD-to-SCLC transformation. Here, we describe biological pathways dysregulated upon transformation and identify potential predictors and potential therapeutic vulnerabilities of NE transformation in the lung. See related commentary by Meador and Lovly, p. 2962. This article is highlighted in the In This Issue feature, p. 2945.