Identification of fusions with potential clinical significance in melanoma.

Though uncommon in melanoma, gene fusions may have therapeutic implications. Next generation sequencing-based clinical assays, designed to detect relevant gene fusions, mutations, and copy number changes, were performed on 750 melanomas (375 primary and 375 metastases) at our institution from 2014-2021. These included 599 (80%) cutaneous, 38 (5%) acral, 11 (1.5%) anorectal, 23 (3%) sinonasal, 27 (3.6%) eye (uveal/ conjunctiva), 11 (1.5%) genital (vulva/penile), and 41 (5.5%) melanomas of unknown primary. Sixteen fusions (2%) were detected in samples from 16 patients: 12/599 (2%) cutaneous, 2/38 (5%) acral, 1/9 (11%) vulva, 1/23(4.3%) sinonasal; and 12/16 (75%) fusions were potentially targetable. We identified two novel rearrangements: NAGS::MAST2 and NOTCH1::GNB1; and two fusions that have been reported in other malignancies but not in melanoma: CANT1::ETV4 (prostate cancer) and CCDC6::RET (thyroid cancer). Additional fusions, previously reported in melanoma, included: EML4::ALK, MLPH::ALK, AGAP3::BRAF, AGK::BRAF, CDH3::BRAF, CCT8::BRAF, DIP2B::BRAF, EFNB1::RAF1, LRCH3::RAF1, MAP4::RAF1, RUFY1::RAF1, and ADCY2::TERT. Fusion positive melanomas harbored recurrent alterations in TERT and CDKN2A, among others. Gene fusions were exceedingly rare (0.2%) in BRAF/RAS/NF1-mutant tumors and were detected in 5.6% of triple wild-type melanomas. Interestingly, gene rearrangements were significantly enriched within the subset of triple wild-type melanomas that harbor TERT promoter mutations (18% versus 2%, p < 0.0001). Thirteen (81%) patients were treated with immunotherapy for metastatic disease or in the adjuvant setting. Six of 12 (50%) patients with potentially actionable fusions progressed on immunotherapy, and 3/6 (50%) were treated with targeted agents (ALK and MEK inhibitors), 2 off-label and 1 as part of a clinical trial. One patient with an AGAP3::BRAF fusion positive melanoma experienced a 30-month long response to trametinib. We show that, detecting fusions, especially in triple wild-type melanomas with TERT promoter mutations, may have a clinically significant impact in patients with advanced disease who have failed front-line immunotherapy.

Implementation and Clinical Adoption of Precision Oncology Workflows Across a Healthcare Network.

BACKGROUND: Precision oncology relies on molecular diagnostics, and the value-proposition of modern healthcare networks promises a higher standard of care across partner sites. We present the results of a clinical pilot to standardize precision oncology workflows. METHODS: Workflows are defined as the development, roll-out, and updating of disease-specific molecular order sets. We tracked the timeline, composition, and effort of consensus meetings to define the combination of molecular tests. To assess clinical impact, we examined order set adoption over a two-year period (before and after roll-out) across all gastrointestinal and hepatopancreatobiliary (GI) malignancies, and by provider location within the network. RESULTS: Development of 12 disease center-specific order sets took ~9 months, and the average number of tests per indication changed from 2.9 to 2.8 (P = .74). After roll-out, we identified significant increases in requests for GI patients (17%; P < .001), compliance with testing recommendations (9%; P < .001), and the fraction of "abnormal" results (6%; P < .001). Of 1088 GI patients, only 3 received targeted agents based on findings derived from non-recommended orders (1 before and 2 after roll-out); indicating that our practice did not negatively affect patient treatments. Preliminary analysis showed 99% compliance by providers in network sites, confirming the adoption of the order sets across the network. CONCLUSION: Our study details the effort of establishing precision oncology workflows, the adoption pattern, and the absence of harm from the reduction of non-recommended orders. Establishing a modifiable communication tool for molecular testing is an essential component to optimize patient care via precision oncology.

Clinicopathologic features of kinase fusion-related thyroid carcinomas: an integrative analysis with molecular characterization.

The discovery of actionable kinase gene rearrangements has revolutionized the therapeutic landscape of thyroid carcinomas. Unsolved challenges include histopathologic recognition of targetable cases, correlation between genotypes and tumor behavior, and evolving resistance mechanisms against kinase inhibitors (KI). We present 62 kinase fusion-positive thyroid carcinomas (KFTC), including 57 papillary thyroid carcinomas (PTC), two poorly differentiated thyroid carcinomas (PDTC), two undifferentiated thyroid carcinomas (ATC), and one primary secretory carcinoma (SC), in 57 adults and 5 adolescents. Clinical records, post-operative histology, and molecular profiles were reviewed. Histologically, all KFTC showed multinodular growth with prominent intratumoral fibrosis. Lymphovascular invasion (95%), extrathyroidal extension, gross and microscopic (63%), and cervical lymph node metastasis (79%) were common. Several kinase fusions were identified: STRN-ALK, EML4-ALK, AGK-BRAF, CUL1-BRAF, MKRN1-BRAF, SND1-BRAF, TTYH3-BRAF, EML4-MET, TFG-MET, IRF2BP2-NTRK1, PPL-NTRK1, SQSTM1-NTRK1, TPR-NTRK1, TPM3-NTRK1, EML4-NTRK3, ETV6-NTRK3, RBPMS-NTRK3, SQSTM1-NTRK3, CCDC6-RET, ERC1-RET, NCOA4-RET, RASAL2-RET, TRIM24-RET, TRIM27-RET, and CCDC30-ROS1. Individual cases also showed copy number variants of EGFR and nucleotide variants and indels in pTERT, TP53, PIK3R1, AKT2, TSC2, FBXW7, JAK2, MEN1, VHL, IDH1, PTCH1, GNA11, GNAQ, SMARCA4, and CDH1. In addition to thyroidectomy and radioactive iodine, ten patients received multi-kinase and/or selective kinase inhibitor therapy, with 6 durable, objective responses and four with progressive disease. Among 47 cases with >6 months of follow-up (median [range]: 41 [6-480] months), persistent/recurrent disease, distant metastasis and thyroid cancer-related death occurred in 57%, 38% and 6%, respectively. In summary, KFTC encompass a spectrum of molecularly diverse tumors with overlapping clinicopathologic features and a tendency for clinical aggressiveness. Characteristic histology with multinodular growth and prominent fibrosis, particularly when there is extensive lymphovascular spread, should trigger molecular testing for gene rearrangements, either in a step-wise manner by prevalence or using a combined panel. Further, our findings provide information on molecular therapy in radioiodine-refractory thyroid carcinomas.

Lymph node metastases develop through a wider evolutionary bottleneck than distant metastases.

Genetic diversity among metastases is poorly understood but contains important information about disease evolution at secondary sites. Here we investigate inter- and intra-lesion heterogeneity for two types of metastases that associate with different clinical outcomes: lymph node and distant organ metastases in human colorectal cancer. We develop a rigorous mathematical framework for quantifying metastatic phylogenetic diversity. Distant metastases are typically monophyletic and genetically similar to each other. Lymph node metastases, in contrast, display high levels of inter-lesion diversity. We validate these findings by analyzing 317 multi-region biopsies from an independent cohort of 20 patients. We further demonstrate higher levels of intra-lesion heterogeneity in lymph node than in distant metastases. Our results show that fewer primary tumor lineages seed distant metastases than lymph node metastases, indicating that the two sites are subject to different levels of selection. Thus, lymph node and distant metastases develop through fundamentally different evolutionary mechanisms.

Clinicopathologic and molecular characterization of NTRK-rearranged thyroid carcinoma (NRTC).

Primary thyroid neoplasms with actionable NTRK rearrangements are rare, and their clinical behavior, histologic characteristics, and molecular landscape are not well understood. We report an institutional series of eleven NTRK-rearranged thyroid carcinomas (NRTC) by performing clinicopathologic review and next-generation sequencing for targeted mutations and gene rearrangements. The NRTC encompass a histomorphologic spectrum of ten papillary thyroid carcinomas (PTC), including one with high-grade features, and one secretory carcinoma (SC), in ten adults and one adolescent. All NRTC were characterized by an unusual multinodular growth pattern, extensive lymphovascular invasion, and cervical lymph node metastases at initial presentation. Immunophenotypically, while most cases were positive for TTF1 and PAX8, the SC case was negative/weak for these markers and instead diffusely expressed GATA3, mammaglobin and S100. Observed gene rearrangements included ETV6-NTRK3 (n = 4, including the SC), TPR-NTRK1 (n = 2), RBPMS-NTRK3 (n = 2), SQSTM1-NTRK1 (n = 1), SQSTM1-NTRK3 (n = 1), and EML4-NTRK3 (n = 1). Mutation profiling revealed a concurrent TERT promotor mutation C228T in two (22%) patients and a novel frameshift MEN1 deletion in one. All patients received total thyroidectomy and radioactive iodine. Despite frequent development of persistent/recurrent disease (9 cases, 82%) and distant metastases (6 cases; 55%), no tumor-related death occurred over a median (range) follow-up of 44 (11 to 471) months. Three patients received NTRK inhibitor therapy, with the SC case showing complete resolution and two other patients experiencing 33% and 69.7% decrease of disease burden. Although the range of features is variable, NRTC appear to be clinically aggressive tumors with high metastatic rate but relatively low mortality with NTRK inhibitor therapy. The histologic findings of multinodular growth and extensive lymphovascular spread, seen in all NRTC, including PTC and SC, may serve as useful histomorphologic clues to prompt NTRK status testing. We also present the first report of concurrent TERT promotor activating mutation which did not appear to confer entrectinib resistance to NRTC.