Undifferentiated Round Cell Sarcoma With CRTC1::SS18 Fusion: Expanding Clinicopathologic Features of a Rare Translocation Sarcoma With Prominent Desmoplastic Stroma.

Undifferentiated round cell sarcomas (URCS) represent a diverse group of tumors, including conventional Ewing sarcoma, round cell sarcoma with EWSR1/FUS-non-ETS fusions, CIC-rearranged sarcoma, and sarcoma with BCOR alterations. Since 2018, 3 cases of URCS with a novel CRTC1::SS18 gene fusion have been reported in the literature. Herein, we report 3 additional cases of CRTC1::SS18 sarcoma, thereby doubling the number of described cases and expanding the clinicopathologic features of this rare translocation sarcoma. Together with the previously reported cases, we show that the male-to-female ratio is 1:2 with a median age of 34 years (range, 12-42 years). Tumors occurred primarily in intramuscular locations involving the lower extremity. Histologically, all tumors contained uniform round-to-epithelioid cells with a moderate amount of eosinophilic cytoplasm growing in sheets and nests with prominent desmoplastic stroma reminiscent of desmoplastic small round cell tumor. Immunohistochemical results were nonspecific, demonstrating variable expression of CD99 (patchy), ALK, GATA3, and cyclin D1. RNA sequencing revealed CRTC1::SS18 gene fusions in all cases, involving exons 1 to 2 of CRTC1 (the 5' partner gene) on chromosome 19 and either exon 2 or exon 4 of SS18 (the 3' partner gene) on chromosome 18. The clinical course was variable. Although 1 previously reported case demonstrated aggressive behavior with a fatal outcome, 2 others had a relatively indolent course with gradual growth for 6 to 7 years prior to resection. Two cases developed metastatic disease, including 1 case with bilateral lung metastasis and 1 with locoregional spread to a lymph node. By analyzing the clinicopathologic features, we aimed to improve recognition of this rare translocation sarcoma to better understand its biologic potential, optimize patient management, and expand the current classification of URCS.

Liquid biopsy-based circulating tumour (ct)DNA analysis of a spectrum of myeloid and lymphoid malignancies yields clinically actionable results.

AIMS: Liquid biopsy (LBx)-based next-generation sequencing (NGS) of circulating tumour DNA (ctDNA) can facilitate molecular profiling of haematopoietic neoplasms (HNs), particularly when tissue-based NGS is infeasible. METHODS AND RESULTS: We studied HN LBx samples tested with FoundationOne Liquid CDx, FoundationOne Liquid, or FoundationACT between July 2016 and March 2022. We identified 271 samples: 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytoses, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukaemia (AML). Among 73.4% with detectable pathogenic alterations, median maximum somatic allele frequency (MSAF) was 16.6%, with AML (36.2%), MDS (19.7%), and MPN (44.5%) having higher MSAFs than DLBCL (3.9%), NHL (8.4%), HL (1.5%), PCN (2.8%), and histiocytoses (1.8%) (P = 0.001). LBx detected characteristic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML. Among 24 samples, the positive percent agreement by LBx was 75.7% for variants present in paired buffy coat, marrow, or tissues. Also, 75.0% of pairs exhibited alterations only present on LBx. These were predominantly subclonal (clonal fraction of 3.8%), reflecting the analytical sensitivity of LBx. CONCLUSION: These data demonstrate that LBx can detect relevant genomic alterations across HNs, including at low clonal fractions, suggesting a potential clinical utility for identifying residual or emerging therapy-resistant clones that may be undetectable in site-specific tissue biopsies.

Precision needle-punch tumor enrichment from paraffin blocks improves the detection of clinically actionable genomic alterations and biomarkers.

Background: While many molecular assays can detect mutations at low tumor purity and variant allele frequencies, complex biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH) require higher tumor purity for accurate measurement. Scalable, quality-controlled, tissue-conserving methods to increase tumor nuclei percentage (TN%) from tumor specimens are needed for complex biomarkers and hence necessary to maximize patient matching to approved therapies or clinical trial enrollment. We evaluated the clinical utility and performance of precision needle-punch enrichment (NPE) compared with traditional razor blade macroenrichment of tumor specimens on molecular testing success. Methods: Pathologist-directed NPE was performed manually on formalin-fixed, paraffin embedded (FFPE) blocks. Quality control of target capture region and quantity of residual tumor in each tissue block was determined via a post-enrichment histologic slide recut. Resultant tumor purity and biomarker status were determined by the computational analysis pipeline component of the FDA-approved next-generation sequencing (NGS) assay, FoundationOne®CDx. Following NPE implementation for real-world clinical samples, assay performance and biomarker (MSI, TMB, gLOH) detection were analyzed. Results: In real-world clinical samples, enrichment rate via NPE was increased to ~50% over a 2.5-year period, exceeding the prior use of razor blade macro-enrichment (<30% of cases) prior to NPE implementation due to proven efficacy in generating high quality molecular results from marginal samples and the ease of use for both pathologist and histotechnologists. NPE was associated with lower test failures, higher computational tumor purity, and higher rates of successful TMB, MSI and gLOH determination when stratified by pre-enriched (incipient) tumor nuclei percentage. In addition, challenging cases in which tumor content was initially insufficient for testing were salvaged for analysis of biomarker status, gene amplification/deletion, and confident mutant or wild-type gene status determination. Conclusions: Pathologist-directed precision enrichment from tissue blocks (aka NPE) increases tumor purity, and consequently, yields a greater number of successful tests and complex biomarker determinations. Moreover, this process is rapid, safe, inexpensive, scalable, and conserves patient surgical pathology material. NPE may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in molecular laboratories.

Pan-cancer Genomic Analysis of AXL Mutations Reveals a Novel, Recurrent, Functionally Activating AXL W451C Alteration Specific to Myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a common soft tissue sarcoma of the elderly that typically shows low tumor mutational burden, with mutations in TP53 and in genes associated with cell cycle checkpoints ( RB1 , CDKN2A ). Unfortunately, no alterations or markers specific to MFS have been identified and, as a consequence, there are no effective targeted therapies. The receptor tyrosine kinase AXL, which drives cellular proliferation, is targetable by new antibody-based therapeutics. Expression of AXL messenger RNA is elevated in a variety of sarcoma types, with the highest levels reported in MFS, but the pathogenic significance of this finding remains unknown. To assess a role for AXL abnormalities in MFS, we undertook a search for AXL genomic alterations in a comprehensive genomic profiling database of 463,546 unique tumors (including 19,879 sarcomas, of which 315 were MFS) interrogated by targeted next-generation DNA and/or RNA sequencing. Notably, the only genomic alterations recurrent in a specific sarcoma subtype were AXL W451C (n = 8) and AXL W450C (n = 2) mutations. The tumors involved predominantly older adults (age: 44 to 81 [median: 72] y) and histologically showed epithelioid and spindle-shaped cells in a variably myxoid stroma, with 6 cases diagnosed as MFS, 3 as undifferentiated pleomorphic sarcoma (UPS), and 1 as low-grade sarcoma. The AXL W451C mutation was not identified in any non-sarcoma malignancy. A review of publicly available data sets revealed a single AXL W451C-mutant case of UPS that clustered with MFS/UPS by methylation profiling. Functional studies revealed a novel activation mechanism: the W451C mutation causes abnormal unregulated dimerization of the AXL receptor tyrosine kinase through disulfide bond formation between pairs of mutant proteins expressing ectopic cysteine residues. This dimerization triggers AXL autophosphorylation and activation of downstream ERK signaling. We further report sarcomas of diverse histologic subtypes with AXL gene amplifications, with the highest frequency of amplification identified in MFS cases without the W451C mutation. In summary, the activating AXL W451C mutation appears highly specific to MFS, with a novel mechanism to drive unregulated signaling. Moreover, AXL gene amplifications and messenger RNA overexpression are far more frequent in MFS than in other sarcoma subtypes. We conclude that these aberrations in AXL are distinct features of MFS and may aid diagnosis, as well as the selection of available targeted therapies.

Intratumoral Escherichia Is Associated With Improved Survival to Single-Agent Immune Checkpoint Inhibition in Patients With Advanced Non-Small-Cell Lung Cancer.

PURPOSE: The impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients with non-small-cell lung cancer (NSCLC) is unknown. Preclinically, intratumoral Escherichia is associated with a proinflammatory tumor microenvironment and decreased metastases. We sought to determine whether intratumoral Escherichia is associated with outcome to ICI in patients with NSCLC. PATIENTS AND METHODS: We examined the intratumoral microbiome in 958 patients with advanced NSCLC treated with ICI by querying unmapped next-generation sequencing reads against a bacterial genome database. Putative environmental contaminants were filtered using no-template controls (n = 2,378). The impact of intratumoral Escherichia detection on overall survival (OS) was assessed using univariable and multivariable analyses. The findings were further validated in an external independent cohort of 772 patients. Escherichia fluorescence in situ hybridization (FISH) and transcriptomic profiling were performed. RESULTS: In the discovery cohort, read mapping to intratumoral Escherichia was associated with significantly longer OS (16 v 11 months; hazard ratio, 0.73 [95% CI, 0.59 to 0.92]; P = .0065) in patients treated with single-agent ICI, but not combination chemoimmunotherapy. The association with OS in the single-agent ICI cohort remained statistically significant in multivariable analysis adjusting for prognostic features including PD-L1 expression (P = .023). Analysis of an external validation cohort confirmed the association with improved OS in univariable and multivariable analyses of patients treated with single-agent ICI, and not in patients treated with chemoimmunotherapy. Escherichia localization within tumor cells was supported by coregistration of FISH staining and serial hematoxylin and eosin sections. Transcriptomic analysis correlated Escherichia-positive samples with expression signatures of immune cell infiltration. CONCLUSION: Read mapping to potential intratumoral Escherichia was associated with survival to single-agent ICI in two independent cohorts of patients with NSCLC.