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Aims: Although molecular tests developed for a growing list of oncogenic alterations have significantly aided in the classification of head and neck carcinomas, tumours in which prototypical histologic and immunophenotypic features are lacking or only partially developed continue to pose diagnostic challenges. Searching for known diagnostic and therapeutic targets by clinical next-generation sequencing (NGS) assays can often lead to new discoveries., Methods and Results: We present our institutional experience in applying targeted RNA NGS in 36 head and neck carcinomas that were morphologically difficult to classify between 2016 and 2023. The patients ranged in age from 5 to 83 years (median, 64), with the majority of tumors occurring in the major salivary glands and the sinonasal tract. Overall, seven (19%) cases showed unusual gene rearrangements, including five novel alterations: MON2::STAT6 in a hard palate adenocarcinoma with mucinous features, POC5::RAF1 in apocrine intraductal carcinoma of the lacrimal gland, EWSR1::CDADC1 fusion in a basaloid carcinoma of the submandibular gland, NFATC2::NUTM2B in myoepithelial carcinoma, and NSD3::NCOA2 fusion in a peculiar high-grade carcinoma with a peritheliomatous growth pattern, and focal myogenic differentiation. Potential therapeutic actionability was identified in three cases (RAF1 and FGFR2 fusions)., Conclusion: These findings broaden the current spectrum of gene rearrangements in head and neck carcinomas and support the utility of clinical NGS in identifying unusual, actionable alterations in diagnostically challenging cases., (© 2024 John Wiley & Sons Ltd.)

Fusions involving CRAF (RAF1) are infrequent oncogenic drivers in pediatric low-grade gliomas, rarely identified in tumors bearing features of pilocytic astrocytoma, and involving a limited number of known fusion partners. We describe recurrent TRAK1::RAF1 fusions, previously unreported in brain tumors, in three pediatric patients with low-grade glial-glioneuronal tumors. We present the associated clinical, histopathologic and molecular features. Patients were all female, aged 8 years, 15 months, and 10 months at diagnosis. All tumors were located in the cerebral hemispheres and predominantly cortical, with leptomeningeal involvement in 2/3 patients. Similar to previously described activating RAF1 fusions, the breakpoints in RAF1 all occurred 5' of the kinase domain, while the breakpoints in the 3' partner preserved the N-terminal kinesin-interacting domain and coiled-coil motifs of TRAK1. Two of the three cases demonstrated methylation profiles (v12.5) compatible with desmoplastic infantile ganglioglioma (DIG)/desmoplastic infantile astrocytoma (DIA) and have remained clinically stable and without disease progression/recurrence after resection. The remaining tumor was non-classifiable; with focal recurrence 14 months after initial resection; the patient remains symptom free and without further recurrence/progression (5 months post re-resection and 19 months from initial diagnosis). Our report expands the landscape of oncogenic RAF1 fusions in pediatric gliomas, which will help to further refine tumor classification and guide management of patients with these alterations., (© 2023 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Among mesenchymal tumors, MAML2 gene rearrangements have been described in a subset of composite hemangioendothelioma and myxoinflammatory fibroblastic sarcoma (MIFS). However, we have recently encountered MAML2-related fusions in a group of seven undifferentiated malignant epithelioid neoplasms that do not fit well to any established pathologic entities. The patients included five males and two female, aged 41-71 years old (median 65 years). The tumors involved the deep soft tissue of extremities (hip, knee, arm, hand), abdominal wall, and the retroperitoneum. Microscopically, the tumors consisted of solid sheets of atypical epithelioid to histiocytoid cells with abundant cytoplasm. Prominent mitotic activity and necrosis were present in 4 cases. In 3 cases, the cells displayed hyperchromatic nuclei or conspicuous macronucleoli, and were admixed with background histiocytoid cells and a lymphoplasmacytic infiltrate. By immunohistochemistry (IHC), the neoplastic cells had a nonspecific phenotype. On targeted RNA sequencing, MAML2 was the 3' partner and fused to YAP1 (4 cases), ARHGAP42 (2 cases), and ENDOD1 (1 case). Two cases with YAP1::MAML2 harbored concurrent RAF kinase fusions (RBMS3::RAF1 and AGK::BRAF, respectively). In 2 cases with targeted DNA sequencing, mutations in TP53, RB1 and PTEN were detected in 1 case, and PDGFRB mutations, CCNE1 amplifications and CDKN2A/2B deletion were detected in another case, which showed strong and diffuse PDGFRB expression by IHC. Of the 4 cases with detailed clinical history (median follow-up period 8 months), three developed distant metastatic disease (one of which died of disease); one case remained free of disease 3 years following surgical excision. In conclusion, we describe a heterogeneous series of MAML2-rearranged undifferentiated malignant epithelioid neoplasms, a subset of which may overlap with a recently described MIFS variant with YAP1::MAML2 fusions, further expanding the clinicopathologic spectrum of mesenchymal neoplasms with recurrent MAML2 gene rearrangements., (© 2022 Wiley Periodicals LLC.)

Anaplastic lymphoma kinase (ALK) fusions are oncogenic drivers in diverse cancer types. Although well established in inflammatory myofibroblastic tumor (IMT) and epithelioid fibrous histiocytoma (EFH), ALK rearrangements also occur in the emerging family of kinase fusion-positive mesenchymal neoplasms. We investigated 9 ALK-rearranged mesenchymal neoplasms (exclusive of IMT and EFH) arising in 6 males and 3 females with a wide age range of 10 to 78 years old (median 42 years). Tumors involved superficial and deep soft tissue (6) and viscera (3). Three were myxoid or collagenous low-grade paucicellular tumors with haphazardly arranged spindled cells. Three were cellular tumors with spindled cells in intersecting short fascicles or solid sheets. Three cases consisted of uniform epithelioid cells arranged in nests or solid sheets, with prominent mitotic activity and necrosis. Band-like stromal hyalinization was present in 6 cases. All tumors expressed ALK; four were positive for S100 and five were positive for CD34, while all were negative for SOX10. By targeted RNA sequencing, the breakpoints involved ALK exon 20; the 5' partners included KLC1, EML4, DCTN1, PLEKHH2, TIMP3, HMBOX1, and FMR1. All but two patients presented with localized disease. One patient had distant lung metastases; another had diffuse pleural involvement. Of the six cases with treatment information, five were surgically excised [one also received neoadjuvant radiation therapy (RT)], and one received RT and an ALK inhibitor. Of the four patients with follow-up (median 5.5 months), one remained alive with stable disease and three were alive without disease. We expand the clinicopathologic spectrum of ALK-fused mesenchymal neoplasms, including a low-grade malignant peripheral nerve sheath tumor-like subset and another subset characterized by epithelioid and high-grade morphology., (© 2022 Wiley Periodicals LLC.)

Mesothelioma is a rare, aggressive malignant neoplasm of mesothelial origin. A small subset of peritoneal mesothelioma is driven by recurrent gene fusions, mostly EWSR1/FUS::ATF1 fusions, with predilection for young adults. To date, only two cases of mesothelioma harboring EWSR1::YY1 fusions have been described. We present three additional cases of EWSR1::YY1-fused peritoneal mesotheliomas, two localized and one diffuse, all occurring in the peritoneum of middle-aged adults (2 females and 1 male), and discovered incidentally by imaging or during surgery performed for unrelated reasons. None presented with symptoms or had a known history of asbestos exposure. All three cases were cellular epithelioid neoplasms with heterogeneous architectural patterns comprising mostly solid nests and sheets with variably papillary and trabecular areas against collagenous stroma. Cytologically, the cells were monomorphic, polygonal, epithelioid cells with dense eosinophilic cytoplasm and centrally located nuclei. Overt mitotic activity or tumor necrosis was absent. All cases showed strong diffuse immunoreactivity for pancytokeratin, CK7, and nuclear WT1, patchy to negative calretinin, retained BAP1 expression, and were negative for Ber-EP4 and MOC31. RNA-sequencing confirmed in-frame gene fusion transcripts involving EWSR1 exon 7/8 and YY1 exon 2/3. By unsupervised clustering analysis, the methylation profiles of EWSR1::YY1-fused mesotheliomas clustered similarly with EWSR1/FUS::ATF1-fused mesotheliomas and conventional mesotheliomas, suggesting a mesothelioma epigenetic signature. All three patients underwent surgical resection or cytoreductive surgery of the masses. On follow-up imaging, no recurrence or progression of disease was identified. Our findings suggest that EWSR1::YY1-fusion defines a small subset of peritoneal epithelioid mesothelioma in middle-aged adults without history of asbestos exposure., (© 2022 Wiley Periodicals LLC.)

Herein we described the clinical, radiological, histological, and molecular characteristics of seven soft tissue aneurysmal bone cysts (STABCs) diagnosed and managed at a tertiary cancer center and to elucidate their relationship with myositis ossificans (MO). All cases had established imaging and histopathological diagnosis of STABC and were subject to fluorescence in situ hybridization (FISH) for USP6 rearrangement and Archer® FusionPlex® targeted RNA sequencing (RNASeq) analysis to identify the fusion partner. A thorough literature review of STABC and MO was conducted. The patients presented with painful masses unpreceded by trauma, occurring most commonly in the deep soft tissue of the thigh/gluteus (4/7), and also in the supraclavicular region, the axilla, and the hand. On imaging, the lesions were frequently associated with peripheral calcification on conventional radiographs and CT (6/7), cystic components on ultrasound, as well as perilesional edema (7/7) and fluid levels (3/7) on MRI. Bone scan (1/1) showed intense radiotracer uptake. Histologically, 6/7 cases demonstrated zonal arrangements reminiscent of MO. USP6 rearrangement was found in all seven cases by FISH and/or RNASeq. RNASeq further detected COL1A1-USP6 fusion in six cases and a novel ANGPTL2-USP6 fusion in one case. Four patients underwent resection of the tumors and were disease free at their last follow-up. Three patients who underwent incisional or needle biopsies had no evidence of disease progression on imaging studies. In conclusion, the clinical, radiological, and pathological overlap between STABC and MO suggests that they are closely related entities. A novel fusion ANGPTL2-USP6 is associated with distinct clinical and pathological presentation.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

An additional copy of the beta-amyloid precursor protein (APP) gene causes early-onset Alzheimer's disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or beta-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (betaCTF), all of which elevate the levels of betaCTFs. Expression of a mutant form of APP that cannot undergo beta-cleavage had no effect on endosomes. Pharmacological inhibition of APP gamma-secretase, which markedly reduced Abeta production but raised betaCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the betaCTF of APP, and exclude Abeta and the alphaCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.