Expanding the Spectrum of NR4A3 Fusion-Positive Gynecologic Leiomyosarcomas.

Recurrent gene fusions have been observed in epithelioid and myxoid variants of uterine leiomyosarcoma. PGR::NR4A3 fusions were recently described in a subset of epithelioid leiomyosarcomas exhibiting rhabdoid morphology. In this study, we sought to expand the clinical, morphologic, immunohistochemical, and genetic features of gynecologic leiomyosarcomas harboring NR4A3 rearrangements with PGR and novel fusion partners. We identified 9 gynecologic leiomyosarcomas harboring PGR::NR4A3, CARMN::NR4A3, ACTB::NR4A3, and possible SLCO5A1::NR4A3 fusions by targeted RNA sequencing. Tumors frequently affected premenopausal women, involving the uterine corpus, uterine cervix, or pelvis. All were similarly characterized by lobules of monomorphic epithelioid and/or spindled cells arranged in sheets, cords, trabeculae, and micro- and macrocysts associated with abundant myxoid matrix and hemorrhage, creating labyrinth-like or pulmonary edema-like architecture. Myogenic differentiation with frequent estrogen receptor and progesterone receptor staining and no CD10 expression characterized all tumors. All cases showed high NR4A3 RNA expression levels and NOR1 (NR4A3) nuclear staining similar to salivary gland acinic cell carcinomas and a subset of extraskeletal myxoid chondrosarcomas harboring NR4A3 rearrangements. NOR1 (NR4A3) immunohistochemistry may serve as a useful diagnostic marker of NR4A3 fusion-positive gynecologic leiomyosarcomas.

FGFR1/2/3-rearranged carcinoma of the head and neck: expanded histological spectrum crossing path with high-risk HPV in the sinonasal tract.

AIMS: Oncogenic FGFR1/2/3 rearrangements are found in various cancers. Reported cases in head and neck (HN) are mainly squamous cell carcinomas (SCCs) with FGFR3::TACC3 fusions, a subset of which also harbour high-risk human papillomavirus (HPV). However, the knowledge of the clinicopathological spectrum of FGFR-rearranged head and neck carcinomas (FHNC) is limited. METHODS AND RESULTS: A retrospective MSK-fusion clinical sequencing cohort 2016-23 was searched to identify malignant tumours in the HN region harbouring FGFR1/2/3 fusion. FHNC were characterised by histological examination, immunohistochemistry and molecular analysis. Electronic medical records were reviewed. Three FHNC were identified. Two cases (cases 1 and 2) involved sinonasal tract and were high-grade carcinomas with squamous, basaloid, glandular and/or ductal-myoepithelial features. Case 1 arose in a 79-year-old man and harboured FGFR2::KIF1A fusion. Case 2 arose in a 58-year-old man, appeared as HPV-related multiphenotypic sinonasal carcinoma (HMSC), and was positive for FGFR2::TACC2 fusion and concurrent high-risk HPV, non-type 16/18. Case 3 was FGFR3::TACC3 fusion-positive keratinising SCCs arising in the parotid of a 60-year-old man. All three cases presented at stage T4. Clinical follow-up was available in two cases; case 1 remained disease-free for 41 months post-treatment and case 3 died of disease 2 months after the diagnosis. CONCLUSIONS: FHNC include a morphological spectrum of carcinomas with squamous features and may occur in different HN locations, such as parotid gland and the sinonasal tract. Sinonasal cases can harbour FGFR2 rearrangement with or without associated high-risk HPV. Timely recognition of FHNC could help select patients potentially amenable to targeted therapy with FGFR inhibitors. Further studies are needed (1) to determine if FGFR2 rearranged/HPV-positive sinonasal carcinomas are biologically distinct from HMSC, and (2) to elucidate the biological and clinical significance of FGFR2 rearrangement in the context of high-risk HPV.

Detection of GRM1 gene rearrangements in chondromyxoid fibroma: a comparison of fluorescence in-situ hybridisation, RNA sequencing and immunohistochemical analysis.

AIMS: Chondromyxoid fibroma (CMF) is a rare, benign bone tumour which arises primarily in young adults and is occasionally diagnostically challenging. Glutamate metabotropic receptor 1 (GRM1) gene encodes a metabotropic glutamate receptor and was recently shown to be up-regulated in chondromyxoid fibroma through gene fusion and promoter swapping. The aim of this study was to interrogate cases of CMF for the presence of GRM1 gene rearrangements, gene fusions and GRM1 protein overexpression. METHODS AND RESULTS: Selected cases were subjected to testing by fluorescent in-situ hybridisation (FISH) with a GRM1 break-apart probe, a targeted RNA sequencing method and immunohistochemical study with an antibody to GRM1 protein. Two cases were subjected to whole transcriptomic sequencing. In 13 of 13 cases, GRM1 protein overexpression was detected by immunohistochemistry using the GRM1 antibody. Of the 12 cases successfully tested by FISH, nine of 12 showed GRM1 rearrangements by break-apart probe assay. Targeted RNA sequencing analysis did not detect gene fusions in any of the eight cases tested, but there was an increase in GRM1 mRNA expression in all eight cases. Two cases subjected to whole transcriptomic sequencing (WTS) showed elevated GRM1 expression and no gene fusions. CONCLUSION: GRM1 gene rearrangements can be detected using FISH break-apart probes in approximately 75% of cases, and immunohistochemical detection of GRM1 protein over-expression is a sensitive diagnostic method. The gene fusion was not detected by targeted RNA sequencing, due most probably to the complexity of fusion mechanism, and is not yet a reliable method for confirming a diagnosis of CMF in the clinical setting.

ALK-rearranged Mesenchymal Neoplasms: A Report of 9 cases Further Expanding the Clinicopathologic Spectrum of Emerging Kinase Fusion Positive Group of Tumors.

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.

Malignant undifferentiated epithelioid neoplasms with MAML2 rearrangements: A clinicopathologic study of seven cases demonstrating a heterogenous entity.

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.

EWSR1::YY1 fusion positive peritoneal epithelioid mesothelioma harbors mesothelioma epigenetic signature: Report of 3 cases in support of an emerging entity.

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.

Androgen receptor splice variant-7 in breast cancer: clinical and pathologic correlations.

Androgen receptor (AR) inhibitor therapy is a developing treatment for AR-positive breast cancer (BC) with ongoing clinical trials. AR splice variant-7 (AR-V7) is a truncated variant of AR that leads to AR inhibitor therapy resistance in prostate cancer; recent studies have identified AR-V7 in BC and theorized that AR-V7 can have a similar impact. This study assessed the prevalence and clinicopathologic features associated with AR-V7 in a large BC cohort. BC samples were evaluated by MSK-Fusion targeted RNAseq for AR-V7 detection and MSK-IMPACT targeted DNAseq, including triple-negative tumors with no driver alteration and estrogen receptor-positive/ESR1 wildtype tumors progressing on therapy. Among 196 primary and metastatic/recurrent cases (196 RNAseq, 194DNAseq), 9.7% (19/196) were AR-V7 positive and 90.3% (177/196) AR-V7 negative. All AR-V7 positive BC were AR-positive by immunohistochemistry (19/19). The prevalence of AR-V7 by receptor subtype (N = 189) was: 18% (12/67) in ER-/PgR-/HER2-negative BC, 3.7% (4/109) in ER-positive/HER2-negative BC, and 15.4% (2/13) in HER2-positive BC; AR-V7 was detected in one ER-positive/HER2-unknown BC. Apocrine morphology was observed in 42.1% (8/19) of AR-V7 positive BC and 3.4% (6/177) AR-V7 negative BC (P < 0.00001). Notably, AR-V7 was detected in 2 primary BC and 7 metastatic/recurrent BC patients with no prior endocrine therapy. We conclude that positive AR IHC and apocrine morphology are pathologic features that may indicate testing for AR-V7 is warranted in both primary and metastatic BC in the appropriate clinical context. The study findings further encourage the assessment of AR-V7 as a predictive biomarker for AR antagonist benefit in ongoing clinical BC trials.

Myositis ossificans-like soft tissue aneurysmal bone cyst: a clinical, radiological, and pathological study of seven cases with COL1A1-USP6 fusion and a novel ANGPTL2-USP6 fusion.

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.

NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.

With the FDA approval of larotrectinib, NTRK fusion assessment has recently become a standard part of management for patients with locally advanced or metastatic cancers. Unlike somatic mutation assessment, the detection of NTRK fusions is not straightforward, and various assays exist at the DNA, RNA, and protein level. Here, we investigate the performance of immunohistochemistry and DNA-based next-generation sequencing to indirectly or directly detect NTRK fusions relative to an RNA-based next-generation sequencing approach in the largest cohort of NTRK fusion positive solid tumors to date. A retrospective analysis of 38,095 samples from 33,997 patients sequenced by a targeted DNA-based next-generation sequencing panel (MSK-IMPACT), 2189 of which were also examined by an RNA-based sequencing assay (MSK-Fusion), identified 87 patients with oncogenic NTRK1-3 fusions. All available institutional NTRK fusion positive cases were assessed by pan-Trk immunohistochemistry along with a cohort of control cases negative for NTRK fusions by next-generation sequencing. DNA-based sequencing showed an overall sensitivity and specificity of 81.1% and 99.9%, respectively, for the detection of NTRK fusions when compared to RNA-based sequencing. False negatives occurred when fusions involved breakpoints not covered by the assay. Immunohistochemistry showed overall sensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusions and lower sensitivity for NTRK3 fusions (79%). Specificity was 100% for carcinomas of the colon, lung, thyroid, pancreas, and biliary tract. Decreased specificity was seen in breast and salivary gland carcinomas (82% and 52%, respectively), and positive staining was often seen in tumors with neural differentiation. Both sensitivity and specificity were poor in sarcomas. Selection of the appropriate assay for NTRK fusion detection therefore depends on tumor type and genes involved, as well as consideration of other factors such as available material, accessibility of various clinical assays, and whether comprehensive genomic testing is needed concurrently.

Diagnosis of known sarcoma fusions and novel fusion partners by targeted RNA sequencing with identification of a recurrent ACTB-FOSB fusion in pseudomyogenic hemangioendothelioma.

Integration of morphological, immunohistochemical, and molecular methods is often necessary for the precise diagnosis and optimal clinical management of sarcomas. We have validated and implemented a clinical molecular diagnostic assay, MSK- Fusion Solid, for detection of gene fusions in solid tumors, including sarcomas. Starting with RNA extracted from formalin-fixed paraffin-embedded tumor material, this targeted RNA sequencing assay utilizes anchored multiplex PCR to detect oncogenic fusion transcripts involving 62 genes known to be recurrently rearranged in solid tumors including sarcomas without prior knowledge of fusion partners. From 1/2016 to 1/2018, 192 bone and soft tissue tumors were submitted for MSK- Fusion Solid analysis and 96% (184/192) successfully passed all the pre-sequencing quality control parameters and sequencing steps. These sarcomas encompass 24 major tumor types, including 175 soft tissue tumors and 9 osteosarcomas. Ewing and Ewing-like sarcomas, rhabdomyosarcoma, and sarcoma-not otherwise specified were the three most common tumor types. Diagnostic in-frame fusion transcripts were detected in 43% of cases, including 3% (6/184) with novel fusion partners, specifically TRPS1-PLAG1, VCP-TFE3, MYLK-BRAF, FUS-TFCP2, and ACTB-FOSB, the latter in two cases of pseudomyogenic hemangioendothelioma, representing a novel observation in this sarcoma. Our experience shows that this targeted RNA sequencing assay performs in a robust and sensitive fashion on RNA extracted from most routine clinical specimens of sarcomas thereby facilitating precise diagnosis and providing opportunities for novel fusion partner discovery.

Impact of Rare and Multiple Concurrent Gene Fusions on Diagnostic DNA Methylation Classifier in Brain Tumors.

DNA methylation is an essential molecular assay for central nervous system (CNS) tumor diagnostics. While some fusions define specific brain tumors, others occur across many different diagnoses. We performed a retrospective analysis of 219 primary CNS tumors with whole genome DNA methylation and RNA next-generation sequencing. DNA methylation profiling results were compared with RNAseq detected gene fusions. We detected 105 rare fusions involving 31 driver genes, including 23 fusions previously not implicated in brain tumors. In addition, we identified 6 multi-fusion tumors. Rare fusions and multi-fusion events can impact the diagnostic accuracy of DNA methylation by decreasing confidence in the result, such as BRAF, RAF, or FGFR1 fusions, or result in a complete mismatch, such as NTRK, EWSR1, FGFR, and ALK fusions. IMPLICATIONS: DNA methylation signatures need to be interpreted in the context of pathology and discordant results warrant testing for novel and rare gene fusions.

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.

Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition.

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.

Recurrent TRAK1::RAF1 Fusions in pediatric low-grade gliomas.

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.

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.

A Performance Comparison of Commonly Used Assays to Detect RET Fusions.

PURPOSE: Selpercatinib and pralsetinib induce deep and durable responses in patients with advanced RET fusion-positive lung and thyroid cancer. RET fusion testing strategies with rapid and reliable results are critical given recent FDA approval. Here, we assess various clinical assays in a large pan-cancer cohort. EXPERIMENTAL DESIGN: Tumors underwent DNA-based next-generation sequencing (NGS) with reflex to RNA-based NGS if no mitogenic driver or if a RET structural variant of unknown significance (SVUS) were present. Canonical DNA-level RET fusions and RNA-confirmed RET fusions were considered true fusions. Break-apart FISH and IHC performance were assessed in subgroups. RESULTS: A total of 171 of 41,869 patients with DNA NGS harbored RET structural variants, including 139 canonical fusions and 32 SVUS. Twelve of 32 (37.5%) SVUS were transcribed into RNA-level fusions, resulting in 151 oncogenic RET fusions. The most common RET fusion-positive tumor types were lung (65.6%) and thyroid (23.2%). The most common partners were KIF5B (45%), CCDC6 (29.1%), and NCOA4 (13.3%). DNA NGS showed 100% (46/46) sensitivity and 99.6% (4,459/4,479) specificity. FISH showed 91.7% (44/48) sensitivity, with lower sensitivity for NCOA4-RET (66.7%, 8/12). A total of 87.5% (7/8) of RET SVUS negative for RNA-level fusions demonstrated rearrangement by FISH. The sensitivity of IHC varied by fusion partner: KIF5B sensitivity was highest (100%, 31/31), followed by CCDC6 (88.9%, 16/18) and NCOA4 (50%, 6/12). Specificity of RET IHC was 82% (73/89). CONCLUSIONS: Although DNA sequencing has high sensitivity and specificity, RNA sequencing of RET SVUS is necessary. Both FISH and IHC demonstrated lower sensitivity for NCOA4-RET fusions.

A Pan-Cancer Study of Somatic TERT Promoter Mutations and Amplification in 30,773 Tumors Profiled by Clinical Genomic Sequencing.

TERT gene promoter mutations are known in multiple cancer types. Other TERT alterations remain poorly characterized. Sequencing data from 30,773 tumors analyzed by a hybridization capture next-generation sequencing assay (Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets) were analyzed for the presence of TERT alterations. Promoter rearrangements (500 bases upstream of the transcriptional start site), hypermethylation (n = 57), and gene expression (n = 155) were evaluated for a subset of cases. Mutually exclusive and recurrent promoter mutations were identified at three hot spots upstream of the transcriptional start site in 11.3% of cases (-124: 74%; -146: 24%; and -138: <2%). Mutually exclusive amplification events were identified in another 2.3% of cases, whereas mutually exclusive rearrangements proximal to the TERT gene were seen in 24 cases. The highest incidence of TERT promoter mutations was seen in cutaneous melanoma (82%), whereas amplification events significantly outnumbered promoter mutations in well-differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%). Gene expression analysis suggests that the highest levels of gene expression are seen in cases with amplifications and rearrangements. Hypermethylation events upstream of the TERT coding sequence were not mutually exclusive with known pathogenic alterations. Studies aimed at defining the prevalence and prognostic impact of TERT alterations should incorporate other pathogenic TERT alterations as these may impact telomerase function.

Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects.

Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains.

Undifferentiated Uterine Sarcomas Represent Under-Recognized High-grade Endometrial Stromal Sarcomas.

Undifferentiated uterine sarcoma is a diagnosis of exclusion with limited molecular genetic data available. Recent recognition of high-grade endometrial stromal sarcomas with diverse genotypes suggests that some tumors classified as undifferentiated uterine sarcomas may represent misdiagnosed high-grade endometrial stromal sarcomas. Archival material from 10 tumors diagnosed as undifferentiated uterine sarcomas in 2009 to 2017 were collected. BCOR immunohistochemistry and fluorescence in situ hybridization (FISH) using break-apart probes flanking BCOR, ZC3H7B, CCNB3, YWHAE, NUTM2, JAZF1, and BCORL1 were performed. Tumors lacking or harboring gene rearrangement with no known fusion partner by FISH were subjected to targeted RNA sequencing. Morphology was correlated with FISH and sequencing results. BCOR expression was moderate to strong in ≥50% of cells in 8 tumors, while weak in <5% cells and negative in 2. FISH detected mutually exclusive ZC3H7B-BCOR and YWHAE-NUTM2 fusions in 3 uniform undifferentiated uterine sarcomas; 2 pleomorphic tumors harbored YWHAE rearrangement with no known partner. Targeted RNA sequencing of 5 FISH-negative uniform undifferentiated uterine sarcomas detected BRD8-PHF1 and YWHAE-NUTM2B fusions and BCOR internal tandem duplication in 4 of them. Tumors with YWHAE-NUTM2 fusions and BCOR genetic abnormalities showed morphology characteristic of high-grade endometrial stromal sarcomas. No fusions were detected by sequencing in the tumor with YWHAE rearrangement only by FISH. Most tumors classified as undifferentiated uterine sarcomas represent misdiagnosed high-grade endometrial stromal sarcomas. BCOR expression in ≥50% of cells may help triage tumors for molecular confirmation of high-grade endometrial stromal sarcoma-related genetic abnormalities. Novel YWHAE rearrangements may define a subset of true undifferentiated pleomorphic sarcomas.

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden.

PURPOSE: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver-negative lung cancers. EXPERIMENTAL DESIGN: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq. RESULTS: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver-negative cases with low TMB [0-5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001). CONCLUSIONS: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver-negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.See related commentary by Davies and Aisner, p. 4586.