Comprehensive clinicopathological, molecular, and methylation analysis of mesenchymal tumors with NTRK and other kinase gene aberrations.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Branchioma: immunohistochemical and molecular genetic study of 23 cases highlighting frequent loss of retinoblastoma 1 immunoexpression.

Branchioma is an uncommon benign neoplasm with an adult male predominance, typically occurring in the lower neck region. Different names have been used for this entity in the past (ectopic hamartomatous thymoma, branchial anlage mixed tumor, thymic anlage tumor, biphenotypic branchioma), but currently, the term branchioma has been widely accepted. Branchioma is composed of endodermal and mesodermal lineage derivatives, in particular epithelial islands, spindle cells, and mature adipose tissue without preexistent thymic tissue or evidence of thymic differentiation. Twenty-three branchiomas were evaluated morphologically. Eighteen cases with sufficient tissue were assessed by immunohistochemistry, next-generation sequencing (NGS) using the Illumina Oncology TS500 panel, and fluorescence in situ hybridization (FISH) using an RB1 dual-color probe. All cases showed a biphasic morphology of epithelial and spindle cells with intermingled fatty tissue. Carcinoma arising in branchioma was detected in three cases. The neoplastic cells showed strong AE1/3 immunolabeling (100%), while the spindle cells expressed CD34, p63, and SMA (100%); AR was detected in 40-100% of nuclei (mean, 47%) in 14 cases. Rb1 showed nuclear loss in ≥ 95% of neoplastic cells in 16 cases (89%), while two cases revealed retained expression in 10-20% of tumor cell nuclei. NGS revealed a variable spectrum of likely pathogenic variants (n = 5) or variants of unknown clinical significance (n = 6). Loss of Rb1 was detected by FISH in two cases. Recent developments support branchioma as a true neoplasm, most likely derived from the rudimental embryological structures of endoderm and mesoderm. Frequent Rb1 loss by immunohistochemistry and heterozygous deletion by FISH is a real pitfall and potential confusion with other Rb1-deficient head and neck neoplasms (i.e., spindle cell lipoma), especially in small biopsy specimens.

Lynch syndrome-associated upper tract urothelial carcinoma frequently occurs in patients older than 60 years: an opportunity to revisit urology clinical guidelines.

Upper tract urothelial carcinoma (UTUC) is the third most common malignancy associated with Lynch syndrome (LS). The current European urology guidelines recommend screening for LS in patients with UTUC up to the age of 60 years. In this study, we examined a cohort of patients with UTUC for potential association with LS in order to establish the sensitivity of current guidelines in detecting LS. A total of 180 patients with confirmed diagnosis of UTUC were enrolled in the study during a 12-year period (2010-2022). Loss of DNA-mismatch repair proteins (MMRp) expression was identified in 15/180 patients (8.3%). Germline analysis was eventually performed in 8 patients confirming LS in 5 patients (2.8%), including 4 germline mutations in MSH6 and 1 germline mutation in MSH2. LS-related UTUC included 3 females and 2 males, with a mean age of 66.2 years (median 71 years, range 46-75 years). Four of five LS patients (all with MSH6 mutation) were older than 65 years (mean age 71.3, median 72 years). Our findings indicate that LS-associated UTUCs can occur in patients with LS older than 60 years. In contrast to previous studies which used mainly highly pre-selected populations with already diagnosed LS, the most frequent mutation in our cohort involved MSH6 gene. All MSH6 mutation carriers were > 65 years, and UTUC was the first LS manifestation in 2/4 patients. Using current screening guidelines, a significant proportion of patients with LS-associated UTUC may be missed. We suggest universal immunohistochemical MMRp screening for all UTUCs, regardless of age and clinical history.

Poorly differentiated extra-axial extraskeletal chordoma diagnosed by methylation profiling: case report and analysis of brachyury expression in SWI/SNF-deficient tumors.

Chordoma is a rare malignant tumor with notochordal differentiation, usually affecting the axial skeleton of young patients. We report a case of a high-grade epithelioid tumor involving the synovium and soft tissues of the knee in a 74-year-old male patient. The preliminary biopsy was inconclusive, but a diagnosis of metastatic clear-cell carcinoma of unknown origin was suggested. However, imaging studies did not reveal any primary lesions. The resection specimen consisted of nests and sheets of oval to polygonal cells with discernible cell borders, clear or lightly amphophilic cytoplasm, and round to oval nuclei with occasional well-visible eosinophilic nucleoli. Rare atypical mitoses, necrotic areas, and bizarre nuclei were noted. The biopsy and resection specimens underwent a wide molecular genetic analysis which included methylation profiling. The DKFZ sarcoma classifier assigned the methylation class chordoma (dedifferentiated) with a calibrated score of 0.96, and additionally, a loss of SMARCB1 locus was noted in the copy number variation plot. To verify these findings, T-brachyury and SMARCB1 immunostaining was performed afterward, showing diffuse nuclear positivity and complete loss in the tumor cells, respectively. To assess the prevalence of T-brachyury immunopositivity among SWI/SNF-deficient tumors and to evaluate its specificity for poorly differentiated chordoma, we analyzed a series of 23 SMARCB1- or SMARCA4-deficient tumors, all of which were negative. After incorporating all the available data, including the absence of any morphological features of conventional chordoma, the case was diagnosed as poorly differentiated chordoma. As illustrated herein, the utilization of methylation profiling in the diagnostic process of some carefully selected unclassifiable soft tissue neoplasms may lead to an increased detection rate of such extremely rare soft tissue tumors and enable their better characterization.

TFG::MET-rearranged soft tissue tumor: A rare infantile neoplasm with a distinct low-grade triphasic morphology.

This article presents 2 cases of TFG::MET-rearranged mesenchymal tumor, an extremely rare molecular subset among an emerging group of mesenchymal neoplasms with kinase gene (NTRK, BRAF, RET and others) alterations. Both tumors were congenital, occurred in female patients and presented as huge masses on the trunk and thigh, measuring 18 and 20 cm in the largest dimension. Both cases showed identical areas with a distinctive triphasic morphology resembling fibrous hamartoma of infancy (FHI), consisting of haphazardly arranged ovoid to spindled cells traversed by variably cellular and hyalinized fascicles admixed with (most likely non-neoplastic) adipose tissue. In other areas, a high-grade infantile fibrosarcoma/malignant peripheral nerve sheath tumor-like (IFS/MPNST-like) morphology was present in both cases. While the first case co-expressed CD34 and S100 protein, the other case did not. When combined with the three previously reported MET-rearranged cases (of which two harbored TFG::MET fusion), 3/5 and 3/4 of MET-rearranged and TFG::MET fusion-associated tumors, respectively exhibited similar triphasic FHI-like low-grade morphology. This points toward the existence of a relatively distinct morphological subset among kinase-fusion-associated tumors which seems to be strongly associated with MET fusions. It seems some of these low-grade cases may transform into a high-grade variant with IFS/MPNST-like morphology as has been observed in other tumors with kinase gene fusions. While most cases seem to follow an indolent clinical course, the recognition of these tumors is clinically relevant as MET tyrosine kinase inhibitors might represent an effective treatment option for clinically aggressive or unresectable cases.

Extraskeletal myxoid chondrosarcoma: A study of 17 cases focusing on the diagnostic utility of INSM1 expression and presenting rare morphological variants associated with non-EWSR1::NR4A3 fusions.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain lineage. Insulinoma-associated protein 1 (INSM1) has recently been described as a highly specific and sensitive immunohistochemical marker for EMC. The goal of this study was to evaluate the diagnostic significance of INSM1 immunohistochemistry in EMC. Furthermore, correlations between molecular and morphological findings were performed. Sixteen of 17 EMC cases were stained with the INSM1 antibody. Tumors with at least 5% INSM1-positive cells and any staining intensity were considered positive. Molecular testing was successfully performed in 12/17 cases. The immunohistochemical analysis detected 13 INSM1-positive (81%) and 3 INSM1-negative tumors (19%). The extent of the staining was classified as 1+ in 7 cases (44%), 2+ in 2 cases (13%), 3+ in 2 cases (13%) and 4+ in 2 cases (13%). Intensity of immunostaining was weak in 5 cases (31%), moderate in 2 cases (13%) and strong in 6 cases (38%). Molecular assays revealed 8 EWSR1::NR4A3 positive tumors (67%), 2 TAF15::NR4A3 positive tumors (17%), 1 TCF12::NR4A3 positive tumor (8%) and 1 NR4A3 positive tumor (8%) in which no other gene alteration was identified. Two of them, namely TCF12 positive and one TAF15 positive tumors, were highly cellular and partially associated with pseudopapillary architecture. Our study found that moderate/strong expression of INSM1 in more than 25% of tumor cells was present in only 31% of cases. Thus, the diagnostic utility of INSM1 is rather low. Two morphologically unique cases of non-EWSR1 rearranged EMC with an extremely rare pseudopapillary growth pattern are also reported.

Epithelioid fibrous histiocytoma: three diagnostically challenging cases with novel ALK gene fusions, unusual storiform growth pattern, and a prominent spindled morphology.

Epithelioid fibrous histiocytoma (EFH) is a distinctive cutaneous neoplasm with a relatively variable morphological appearance. Recently, it has been shown that this tumor is molecularly characterized by ALK gene fusions. We report three EFHs with unusual histological presentation represented by a prominent/predominant spindle cell proliferation arranged in a variably storiform/whirling architectural pattern with or without stromal sclerosis. One of the cases closely resembled cellular fibrous histiocytoma. All three cases were immunohistochemically ALK-positive and were analyzed for ALK gene rearrangements using a next-generation sequencing-based assay (FusionPlex Sarcoma Kit, ArcherDx). Three novel fusions, namely AP3D1::ALK, COL1A::ALK, and LRRFIP2::ALK, were detected and further confirmed by FISH in all 3 cases and RT-PCR in 1 case. All patients were elderly (62-63 years) and presented with a solitary polypoid lesion on the extremities. The awareness of these morphological variants is important since it entertains a wide and slightly different differential diagnosis than conventional EFH. We also presented evidence that a clear separation of EFH from BFH in all cases may not be as straightforward as previously thought. The consistent ALK immunoexpression and the continually expanding scale of ALK gene rearrangements provide a useful tool to distinguish EFH from its histologic mimics.

TIMP3::ALK fusions characterize a distinctive myxoid fibroblastic tumor of the vocal cords: a report of 7 cases.

We report 7 cases of an indolent, variably myxoid tumor of the vocal cords, characterized by overt cellular atypia with large cells containing intranuclear and intracytoplasmic vacuoles, delicate curvilinear vessels, and sparse inflammatory infiltrate. Six patients were male, aged 15 to 65 years, and 1 patient was a 54-year-old female. All tumors were located in the superficial portion of the vocal cord. One patient suffered a recurrence that was completely resected; all patients with available follow-up data currently have no evidence of disease. The tumors contained alternating areas with myxoid stroma and more compacted regions with tumor cells organized in short fascicles, interwoven with delicate curvilinear vasculature. Overt cellular atypia with large cells containing intranuclear and intracytoplasmic vacuoles or resembling ganglion cells was present in all cases but mitoses and necrosis were absent. ALK immunostaining was positive in all cases, while most tumors were negative for smooth muscle actin. Targeted RNA-sequencing revealed an identical TIMP3::ALK fusion with exon 1 of TIMP3 gene being fused with exon 12 of ALK gene in all analyzable cases. For various reasons discussed, it remains unclear whether this tumor represents a mere subtype of IMT or a separate entity. Nevertheless, it is a morphologically distinct and diagnostically challenging lesion that needs to be recognized by surgical pathologists in order to prevent overdiagnosis in this clinically very delicate area.

RNA-sequencing of myxoinflammatory fibroblastic sarcomas reveals a novel SND1::BRAF fusion and 3 different molecular aberrations with the potential to upregulate the TEAD1 gene including SEC23IP::VGLL3 and TEAD1::MRTFB gene fusions.

Myxoinflammatory fibroblastic sarcoma (MIFS) has been shown to harbor various recurrent molecular aberrations; most of which, however, seem to be present in only a minority of cases. In order to better characterize the molecular underpinnings of MIFS, fourteen cases were analyzed by targeted RNA-sequencing (RNA-seq), VGLL3 enumeration FISH probe, and BRAF break-apart and enumeration probes. Neither t(1;10)(p22;q24) nor BRAF gene amplifications were found. However, VGLL3 gene amplification was detected in 5 cases by FISH which corresponded with an increase in VGLL3 expression detected by RNA-seq. In 1 of these cases, RNA-seq additionally revealed a novel SND1::BRAF fusion. Two of the 9 cases lacking VGLL3 amplification harbored either a SEC23IP::VGLL3 or a TEAD1::MRTFB rearrangement by RNA-seq, both confirmed by RT-PCR and Sanger sequencing. The detected molecular aberrations have a potential to either activate the expression of genes regulated by the transcription factors of the TEAD family, which are involved in tumor initiation and progression, or switch on the MEK/ERK signaling cascade, which plays an important role in cell cycle progression. Our results broaden the molecular genetic spectrum of MIFS and point toward the importance of the VGLL3-TEAD interaction, as well as the deregulation of the MEK/ERK pathway in the pathogenesis of MIFS, and may represent a potential target for therapy of recurrent or advanced disease.

Misleading Morphologic and Phenotypic Features (Transdifferentiation) in Solitary Fibrous Tumor of the Head and Neck: Report of 3 Cases and Review of the Literature.

Solitary fibrous tumor (SFT) is a rare fibroblastic neoplasm with potentially malignant behavior that may develop in any anatomic site and may involve the head and neck (H&N) region as well. Although typical SFT has a relatively characteristic morphology, its morphologic spectrum is extraordinarily broad and also includes rare cases with dedifferentiation or transdifferentiation which result in aberrant morphologic and/or immunohistochemical features. However, since virtually all cases are molecularly characterized by NAB2::STAT6 gene fusions, molecular genetic methods or STAT6 immunohistochemistry can be effectively used in confirming the diagnosis. Herein, we report 3 diagnostically challenging H&N SFT cases with an unusual morphology and/or phenotypes closely mimicking other well-known H&N entities. The tumors originated in the oral minor salivary glands, the base of the tongue, and sinonasal tract and closely resembled hyalinizing clear cell carcinoma of the salivary gland, adenocarcinoma not otherwise specified and biphenotypic sinonasal sarcoma, respectively. All cases were positive for cytokeratins, variably expressed S100 protein, showed diffuse nuclear STAT6 positivity, and harbored NAB2::STAT6 gene fusions.

Clear cell renal cell carcinoma with prominent microvascular hyperplasia: Morphologic, immunohistochemical and molecular-genetic analysis of 7 sporadic cases.

Clear cell renal cell carcinoma (CCRCC) is well known for intratumor heterogeneity. An accurate mapping of the tumor is crucial for assessing prognosis, and perhaps this can be linked to potential success/failure of targeted therapies. We assembled a cohort of 7 CCRCCs with prominent vasculature and microvascular hyperplasia (ccRCCPV), resembling those seen in high grade gliomas. A control group of classic CCRCC with no variant morphologies was also included. Both groups were analyzed for clinicopathologic, morphologic, immunohistochemical, and molecular genetic features. No statistically significant differences in mRNA expression of studied genes between the two groups were found. Using NGS panel Trusight Oncology 500 (TSO500), only one clinically significant gene mutation, VHL c.263G > A, p. (Trp88Ter), was found. TMB (Tumor Mutation Burden) and MSI (MicroSatellite Instability) were low, and no copy number variations (CNVs) were detected in the study cohort. Prominent microvascular hyperplasia in CCRCC is a rare phenomenon. From molecular genetic point of view, these tumors do not appear to be different from classic CCRCC. Prognostically, they also demonstrated similar clinical behaviors.

Epithelioid Soft Tissue Neoplasm of the Soft Palate with a PTCH1-GLI1 Fusion: A Case Report and Review of the Literature.

GLI1 fusions involving ACTB, MALAT1, PTCH1 and FOXO4 genes have been reported in a subset of malignant mesenchymal tumors with a characteristic nested epithelioid morphology and frequent S100 positivity. Typically, these multilobulated tumors consist of uniform epithelioid cells with bland nuclei and are organized into distinct nests and cords with conspicuously rich vasculature. We herein expand earlier findings by reporting a case of a 34-year-old female with an epithelioid mesenchymal tumor of the palate. The neoplastic cells stained positive for S100 protein and D2-40, whereas multiple other markers were negative. Genetic alterations were investigated by targeted RNA sequencing, and a PTCH1-GLI1 fusion was detected. Epithelioid mesenchymal tumors harboring a PTCH1-GLI1 fusion are vanishingly rare with only three cases reported so far. Due to the unique location in the mucosa of the soft palate adjacent to minor salivary glands, multilobulated growth, nested epithelioid morphology, focal clearing of the cytoplasm, and immunopositivity for S100 protein and D2-40, the differential diagnoses include primary salivary gland epithelial tumors, in particular myoepithelioma and myoepithelial carcinoma. Another differential diagnostic possibility is the ectomesenchymal chondromyxoid tumor. Useful diagnostic clues for tumors with a GLI1 rearrangement include a rich vascular network between the nests of neoplastic cells, tumor tissue bulging into vascular spaces, and absence of SOX10, GFAP and cytokeratin immunopositivity. Identifying areas with features of GLI1-rearranged tumors should trigger subsequent molecular confirmation. This is important for appropriate treatment measures as PTCH1-GLI1 positive mesenchymal epithelioid neoplasms have a propensity for locoregional lymph node and distant lung metastases.

Various fatal complications of urolithiasis.

This article describes two cases of fatal complications of urolithiasis. The first case discusses the development of fatal urosepsis in a patient with a known mental health issue. The patient had self-introduced a thermometer into her bladder, which led to stone formation around the thermometer and consequently to fatal urosepsis. The second case deals with a motor vehicle accident in which a passenger with pre-existing hydronephrosis suffered a kidney injury leading to fatal bleeding into the surrounding adipose tissue. To provide a wider context, the paper also discusses the fatal complications and origin of urolithiasis.

Renal cell carcinomas with tubulopapillary architecture and oncocytic cells: Molecular analysis of 39 difficult tumors to classify.

So-called oncocytic papillary renal cell carcinoma (OPRCC) is a poorly defined variant of papillary renal cell carcinoma. Since its first description, several studies were published with conflicting results, and thus precise definition is lacking. A cohort of 39 PRCCs composed of oncocytic cells were analyzed. Cases were divided into 3 groups based on copy number variation (CNV) pattern. The first group consisted of 23 cases with CNV equal to renal oncocytoma. The second group consisted of 7 cases with polysomy of chromosomes 7 and 17 and the last group of 9 cases included those with variable CNV. Epidemiologic, morphologic and immunohistochemical features varied among the groups. There were not any particular histomorphologic features correlating with any of the genetic subgroups. Further, a combination of morphologic, immunohistochemical, and molecular-genetic features did not allow to precisely predict biologic behavior. Owing to variable CNV pattern in OPRCC, strict adherence to morphology and immunohistochemical profile is recommended, particularly in limited samples (i.e., core biopsy). Applying CNV pattern as a part of a diagnostic algorithm can be potentially misleading. OPRCC is a highly variable group of tumors, which might be misdiagnosed as renal oncocytoma. Using the term OPRCC as a distinct diagnostic entity is, thanks to its high heterogeneity, questionable.

EWSR1-PATZ1-rearranged sarcoma: a report of nine cases of spindle and round cell neoplasms with predilection for thoracoabdominal soft tissues and frequent expression of neural and skeletal muscle markers.

The knowledge of clinical features and, particularly, histopathological spectrum of EWSR1-PATZ1-rearranged spindle and round cell sarcomas (EPS) remains limited. For this reason, we report the largest clinicopathological study of EPS to date. Nine cases were collected, consisting of four males and five females ranging in age from 10 to 81 years (average: 49 years). Five tumors occurred in abdominal wall soft tissues, three in the thorax, and one in the back of the neck. Tumor sizes ranged from 2.5 to 18 cm (average 6.6 cm). Five patients had follow-up with an average of 38 months (range: 18-60 months). Two patients had no recurrence or metastasis 19 months after diagnosis. Four patients developed multifocal pleural or pulmonary metastasis and were treated variably by surgery, radiotherapy, and chemotherapy. The latter seemed to have little to no clinical benefit. One of the four patients was free of disease 60 months after diagnosis, two patients were alive with disease at 18 and 60 months, respectively. Morphologically, low, intermediate, and high-grade sarcomas composed of a variable mixture of spindled, ovoid, epithelioid, and round cells were seen. The architectural and stromal features also varied, resulting in a broad morphologic spectrum. Immunohistochemically, the following markers were most consistently expressed: S100-protein (7/9 cases), GFAP (7/8), MyoD1 (8/9), Pax-7 (4/5), desmin (7/9), and AE1/3 (4/9). By next-generation sequencing, all cases revealed EWSR1-PATZ1 gene fusion. In addition, 3/6 cases tested harbored CDKN2A deletion, while CDKN2B deletion and TP53 mutation were detected in one case each. Our findings confirm that EPS is a clinicopathologic entity, albeit with a broad morphologic spectrum. The uneventful outcome in some of our cases indicates that a subset of EPS might follow a more indolent clinical course than previously appreciated. Additional studies are needed to validate whether any morphological and/or molecular attributes have a prognostic impact.

Primary Cutaneous Desmoplastic Melanoma With Collagen Rosettes and Pseudoglandular Features.

ABSTRACT: Primary cutaneous desmoplastic melanoma (DM) is a group of rare melanocytic tumors arising on severely sun-damaged skin, histologically characterized by the proliferation of spindled melanocytes in a prominent desmoplastic stroma, with a range of morphological presentations. In this article, we report a unique case of primary cutaneous DM composed of a nodular proliferation of highly pleomorphic spindled and epithelioid cells, pseudoglandular structures, clear cell change, and unusual collagen rosettes. Immunohistochemical analysis showed a strong and diffuse positivity for S-100 protein, SOX-10, nestin, p75 (nerve growth factor receptor), WT1, and p53. Molecular analysis detected a mutation in the NF1 gene [c.4084C > T, p.(Arg1362Ter)], 2 different pathogenic mutations in TP53 [c.742C > T, p.(Arg248Trp), AF:12%, COSM1640831 and c.528C > G, p.(Cys176Trp), AF:12%, COSM11114], and a mutation in GNAS [c.601C > T, p.(Arg201Cys), AF: 9%, COSM123397]. To the best of our knowledge, this is the first case reporting collagen rosettes and pseudoglandular features in primary cutaneous DM.

A Clinicopathological Study of 29 Spitzoid Melanocytic Lesions With ALK Fusions, Including Novel Fusion Variants, Accompanied by Fluorescence In Situ Hybridization Analysis for Chromosomal Copy Number Changes, and Both TERT Promoter and Next-Generation Sequencing Mutation Analysis.

ALK-fused spitzoid neoplasms represent a distinctive group of melanocytic lesions. To date, few studies addressed genetic and chromosomal alterations in these lesions beyond the ALK rearrangements. Our objective was to study genetic alterations, including ALK gene fusions, telomerase reverse transcriptase promoter (TERT-p) mutations, chromosomal copy number changes, and mutations in other genes. We investigated 29 cases of Spitz lesions (11 Spitz nevi and 18 atypical Spitz tumors), all of which were ALK immunopositive. There were 16 female and 13 male patients, with age ranging from 1 to 43 years (mean, 18.4 years). The most common location was the lower extremity. Microscopically, all neoplasms were polypoid or dome shaped with a plexiform, predominantly dermally located proliferation of fusiform to spindled melanocytes with mild to moderate pleomorphism. The break-apart test for ALK was positive in 17 of 19 studied cases. ALK fusions were detected in 23 of 26 analyzable cases by Archer FusionPlex Solid Tumor Kit. In addition to the previously described rearrangements, 3 novel fusions, namely, KANK1-ALK, MYO5A-ALK, and EEF2-ALK, were found. Fluorescence in situ hybridization for copy number changes yielded one case with the loss of RREB1 among 21 studied cases. TERT-p hotspot mutation was found in 1 of 23 lesions. The mutation analysis of 271 cancer-related genes using Human Comprehensive Cancer Panel was performed in 4 cases and identified in each case mutations in several genes with unknown significance, except for a pathogenic variant in the BLM gene. Our study confirms that most ALK fusion spitzoid neoplasms can be classified as atypical Spitz tumors, which occurs in young patients with acral predilection and extends the spectrum of ALK fusions in spitzoid lesions, including 3 hitherto unreported fusions. TERT-p mutations and chromosomal copy number changes involving 6p25 (RRB1), 11q13 (CCND1), 6p23 (MYB), 9p21 (CDKN2A), and 8q24 (MYC) are rare in these lesions. The significance of mutation in other genes remains unknown.

Targeted next generation sequencing of MLH1-deficient, MLH1 promoter hypermethylated, and BRAF/RAS-wild-type colorectal adenocarcinomas is effective in detecting tumors with actionable oncogenic gene fusions.

Oncogenic gene fusions represent attractive targets for therapy of cancer. However, the frequency of actionable genomic rearrangements in colorectal cancer (CRC) is very low, and universal screening for these alterations seems to be impractical and costly. To address this problem, several large scale studies retrospectivelly showed that CRC with gene fusions are highly enriched in groups of tumors defined by MLH1 DNA mismatch repair protein deficiency (MLH1d), and hypermethylation of MLH1 promoter (MLH1ph), and/or the presence of microsatellite instability, and BRAF/KRAS wild-type status (BRAFwt/KRASwt). In this study, we used targeted next generation sequencing (NGS) to explore the occurence of potentially therapeutically targetable gene fusions in an unselected series of BRAFwt/KRASwt CRC cases that displayed MLH1d/MLH1ph. From the initially identified group of 173 MLH1d CRC cases, 141 cases (81.5%) displayed MLH1ph. BRAFwt/RASwt genotype was confirmed in 23 of 141 (~16%) of MLH1d/MLH1ph cases. Targeted NGS of these 23 cases identified oncogenic gene fusions in nine patients (39.1%; CI95: 20.5%-61.2%). Detected fusions involved NTRK (four cases), ALK (two cases), and BRAF genes (three cases). As a secondary outcome of NGS testing, we identified PIK3K-AKT-mTOR pathway alterations in two CRC cases, which displayed PIK3CA mutation. Altogether, 11 of 23 (~48%) MLH1d/MLH1ph/BRAFwt/RASwt tumors showed genetic alterations that could induce resistance to anti-EGFR therapy. Our study confirms that targeted NGS of MLH1d/MLH1ph and BRAFwt/RASwt CRCs could be a cost-effective strategy in detecting patients with potentially druggable oncogenic kinase fusions.

Papillary thyroid carcinoma with prominent myofibroblastic stromal component: clinicopathologic, immunohistochemical and next-generation sequencing study of seven cases.

Papillary thyroid carcinoma with desmoid-type fibromatosis or nodular fasciitis-like stroma is an extremely unusual and poorly understood subtype of papillary thyroid cancer. Although prior studies have demonstrated alterations in the Wnt/ß-catenin signaling pathway in some of these tumors, controversy still exists regarding the nature of the stromal spindle component. We have studied seven cases of papillary thyroid carcinoma with prominent myofibroblastic stroma, including six men and one woman aged 20-65 years (mean age = 44). All cases displayed areas consistent with conventional papillary thyroid carcinoma embedded in abundant myofibroblastic-like stroma. The myofibroblastic stroma in six cases resembled desmoid-type fibromatosis and in one case it more closely resembled nodular fasciitis. By immunohistochemical staining, the stromal spindle component showed positivity for SMA and low MIB1 proliferation index in all cases, and there was at least patchy strong nuclear positivity for beta-catenin in six/seven cases. Stains for cytokeratin AE1/AE3 and PAX8 were positive in the epithelial elements but negative in the stromal component. Next-generation sequencing was performed on six of seven cases. CTNNB1 gene mutations were identified in six/seven cases. The epithelial component showed BRAF mutations in two cases and an NRAS mutation in one case. The case with fasciitis-like stroma was negative for beta-catenin by sequencing and immunostaining as well as negative for USP6 gene rearrangement. Our findings indicate that papillary thyroid carcinoma with prominent myofibroblastic stroma may represent more than one category of lesions.