Uterine mesenchymal tumours: recent advances

Almost all uterine mesenchymal tumours have been historically classified as either smooth muscle or endometrial stromal neoplasms. Recent application of molecular techniques has identified numerous lesions with distinctive genetic abnormalities and clinicopathological characteristics. Newly discovered uterine sarcoma subtypes include high-grade endometrial stromal sarcomas with BCOR genetic abnormalities, fibrosarcoma-like uterine sarcomas with NTRK rearrangements and COL1A–PDGFRB fusions, as well as undifferentiated uterine sarcomas with SMARCA4 mutations. Novel PLAG1 and PGR fusions have been identified in subsets of myxoid and epithelioid leiomyosarcomas. Some uterine tumours resembling ovarian sex-cord tumour harbour GREB1 and ESR1 rearrangements. Histological and immunophenotypical features as well as underlying genetic abnormalities defining these lesions are discussed.     

Emerging soft tissue tumors with kinase fusions: An overview of the recent literature with an emphasis on diagnostic criteria

A recent breakthrough in the classification of soft tissue tumors (STT) has been a significant expansion in the number of neoplasms associated with NTRK and other kinase related fusions. This is important not only for diagnostic purposes, but also because it opens new avenues for targeted therapy. Indeed, recent clincal trials have shown significant benefit across multiple tumor‐types, prompting approval of NTRK inhibitors for clinical use in the setting of advanced/metastatic NTRK‐rearranged neoplasms. Despite these therapeutic oportunities, diagnostic challenges have transpired in recognizing these emerging new histologic subtypes of kinase fusions positive‐STT prospectively. This, in part, is attributable to their wide morphologic spectrum, variable risk of malignancy, and non‐specific immunoprofile. As such, recommendations for pathologic criteria and immunohistochemical testing are needed to improve classification and streamline the small subset of potential candidates for further molecular validation. This overview summarizes the key histologic features of various STT associated with NTRK and other kinase fusions, which appear to share a similar morphologic spectrum. Immunohistochemically, many of these tumors, regardless of the kinase fusion type, notably show co‐expression of S100 and CD34; issues related to the utility of pan‐NTRK and NTRK1 immunostaining are therefore summarized. Finally, I discuss the role of confirmatory molecular testing and how, in some instances, this may also be of prognostic value. This review is intended as a critical summary of the current literature to emphasize pathologic criteria for improving recognition of this emerging and complex group of kinase fusion associated STT.     

NTRK testing: First results of the QuiP‐EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA‐based NGS assays

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next‐generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA‐/DNA‐based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH‐based and 18 in the NGS‐based round robin test, the latter additionally subdivided into low‐input and high‐input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin‐fixed and paraffin‐embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS‐based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH‐ and tNGS‐based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA‐based tNGS panels.     

NTRK3 kinase fusions in Spitz tumours

Oncogenic fusions in TRK family receptor tyrosine kinases have been identified in several cancers and can serve as therapeutic targets. We identified ETV6–NTRK3, MYO5A–NTRK3 and MYH9–NTRK3 fusions in Spitz tumours, and demonstrated that NTRK3 fusions constitutively activate the mitogen‐activated protein kinase, phosphoinositide 3‐kinase and phospholipase Cγ1 pathways in melanocytes. This signalling was inhibited by DS‐6051a, a small‐molecule inhibitor of NTRK1/2/3 and ROS1. NTRK3 fusions expand the range of oncogenic kinase fusions in melanocytic neoplasms and offer targets for a small subset of melanomas for which no targeted options currently exist. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Paediatric and adult soft tissue sarcomas with NTRK1 gene fusions: a subset of spindle cell sarcomas unified by a prominent myopericytic/haemangiopericytic pattern

Neoplasms with a myopericytomatous pattern represent a morphological spectrum of lesions encompassing myopericytoma of the skin and soft tissue, angioleiomyoma, myofibromatosis/infantile haemangiopericytoma and putative neoplasms reported as malignant myopericytoma. Lack of reproducible phenotypic and genetic features of malignant myopericytic neoplasms have prevented the establishment of myopericytic sarcoma as an acceptable diagnostic category. Following detection of a LMNA–NTRK1 gene fusion in an index case of paediatric haemangiopericytoma‐like sarcoma by combined whole‐genome and RNA sequencing, we identified three additional sarcomas harbouring NTRK1 gene fusions, termed 'spindle cell sarcoma, NOS with myo/haemangiopericytic growth pattern'. The patients were two children aged 11 months and 2 years and two adults aged 51 and 80 years. While the tumours of the adults were strikingly myopericytoma‐like, but with clear‐cut atypical features, the paediatric cases were more akin to infantile myofibromatosis/haemangiopericytoma. All cases contained numerous thick‐walled dysplastic‐like vessels with segmental or diffuse nodular myxohyaline myo‐intimal proliferations of smooth muscle actin‐positive cells, occasionally associated with thrombosis. Immunohistochemistry showed variable expression of smooth muscle actin and CD34, but other mesenchymal markers, including STAT6, were negative. This study showed a novel variant of myo/haemangiopericytic sarcoma with recurrent NTRK1 gene fusions. Given the recent introduction of a novel therapeutic approach targeting NTRK fusion‐positive neoplasms, recognition of this rare but likely under‐reported sarcoma variant is strongly encouraged. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

An addition to the evolving spectrum of lipofibromatosis and lipofibromatosis-like neural tumor: Molecular findings in an unusual phenotype aid in accurate classification

Lipofibromatosis (LPF) and lipofibromatosis-like neural tumor (LPF-NT) are histologically and prognostically similar neoplasms having differences in immunophenotype as well as molecular biology. In most cases, LPF-NT is driven by fusions in the NTRK gene, whereas LPF has been associated with fusions in a variety of receptor tyrosine kinases. The distinction between the driver fusion event holds clinical significance because of the profound clinical response to tropomyosin receptor kinase (Trk) inhibitors (larotrectinib) in the NTRK-driven tumors. Immunohistochemically, and consistent with its namesake, to-date all reported cases classified as LPF-NT have shown positivity for S100-protein staining. Consequently, as S100-protein staining is widely available, it represents a cost-effective screening tool for LPF-NT where the more specific studies such as the pan-Trk stain or fluorescence in situ hybridization for NTRK rearrangement are not available. Herein, we present a case of presumed LPF-NT harboring the recurrent NTRK1-LMNA fusion, but which was negative for S100-protein immunostaining and was previously classified as classical LPF. This case reveals a potential pitfall in distinguishing these rare subcutaneous tumors by S100-protein staining and highlights the challenges in reconciling the rapid and novel discoveries made in the field of diagnostic pathology.     

Soft tissue tumors characterized by a wide spectrum of kinase fusions share a lipofibromatosis‐like neural tumor pattern

Gene fusions resulting in oncogenic activation of various receptor tyrosine kinases, including NTRK1‐3, ALK, and RET, have been increasingly recognized in soft tissue tumors (STTs), displaying a wide morphologic spectrum and therefore diagnostically challenging. A subset of STT with NTRK1 rearrangements were recently defined as lipofibromatosis‐like neural tumors (LPFNTs), being characterized by mildly atypical spindle cells with a highly infiltrative growth in the subcutis and expression of S100 and CD34 immunostains. Other emerging morphologic phenotypes associated with kinase fusions include infantile/adult fibrosarcoma and malignant peripheral nerve sheath tumor‐like patterns. In this study, a large cohort of 73 STT positive for various kinase fusions, including 44 previously published cases, was investigated for the presence of an LPFNT phenotype, to better define the incidence of this distinctive morphologic pattern and its relationship with various gene fusions. Surprisingly, half (36/73) of STT with kinase fusions showed at least a focal LPFNT component defined as >10%. Most of the tumors occurred in the subcutaneous tissues of the extremities (n = 25) and trunk (n = 9) of children or young adults (<30 years old) of both genders. Two‐thirds (24/36) of these cases showed hybrid morphologies with alternating LPFNT and solid areas of monomorphic spindle to ovoid tumor cells with fascicular or haphazard arrangement, while one‐third (12/36) had pure LPFNT morphology. Other common histologic findings included lymphocytic infiltrates, staghorn‐like vessels, and perivascular or stromal hyalinization, especially in hybrid cases. Mitotic activity was generally low (<4/10 high power fields in 81% cases), being increased only in a minority of cases. Immunoreactivity for CD34 (92% in hybrid cases, 89% in pure cases) and S100 (89% in hybrid cases, 64% in pure cases) were commonly present. The gene rearrangements most commonly involved NTRK1 (75%), followed by RET (8%) and less commonly NTRK2, NTRK3, ROS1, ALK, and MET.     

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.     

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.     

NTRK-Fusions – A new kid on the block

The neurotrophic tyrosine receptor kinases (NTRK) play an important role in the development and function of the nervous system. Fusions involving NTRK and a wide range of genes that act as fusion partners are oncogenic and activate well-known signal transduction pathways like the MAPK-ERK pathway. NTRK fusions occur in many very different tumor entities in children and youth as well as in adults. There are a few tumors like secretory breast cancer and congenital fibrosarcoma for which NTRK fusions are pathognomonic. At the same time there a large number of tumors in which NTRK fusions occur in very rare frequency (e.g., lung cancer). TRK inhibitors offer now the possibility to use NTRK fusion as antitumorigenic targets in a tumor agnostic fashion regardless of the basic histology. It is the task of modern pathology to identify such targetable fusions in a highly effective and efficient manner.     

The genetics of vascular tumours: an update

Recent molecular advances have shed significant light on the classification of vascular tumours. Except for haemangiomas, vascular lesions remain difficult to diagnose, owing to their rarity and overlapping clinical, radiographic and histological features across malignancies. In particular, challenges still remain in the differential diagnosis of epithelioid vascular tumours, including epithelioid haemangioma and epithelioid haemangioendothelioma at the benign/low-grade end of the spectrum, and epithelioid angiosarcoma at the high-grade end. Historically, the classification of vascular tumours has been heavily dependent on the clinical setting and histological features, as traditional immunohistochemical markers across the group have often been non-discriminatory. The increased application of next-generation sequencing in clinical practice, in particular targeted RNA sequencing (such as Archer, Illumina), has led to numerous novel discoveries, mainly recurrent gene fusions (e.g. those involving FOS, FOSB, YAP1, and WWTR1), which have resulted in refined tumour classification and improved diagnostic reproducibility for vascular tumours. However, other molecular alterations besides fusions have been discovered in vascular tumours, including somatic mutations (e.g. involving GNA family and IDH genes) in a variety of haemangiomas, as well as copy number alterations in high-grade angiosarcomas (e.g. MYC amplifications). Moreover, the translation of these novel molecular abnormalities into diagnostic ancillary markers, either fluorescence in-situ hybridisation probes or surrogate immunohistochemical markers (FOSB, CAMTA1, YAP1, and MYC), has been remarkable. This review will focus on the latest molecular discoveries covering both benign and malignant vascular tumours, and will provide practical diagnostic algorithms, highlighting frequently encountered pitfalls and challenges in the diagnosis of vascular lesions.     

Dramatic response to entrectinib in a patient with malignant peripheral nerve sheath tumor harboring novel SNRNP70-NTRK3 fusion gene

Neurotropic tropomyosin receptor kinase (NTRK) gene rearrangements have been reported in limited cases of sarcomas; however, to date, there has been only one report of such rearrangements in malignant peripheral nerve sheath tumors (MPNSTs). Herein, we describe a 51-year-old male patient with a buttock tumor arising from the sciatic nerve, which was diagnosed as MPNST with positive S-100 staining, negative SOX10 staining, and loss of trimethylation at lysine 27 of histone H3 (H3K27me3) confirmed by immunohistochemistry. Soon after the resection of the primary tumor, the patient was found to have pulmonary and lymph node metastases. Chemotherapy with eribulin and trabectedin showed limited effects. However, the patient responded rapidly to pazopanib, but severe side effects caused discontinuation of the treatment. RNA panel testing revealed a novel fusion gene between Small Nuclear Ribonucleoprotein U1 Subunit 70 (SNRNP70) gene and NTRK3 gene. Furthermore, loss of NF1, SUZ12, and CDKN2A genes was confirmed by DNA panel testing, which is compatible with a histological diagnosis of MPNST. SNRNP70 possesses a coiled-coiled domain and seems to induce constitutive activation of NTRK3 through dimerization. In fact, immunohistochemistry revealed diffuse staining of pan-TRK within tumor cells. Treatment with entrectinib, which is an NTRK inhibitor, showed a quick and durable response for 10 months. Although NTRK rearrangements are very rare in MPNST, this case highlights the importance of genetic testing in MPNST, especially using an RNA panel for the detection of rare fusion genes.     

Functional characterization of NTRK1 mutations identified in melanoma

Cutaneous melanoma is the most aggressive form of skin cancer, with a complex and heterogeneous aetiology. Deregulation of the mitogen activated protein kinase cascade is common in melanoma, due to activating mutations in the BRAF and NRAS genes. Genetic studies and high‐throughput screening technologies have recently identified several somatic mutations affecting different receptor tyrosine kinase (RTK) genes. For the majority of these, however, the contribution to the complexity of melanoma biology has not been assessed. Among these, two novel missense somatic mutations (M379I and R577G) have recently been identified in the gene encoding the neurotrophic RTK NTRK1. The NTRK1 melanoma‐associated point mutations were introduced in a NTRK1 expression plasmid. Functional characterization of mutants was assessed after transient and stable transfection in HeLa and NIH3T3 cells, respectively. We showed that M379I and R577G NTRK1 receptors do not display the kinase as constitutively activated and are functionally indistinguishable from the wild‐type NTRK1 receptor. Our results indicate that a causative role for M379I and R577G NTRK1 mutations in melanoma development is highly unlikely. This supports the issue that, in parallel to systematic large scale cancer genome screening, functional studies are required to distinguish between mutations that play a causative role in tumor development and others that may only be passenger changes. © 2014 Wiley Periodicals, Inc.     

Gastrointestinal stromal tumours: from KIT to succinate dehydrogenase

The discovery of activating mutations in the tyrosine kinase receptor genes KIT and PDGFRA has led to the development of effective targeted therapies for gastrointestinal stromal tumours (GISTs). Specific genotypes, in part, predict the response to treatment with tyrosine kinase inhibitors. However, ~10% of GISTs lack such mutations (often referred to as ‘wild‐type’ GISTs). Recent insights into the biology of ‘wild‐type’ GISTs have resulted in clinically significant subclassification of this heterogeneous group of tumours, a large subset of which are now known to represent succinate dehydrogenase‐deficient GISTs. Recognition of this distinctive class of tumours has critical implications for prognosis, therapy, clinical follow‐up, and genetic counselling. Other uncommon genetic groups include neurofibromatosis type I‐associated and BRAF‐mutant GISTs. This review provides an update on the diagnosis and pathogenesis of these less common classes of GISTs, summarizes the clinical and pathological features associated with particular genotypes, and discusses mechanisms of resistance to targeted therapies.