Pan-TRK immunohistochemistry in gynaecological mesenchymal tumours: diagnostic implications and pitfalls.

AIMS: NTRK-rearranged sarcomas of the female genital tract mainly occur in the uterus (more commonly cervix than corpus) and are characterized by a "fibrosarcoma-like" morphology and NTRK gene rearrangements. These neoplasms may exhibit histological overlap with other entities and can present diagnostic difficulties without molecular confirmation. Pan-TRK immunohistochemistry was developed to identify tumours harbouring NTRK rearrangements. The aim of this study was to characterize pan-TRK immunohistochemical expression in a large cohort of gynaecological mesenchymal neoplasms and investigate the utility of pan-TRK immunohistochemistry to distinguish NTRK-rearranged sarcoma from its mimics. METHODS AND RESULTS: A total of 473 gynaecological mesenchymal tumours (461 without known NTRK fusions and 12 NTRK-rearranged sarcomas) were selected. Pan-TRK immunohistochemistry (EPR17341, Abcam) was performed on whole tissue sections and tissue microarrays. Molecular interrogation of pan-TRK positive tumours was performed by RNA sequencing or fluorescence in situ hybridization (FISH). Of the 12 NTRK-rearranged sarcomas, 11 (92%) exhibited diffuse (≥70%) cytoplasmic pan-TRK staining with moderate/marked intensity, while the other was negative. Eleven (2.4%) additional tumours also exhibited pan-TRK immunohistochemical expression: three low-grade endometrial stromal sarcomas, seven high-grade endometrial stromal sarcomas, and an undifferentiated uterine sarcoma. Molecular confirmation of the absence of NTRK rearrangements was possible in nine of these tumours. Of these nine neoplasms, seven exhibited focal/multifocal (<70%) pan-TRK cytoplasmic staining with weak/moderate intensity. CONCLUSION: Even though pan-TRK immunohistochemical expression is not entirely sensitive or specific for NTRK-rearranged sarcomas, these neoplasms tend to exhibit diffuse staining of moderate/strong intensity, unlike its mimics. Pan-TRK should be performed in monomorphic uterine (corpus and cervix) spindle cell neoplasms that are negative for smooth muscle markers and hormone receptors and positive for CD34 and/ or S100. Ultimately, the diagnosis requires molecular confirmation.

SMARCB1 (INI1) Deficient Tumours of the Uterine Cervix: Report of Two Cases, Including One Associated With an NTRK Fusion.

Pathogenic variants (mutations) and other molecular events involving subunits of the SWItch/Sucrose Non-Fermentable chromatin remodelling complex are common in a wide variety of malignancies. Many of these neoplasms are characterized by undifferentiated morphology. They arise at a variety of sites in the female genital tract but have rarely been reported in the uterine cervix. We report 2 primary cervical neoplasms arising in young women (ages 28 and 29 yr) exhibiting loss of nuclear immunoreactivity with SMARCB1 (INI1). In one case, which had a mixture of epithelioid and spindle cells, molecular studies revealed no SMARCB1 pathogenic variant, but showed a SPECCL1::NTRK 3 fusion, in keeping with an NTRK fusion sarcoma. The second case exhibited rhabdoid morphology and molecular testing confirmed a SMARCB1 pathogenic variant (c.425 T>G:p.(Leu142Ter) which, interpreted in conjunction with the morphology and immunohistochemistry, resulted in classification as a proximal-type epithelioid sarcoma. To our knowledge, this is the first reported cervical neoplasm exhibiting a SMARCB1 pathogenic variant and the first NTRK fusion sarcoma showing SMARCB1 protein loss. We discuss the diagnostic challenges and complexities of the molecular findings.

The KAT6B::KANSL1 Fusion Defines a New Uterine Sarcoma With Hybrid Endometrial Stromal Tumor and Smooth Muscle Tumor Features.

Neoplasms harboring a KAT6B/A::KANSL1 fusion were initially reported as benign (leiomyomas) and malignant (leiomyosarcomas, low-grade endometrial stromal sarcomas [LG-ESSs]) uterine neoplasms. However, they may represent an emerging entity characterized by clinical aggressiveness contrasting with a rather reassuring microscopic appearance. Here, we aimed to confirm that this neoplasm is a distinct clinicopathologic and molecular sarcoma and identify criteria that should alert pathologists and lead to KAT6B/A::KANSL1 fusion testing in routine practice. Therefore, we conducted a comprehensive clinical, histopathologic, immunohistochemical, and molecular study, including array comparative genomic hybridization, whole RNA-sequencing, unsupervised clustering, and cDNA mutational profile analyses of 16 tumors with KAT6B::KANSL1 fusion from 12 patients. At presentation, patients were peri-menopausal (median, 47.5 years), and the primary tumors were located in the uterine corpus (12/12, 100%), with an additional prevesical location in 1 (8.3%) of 12 cases. The relapse rate was 33.3% (3/9). All tumors (16/16, 100%) showed morphologic and immunohistochemical features overlapping between leiomyoma and endometrial stromal tumors. A whirling recurrent architecture (resembling fibromyxoid-ESS/fibrosarcoma) was found in 13 (81.3%) of 16 tumors. All tumors (16/16, 100%) exhibited numerous arterioliform vessels, and 13 (81.3%) of 18 had large hyalinized central vessels and collagen deposits. Estrogen and progesterone receptors were expressed in 16 (100%) of 16 and 14 (87.5%) of 16 tumors, respectively. Array comparative genomic hybridization performed on 10 tumors classified these neoplasms as simple genomic sarcomas. Whole RNA-sequencing on 16 samples and clustering analysis on primary tumors found that the KAT6B::KANSL1 fusion always occurred between exons 3 of KAT6B and 11 of KANSL1; no pathogenic variant was identified on cDNA, all neoplasms clustered together, close to LG-ESS, and pathway enrichment analysis showed cell proliferation and immune infiltrate recruitment pathway involvement. These results confirm that the sarcomas harboring a KAT6B/A::KANSL1 fusion represent a distinct clinicopathologic entity, close to LG-ESS but different, with clinical aggressiveness despite a reassuring morphology, for which the KAT6B/A::KANSL1 fusion is the molecular driver alteration.

NTRK-rearranged spindle cell neoplasms are ubiquitous tumours of myofibroblastic lineage with a distinct methylation class.

AIMS: NTRK gene fusions have been described in a wide variety of central nervous system (CNS) and soft tissue tumours, including the provisional tumour type 'spindle cell neoplasm, NTRK-rearranged' (SCN-NTRK), added to the 2020 World Health Organisation Classification of Soft Tissue Tumours. Because of histopathological and molecular overlaps with other soft tissue entities, controversy remains concerning the lineage and terminology of SCN-NTRK. METHODS AND RESULTS: This study included 16 mesenchymal tumours displaying kinase gene fusions (NTRK fusions and one MET fusion) initially diagnosed as infantile fibrosarcomas (IFS), SCN-NTRK and adult-type fibrosarcomas from the soft tissue, viscera and CNS. We used immunohistochemistry, DNA methylation profiling, whole RNA-sequencing and ultrastructural analysis to characterise them. Unsupervised t-distributed stochastic neighbour embedding analysis showed that 11 cases (two CNS tumours and nine extra-CNS) formed a unique and new methylation cluster, while all tumours but one, initially diagnosed as IFS, clustered in a distinct methylation class. All the tumours except one formed a single cluster within the hierarchical clustering of whole RNA-sequencing data. Tumours from the novel methylation class co-expressed CD34 and S100, had variable histopathological grades and frequently displayed a CDKN2A deletion. Ultrastructural analyses evidenced a myofibroblastic differentiation. CONCLUSIONS: Our findings confirm that SCN-NTRK share similar features in adults and children and in all locations combine an infiltrative pattern, distinct epigenetic and transcriptomic profiles, and ultrastructural evidence of a myofibroblastic lineage. Further studies may support the use of new terminology to better describe their myofibroblastic nature.

Detection of sarcoma fusions by a next-generation sequencing based-ligation-dependent multiplex RT-PCR assay.

Morphological, immunohistochemical, and molecular methods often need to be combined for accurate diagnosis and optimal clinical management of sarcomas. Here, we have developed, a new molecular diagnostic assay, for the detection of gene fusions in sarcomas. This targeted multiplexed next-generation sequencing (NGS)-based method utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR-NGS) to detect oncogenic fusion transcripts involving 137 genes, leading to 139 gene fusions known to be recurrently rearranged in soft-tissue and bone tumors. 158 bone and soft-tissue tumors with previously identified fusion genes by fluorescent in situ hybridization (FISH) or RT-PCR were selected to test the specificity and the sensitivity of this assay. RNA were extracted from formalin-fixed paraffin-embedded (n = 143) or frozen (n = 15) material (specimen; n = 42 or core needle biopsies; n = 116). Tested tumors encompassed 23 major translocation-related sarcomas types, including Ewing and Ewing-like sarcomas, rhabdomyosarcomas, desmoplastic small round-cell tumors, clear-cell sarcomas, infantile fibrosarcomas, endometrial stromal sarcomas, epithelioid hemangioendotheliomas, alveolar soft-part sarcomas, biphenotypic sinonasal sarcomas, extraskeletal myxoid chondrosarcomas, myxoid/round-cell liposarcomas, dermatofibrosarcomas protuberans and solitary fibrous tumors. In-frame fusion transcripts were detected in 98.1% of cases (155/158). Gene fusion assay results correlated with conventional techniques (FISH and RT-PCR) in 155/158 tumors (98.1%). These data demonstrate that this assay is a rapid, robust, highly sensitive, and multiplexed targeted RNA sequencing assay for the detection of recurrent gene fusions on RNA extracted from routine clinical specimens of sarcomas (formalin-fixed paraffin-embedded or frozen). It facilitates the precise diagnosis and identification of tumors with potential targetable fusions. In addition, this assay can be easily customized to cover new fusions.

Cervical Small Cell Variant of Paraganglioma With Sarcomatous Transformation: Report of a Unique Case.

We report a unique primary cervical neoplasm in a 44-yr-old woman which we believe, based on the morphology and immunophenotype, represents an extremely unusual small cell variant of paraganglioma. This represents the first report of a primary cervical paraganglioma. Following chemoradiation treatment, the tumor underwent malignant transformation into an S100 and SOX10 positive sarcoma, morphologically and immunohistochemically resembling a malignant peripheral nerve sheath tumor, which we believe represents a sarcoma derived from the sustentacular cells of the paraganglioma. Mutational analysis detected a nonsense mutation of NF1 gene in the sarcoma. This further supports the diagnosis as both somatic and germline NF1 mutations have been associated with paragangliomas and malignant peripheral nerve sheath tumors. Targeted RNA sequencing (ARCHER, expanded sarcoma panel) covering many known genes implicated in sarcoma development, did not reveal any other molecular alteration (fusion or internal tandem duplication).

NTRK and other recently described kinase fusion positive uterine sarcomas: A review of a group of rare neoplasms.

The landscape of uterine sarcomas has greatly expanded in recent years to include neoplasms with recurrent gene fusions, such as BCOR and YWHAE translocated high-grade endometrial stromal sarcomas. Sophisticated molecular techniques have also resulted in the description of "new" entities associated with recurrent kinase fusions involving NTRK and RET as well as COL1A1-PDGFB rearranged uterine sarcomas. These rare neoplasms will be discussed in this review, highlighting that some of the underlying molecular events are clinically actionable and potentially susceptible to targeted therapy. While relatively few of these neoplasms have been described to date, likely being previously lumped under the spectrum of undifferentiated uterine sarcoma, the number of cases will expand in the future given their recognition and the increasing availability of molecular testing. These neoplasms have overlapping morphology (often with a "fibrosarcoma-like" appearance) and immunohistochemical features, and are characterized by variable clinical outcomes. Although immunohistochemistry may assist in some cases, a definitive subclassification requires confirmatory molecular studies. As these molecular assays may not be routinely available in most laboratories, referral to reference centers may be needed. In order to assist the pathologist, we suggest a diagnostic algorithm for routine practice when dealing with a malignant or potentially malignant uterine spindle cell neoplasm.

Uterine and vaginal sarcomas resembling fibrosarcoma: a clinicopathological and molecular analysis of 13 cases showing common NTRK-rearrangements and the description of a COL1A1-PDGFB fusion novel to uterine neoplasms.

Mesenchymal neoplasms of the uterus (corpus and cervix) encompass a heterogeneous group of tumors with differing morphologies, immunophenotypes and molecular alterations. With the advent of modern molecular techniques, such as next generation sequencing, newly defined genetic abnormalities are being reported in this group of neoplasms. Herein we report the clinicopathological and molecular features of a series of 13 spindle cell sarcomas of the uterus and vagina (10 cervix, 2 uterine corpus, 1 vagina) with morphology resembling fibrosarcoma. After targeted RNA-sequencing, dual FISH fusion and array-CGH analysis, 7 of 13 tumors exhibited NTRK rearrangements (6 TPM3-NTRK1 and 1 EML4-NTRK3) and 3 a COL1A1-PDGFB fusion; in the other 3 neoplasms, all of which were positive with S100 (2 diffuse, 1 focal), we identified no rearrangement. All the NTRK fusion-positive sarcomas were located in the cervix and exhibited diffuse staining with Trk while all the other neoplasms were negative. CD34 was diffusely positive in all 3 of the COL1A1-PDGFB fusion sarcomas. The latter molecular abnormality is identical to that commonly found in dermatofibrosarcoma protuberans and has not been reported previously in uterine mesenchymal neoplasms. We suggest that uterine sarcomas with a morphology resembling fibrosarcoma (and in which leiomyosarcoma and the known molecularly confirmed high-grade endometrial stromal sarcomas have been excluded) can be divided into 3 groups:- an NTRK fusion group, a COL1A1-PDGFB fusion group and a group containing neither of these molecular abnormalities which, on the basis of positive staining with S100, could be tentatively classified as malignant peripheral nerve sheath tumor, although additional molecular studies may identify specific genetic alterations necessitating a nomenclature change. We suggest a diagnostic algorithm when reporting such neoplasms. Identification of these newly described fusion-associated sarcomas is important given the potential for targeted treatments.

Uterine Tumor Resembling Ovarian Sex Cord Tumor (UTROSCT) Commonly Exhibits Positivity With Sex Cord Markers FOXL2 and SF-1 but Lacks FOXL2 and DICER1 Mutations.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare neoplasm which morphologically and immunohistochemically exhibits overlap with an ovarian sex cord tumor. Although many of these neoplasms are positive with markers of ovarian sex cord-stromal tumors, staining is often limited and the pathogenesis of UTROSCT is unknown. To further explore the sex cord lineage of UTROSCT, we studied 19 of these neoplasms and examined the expression of 2 recently described markers of ovarian sex cord-stromal tumors, FOXL2, and steroidogenic factor-1. We also undertook FOXL2 and DICER1 mutation analysis in these cases; a somatic missense mutation in codon C134W (402C→G) of FOXL2 gene has been demonstrated in the vast majority (>95%) of ovarian adult granulosa cell tumors and somatic DICER1 mutations are found in approximately 60% of ovarian Sertoli-Leydig cell tumors. Ten of 19 cases (53%) exhibited nuclear immunoreactivity with FOXL2 and 11 of 19 (58%) exhibited nuclear staining with steroidogenic factor-1. Neither FOXL2 nor DICER1 mutations were identified in any case where there was sufficient tumor tissue for analysis (18 and 9 cases, respectively). Despite exhibiting an immunophenotype characteristic of a sex cord-stromal tumor, mutations in FOXL2 and DICER1, the 2 most common mutations hitherto reported in ovarian sex cord-stromal tumors, are not a feature of UTROSCT.

Impact of chromosomal instability on colorectal cancer progression and outcome.

BACKGROUND: It remains presently unclear whether disease progression in colorectal carcinoma (CRC), from early, to invasive and metastatic forms, is associated to a gradual increase in genetic instability and to a scheme of sequentially occurring Copy Number Alterations (CNAs). METHODS: In this work we set to determine the existence of such links between CRC progression and genetic instability and searched for associations with patient outcome. To this aim we analyzed a set of 162 Chromosomal Instable (CIN) CRCs comprising 131 primary carcinomas evenly distributed through stage 1 to 4, 31 metastases and 14 adenomas by array-CGH. CNA profiles were established according to disease stage and compared. We, also, asked whether the level of genomic instability was correlated to disease outcome in stage 2 and 3 CRCs. Two metrics of chromosomal instability were used; (i) Global Genomic Index (GGI), corresponding to the fraction of the genome involved in CNA, (ii) number of breakpoints (nbBP). RESULTS: Stage 1, 2, 3 and 4 tumors did not differ significantly at the level of their CNA profiles precluding the conventional definition of a progression scheme based on increasing levels of genetic instability. Combining GGI and nbBP,we classified genomic profiles into 5 groups presenting distinct patterns of chromosomal instability and defined two risk classes of tumors, showing strong differences in outcome and hazard risk (RFS: p = 0.012, HR = 3; OS: p < 0.001, HR = 9.7). While tumors of the high risk group were characterized by frequent fractional CNAs, low risk tumors presented predominantly whole chromosomal arm CNAs. Searching for CNAs correlating with negative outcome we found that losses at 16p13.3 and 19q13.3 observed in 10% (7/72) of stage 2-3 tumors showed strong association with early relapse (p < 0.001) and death (p < 0.007, p < 0.016). Both events showed frequent co-occurrence (p < 1x10-8) and could, therefore, mark for stage 2-3 CRC susceptible to negative outcome. CONCLUSIONS: Our data show that CRC disease progression from stage 1 to stage 4 is not paralleled by increased levels of genetic instability. However, they suggest that stage 2-3 CRC with elevated genetic instability and particularly profiles with fractional CNA represent a subset of aggressive tumors.

Superficial CD34-Positive Fibroblastic Tumor.

Superficial CD34-positive fibroblastic tumor is a mesenchymal neoplasm of "intermediate malignancy" recently included in the fifth edition of the World Health Organization classification of soft tissue and bone tumors. In this review, we summarize the current knowledge on this rare entity with a special focus on its clinicopathological features, morphologic spectrum, and differential diagnosis. We also provide data regarding recent discoveries on its molecular profile and discuss its prognosis and management.

YWHAE rearrangement identified by FISH and RT-PCR in endometrial stromal sarcomas: genetic and pathological correlations.

Endometrial stromal sarcomas represent the second most common mesenchymal uterine tumor. The 2003 WHO classification distinguishes low-grade and undifferentiated endometrial stromal sarcomas with different prognoses. Endometrial stromal sarcomas are a genetically heterogeneous group of sarcomas harboring different cytogenetic anomalies. Recently, a fusion between the YWHAE and FAM22A/B genes subsequent to a t(10;17) (q22;p13) has been described in endometrial sarcomas with high-grade histology. We examined YWHAE rearrangements by FISH break-apart and RT-PCR in a series of 27 undifferentiated uterine stromal sarcoma without JAZF1 rearrangements. Immunohistochemistry (IHC) was carried out with a panel of antibodies (estrogen (ER) and progesterone (PR) receptors, CD10, Cyclin D1, ß-catenin, p53, and Ki-67). We identified a subgroup of endometrial sarcomas with high-grade histology and uniform morphology harboring YWHAE rearrangements. FISH break-apart was interpretable in 20 cases (74%). Twelve cases (60%) showed <10% of tumor cells with a YWHAE rearrangement, 4 cases (20%) showed between 10 and ≤20%, and 4 (20%) >20%. RT-PCR was tested on 24/27 cases (88%) and 19 cases were interpretable (79%). Five cases (26%) showed a specific fusion transcript YWHAE-FAM22A/B sequence. The best concordance rate between FISH and RT-PCR (94%) was obtained with the threshold of 20% of cells with a YWHAE rearrangement. The YWHAE-rearranged cases showed high-grade morphology with uniform appearance, spindle or round epithelioid cells, low ER and PR, CD10 expression, and a high and diffuse positivity for Cyclin D1, p53, and nuclear ß-catenin negativity. Cyclin D1 was the most sensitive marker for high-grade endometrial sarcomas with YWHAE rearrangement. All undifferentiated uterine sarcomas with pleomorphic appearances did not harbor any YWHAE rearrangements, except for one case. Overall, for endometrial sarcoma cases with high-grade morphology we recommend to test for YWHAE rearrangements by FISH break-apart, a cost- and time-efficient method, and to complete the investigation by RT-PCR in borderline cases.

SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

BACKGROUND: Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with "gold standard FISH" for evaluation of HER2 amplification status. METHODS: This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. RESULTS: The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. CONCLUSION: These alternative techniques showed an excellent concordance with FISH in core biopsies allowing their use in routine clinical practice. This newly designed qPCR on paraffin-embedded core biopsies deserves special attention, as it is reliable, easy to perform and less expensive than ISH tests.

"PRRX1-rearranged mesenchymal tumors": expanding the immunohistochemical profile and molecular spectrum of a recently described entity with the proposed revision of nomenclature.

Since the publication of the 2020 World Health Organization classification of soft tissue and bone tumors, the classification of "fibroblastic" tumors has expanded to include a novel subset of tumors characterized by PRRX1::NCOA1/2 gene fusions. These tumors defy conventional classification and are morphologically distinct, characterized by a multi-nodular growth of bland spindle cells suspended in a myxo-collagenous stroma with mild cytologic atypia, "staghorn-like" vessels, and variable perivascular hyalinization. Mitotic activity is rare, and necrosis is not identified. Herein, we present six additional cases of PRRX1-rearranged mesenchymal tumors, including five cases with PRRX1::NCOA1 fusion and one case with PRRX1::KMT2D fusion. Three cases (3/6, 50%) demonstrated focal co-expression of S100 protein and SOX10, thereby expanding the immunohistochemical profile of this emerging entity. Like prior reported cases, there was no evidence of malignant behavior on short-term follow-up. The novel fusion, PRRX1::KMT2D, further expands the molecular spectrum of this entity and leads to a proposed revision of the provisional nomenclature to "PRRX1-rearranged mesenchymal tumor" to both accommodate non-NCOA1/2 fusion partners and allow for the possibility of partial neural or neuroectodermal differentiation.

Giant Cell Tumors With HMGA2::NCOR2 Fusion : Clinicopathologic, Molecular, and Epigenetic Study of a Distinct Entity.

Giant cell tumors (GCTs) with high mobility group AT-Hook 2 ( HMGA2 )::nuclear receptor corepressor 2 ( NCOR2 ) fusion are rare mesenchymal tumors of controversial nosology, which have been anecdotally reported to respond to CSFR1 inhibitors. Here, we performed a comprehensive study of 6 GCTs with HMGA2::NCOR2 fusion and explored their relationship with other giant cell-rich neoplasms. Tumors occurred in 4 females and 2 males ranging in age from 17 to 32 years old (median 24). Three lesions originated in subcutaneous soft tissue and 3 in bone. Tumor size ranged from 20 to 33 mm (median 27 mm). The lesions had a nodular/multinodular architecture and were composed of sheets of mononuclear "histiocytoid" cells with uniform nuclei intermingled with multinucleated giant cells. Mitotic activity was low and nuclear atypia and metaplastic bone were absent. Variable findings included necrosis, cystic degeneration, lymphocytic infiltrate (sometimes forming nodules), and xanthogranulomatous inflammation. On immunohistochemistry, all cases focally expressed pan-keratin and were negative with SATB2 and H3.3G34W. Whole RNA-sequencing was performed in all cases of GCT with HMGA2::NCOR2 fusion and a subset of giant cell-rich tumors (tenosynovial-GCT, n = 19 and "wild-type" GCT of soft tissue, n = 9). Hierarchical clustering of RNA-sequencing data showed that GCT with HMGA2::NCOR2 fusion formed a single cluster, independent of the other 2 entities. Methylome profiling showed similar results, but the distinction from "wild-type" GCT of soft tissue was less flagrant. Gene expression analysis showed similar levels of expression of the CSF1/CSFR1 axis between GCT with HMGA2::NCOR2 fusion and tenosynovial-GCT, supporting their potential sensitivity to CSFR1 inhibitors. Clinical follow-up was available for 5 patients (range: 10 to 64 mo; median 32 mo). Three patients (60%) experienced local recurrences, whereas none had distant metastases or died of disease. Overall, our study confirms and expands previous knowledge on GCT with HMGA2::NCOR2 fusion and supports its inclusion as an independent entity.

Deep learning predicts patients outcome and mutations from digitized histology slides in gastrointestinal stromal tumor.

Risk assessment of gastrointestinal stromal tumor (GIST) according to the AFIP/Miettinen classification and mutational profiling are major tools for patient management. However, the AFIP/Miettinen classification depends heavily on mitotic counts, which is laborious and sometimes inconsistent between pathologists. It has also been shown to be imperfect in stratifying patients. Molecular testing is costly and time-consuming, therefore, not systematically performed in all countries. New methods to improve risk and molecular predictions are hence crucial to improve the tailoring of adjuvant therapy. We have built deep learning (DL) models on digitized HES-stained whole slide images (WSI) to predict patients' outcome and mutations. Models were trained with a cohort of 1233 GIST and validated on an independent cohort of 286 GIST. DL models yielded comparable results to the Miettinen classification for relapse-free-survival prediction in localized GIST without adjuvant Imatinib (C-index=0.83 in cross-validation and 0.72 for independent testing). DL splitted Miettinen intermediate risk GIST into high/low-risk groups (p value = 0.002 in the training set and p value = 0.29 in the testing set). DL models achieved an area under the receiver operating characteristic curve (AUC) of 0.81, 0.91, and 0.71 for predicting mutations in KIT, PDGFRA and wild type, respectively, in cross-validation and 0.76, 0.90, and 0.55 in independent testing. Notably, PDGFRA exon18 D842V mutation, which is resistant to Imatinib, was predicted with an AUC of 0.87 and 0.90 in cross-validation and independent testing, respectively. Additionally, novel histological criteria predictive of patients' outcome and mutations were identified by reviewing the tiles selected by the models. As a proof of concept, our study showed the possibility of implementing DL with digitized WSI and may represent a reproducible way to improve tailoring therapy and precision medicine for patients with GIST.

CTNNB1 mutation analysis is a useful tool for the diagnosis of desmoid tumors: a study of 260 desmoid tumors and 191 potential morphologic mimics.

Desmoid tumors are benign monoclonal fibroblastic or myofibroblastic neoplasms, characterized by local invasiveness and high rates of recurrence. Desmoid tumors must be distinguished from benign fibroblastic and myofibroblastic lesions, as well as from low-grade sarcoma, which can appear histologically similar to desmoid tumors. This differential diagnosis can be very difficult, especially when diagnosis is based on a core needle biopsy. On the molecular level, most sporadic desmoid tumors are associated with mutations of the ß-catenin gene (CTNNB1). A minority of desmoid tumors are associated with Gardner syndrome and mutations of the familial adenomatous polyposis gene. We identified the common CTNNB1 mutations associated with sporadic desmoid tumors by direct sequencing: in (i) 260 cases of typical desmoid tumors; and (ii) in 191 cases of spindle cell lesions, which can morphologically 'mimic' desmoid tumors. Formalin-fixed paraffin-embedded tissues were obtained via core needle biopsy (n=150) or open biopsy/surgical excision (n=301). Only 16 cases (4%) were not analyzable (Bouin's fixed tissue). CTNNB1 mutations were observed in 223 of 254 (88%) of sporadic desmoid tumors. No CTNNB1 mutations were detected in all other lesions (n=175) studied. CTNNB1 sequencing can be easily and reliably done using tissues obtained via core needle biopsy. Detection of CTNNB1 mutations in formalin-fixed paraffin-embedded tissues among spindle cell lesions is proposed as a specific diagnostic tool for the diagnosis of desmoid tumors. This result has significant implications for patient care and management.

[KRAS analysis management: Process and delays]. / Prise en charge d'une analyse KRAS : processus et délais.

INTRODUCTION: The prescription of some anti-cancer therapies is now based on the detection of specific genetic alterations that should be determined as early as possible not to put patients at a disadvantage. In 2009, the 'Aquitaine platform of molecular tumour genetics' (PGMC) developed a programme to evaluate and to improve the organisation of molecular cancer analyses, particularly the analysis of the KRAS gene. The objective was to describe the analysis process, the organization of pathology laboratories and the delays between the different phases of the process. METHODS: We established a working group to describe the different steps between the prescription of molecular biology analyses and the analysis report. A retrospective study based on the first quarter of 2009 allowed us to measure management delays. In addition, a pathology laboratory organisational questionnaire allowed us to identify organisational features hindering rapid delivery. RESULTS: The median delay between the analysis prescription and the results was 15 days (range: 7-78 days). Practices explaining longer delays were highlighted not only in the pathology laboratories (for example, pending of the prescription before sending the analysis, waiting for several cases before sending the material, sample slicing, sending by standard mail), but also within the PGMC (for example, sample testing by another technique or new extraction for non-contributory samples). CONCLUSION: The results of this study emphasise the necessity of speeding up pre-analytical phases, and of creating an electronic procedure and regional facilities in order to provide results more rapidly to clinicians.