Unraveling the Molecular Landscape of Uterine Tumor Resembling Ovarian Sex Cord Tumor: Insights From A Clinicopathological, Morphologic, Immunohistochemical, and Molecular Analysis of 35 Cases.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare tumor of uncertain lineage and low malignant potential. Most tumors behave in a benign manner, but a subset of UTROSCT exhibit an aggressive clinical course with recurrences and metastases. The recurrent molecular alterations in UTROSCT mostly represent gene fusions involving NCOA1-3. We performed a comprehensive clinicopathological, morphologic, immunohistochemical, and molecular analysis on a cohort of 35 UTROSCT. The tumors exhibited various architectural patterns (diffuse, corded/trabecular, tubular, sertoliform, fascicular, whorled, nested, microfollicular, and pseudoglandular), often in combination. The immunohistochemical analysis confirmed the polyphenotypic immunoprofile, often with coexpression of sex cord-stromal, smooth muscle, and epithelial markers, as well as hormone receptors. Next-generation sequencing RNA analysis revealed recurrent NCOA1-3 gene fusions in 22/32 analyzed cases (69%), including ESR1::NCOA3 (11/22), GREB1::NCOA2 (7/22), ESR1::NCOA2 (3/22), and GREB1::NCOA1 (1/22). Tumor mutation burden was low in all cases. The fusion-positive cases exhibited statistically significant association with whorled architecture, conversely necrosis was associated with fusion-negative status. We did not find a significant relationship between any architectural pattern and GREB1 alterations, but the NCOA2-altered tumors were associated with pseudoglandular architecture. The GREB1-altered cases occurred in older patients and tended to be more often intramural masses compared with ESR1-altered cases. On the contrary, the ESR1-altered cases presented more often like submucosal or polypoid tumors. Two tumors exhibited aggressive behavior with recurrent disease. Both of these cases harbored a GREB1::NCOA2 fusion. Unsupervised hierarchical cluster analysis of our cohort revealed 2 main clusters. The tumors with GREB1 or NCOA2 fusion cluster together, suggesting that there are underlying molecular differences between these cases and cases with ESR1::NCOA3 fusion or without fusion. Our findings contribute to the growing knowledge about a rare neoplasm with currently uncertain biological behavior.

Parallel DNA/RNA NGS Using an Identical Target Enrichment Panel in the Analysis of Hereditary Cancer Predisposition.

Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.

Primary Mucinous Tumors of the Ovary: An Interobserver Reproducibility and Detailed Molecular Study Reveals Significant Overlap Between Diagnostic Categories.

Primary ovarian mucinous tumors represent a heterogeneous group of neoplasms, and their diagnosis may be challenging. We analyzed 124 primary ovarian mucinous tumors originally diagnosed as mucinous borderline tumors (MBTs) or mucinous carcinomas (MCs), with an emphasis on interobserver diagnostic agreement and the potential for diagnostic support by molecular profiling using a next-generation sequencing targeted panel of 727 DNA and 147 RNA genes. Fourteen experienced pathologists independently assigned a diagnosis from preset options, based on a review of a single digitized slide from each tumor. After excluding 1 outlier participant, there was a moderate agreement in diagnosing the 124 cases when divided into 3 categories (κ = 0.524, for mucinous cystadenoma vs MBT vs MC). A perfect agreement for the distinction between mucinous cystadenoma/MBT as a combined category and MC was found in only 36.3% of the cases. Differentiating between MBTs and MCs with expansile invasion was particularly problematic. After a reclassification of the tumors into near-consensus diagnostic categories on the basis of the initial participant results, a comparison of molecular findings between the MBT and MC groups did not show major and unequivocal differences between MBTs and MCs or between MCs with expansile vs infiltrative pattern of invasion. In contrast, HER2 overexpression or amplification was found only in 5.3% of MBTs and in 35.3% of all MCs and in 45% of MCs with expansile invasion. Overall, HER2 alterations, including mutations, were found in 42.2% of MCs. KRAS mutations were found in 65.5% and PIK3CA mutations in 6% of MCs. In summary, although the diagnostic criteria are well-described, diagnostic agreement among our large group of experienced gynecologic pathologists was only moderate. Diagnostic categories showed a molecular overlap. Nonetheless, molecular profiling may prove to be therapeutically beneficial in advanced-stage, recurrent, or metastatic MCs.

Giant cell fibroblastoma: a case report.

Giant cell fibroblastoma is a rare locally aggressive tumor of subcutaneous mesenchymal tissue, occurring mostly on the trunk in young individuals with maximal incidence in the first decade of life. Local recurrences of giant cell fibroblastoma are common if marginally excised, however, distant metastases do not occur. Giant cell fibroblastoma was labelled as a juvenile variant of dermatofibrosarcoma protuberans (DFSP) due to quite frequent combination of both lesions, morphological similarities, identical immunoprofile, and shared gene fusion t(17;22) COL1A1-PDGFB. In this paper, we report a case of a young man with a slowly growing subcutaneous tumor in the groin. The tumor was excised and histological examination identified a mesenchymal tumor with variable cellularity, presence of multinucleated giant cells and pleomorphic spindle cells, which lined pseudovascular or angiectoid spaces. The CD34 immunohistochemistry showed strong positivity in all of these cells, whereas ERG was positive only in endothelial cells in true vessels. These findings led to a suspicion on giant cell fibroblastoma. Because of its borderline malignant behaviour and positive surgical margins, the lesion was subsequently reexcised. The molecular analysis identified the transcription product of gene fusion COL1A1-PDGFB and thus, final diagnosis was confirmed. The article includes review of the literature and brief historical overview of giant cell fibroblastoma concept as an unique entity.

Gynecological lesions in hereditary cancer predisposition syndromes.

Hereditary tumor syndromes with a possible manifestation in the female internal genital tract represent a heterogeneous group of diseases. The two most common entities are the hereditary breast and ovarian cancer syndrome, and the Lynch syndrome. The less common syndromes include the rhabdoid tumor predisposition syndrome, Cowden syndrome, tuberous sclerosis complex, DICER1 syndrome, nevoid basal cell carcinoma syndrome, Peutz-Jeghers syndrome, von Hippel-Lindau disease, and hereditary leiomyomatosis and renal cell cancer syndrome. The goal of this manuscript is to provide a comprehensive overview of those hereditary tumor syndromes which can manifest in the area of the female genital system, with an emphasis on their summary, the characteristics of the tumors which can develop in association with these syndromes, and the approach to the processing of prophylactically removed tissues and organs. The issue of Lynch syndrome screening is also discussed.

Microscopic extraovarian sex cord proliferation: report of a case with bilateral Fallopian tube involvement and a comprehensive molecular analysis.

We report a case of a 58-year-old female with microscopic extraovarian sex cord proliferations affecting both Fallopian tubes. Molecular analysis showed likely pathogenic germline missense mutations of the KDM5A and KMT2D genes. However, mutations of other genes, including FOXL2 and STK11, were not detected. Our case represents the 12th case of extraovarian sex cord proliferation reported in the literature to date. This is the first time that a molecular genetic analysis of the lesion has been performed, and it showed a wild-type FOXL2 gene, which represents another argument supporting the estimated benign nature of these rare lesions.

Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer.

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10-14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10-14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10-4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification.

High tumour mutational burden is associated with strong PD-L1 expression, HPV negativity, and worse survival in penile squamous cell carcinoma: an analysis of 165 cases.

Penile squamous cell carcinoma (pSCC) is a rare tumour with a variable prognosis. More prognostic markers linked to mutational signatures and the tumour immune microenvironment are needed. A cohort made up of 165 invasive pSCC was retrospectively analysed using formalin-fixed, paraffin-embedded tumour tissue, focusing on tumour mutational burden (TMB), programmed death ligand 1 (PD-L1) expression, microsatellite instability (MSI), the number of tumour infiltrating lymphocytes (TILs) expressing cytotoxic T-lymphocyte-associated protein 4 (CTLA4), HPV status determined by p16 immunohistochemistry, and several traditional histopathological variables. High TMB (>10 mut/Mb) was associated with high PD-L1 expression (TPS 50-100%), and HPV-negative status. High PD-L1 expression was linked to HPV negativity, a high number of intratumoural CTLA4+ cells, and brisk lymphocytic infiltrate. High TMB was a significant predictor of shorter overall survival (OS) in both univariate and multivariate analysis when using a median cut-off value of 4.3 mut/Mb, but not when using an arbitrary cut-off of 10 mut/Mb. Low CTLA4+ cell infiltration at the tumour invasion front was a marker of shorter OS and cancer-specific survival in both univariate and multivariate analysis. PD-L1 expression had no significant impact on prognosis. Only two cases were MSI high. The results support the hypothesis of two aetiological pathways in pSCC cancerogenesis: (1) SCC linked to HPV infection characterised by low TMB, less common PD-L1 expression, and a lower number of TILs; and (2) SCC linked to chronic inflammation leading to a high number of acquired mutations (high TMB), HPV negativity, increased neoantigen production (i.e., PD-L1), and high immune cell infiltration.

The Spectrum of Fusions Occurring in Non-Smooth Muscle Mesenchymal Uterine Tumors: A Review of the Current Knowledge.

CONTEXT.­: Non-smooth muscle uterine sarcomas are mostly represented by low-grade endometrial stromal sarcoma. However, several other rare, distinct types of uterine sarcoma are recognized, including high-grade endometrial stromal sarcoma, tumors with kinase fusions, uterine tumors resembling ovarian sex cord tumors, soft tissue-type sarcoma, and emerging entities such as KAT6A/B-rearranged tumors. The landscape of uterine sarcomas has changed, mostly because of the increasing knowledge concerning their molecular aberrations. OBJECTIVE.­: To offer a comprehensive review of the literature focusing on fusions occurring in other than smooth muscle mesenchymal uterine tumors with respect to their type, frequency, and overlap between diagnostic categories and entities. DATA SOURCES.­: The data were mined from the PubMed/MEDLINE database covering the time period from January 1988 to June 2023. In total, 156 studies focusing on the problematics of fusions occurring in non-smooth muscle mesenchymal uterine tumors were selected, and thus became the basis for this review. CONCLUSIONS.­: One hundred ten fusions were identified in 703 tumors. The diagnostic significance of the molecular aberrations occurring in these tumors can be unclear in some cases. This can be related to the rare aberrations with a limited number of reported cases. Additionally, even well-known aberrations considered as specific for a certain distinct entity can occur in more lesions, the biological behavior and clinical significance of which can differ substantially.

Somatic Genomic and Transcriptomic Characterization of Primary Ovarian Serous Borderline Tumors and Low-Grade Serous Carcinomas.

Low-grade serous carcinoma (LGSC) may develop from serous borderline tumor (SBT) tissue, where the micropapillary type (mSBT) presents the highest risk for progression. The sensitivity of LGSC to standard chemotherapy is limited, so alternative therapeutic approaches, including targeted treatment, are needed. However, knowledge about the molecular landscape of LGSC and mSBT is limited. A sample set of 137 pathologically well-defined cases (LGSC, 97; mSBT, 40) was analyzed using capture DNA next-generation sequencing (727 genes) and RNA next-generation sequencing (147 genes) to show the landscape of somatic mutations, gene fusions, expression pattern, and prognostic and predictive relevance. Class 4/5 mutations in the main driver genes (KRAS, BRAF, NRAS, ERBB2, USP9X) were detected in 48% (14/29) of mSBT cases and 63% (47/75) of LGSC cases. The USP9X mutation was detected in only 17% of LGSC cases. RNA next-generation sequencing revealed gene fusions in 6 of 64 LGSC cases (9%) and 2 of 33 mSBT cases (9%), and a heterogeneous expression profile across LGSC and mSBT. No molecular characteristics were associated with greater survival. The somatic genomic and transcriptomic profiles of 35 mSBT and 85 LGSC cases are compared for the first time. Candidate oncogenic gene fusions involving BRAF, FGFR2, or NF1 as a fusion partner were identified. Molecular testing of LGSC may be used in clinical practice to reveal therapeutically significant targets.

A comprehensive study evaluating germline FANCG variants in predisposition to breast and ovarian cancer.

BACKGROUND: Monoallelic germline pathogenic variants (GPVs) in five Fanconi anemia (FA) genes (BRCA1/FANCS, BRCA2/FANCD1, PALB2/FANCN, BRIP1/FANCJ, and RAD51C/FANCO) confer an increased risk of breast (BC) and/or ovarian (OC) cancer, but the role of GPVs in 17 other FA genes remains unclear. METHODS: Here, we investigated the association of germline variants in FANCG/XRCC9 with BC and OC risk. RESULTS: The frequency of truncating GPVs in FANCG did not differ between BC (20/10,204; 0.20%) and OC (8/2966; 0.27%) patients compared to controls (6/3250; 0.18%). In addition, only one out of five tumor samples showed loss-of-heterozygosity of the wild-type FANCG allele. Finally, none of the nine functionally tested rare recurrent missense FANCG variants impaired DNA repair activities (FANCD2 monoubiquitination and FANCD2 foci formation) upon DNA damage, in contrast to all tested FANCG truncations. CONCLUSION: Our study suggests that heterozygous germline FANCG variants are unlikely to contribute to the development of BC or OC.

Soft tissue sarcoma with ZC3H7B::BCOR fusion in a male mimicking low-grade fibromyxoid sarcoma - A case report.

Diagnosis of soft tissue tumors is often challenging, given the large number of entities, often with non-specific or overlapping morphology. Although morphology still plays an important part in diagnostic process, additional studies including immunohistochemistry and molecular genetics are often needed to arrive at correct diagnosis. We report a case of 61-year-old male with subcutaneous tumor in right hip area, that was surgically removed. The tumor was composed of uniform bland spindle cells in mild to moderately cellular myxoid nodules, with limited areas of collagenization and the diagnosis of low grade fibromyxoid sarcoma was made. The tumor recurred 3 years after the initial diagnosis and the new sample showed a high-grade round cell sarcoma with limited residual low-grade areas and non-specific immunoprofile after extended immunohistochemical work-up. Molecular analysis demonstrated ZC3H7B::BCOR fusion. Sarcomas with ZC3H7B::BCOR fusion occurring outside of uterus are exceedingly rare. A comprehensive review of previously published cases and a short discussion about classification of the entity is provided, together with data about morphology and immunoprofile of the lesions. The case also underscores the necessity of extended work up of soft tissue tumors with unusual immunohistochemical or morphological features in order to accurately assess their biological potential.

Immune cell infiltration, tumour budding, and the p53 expression pattern are important predictors in penile squamous cell carcinoma: a retrospective study of 152 cases.

Penile squamous cell carcinoma (pSCC) is a rare malignancy with a slowly increasing incidence and variable prognosis. Regional lymph node involvement signifies poor prognosis but represents a late sign, and more prognostic markers for effective patient risk stratification are urgently needed. In this retrospective study, 152 tumour samples with formalin-fixed, paraffin-embedded tissue were analysed for traditional pathological variables, tumour budding, p53, p16, and mismatch repair proteins (MMR) immunohistochemistry. The density of tumour lymphocytic infiltrate was also determined, using subjective evaluation by two pathologists (brisk/non-brisk/absent) and also using the immunoscore method, which categorised the cohort into five immunoscore groups according to the number of CD3+ and CD8+ T-cells in both the tumour centre and tumour invasion front. Only one case (0.6%) was MMR-deficient. Tumour budding count ≥5 tumour buds/20× power field and non-brisk/absent lymphocytic infiltrate were significant negative predictors of both the overall survival (OS) and cancer-specific survival (CSS), whereas a low immunoscore was a significant marker of shorter OS but not CSS. Advanced pT stage (3+4) was a significant marker of shorter CSS but not OS. In the multivariate analysis, high-grade budding was a significant parameter if adjusted for the patient's age and associated variables, except for the pN stage. The lymphocytic infiltrate retained its prognostic significance if adjusted for age and associated variables. The negative prognostic significance of the previously described parameters (lymphatic, venous, and perineural invasion, regional lymph node metastasis, and p53 mutated profile) were confirmed in our study. Grade, histological subtype, and HPV status (as determined by p16 immunohistochemistry) showed, surprisingly, little or no prognostic significance.

Uterine leiomyoma with RAD51B::NUDT3 fusion: a report of 2 cases.

Three main uterine leiomyoma molecular subtypes include tumors with MED12 mutation, molecular aberrations leading to HMGA2 overexpression, and biallelic loss of FH. These aberrations are mutually exclusive and can be found in approximately 80-90% of uterine leiomyoma, in which they seem to be a driver event. Approximately 10% of uterine leiomyoma, however, does not belong to any of these categories. Uterine leiomyoma with HMGA2 overexpression is the most common subtype in cellular and second most common category of usual leiomyoma. In some of these tumors, rearrangement of HMGA2 gene is present. The most common fusion partner of HMGA2 gene is RAD51B. Limited data suggests that RAD51B fusions with other genes may be present in uterine leiomyoma. In our study, we described two cases of uterine leiomyoma with RAD51B::NUDT3 fusion, which occur in one case of usual and one case of highly cellular leiomyoma. In both cases, no other driver molecular aberrations were found. The results of our study showed that RAD51::NUDT3 fusion can occur in both usual and cellular leiomyoma. RAD51B may be a fusion partner of multiple genes other than HMGA2 and HMGA1. In these cases, RAD51B fusion seems to be mutually exclusive with other driver aberrations defining molecular leiomyoma subtypes. RAD51B::NUDT3 fusion should be added to the spectrum of fusions which may occur in uterine leiomyoma, which can be of value especially in cellular leiomyoma in the context of differential diagnosis against endometrial stromal tumors.

HER2 status as a potential predictive biomarker for ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian carcinoma characterized by unique biological features and highly malignant characteristics including low chemosensitivity. Therefore, new therapeutic targets are needed. These could include the downstream pathways of receptor tyrosine kinases, especially the human epidermal growth factor receptor 2 (HER2). Our main objective was to characterize the HER2 status using immunohistochemistry (IHC) and FISH on 118 OCCCs, also considering the novel paradigm of HER2-zero and HER2-low status. Other aims included determination of the association between HER2 status and survival, HER2 gene DNA and RNA NGS analysis, HER2 gene expression analysis, and correlation between IHC and gene expression in HER2-zero and HER2-low cases. Cases with HER2 overexpression/amplification accounted for 5.1% (6/118), with additional 3% harbouring HER2 gene mutation. The remaining 112 (94.9%) cases were HER2-negative. Of these, 75% were classified as HER2-zero and 25% as HER2-low. This percentage of HER2 aberrations is significant concerning their possible therapeutic influence. Cases from the HER2-zero group showed significantly better survival. Although this relationship lost statistical significance in multivariate analysis, the results have potential therapeutic significance. HER2 gene expression analysis showed a significant correlation with HER2 IHC status in the entire cohort (HER2-positive vs. HER2-negative), while in the cohort of only HER2-negative cases, the results did not reach statistical significance, suggesting that gene expression analysis would not be suitable to confirm the subdivision into HER2-low and HER2-zero. Our results also emphasize the need for standardized HER2 testing in OCCC to determine the best predictor of clinical response.

Refined criteria for p53 expression in ovarian mucinous tumours are highly concordant with TP53 mutation status, but p53 expression/TP53 status lack prognostic significance.

In gynecological neoplasms, immunohistochemical (IHC) expression of p53 is generally an accurate predictor of TP53 mutation status if correctly interpreted by the pathologist. However, the literature concerning cut-offs, frequency and prognostic significance of p53 staining in ovarian mucinous tumours is limited and heterogeneous. We performed an analysis of 123 primary ovarian mucinous tumours including mucinous borderline tumours (MBT), mucinous carcinomas (MC), and tumours with equivocal features between MBT and MC. We assessed p53 expression for the three recognised patterns of aberrant staining in ovarian carcinoma [overexpression ('all'), null and cytoplasmic] but using a recently suggested cut-off for aberrant overexpression in ovarian mucinous tumours (strong nuclear p53 staining in ≥12 consecutive tumour cells) and correlated the results with next generation sequencing (NGS) in all qualitatively sufficient cases (92/123). Aberrant p53 expression was present in 25/75 (33.3%) MBT, 23/33 (69.7%) MC (75% of MC with expansile invasion and 61.5% with infiltrative invasion), and 10/15 (66.7%) tumours equivocal between MBT and MC. Regarding the 92 tumours with paired IHC and mutation results, 86 showed concordant results and six cases were discordant. Three discordant MBT cases showed aberrant expression but were TP53 wild-type on sequencing. Three cases had normal p53 expression but contained a TP53 mutation. Overall, IHC predicted the TP53 mutation status with high sensitivity (94.1%) and specificity (92.7%). The accuracy of IHC was 93.5% with a positive predictive value of 94.1% and a negative predictive value of 92.7%. When comparing MC cases with wild-type TP53 versus those with TP53 mutation, there were no significant differences concerning disease-free survival, local recurrence-free survival, or metastases-free survival (p>0.05). In the MBT subgroup, there were no events for survival analyses. In conclusion, using an independent large sample set of ovarian mucinous tumours, the results of our study confirm that the suggested refined cut-off of strong nuclear p53 staining in ≥12 consecutive tumour cells reflect high accuracy, sensitivity and specificity for an underlying TP53 mutation but the TP53 mutation status has no prognostic significance in either MC or MBT.

Microsatellite instability in non-endometrioid ovarian epithelial tumors: a study of 400 cases comparing immunohistochemistry, PCR, and NGS based testing with mutation status of MMR genes.

Testing of microsatellite instability is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. The aim of this study was to assess the frequency of mismatch repair deficiency (MMR-D)/microsatellite instability (MSI) in 400 cases of non-endometrioid ovarian tumors (high-grade serous, low-grade serous, mucinous and clear cell), to compare different methodological approaches of testing, and to assess the optimal approach for next generation sequencing (NGS) MSI testing. For all tumors, we evaluated immunohistochemical (IHC) expression of MMR proteins and assessed microsatellite markers by PCR-based method. Except for high-grade serous carcinoma, we correlated the findings of IHC and PCR with NGS-based MSI testing. We compared the results with somatic and germline mutation in MMR genes. Among the whole cohort, seven MMR-D cases, all clear cell carcinomas (CCC), were found. On PCR analysis, 6 cases were MSI-high and one was MSS. In all cases, mutation of an MMR gene was found; in 2 cases, the mutation was germline (Lynch syndrome). An additional 5 cases with a mutation in MMR gene(s) with MSS status and without MMR-D were identified. We further utilized sequence capture NGS for MSI testing. Employing 53 microsatellite loci provided high sensitivity and specificity. Our study shows that MSI occurs in 7% of CCC while it is rare or absent in other nonendometrioid ovarian neoplasms. Lynch syndrome was present in 2% of patients with CCC. However, some cases with MSH6 mutation can evade all testing methods, including IHC, PCR, and NGS-MSI.

A comprehensive molecular analysis of 113 primary ovarian clear cell carcinomas reveals common therapeutically significant aberrations.

BACKGROUND: Molecular aberrations occurring in primary ovarian clear cell carcinoma (OCCC) can be of diagnostic, predictive, and prognostic significance. However, a complex molecular study including genomic and transcriptomic analysis of large number of OCCC has been lacking. METHODS: 113 pathologically confirmed primary OCCCs were analyzed using capture DNA NGS (100 cases; 727 solid cancer related genes) and RNA-Seq (105 cases; 147 genes) in order to describe spectra and frequency of genomic and transcriptomic alterations, as well as their prognostic and predictive significance. RESULTS: The most frequent mutations were detected in genes ARID1A, PIK3CA, TERTp, KRAS, TP53, ATM, PPP2R1A, NF1, PTEN, and POLE (51,47,27,18,13,10,7,6,6, and 4%, respectively). TMB-High cases were detected in 9% of cases. Cases with POLEmut and/or MSI-High had better relapse-free survival. RNA-Seq revealed gene fusions in 14/105 (13%) cases, and heterogeneous expression pattern. The majority of gene fusions affected tyrosine kinase receptors (6/14; four of those were MET fusions) or DNA repair genes (2/14). Based on the mRNA expression pattern, a cluster of 12 OCCCs characterized by overexpression of tyrosine kinase receptors (TKRs) AKT3, CTNNB1, DDR2, JAK2, KIT, or PDGFRA (p < 0.00001) was identified. CONCLUSIONS: The current work has elucidated the complex genomic and transcriptomic molecular hallmarks of primary OCCCs. Our results confirmed the favorable outcomes of POLEmut and MSI-High OCCC. Moreover, the molecular landscape of OCCC revealed several potential therapeutical targets. Molecular testing can provide the potential for targeted therapy in patients with recurrent or metastatic tumors.

Epithelioid sarcoma with retained INI1 expression as a cause of a chronic leg ulcer.

Epithelioid sarcoma is a rare soft-tissue sarcoma typically presenting itself as a subcutaneous or deep dermal mass in distal portions of the extremities of adolescents and young adults. They are frequently mistaken for ulcers, abscesses, or infected warts resistant to standard medical treatment. Patients often develop multiple local recurrences with subsequent metastases. We report a case of a 66-year-old patient with chronic leg ulcer who died of generalization of an epithelioid sarcoma.

Comprehensive quantitative analysis of alternative splicing variants reveals the HNF1B mRNA splicing pattern in various tumour and non-tumour tissues.

Hepatocyte nuclear factor-1-beta (HNF1B) is a transcription factor and putative biomarker of solid tumours. Recently, we have revealed a variety of HNF1B mRNA alternative splicing variants (ASVs) with unknown, but potentially regulatory, functions. The aim of our work was to quantify the most common variants and compare their expression in tumour and non-tumour tissues of the large intestine, prostate, and kidney. The HNF1B mRNA variants 3p, Δ7, Δ7-8, and Δ8 were expressed across all the analysed tissues in 28.2-33.5%, 1.5-2%, 0.8-1.7%, and 2.3-6.9% of overall HNF1B mRNA expression, respectively, and occurred individually or in combination. The quantitative changes of ASVs between tumour and non-tumour tissue were observed for the large intestine (3p, Δ7-8), prostate (3p), and kidney samples (Δ7). Decreased expression of the overall HNF1B mRNA in the large intestine and prostate cancer samples compared with the corresponding non-tumour samples was observed (p = 0.019 and p = 0.047, respectively). The decreased mRNA expression correlated with decreased protein expression in large intestine carcinomas (p < 0.001). The qualitative and quantitative pattern of the ASVs studied by droplet digital PCR was confirmed by next-generation sequencing, which suggests the significance of the NGS approach for further massive evaluation of the splicing patterns in a variety of genes.