Kvetoslava Michalova

Next-generation sequencing in the molecular classification of endometrial carcinomas: Experience with 270 cases suggesting a potentially more aggressive clinical behavior of multiple classifier endometrial carcinomas

Molecular classification of endometrial carcinomas (EC) divides these neoplasms into four distinct subgroups based on their molecular background. Given its clinical significance, genetic examination is becoming integral to the diagnostic process. This study aims to share our experience with the molecular classification of EC using immunohistochemistry (IHC) and next-generation sequencing (NGS). We included all ECs diagnosed at two institutions from 2020 to the present. All cases were prospectively examined by IHC for MMR proteins and p53, followed by NGS using a customized panel covering 18 genes, based on which ECs were classified into four molecular subgroups: POLE mutated, hypermutated (MMR deficient), no specific molecular profile (NSMP), and TP53 mutated. The cohort comprised 270 molecularly classified ECs: 18 (6.6%) POLE mutated, 85 (31.5%) hypermutated, 137 (50.7%) NSMP, and 30 (11.1%) TP53 mutated. Twelve cases (4.4%) were classified as 'multiple classifier' EC. Notably, most of these cases with available follow-up (6/9) behaved aggressively. Within the POLEmut EC group, 3/4 cases had advanced tumors, including one patient who died of the disease. Similarly, in the MMRd/TP53mut group, 3/5 patients with available follow-up had metastatic disease, leading to death of the patient in 1 case. ECs of NSMP showed multiple genetic alterations, with the most common mutations being PTEN (44% within the group of NSMP), followed by PIK3CA (30%), ARID1A (21%), and KRAS (9%). Our findings suggest that combining immunohistochemistry with NGS offers a more reliable classification of ECs, including 'multiple classifier' cases, which, based on our observations, tend to exhibit aggressive behavior. Additionally, our data highlight the complex genetic background of NSMP ECs, which can facilitate further stratification of tumors within this group and potentially help select patients for dedicated clinical trials.

Adult granulosa cell tumours of the testis analogous to ovarian counterparts are exceptionally rare: analysis of a multicentric series and review of the literature

Aims Testicular adult granulosa cell tumours (AGCTs) are rare and show several clinical–pathological differences with their ovarian counterparts. In a limited number of prior studies, FOXL2 p.Cys134Trp, the hallmark molecular alteration of ovarian AGCT, appeared to be infrequent in testicular AGCTs. However, the number of cases analysed to date is relatively small. Methods and results Twenty testicular AGCTs were analysed de novo using two different next‐generation sequencing ( NGS ) panels that cover sex cord‐stromal tumour ( SCST )–relevant genes, including FOXL2 , CTNNB1 , FH and DICER1 . Among 12 tumours (12/20; 60%) that were sequenced successfully, none harboured FOXL2 mutations. Eight tumours (8/12, 66.7%) showed a wild‐type ( WT ) status for all genes assessed with the panels. Three tumours harboured pathogenic or likely pathogenic CTNNB1 alterations. One of these exhibited predominant spindle cell morphology, while the other two showed focal tubular architecture. Immunohistochemistry performed in one of these tumours with available material showed β‐catenin expression in ~70% of tumor cell nuclei. The remaining AGCTs showed variants of uncertain significance (likely benign) in KIT and MED12 . Considering the tumors asseseed in this study and those previously reported in the literature, only 2 of 29 neoplasms classified as testicular AGCTs have shown a FOXL2 p. Cys134Trp mutation to date. Conclusions The present study confirms that SCSTs classified as AGCTs differ from their ovarian counterparts in that they largely lack FOXL2 mutations.

Advantages of next-generation sequencing (NGS) in the molecular classification of endometrial carcinomas – our experience with 270 cases

Molecular classification of endometrial carcinomas (EC) divides these neoplasms into four distinct subgroups defined by a molecular background. Given its proven clinical significance, genetic examination is becoming an integral component of the diagnostic procedure. Recommended diagnostic algorithms comprise molecular genetic testing of the POLE gene, whereas the remaining parameters are examined solely by immunohistochemistry. The aim of this study is to share our experiences with the molecular classification of EC, which has been conducted using immunohistochemistry and next-generation sequencing (NGS) at our department. This study includes all cases of EC diagnosed at Šikl's Department of Pathology and Biopticka Laboratory Ltd. from 2020 to the present. All ECs were prospectively examined by immunohistochemistry (MMR, p53), fol lowed by NGS examination using a customized Gyncore panel (including genes POLE, POLD1, MSH2, MSH6, MLH1, PMS2, TP53, PTEN, ARID1A, PIK3CA, PIK3R1, CTNNB1, KRAS, NRAS, BRCA1, BRCA2, BCOR, ERBB2), based on which the ECs were classified into four molecularly distinct groups [POLE mutated EC (type 1), hypermutated (MMR deficient, type 2), EC with no specific molecular profile (type 3), and TP53 mutated ("copy number high", type 4)]. The cohort comprised a total of 270 molecularly classified ECs. Eighteen cases (6.6%) were classified as POLE mutated EC, 85 cases (31.5%) as hypermutated EC (MMR deficient), 137 cases (50.7%) as EC of no specific molecular profile, and 30 cases (11.1%) as TP53 mutated EC. Twelve cases (4.4%) were classified as "multiple classifier" endometrial carcinoma. ECs of no specific molecular profile showed multiple genetic alterations, with the most common mutations being PTEN (44% within the group of NSMP), fol lowed by PIK3CA (30%), ARID1A (21%), and KRAS (9%). In comparison with recommended diagnostic algorithms, NGS provides a more reliable classification of EC into particular molecular subgroups. Furthermore, NGS reveals the complex molecular genetic background in individual ECs, which is especially significant within ECs with no specific molecular profile. These data can serve as a springboard for the research of therapeutic programs committed to targeted therapy in this type of tumor.