Decreased HER2 expression in endometrial cancer following anti-HER2 therapy.

Trastuzumab has demonstrated clinical efficacy in the treatment of HER2-positive serous endometrial cancer (EC), which led to its incorporation into standard-of-care management of this aggressive disease. Acquired resistance remains an important challenge, however, and its underlying mechanisms in EC are unknown. To define the molecular changes that occur in response to anti-HER2 therapy in EC, targeted next-generation sequencing (NGS), HER2 immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) were performed on pre- and post-treatment tumour samples from 14 patients with EC treated with trastuzumab or trastuzumab emtansine. Recurrent tumours after anti-HER2 therapy acquired additional genetic alterations compared with matched pre-treatment ECs and frequently showed decreased HER2 protein expression by IHC (7/14, 50%). Complete/near-complete absence of HER2 protein expression (score 0/1+) observed post-treatment (4/14, 29%) was associated with retained HER2 gene amplification (n = 3) or copy number neutral status (n = 1). Whole-exome sequencing performed on primary and recurrent tumours from the latter case, which exhibited genetic heterogeneity of HER2 amplification in the primary tumour, revealed selection of an early HER2-non-amplified clone following therapy. Our findings demonstrate that loss of target expression, by selection of HER2-non-amplified clones or, more commonly, by downregulation of expression, may constitute a mechanism of resistance to anti-HER2 therapy in HER2-positive EC. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expanding the Spectrum of NR4A3 Fusion-Positive Gynecologic Leiomyosarcomas.

Recurrent gene fusions have been observed in epithelioid and myxoid variants of uterine leiomyosarcoma. PGR::NR4A3 fusions were recently described in a subset of epithelioid leiomyosarcomas exhibiting rhabdoid morphology. In this study, we sought to expand the clinical, morphologic, immunohistochemical, and genetic features of gynecologic leiomyosarcomas harboring NR4A3 rearrangements with PGR and novel fusion partners. We identified 9 gynecologic leiomyosarcomas harboring PGR::NR4A3, CARMN::NR4A3, ACTB::NR4A3, and possible SLCO5A1::NR4A3 fusions by targeted RNA sequencing. Tumors frequently affected premenopausal women, involving the uterine corpus, uterine cervix, or pelvis. All were similarly characterized by lobules of monomorphic epithelioid and/or spindled cells arranged in sheets, cords, trabeculae, and micro- and macrocysts associated with abundant myxoid matrix and hemorrhage, creating labyrinth-like or pulmonary edema-like architecture. Myogenic differentiation with frequent estrogen receptor and progesterone receptor staining and no CD10 expression characterized all tumors. All cases showed high NR4A3 RNA expression levels and NOR1 (NR4A3) nuclear staining similar to salivary gland acinic cell carcinomas and a subset of extraskeletal myxoid chondrosarcomas harboring NR4A3 rearrangements. NOR1 (NR4A3) immunohistochemistry may serve as a useful diagnostic marker of NR4A3 fusion-positive gynecologic leiomyosarcomas.

Concurrent POLE hotspot mutations and mismatch repair deficiency/microsatellite instability in endometrial cancer: A challenge in molecular classification.

OBJECTIVE: Endometrial carcinoma (EC) has different molecular subtypes associated with varied prognosis. We sought to characterize the molecular features of ECs with POLE hotspot mutations and concurrent mismatch repair (MMR) deficiency/high microsatellite instability (MSI). METHODS: We identified POLE-mutated (POLEmut), MMR-deficient (MMRd)/MSI-high (MSI-H), or combined POLEmut/MMRd ECs subjected to clinical tumor-normal panel sequencing between 2014 and 2023. Clonality of somatic mutations, MSI scoring, tumor mutational burden (TMB), proportion of somatic insertions and deletions (indels), and single base substitution (SBS) mutational signatures were extracted. RESULTS: We identified 41 ECs harboring POLE exonuclease domain hotspot mutations, 138 MMRd and/or MSI-H ECs, and 14 POLEmut/MMRd ECs. Among the 14 POLEmut/MMRd ECs, 11 (79 %) exhibited clonal POLE hotspot mutations; 4 (29 %) had a dominant POLE-related mutational signature, 4 (29 %) displayed dominant MMRd-related signatures, and 6 (43 %) had mixtures of POLE, aging/clock, MMRd, and POLEmut/MMRd-related SBS mutational signatures. The number of single nucleotide variants was higher in POLEmut/MMR-proficient (MMRp) and in POLEmut/MMRd ECs compared to POLE wild-type (wt)/MMRd EC (both p < 0.001). Small indels were enriched in POLEwt/MMRd ECs (p < 0.001). TMB was highest in POLEmut/MMRd EC compared to POLEmut/MMRp and POLEwt/MMRd ECs (both p < 0.001). Of 14 patients with POLEmut/MMRd EC, 21 % had a recurrence, versus 10 % of those with POLEmut/MMRp EC. Similar findings were noted in 3 POLEmut ECs in patients with Lynch syndrome; akin to somatic POLEmut ECs, these tumors had high TMB. CONCLUSION: POLEmut/MMRd ECs may be genetically distinct. Further studies are needed to assess the impact on outcomes and treatment response within this population.

Lymph node metastases in endometrial carcinoma: A modern assessment in the era of sentinel lymph node mapping and molecular subtyping.

OBJECTIVE: To examine the risk of sentinel lymph node (SLN) metastases in apparent uterine-confined endometrial cancer (EC) using molecular classification with clinicopathologic features and assess oncologic outcomes by molecular subtypes with micro- or macro-metastases in SLN. METHODS: Patients undergoing surgical staging for presumed uterine-confined EC of any histology, with successful bilateral SLN mapping were included. Primary tumors were assigned molecular subtypes using a published algorithm. SLN pathology was categorized as negative, isolated tumor cells (ITCs), or micro- or macro-metastases. RESULTS: Overall, 756 patients were included; 80 (10 %) had micro- or macro-metastases and 51 (7 %) had ITCs. On multivariate multinomial logistic regression, risk of micro- or macro-metastases versus negative SLN was higher for ECs with copy number-high (CN-H)/TP53abn (OR 3.1; 95 % CI 1.3-7), lymphovascular space invasion ([LVSI]; OR 8.0; 95 % CI 4-16), and deep myoinvasion (≥50 %; OR 3.33; 95 % CI 1.9-6.04). Three-year PFS rates by subtype for 68 patients with macro-metastases were 38 % (95 % CI 10-67 %) CN-low/no specific molecular subtype (CN-L/NSMP), 66 % (95 % CI 44-82 %) microsatellite instability-high (MSI-H), and 23 % (95 % CI 10-40 %) CN-H/TP53abn (p = 0.006). Three-year OS rates were 55 % (95 % CI 20-80 %) CN-L/NSMP, 83 % (95 % CI 61-93 %) MSI-H, and 55 % (95 % CI 34-71 %) CN-H/TP53abn (p = 0.048). CONCLUSIONS: Integrating molecular subtype with uterine risk factors (LVSI and myoinvasion) further stratifies risk of occult SLN metastases in patients undergoing surgical staging for early-stage EC. No molecular subgroup had exceedingly low SLN metastases detected, supporting continued universal SLN assessment. Patients with macro-metastases and CN-L/NSMP or CN-H/TP53abn EC had worse outcomes than those with MSI-H EC.

The Significance of International Federation of Gynecology and Obstetrics Grading in Microsatellite Instability-High and POLE-Mutant Endometrioid Endometrial Carcinoma.

With the advancement of diagnostic molecular technology and the molecular classification of endometrial endometrioid carcinoma (EEC), it remains to be seen whether conventional International Federation of Gynecology and Obstetrics (FIGO) grading retains clinical significance in certain molecular subtypes of EECs. In this study, we explored the clinical significance of FIGO grading in microsatellite instability-high (MSI-H) and POLE-mutant EECs. A total of 162 cases of MSI-H EECs and 50 cases of POLE-mutant EECs were included in the analysis. Significant differences in tumor mutation burden (TMB), progression-free survival, and disease-specific survival were seen between the MSI-H and POLE-mutant cohorts. Within the MSI-H cohort, there were statistically significant differences in TMB and stage at presentation across FIGO grades, but not survival. Within the POLE-mutant cohort, there was significantly greater TMB with increasing FIGO grade, but there were no significant differences in stage or survival. In both the MSI-H and POLE-mutant cohorts, log-rank survival analysis showed no statistically significant difference in progression-free and disease-specific survival across FIGO grades. Similar findings were also seen when a binary grading system was utilized. Since FIGO grade was not associated with survival, we conclude that the intrinsic biology of these tumors, characterized by their molecular profile, may override the significance of FIGO grading.

Molecular-Based Immunohistochemical Algorithm for Uterine Leiomyosarcoma Diagnosis.

The morphologic assessment of uterine leiomyosarcoma (LMS) may be challenging, and diagnostic immunohistochemical (IHC) analysis is currently lacking. We evaluated the genomic landscape of 167 uterine LMS by targeted next-generation sequencing (NGS) to identify common genomic alterations. IHC analyses corresponding to these genomic landmarks were applied to a test cohort of 16 uterine LMS, 6 smooth muscle tumors of uncertain malignant potential (STUMP), and 6 leiomyomas with NGS data and a validation cohort of 8 uterine LMS, 12 STUMP, 21 leiomyomas and leiomyoma variants, 7 low-grade endometrial stromal sarcomas, and 2 diagnostically challenging uterine smooth muscle tumors. IHC results were individually interpreted by 3 pathologists blinded to NGS data. Overall, 94% of LMS showed ≥1 genomic alteration involving TP53, RB1, ATRX, PTEN, CDKN2A, or MDM2, with 80% showing alterations in ≥2 of these genes. In the test cohort, an initial panel of p53, Rb, PTEN, and ATRX was applied, followed by a panel of DAXX, MTAP, and MDM2 in cases without abnormalities. Abnormal p53, Rb, PTEN, and ATRX IHC expression was seen in 75%, 88%, 44%, and 38% of LMS, respectively, in the test cohort. Two or more abnormal IHC results among these markers were seen in 81% of LMS. STUMPs demonstrated only 1 IHC abnormality involving these markers. No IHC abnormalities were seen in leiomyomas. In the validation cohort, abnormal p53, Rb, and PTEN IHC results were seen in LMS, whereas rare STUMP or leiomyomas with bizarre nuclei showed IHC abnormalities involving only 1 of the markers. Abnormalities in ≥2 markers were present in both diagnostically challenging smooth muscle tumors, confirming LMS. Concordance was excellent among pathologists in the interpretation of IHC (κ = 0.97) and between IHC and NGS results (κ = 0.941). Uterine LMS exhibit genomic landmark alterations for which IHC surrogates exist, and a diagnostic algorithm involving molecular-based IHC may aid in the evaluation of unusual uterine smooth muscle tumors.

Landscape of chromatin remodeling gene alterations in endometrial carcinoma.

OBJECTIVE: Chromatin remodeling genes (CRGs) encode components of epigenetic regulatory mechanisms and alterations in these genes have been identified in several tumor types, including gynecologic cancers. In this study, we sought to investigate the prevalence and clinicopathological associations of CRG alterations in endometrial carcinoma (EC). METHODS: We performed a retrospective analysis of 660 ECs sequenced using a clinical massively parallel sequencing assay targeting up to 468 genes, including 25 CRGs, and defined the presence of somatic CRG alterations. Clinicopathologic features were obtained for all cases. Immunohistochemical interrogation of ARID1A and PTEN proteins was performed in a subset of samples. RESULTS: Of the 660 ECs sequenced, 438 (66.4%) harbored CRG alterations covered by our panel. The most commonly altered CRG was ARID1A (46%), followed by CTCF (21%), KMT2D (18%), KMT2B (17%), BCOR (16%), ARID1B (12%) and SMARCA4 (11%). We found that ARID1A genetic alterations were preferentially bi-allelic and often corresponded to altered ARID1A protein expression in ECs. We further observed that ARID1A alterations were often subclonal when compared to PTEN alterations, which were primarily clonal in ECs harboring both mutations. Finally, CRG alterations were associated with an increased likelihood of myometrial and lymphovascular invasion in endometrioid ECs. CONCLUSION: CRG alterations are common in EC and are associated with clinicopathologic features and likely play a crucial role in EC.

TERT promoter mutations and gene amplification in endometrial cancer.

OBJECTIVE: To assess the clinicopathologic, molecular profiles, and survival outcomes of patients with endometrial carcinomas (ECs) harboring telomerase reverse transcriptase (TERT) hotspot mutations or gene amplification. METHODS: ECs harboring somatic TERT promoter hotspot mutations or gene amplification (TERT-altered) were identified from 1944 ECs that underwent clinical tumor-normal sequencing from 08/2016-12/2021. Clinicopathologic variables, somatic mutation profiles, and survival outcomes of TERT-alt and TERT-wild-type EC were assessed. RESULTS: We identified 66 TERT-altered ECs (43 TERT-mutated and 23 TERT-amplified), representing 3% of the unselected ECs across histologic subtypes. Most TERT-altered ECs were of copy number (CN)-high/TP53abn molecular subtype (n = 40, 60%), followed by microsatellite-unstable (MSI-H) or CN-low/no specific molecular profile (NSMP)(n = 13, 20% each). TERT-amplified and TERT-mutated ECs were molecularly distinct, with TERT-amplified ECs being more genomically instable and more frequently harboring TP53 and PPP2R1A alterations (q < 0.1). Compared to TERT-wild-type ECs, TERT-altered ECs were more commonly of CN-H/TP53abn molecular subtype (31% vs 57%, p = 0.001), serous histology (10% vs 26%, p = 0.004), and were significantly enriched for TP53, CDKN2A/B, and DROSHA somatic genetic alterations (q < 0.1). Median progression-free survival was 18.7 months (95% CI 11.8-not estimable [NE]) for patients with TERT-altered EC and 80.9 months (65.8-NE) for patients with TERT-wild-type EC (HR 0.33, 95% CI 0.21-0.51, p < 0.001). Similarly, median overall survival was 46.7 months (95% CI 30-NE) for TERT-altered EC patients and not reached for TERT-wild-type EC patients (HR 0.24, 95% CI 0.13-0.44, p < 0.001). CONCLUSION: TERT-altered ECs, although rare, are enriched for CN-high/TP53abn tumors, TP53, CDKN2A/B and DROSHA somatic mutations, and independently predict worse survival outcomes.

Integration of clinical sequencing and immunohistochemistry for the molecular classification of endometrial carcinoma.

PURPOSE: Using next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data. EXPERIMENTAL DESIGN: All patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution. RESULTS: Utilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949-0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC. CONCLUSIONS: The integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification.

Molecular landscape of vulvovaginal squamous cell carcinoma: new insights into molecular mechanisms of HPV-associated and HPV-independent squamous cell carcinoma.

Squamous cell carcinomas of the lower female genital tract may be human papillomavirus-associated or independent. We studied the HPV status, mutational repertoire, histology, and clinical data of 28 samples from 26 patients, 65% with a vulvar primary and 35% with a vaginal primary. These represented invasive vulvovaginal squamous cell carcinomas that underwent clinical tumor-normal targeted massively parallel sequencing analysis. HPV status was determined using the HPV high-risk RNA ISH assay and/or by MSK-IMPACT. Eleven patients had HPV-associated squamous cell carcinoma (four vulvar and seven vaginal) and 15 patients had HPV-independent SqCC (13 vulvar and 2 vaginal). Well-differentiated squamous cell carcinomas were always HPV-independent. HPV-independent moderately and poorly differentiated carcinomas frequently had alterations in the NOTCH signaling pathway (6/7), which were also associated with increased tumor budding (P: 0.002). HPV-associated vulvovaginal squamous cell carcinoma had PIK3CA activating mutations (7/11, 64%) as the most common genomic event, while TERT gene alterations, mainly TERT promoter mutations (14/15 cases, 93%) featured significantly in HPV-independent carcinomas. Other common abnormalities in HPV-independent tumors were TP53 mutations (13/15, 87%), CDKN2A alterations (10/15, 67%), and NOTCH1 and FAT1 mutations (7/15, 47% each). A subset of both HPV-associated and -independent tumors had NOTCH pathway alterations (6/11, 55% and 10/15, 67% respectively), but different genes in this pathway were altered in these tumors. In summary, TERT, TP53, CDKN2A, and NOTCH1 gene alterations strongly point away from an HPV-driven process (odds ratios: 0.01, 0.07, 0, and 0, respectively with p values < 0.02 for all four genes), while PIK3CA activating mutations without the other mutations strongly favors an HPV-driven tumor (odds ratio: 10.12, p value: 0.016). HPV-independent carcinomas are more likely to be moderately-poorly differentiated with intermediate to high tumor cell budding. Cancer cell fraction analysis of HPV-independent squamous carcinomas suggests that TERT and/or NOTCH1 alterations along with TP53 alterations can be the initiating event in these tumors.

Genomic landscape of endometrial carcinomas of no specific molecular profile.

Endometrial carcinomas (ECs) classified by The Cancer Genome Atlas (TCGA) as copy number-low (also referred to as "no specific molecular profile" [NSMP]) have a prognosis intermediate between POLE-mutated and copy number-high ECs. NSMP-ECs are a heterogeneous group, however, comprising both relatively indolent and aggressive ECs. We identified a total of 472 NSMP-ECs among 1,239 ECs that underwent clinical sequencing of 410-468 cancer-related genes. Somatic mutation and copy number alteration data were subjected to unsupervised hierarchical clustering, which identified three genomic clusters. Random sampling with stratification was used to choose ~80 endometrioid ECs from each cluster, resulting in a study size of 240 endometrioid ECs as well as an additional 44 non-endometrioid NSMP-ECs. Cluster 1 (C1, n = 80) consisted primarily of NSMP-ECs with PTEN and PIK3R1 mutations, Cluster 2 (C2, n = 81) of tumors with PTEN and PIK3CA mutations and Cluster 3 (C3, n = 79) of NSMP-ECs with chromosome 1q high-level gain and lack of PTEN mutations. The majority (72.7%) of non-endometrioid NSMP-ECs mapped to C3. NSMP-ECs from C3 were more likely to be FIGO grade 3 (30%), estrogen receptor-negative/weak (54.5%) and FIGO stages III or IV. In multivariate analysis, molecular clusters were associated with worse overall survival outcomes with C3 tumors having the worst (hazard ratio: 4) and C1 tumors having the best outcome. In conclusion, NSMP-ECs are a heterogenous group of tumors and comprise both aggressive and clinically low-risk ECs that can be identified based on mutation and copy number data.

TSC2-mutant uterine sarcomas with JAZF1-SUZ12 fusions demonstrate hybrid features of endometrial stromal sarcoma and PEComa and are responsive to mTOR inhibition.

Uterine perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasm that occasionally shares morphologic and immunohistochemical overlap with low- and high-grade endometrial stromal sarcoma (LGESS and HGESS). In this study, we sought to characterize the clinical, morphologic, genetic, and epigenetic features of five uterine sarcomas that display histologic features of LGESS, HGESS, and PEComa. All tumors demonstrated epithelioid cells often associated with a low-grade spindled component resembling LGESS, with both regions expressing CD10, ER, PR, variable HMB45, and Melan-A immunoreactivity, and strong cathepsin K and pS6 expression. Targeted massively parallel sequencing analysis revealed the presence of somatic TSC2 mutations in all five cases, of which four harbored concurrent or consecutive JAZF1-SUZ12 gene fusions. Unsupervised hierarchical clustering analysis of methylation profiles of TSC2-mutant uterine sarcomas (n = 4), LGESS (n = 10), and HGESS (n = 12) demonstrated two clusters consisting of (1) all LGESS and TSC2-mutant uterine sarcomas and (2) all HGESS. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, calcium, and Rap1 signaling. TSC2-mutant uterine sarcomas were responsive to hormone suppression, and mTOR inhibition demonstrated clinical benefit in four patients with these neoplasms. Our results suggest that these tumors represent histologically distinctive LGESS with TSC2 mutations. TSC2 mutations and JAZF1-SUZ12 fusion may help diagnose these tumors and possibly direct effective treatment.

Mullerian adenosarcoma: clinicopathologic and molecular characterization highlighting recurrent BAP1 loss and distinctive features of high-grade tumors.

Mullerian adenosarcoma is an uncommon mesenchymal tumor of the gynecologic tract. Most cases are low-grade, while high-grade adenosarcomas are rare and not well studied. Herein, we characterize the clinicopathologic and molecular features of 27 adenosarcomas of gynecologic origin, enriched for high-grade tumors subjected to targeted panel sequencing. Sarcomatous overgrowth was more frequently seen in high-grade compared to low-grade tumors (12/17, 71%, vs 1/10, 10%, p = 0.004) and heterologous elements were exclusive to high-grade cases (n = 7, p = 0.03). All deaths were from high-grade disease (advanced primary, n = 2, or recurrence, n = 5). Genetic alterations specific to high-grade adenosarcomas have known associations with chromosome instability, including TP53 mutations (n = 4) and amplifications of MDM2 (n = 2) and CCNE1 (n = 2). Somatic ATRX frameshift mutations were found in 2 patients with high-grade recurrences following a primary low-grade adenosarcoma and ATRX deletion in 1 high-grade adenosarcoma with an adjacent low-grade component. The fraction of genome altered by copy number alterations was significantly higher in high-grade compared to low-grade adenosarcomas (P = 0.001). Other recurrent genetic alterations across the entire cohort included BAP1 homozygous deletions (n = 4), DICER1 mutations (n = 4), ARID1A mutations (n = 3), TERT promoter mutations (n = 2) and amplification (n = 1), as well as alterations involving members of the PI3K and MAPK signaling pathways. One tumor harbored an ESR1-NCOA3 fusion and another had an MLH1 homozygous deletion. Immunohistochemical analysis for BAP1 revealed loss of nuclear expression in 6/24 (25%) cases, including all four tumors with BAP1 deletions. Notably, out of 196 mesenchymal neoplasms of gynecologic origin, BAP1 homozygous deletion was only found in adenosarcomas (P = 0.0003). This study demonstrates that high-grade adenosarcomas are heterogeneous at the molecular level and are characterized by genomic instability and TP53 mutations; ATRX loss may be involved in high-grade transformation of low-grade adenosarcoma; and BAP1 inactivation appears to be a specific pathogenic driver in a subset of adenosarcomas.

ERBB2 amplification status in 67 salivary duct carcinomas assessed by immunohistochemistry, fluorescence in situ hybridization, and targeted exome sequencing.

Salivary duct carcinoma (SDC) is an aggressive salivary gland malignancy with poor survival. Approximately 30% SDC harbor HER2 amplification and response to trastuzumab has been reported. However, a systematic approach for HER2 status assessment in this tumor type has not been established. A total of 67 tumor samples were evaluated for HER2 protein overexpression or ERBB2 gene amplification using at least 2 methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and/or targeted exome next-generation sequencing (NGS). NGS assessed ERBB2 copy number fold change (FC) and total copy number (TCN). HER2 status was first determined by IHC/FISH according to the 2018 ASCO/CAP breast cancer guidelines. FISH results, the "gold standard", were compared with the NGS results. All (15/15) IHC positive, 35% (6/17) equivocal, and no (0/19) IHC negative SDC were HER2 amplified by FISH. HER2 FISH signal/cell showed a good correlation with FC (Spearman correlation: 0.708, R2: 0.501, p < 0.0001) and TCN (Spearman correlation: 0.763, R2: 0.582, p < 0.0001). Receiver operating characteristics curve estimation showed an area under curve (AUC) of 0.975 for ERBB2 FC. FC cutoff of ≥1.8 corresponded to an accuracy of 95.2% for ERBB2 amplification (Youden's index: 0.84, sensitivity: 89.47%, specificity: 100%). FC < 1.3 could be reliably classified as ERBB2 not amplified and FC ≥ 1.3 and <1.8 as equivocal. TCN estimation showed AUC of 0.981. TCN cutoff of >6.0 corresponded to an accuracy of 92% for HER2 amplification (Youden's index: 0.81, sensitivity: 81.2%, specificity: 100%). TCN < 4 could be reliably classified as ERBB2 not amplified and TCN ≥ 4.0 and ≤6.0 as equivocal. FC and TCN were binarized with respective cutoffs of ≥1.8 and ≥6.0 and the proportion of agreement with FISH were 95% and 92%, respectively. The assessment of ERBB2 copy number by NGS is accurate and reliable with FC or TCN nearly equivalent to FISH in identifying HER2 amplified SDC.

Targeted RNA expression profiling identifies high-grade endometrial stromal sarcoma as a clinically relevant molecular subtype of uterine sarcoma.

High-grade endometrial stromal sarcoma (HGESS) may harbor YWHAE-NUTM2A/B fusion, ZC3H7B-BCOR fusion, and BCOR internal tandem duplication (ITD). NTRK3 upregulation and pan-Trk expression were reported in soft tissue lesions that share similar morphology and genetic abnormalities. To confirm these findings in HGESS, differential expression analysis was performed at gene level comparing 11 HGESS with 48 other uterine sarcomas, including 9 low-grade endometrial stromal sarcomas, 23 undifferentiated uterine sarcomas, and 16 leiomyosarcomas, using targeted RNA sequencing data. Pan-Trk immunohistochemistry was performed on 35 HGESS, including 10 tumors with RNA expression data, with genotypes previously confirmed by targeted RNA sequencing, fluorescence in situ hybridization, and/or genomic PCR. Unsupervised hierarchical clustering of the top 25% of differentially expressed probes identified three molecular groups: (1) high NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1 and low ESR1 expression; (2) low NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1 and high ESR1 expression; and (3) low NTRK3, FGFR3, RET, BCOR, GLI1, PTCH1, and ESR1 expression. Among HGESS, 64% of tumors clustered in group 1, while 27% clustered in group 2. Cytoplasmic and/or nuclear pan-Trk staining of variable extent and intensity was seen in 91% of HGESS regardless of cyclin D1 and/or BCOR positivity. ER and PR expression was seen in 44% of HGESS despite ESR1 downregulation. Two patients with ER and PR positive but ESR1 downregulated stage I HGESS were treated with endocrine therapy, and both recurred at 12 and 36 months after primary resection. By RNA expression, HGESS appear homogenous and distinct from other uterine sarcomas by activation of kinases, including NTRK3, and sonic hedgehog pathway genes along with downregulation of ESR1. Most HGESS demonstrate pan-Trk staining which may serve as a diagnostic biomarker. ESR1 downregulation is seen in some HGESS that express ER and PR which raises implications in the utility of endocrine therapy in these patients.

Characterization of TP53-wildtype tubo-ovarian high-grade serous carcinomas: rare exceptions to the binary classification of ovarian serous carcinoma.

While TP53 mutation is widely considered to be a defining feature of tubo-ovarian high-grade serous carcinoma (HGSC), rare TP53-mutation-negative cases have been reported. To gain further insight into this rare subset, a retrospective review was conducted on 25 TP53-wildtype tubo-ovarian HGSCs, constituting 2.5% of 987 HGSCs profiled by the MSK-IMPACT sequencing platform. Consistent with serous differentiation, positive staining for Pax8 and WT1 was present in virtually all TP53-wildtype HGSCs. Other characteristic features of HGSC, such as serous tubal intraepithelial carcinoma, or genetic alterations of CCNE1 and BRCA1/2 were identified in these tumors, furthering supporting their classification as bona fide HGSC, despite lacking TP53 mutations. Overall, the level of chromosomal instability of TP53-wildtype HGSCs was intermediate between low-grade serous carcinoma (LGSC) and TP53-mutated HGSC. Morphologic assessment by observers blinded to mutation status revealed a significant subset of tumors with Grade 2 nuclear atypia (which exceeds the degree of atypia allowed for LGSC, but less than typically encountered for HGSC) combined with micropapillary features (6/19, 32%, chemotherapy-naive TP53-wildtype HGSCs compared to 0/21, 0%, TP53-mutated HGSCs; p = 0.007). Some TP53-wildtype HGSCs harbored driver mutations in KRAS (n = 3), BRAF (n = 1) or NRAS (n = 2). Overall, 10 (40%) cases had "LGSC-like" morphology (i.e., Grade 2 nuclear atypia and micropapillary features) and/or RAS/RAF mutation, and most of these showed a wildtype p53 pattern of expression by immunohistochemistry (7/9, 78%). The remaining TP53-wildtype HGSCs (n = 15, 60%) exhibited severe nuclear atypia (Grade 3) and were morphologically indistinguishable from conventional TP53-mutated HGSC. Despite lacking genetic alterations of TP53, these "usual HGSC-like" tumors often showed evidence of p53 dysfunction, including downregulation of expression ('null' or equivocal p53 staining in 9/14, 64%) or MDM2 amplification (n = 2). Our results support the existence of TP53-wildtype HGSCs, which comprise a heterogeneous group of tumors which may arise via distinct pathogenic mechanisms.

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.

DNA methylation-based classification of sinonasal undifferentiated carcinoma.

Sinonasal undifferentiated carcinoma (SNUC) is an aggressive malignancy harboring IDH2 R172 mutations in >80% cases. We explored the potential of genome-wide DNA methylation profiling to elucidate tumor biology and improve the diagnosis of sinonasal undifferentiated carcinoma and its histologic mimics. Forty-two cases, including sinonasal undifferentiated, large cell neuroendocrine, small cell neuroendocrine, and SMARCB1-deficient carcinomas and olfactory neuroblastoma, were profiled by Illumina Infinium Methylation EPIC array interrogating >850,000 CpG sites. The data were analyzed using a custom bioinformatics pipeline. IDH2 mutation status was determined by the targeted exome sequencing (MSK-IMPACTTM) in most cases. H3K27 methylation level was assessed by the immunohistochemistry-based H-score. DNA methylation-based semi-supervised hierarchical clustering analysis segregated IDH2 mutants, mostly sinonasal undifferentiated (n = 10) and large cell neuroendocrine carcinomas (n = 4), from other sinonasal tumors, and formed a single cluster irrespective of the histologic type. t-distributed stochastic neighbor embedding dimensionality reduction analysis showed no overlap between IDH2 mutants, SMARCB1-deficient carcinoma and olfactory neuroblastoma. IDH2 mutants demonstrated a global methylation phenotype and an increase in repressive trimethylation of H3K27 in comparison to IDH2 wild-type tumors (p < 0.001). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed no difference in pathway activation between IDH2-mutated sinonasal undifferentiated and large cell neuroendocrine carcinomas. In comparison to SMARCB1-deficient, IDH2-mutated carcinomas were associated with better disease-free survival (p = 0.034) and lower propensity for lung metastasis (p = 0.002). ARID1A mutations were common in small cell neuroendocrine carcinoma but not among IDH2 mutants (3/3 versus 0/18 and p < 0.001). IDH2 mutations in sinonasal carcinomas induce a hypermethylator phenotype and define a molecular subgroup of tumors arising in this location. IDH2-mutated sinonasal undifferentiated carcinoma and large cell neuroendocrine carcinoma likely represent a phenotypic spectrum of the same entity, which is distinct from small cell neuroendocrine and SMARCB1-deficient sinonasal carcinomas. DNA methylation-based analysis of the sinonasal tumors has potential to improve the diagnostic accuracy and classification of tumors arising in this location.

Chorangiomatosis: Evaluation of a placental vascular lesion and related clinical effects.

BACKGROUND: Chorangiomatosis is a unique placental vascular abnormality that can cause growth retardation and even fetal demise in severe cases. In this study we aim to better understand this lesion and the possible clinical implications. METHODS AND MATERIALS: In this study 170 placentas were evaluated, both grossly and microscopically. The patients' charts were reviewed and relevant clinical data were extracted. In the histological examination, presence of placental lesions including chorangiomatosis (focal, multifocal and diffuse) and chorangiosis was determined and possible correlation between placental findings and clinical outcomes investigated. RESULTS: Among the 170 placentas examined, 42 cases of multifocal chorangiomatosis (25.6%), 7 cases with diffuse chorangiomatosis (4.26%), and 56 cases of focal chorangiomatosis (34.1%) were identified. We found that there is a significant correlation between multifocal/diffuse chorangiomatosis and adverse clinical outcomes including lower birth weight and NICU admission. CONCLUSION: Chorangiomatosis can significantly affect the outcomes of pregnancy and more research is needed to better understand this lesion.

Presence and extent of lymphovascular invasion in surgical stage I squamous cell carcinoma of the cervix: a comprehensive, international, multicentre, retrospective clinicopathological study.

The aim of this study was to determine whether the presence and extent of lymphovascular invasion (LVI) is prognostic in surgical stage I cervical squamous cell carcinoma (SCC). All available tumour slides and/or paraffin blocks from 426 patients with stage I cervical SCC treated surgically with curative intent were collected from 18 institutions and retrospectively analysed. Presence and extent of LVI (focal <5 spaces, extensive ≥5 spaces) were assessed on scanning magnification in large haematoxylin and eosin slide sets in 366 cases. Progression-free survival (PFS) was calculated as the time from surgery to first progression or death or last follow-up, whichever occurred first. Overall survival (OS) was defined as the time from surgery to death or last follow-up. Clinicopathological and statistical analyses were performed on 97 patients with the International Federation of Gynecology and Obstetrics (FIGO) 2018 stage IA and 329 patients with stage IB SCC of the cervix. LVI, both focal and extensive, was more frequent in stage IB than in stage IA (p<0.001). Patients with stage IB carcinomas with extensive LVI had worse PFS [hazard ratio (HR) 2.86; 95% confidence interval (CI) 1.49, 5.49; p=0.005] and OS (HR 2.88; 95% CI 1.38, 6.02; p=0.012) than those with focal or no LVI. In stage IA, in contrast, the presence and extent of LVI did not associate with PFS (p=0.926) or OS. Extensive LVI was not statistically correlated with PFS and OS in substages IA1, IA2 or IB2. PFS (HR 3.7; 95% CI 1.61, 8.46; p<0.001) and OS (HR 4.18; 95% CI 1.58, 11.04; p=0.002) in stage IB1, and PFS (HR 7.78; 95% CI 0.87, 69.82; p=0.039) in stage IB3 were diminished in the presence of extensive LVI. In conclusion, in patients with FIGO stage I cervical SCC, the presence and extent of LVI has prognostic significance in stage IB carcinoma, and quantifying LVI is recommended.