M. Herman Chui

Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. Damaging mutations in the SWI/SNF chromatin remodeling complex, such as SMARCA4 (BRG1), are associated with improved response to immune checkpoint blockade; however, the mechanism by which this occurs is unclear. We found that SMARCA4 loss in OC models resulted in increased cancer cell–intrinsic immunogenicity, characterized by up-regulation of long-terminal RNA repeats, increased expression of interferon-stimulated genes, and up-regulation of antigen presentation machinery. Notably, this response was dependent on STING, MAVS, and IRF3 signaling but was independent of the type I interferon receptor. Mouse ovarian and melanoma tumors with SMARCA4 loss demonstrated increased infiltration and activation of cytotoxic T cells, NK cells, and myeloid cells in the tumor microenvironment. These results were recapitulated in BRG1 inhibitor–treated SMARCA4- proficient tumor models, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to overcome cancer immune evasion.

Clinicopathologic Features, Molecular Landscape, and Prognostic Implications of Ovarian Low-grade Serous Tumors with Histologic Transformation

Abstract Purpose: The purpose of this study was to characterize the clinicopathologic features, molecular genetic landscape, and clinical behavior of ovarian low-grade serous tumors with histologic transformation (LGS-HT) to indeterminate/high-grade carcinoma. Experimental Design: LGS-HT were retrospectively identified from an institutional cohort of patients with ovarian cancer and underwent central pathology re-review. Data on clinicopathologic characteristics, including age, stage, surgical outcomes, systemic treatments, and overall survival (OS), were collected. IHC profiling and next-generation sequencing were performed. OS comparisons were performed with our institutional cohorts of ovarian low-grade serous carcinoma (n = 109) and high-grade serous carcinoma (n = 1,672). Results: From 4,371 ovarian serous cancers, 40 (0.9%) LGS-HT were identified: 30 with synchronous low-grade and higher-grade tumor components at initial diagnosis and 10 with an ovarian low-grade serous neoplasm that recurred as a higher-grade carcinoma. The most common somatic driver mutations included TP53 (38.5%), KRAS (21.8%), NF1 (15.6%), BRAF (15.6%), and NRAS (12.5%), with coexisting TP53 and RAS/RAF mutations in 18.8% of cases. Alterations in DNA damage response genes (BRCA2, PALB2, CHEK2, ATM, NBN, and RECQL4) were identified in LGS-HT lacking TP53 genetic alterations. Synchronous low-grade and higher-grade tumor components at initial diagnosis were associated with poorer OS (median, 59.7 months) compared with low-grade serous carcinoma (median, 105.4 months; P = 0.026) and were similar to high-grade serous carcinoma (median, 48.8 months; P = 0.61). Severe nuclear atypia and the absence of RAS/RAF-driver mutations were significant adverse prognostic factors. Conclusions: LGS-HT exhibit both low-grade and high-grade morphologic and molecular features, representing an exception to the dualistic classification of ovarian serous neoplasms. The presence of a definitive high-grade carcinoma component in a low-grade serous tumor portends aggressive clinical behavior.

Early Genetic Divergence of High-Grade Carcinomas Originating from Low-Grade Serous Ovarian Neoplasms

The current paradigm implicates a fallopian tube precursor as the origin of most ovarian high-grade serous carcinomas (HGSCs). However, a rare subset of HGSCs develop via a distinct pathway from low-grade serous ovarian neoplasms (namely, serous borderline tumors and low-grade serous carcinoma). This alternate pathway for the development of HGSC and other poorly differentiated carcinomas of the ovary is not well understood. To elucidate the molecular pathogenesis and evolutionary trajectory of histologic transformation of low-grade serous neoplasms, we performed whole exome sequencing on microdissected low-grade and higher-grade components from 7 cases of serous borderline tumor or low-grade serous carcinoma associated with a synchronous or metachronous indeterminate/high-grade carcinoma. In most cases, there were relatively few somatic mutations shared between matched low-grade and higher-grade tumors compared with private mutations specific to each component (ie, phylogenetic trees with short trunks and long branches). Truncal mutations, present across all tumor samples from a given patient, included known drivers of low-grade serous neoplasms: KRAS (G12D, n = 4), BRAF (G469A, n = 1), NF2 (n = 1), and USP9X (n = 1). Transformation to HGSC was associated with a TP53 mutation with bi-allelic inactivation in 3 cases, all with severe nuclear atypia, and associated with genome-wide copy number alterations and allelic imbalances. TP53-wildtype tumors comprised a morphologic spectrum, which included indeterminate-grade serous carcinomas with moderate nuclear atypia and high mitotic activity, although lacking extensive chromosomal instability (n = 2) and poorly differentiated carcinomas (n = 2, including a high-grade Mullerian carcinoma and an undifferentiated carcinoma with sarcomatoid features). In summary, synchronous and metachronous low-grade serous neoplasms and higher-grade carcinomas are clonally related. Early genetic divergence, most evident in cases with TP53 mutations, suggests that high-grade transformation may be a relatively early molecular event.

Decreased HER2 expression in endometrial cancer following anti‐HER2 therapy

Abstract 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.

ERBB2 amplification is a late event in the pathogenesis of high‐grade endometrial carcinomas with heterogeneous HER2 expression

Abstract Intratumor heterogeneity of ERBB2 amplification/HER2 overexpression is frequently observed in ERBB2 ‐amplified high‐grade endometrial carcinoma (HG‐EC) and contributes to anti‐HER2 therapy resistance. To elucidate the molecular pathogenesis and evolutionary trajectory of HER2‐heterogeneous HG‐ECs, we performed next‐generation sequencing of spatially distinct HER2‐negative (HER2−) and HER2‐positive (HER2+) tumor areas from nine tumors (whole exome, n  = 7; targeted panel, n  = 2). HER2− and HER2+ components shared a high proportion of somatic mutations, particularly clonal mutations, including known EC driver genetic alterations. The 17q12 amplicon, containing the ERBB2 gene, was the only significant recurrent copy number alteration that differed between HER2− and HER2+ components. By unsupervised hierarchical clustering of genome‐wide copy number alterations, samples clustered together at the patient level rather than by HER2 status. Intra‐ and intertumor heterogeneity in ERBB2 amplification/HER2 expression was also observed in metastatic lesions, which likely originated from different tumor subpopulations within the primary tumor. Exploratory spatial transcriptomics analyses revealed gene expression differences associated with HER2 status, including a shift from ‘mesenchymal‐like’ toward epithelial differentiation in HER2+ components for a subset of cases, a finding that warrants further investigation. Our results suggest that HER2 heterogeneity in HG‐EC reflects late acquisition of ERBB2 amplification during tumor evolution. ERBB2 does not appear to drive tumor initiation in HER2‐heterogeneous HG‐EC but likely serves a context‐dependent role in the progression of established tumors. © 2026 The Pathological Society of Great Britain and Ireland.

HER2 Genetic Intratumor Heterogeneity Is Associated With Resistance to Trastuzumab and Trastuzumab Emtansine Therapy in Recurrent High-Grade Endometrial Cancer

Anti-HER2 targeted therapies have recently demonstrated clinical activity in the treatment of high-grade endometrial carcinomas (ECs), particularly serous carcinomas with HER2 amplification and/or overexpression. Intratumor heterogeneity of HER2 amplification or HER2 genetic intratumor heterogeneity (G-ITH) has been associated with resistance to anti-HER2 therapies in breast and gastroesophageal cancers; however, its clinical relevance in EC is unknown. To characterize HER2 G-ITH in EC, archival specimens from a clinically annotated cohort of 57 ECs treated with trastuzumab or trasutuzmab emtansine in the recurrent (n = 38) or adjuvant (n = 19) setting were subjected to central pathology review, HER2 assessment by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and next-generation sequencing. HER2 G-ITH, defined as HER2 amplification in 5% to 50% of tumor cells examined by FISH, was identified in 36% (19/53) of ECs and was associated with lower HER2 copy number and levels of protein expression. HER2 IHC revealed spatially distinct areas of strong expression juxtaposed with areas of low/absent expression in tumors with the "cluster" pattern of G-ITH, whereas the "mosaic" pattern was typically associated with a diffuse admixture of cells with variable levels of HER2 expression. HER2 G-ITH was frequently observed in cases with IHC/FISH or FISH/next-generation sequencing discrepancies and/or with an equivocal/negative FISH result (9/13, 69%). Although the objective response rate to anti-HER2 therapy in recurrent ECs was 52% (13/25) for tumors lacking HER2 G-ITH, none (0%, 0/10) of the patients with HER2 G-ITH achieved a complete or partial response (P = .005). HER2 G-ITH was significantly associated with worse progression-free survival (hazard ratio, 2.88; 95% CI, 1.33-6.27; P = .005) but not overall survival. HER2 IHC score, HER2/CEP17 ratio, HER2 copy number, histologic subtype, and other genetic alterations, including PIK3CA hotspot mutations, were not significantly associated with therapeutic response or survival outcomes. Treatment responses were not restricted to serous carcinomas, supporting consideration of anti-HER2 therapy in patients with HER2-positive high-grade ECs of non-serous histology. Our results demonstrate that HER2 G-ITH is an important determinant of response to trastuzumab and trastuzumab emtansine in EC, providing a rationale for the development of novel therapeutic strategies to target HER2-nonamplified resistant tumor subpopulations, such as HER2 antibody-drug conjugates with bystander effects.

ERBB2 mutations define a subgroup of endometrial carcinomas associated with high tumor mutational burden and the microsatellite instability‐high (MSI‐H) molecular subtype

Anti‐HER2 therapy is indicated for erb‐b2 receptor tyrosine kinase 2 ( ERBB2 )‐amplified/overexpressing endometrial carcinoma (EC). Mutations constitute another mode of ERBB2 activation, but only rare ERBB2 ‐mutated ECs have been reported. We sought to characterize the clinicopathologic and genetic features of ERBB2 ‐mutated EC. From an institutional cohort of 2638 ECs subjected to clinical tumor‐normal panel sequencing, 69 (2.6%) with pathogenic ERBB2 mutation(s) were identified, of which 11 were also ERBB2 ‐amplified. The most frequent ERBB2 hotspot mutations were V842I (38%) and R678Q (25%). ERBB2 mutations were clonal in 87% of evaluable cases. Immunohistochemistry revealed low HER2 protein expression in most ERBB2 ‐mutated ECs (0/1+ in 66%, 2+ in 27%); all 3+ tumors (7.3%) were also ERBB2 ‐amplified. Compared to ERBB2 ‐wildtype ECs (with or without ERBB2 amplification), ERBB2 ‐mutated/non‐amplified ECs were enriched for the microsatellite instability‐high (MSI‐H) and, to a lesser extent, DNA polymerase epsilon, catalytic subunit ( POLE ) molecular subtypes, and associated with high tumor mutational burden and low chromosomal instability. Survival outcomes were similar between patients with ERBB2 ‐mutated/non‐amplified versus wildtype EC, whereas ERBB2 amplification was associated with worse prognosis on univariate, but not multivariate, analyses. In conclusion, ERBB2 mutation defines a rare subgroup of ECs that is pathogenically distinct from ERBB2 ‐wildtype and ERBB2 ‐amplified ECs.

Histopathologic features and molecular genetic landscape of HER2-amplified endometrial carcinomas

HER2 is an established therapeutic biomarker in advanced or recurrent endometrial serous carcinoma. Current clinical guidelines recommend HER2 testing exclusively in this endometrial carcinoma (EC) subtype; however, the full spectrum of ECs harboring HER2 amplification remains ill-defined. The present study characterizes the clinicopathologic and molecular features of HER2-amplified ECs across all histologic subtypes. Retrospective analysis of our institutional cohort of 2,042 ECs subjected to targeted clinical massively parallel sequencing identified 77 (3.8%) cases with HER2 amplification, a group comprised of serous (n = 29), endometrioid (low-grade, n = 2, high-grade, n = 1) and clear cell (n = 4) carcinomas, carcinosarcomas (n = 18) and high-grade ECs with ambiguous features (HGEC, n = 23). A co-existing TP53 mutation was identified in 94% (72/77) of HER2-amplified ECs. Other recurrent genetic alterations included amplification of CCNE1 (22%) and ERBB3 (10%), FBXW7 mutations or deletions (13%), and mutations in PIK3CA (40%) and PPP2R1A (13%). The HER2 immunohistochemistry score was 2+ or 3+ for all evaluable cases (n = 61). Apart from carcinosarcomas, which often showed lower HER2 expression, particularly in the sarcomatous component, HER2 immunohistochemical staining pattern and intensity were similar across EC subtypes. Intratumor heterogeneity in HER2 expression was common and correlated with genetic heterogeneity as detected by fluorescence in-situ hybridization. These results demonstrate the frequent co-occurrence of HER2 amplification with TP53 mutation and high-grade histology, rather than being specific to serous carcinoma, per se. Overall, these findings suggest that HER2 targeted therapy may be more broadly applicable to all high-grade EC histotypes and consideration should be given to expanding therapeutic eligibility.

Mixed Ovarian Neoplasms With Gastrointestinal-type Mucinous and Mullerian Epithelial Components

Primary mucinous ovarian neoplasms, gastrointestinal-type (GI-type), are composed of mucin-producing tumor cells resembling intestinal goblet cells or gastric foveolar epithelium. In contrast to seromucinous tumors, which exhibit endocervical-type mucinous differentiation and are thought to be derived from endometriosis, the cell/tissue-of-origin of most GI-type mucinous ovarian tumors is unknown. We identified 8 GI-type mucinous ovarian tumors (cystadenomas, n=4; borderline tumor/carcinoma, n=4) with spatially distinct areas that showed morphologic features of Mullerian-type epithelial differentiation (ciliated cells or endometrioid-type glands). Immunohistochemistry for cell lineage markers and Alcian blue (pH 2.5)/Periodic Acid-Schiff staining were performed. Morphologically distinct components were isolated by microdissection, from which extracted DNA was analyzed by targeted next-generation sequencing. In all cases, immunohistochemistry demonstrated mucin-producing cells to be positive for at least one GI marker (CK20 or CDX2), while areas with morphologic features of Mullerian differentiation were positive for PAX8, ER and/or PR, and lacked expression of CK20 and CDX2; CK7 was strongly and diffusely positive in all tumor cells. Tumor cells with a gastric-type phenotype produced neutral mucin, while acidic mucin was present within intestinal-type goblet cells. Targeted sequencing revealed ARID1A mutations in all mixed borderline tumors/carcinomas (n=4); other recurrent genetic alterations included KRAS (n=2) and TP53 mutations (n=2). Shared mutations were present in paired Mullerian and GI-type mucinous tumor components in 4 mixed borderline tumors/carcinomas, with more shared mutations between components than private mutations specific to each component. All mixed borderline tumors/carcinomas were associated with endometriosis (n=3) or Mullerian inclusion cysts (n=1); mutation or loss of ARID1A expression was seen in these putative precursor lesions in 2 cases. Hence, ovarian neoplasms composed of clonally related GI-type mucinous and Mullerian-type epithelial components harbor ARID1A mutations and are frequently associated with endometriosis. The existence of a Mullerian stem/progenitor cell with the capacity to differentiate toward cell lineages within the GI-tract may be involved in the pathogenesis of at least a subset of GI-type mucinous ovarian neoplasms.

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.

Oncogenic BRAF and KRAS mutations in endosalpingiosis

AbstractEndosalpingiosis, a microscopic lesion composed of ectopic Fallopian tube epithelium, frequently involves the peritoneum and lymph nodes in patients with ovarian serous borderline tumour or low‐grade serous carcinoma, but its pathogenic significance remains unclear. Using laser‐capture microdissection and droplet digital PCR, we investigated whether endosalpingiosis harbours the driver mutations in BRAF and KRAS that characterise ovarian low‐grade serous neoplasms. Somatic mutations were detected in 14 (33%) of 43 endosalpingiotic lesions analysed. Of 21 women with endosalpingiosis associated with a synchronous or metachronous ovarian low‐grade serous tumour, mutations were identified in endosalpingiotic lesions from 11 (52%) women, with most cases (10/11, 91%) demonstrating identical mutations in both tumour and endosalpingiosis. In contrast, of 13 cases of endosalpingiosis not associated with an ovarian tumour, only one harboured a KRAS mutation. The proliferative activity as assessed by Ki‐67 immunohistochemistry was lower in endosalpingiosis than in low‐grade serous tumours, and endosalpingiosis with either a BRAF or KRAS mutation had a significantly lower Ki‐67 index than those without. Ectopic expression of KRASG12V in Fallopian tube epithelial cells led to ERK phosphorylation, p21 induction, growth arrest and cellular senescence. In conclusion, we demonstrate that endosalpingiosis represents an interesting example of cancer driver mutations in deceptively normal‐appearing cells, which may be prone to neoplastic transformation upon bypass of endogenous oncosuppressive mechanisms. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Spectrum ofBRAFMutations and Gene Rearrangements in Ovarian Serous Carcinoma

PURPOSELow-grade serous carcinoma (LGSC) is a rare type of ovarian cancer, which commonly arises from serous borderline tumor (SBT) and is characterized by frequent activating mutations in the mitogen-activated protein kinase pathway, including BRAF. The BRAFV600Emutation is associated with improved prognosis in SBT and LGSC, and responses to BRAF inhibitor therapy have been reported. We sought to characterize the clinicopathologic and molecular features of BRAF-driven tubo-ovarian and primary peritoneal serous tumors.METHODSRetrospective analysis of our institutional cohort of SBTs (n = 22), LGSCs (n = 119) and high-grade serous carcinomas (HGSCs, n = 1,290) subjected to targeted massively parallel sequencing was performed to identify cases with BRAF genetic alterations. Putative BRAF rearrangements were confirmed using targeted RNA sequencing and/or fluorescence in situ hybridization (FISH). BRAFV600Eoncoprotein expression was assessed by immunohistochemistry on selected cases.RESULTSBRAF somatic genetic alterations were identified in 29 of 1,431 (2%) serous tumors and included mutations (n = 24), gene rearrangements (n = 3), and amplification (n = 2). BRAF mutations were more frequent in SBTs (7 of 22; 32%) compared with LGSCs (11 of 119; 9%, P = .009) and HGSCs (6 of 1,290; 0.5%; P < .0001, SBT/LGSC v HGSC). The BRAFV600Ehotspot mutation was most common (n = 16); however, other BRAF driver mutations were also detected (n = 8). BRAF mutations were often clonal or truncal in SBTs and LGSCs, but subclonal in most HGSCs. Pathogenic BRAF gene fusions were identified in LGSCs (n = 2) and HGSC (n = 1) and involved distinct fusion partners ( AGK, MKRN1, and AGAP3). Three patients with BRAF-mutant LGSC were treated with targeted mitogen-activated protein kinase inhibitors, one of whom was maintained on therapy for over 3 years with clinical benefit.CONCLUSIONRecognition of BRAF alterations beyond V600E mutation in LGSC may have clinical implications for appropriate targeted therapy selection.

Advancements in Low-Grade Serous Carcinoma of the Ovary and Peritoneum

Low-grade serous ovarian cancer (LGSOC) is a rare form of epithelial ovarian cancer that generally exhibits a protracted course and is less sensitive to chemotherapy than high-grade serous ovarian cancer. Over the past decade, it has become clear that patients with LGSOC have a clinically distinct course and are molecularly and histologically unique from patients with high-grade serous ovarian cancer. Endocrine therapy is frequently used for the treatment of patients with recurrent LGSOC and is now also part of the standard upfront treatment of this disease, with an ongoing phase III clinical trial seeking to determine if chemotherapy can be eliminated altogether from the initial treatment of LGSOC. Tumors are frequently found to exhibit alterations affecting the mitogen-activated protein kinase (MAPK) pathway, recently leading to developments in the use of targeted treatments for those patients with recurrent disease. LGSOC is a clinically, histologically, and molecularly unique form of epithelial ovarian cancer. Recent advances in the understanding of endocrine and molecular drivers of this disease have led to changes in both the treatment of newly diagnosed and recurrent disease, with ongoing studies focused on refining upfront therapy and seeking novel targeted combinations for those patients with recurrent disease.

MAPK Pathway Genetic Alterations Are Associated with Prolonged Overall Survival in Low-Grade Serous Ovarian Carcinoma

Abstract Purpose: To characterize the somatic mutational landscape, investigate associations between genetic alterations and clinical outcomes, and determine the prevalence of pathogenic germline mutations in low-grade serous ovarian carcinomas (LGSC). Experimental Design: Patients with LGSC tumors who underwent panel-based sequencing of up to 505 genes were identified. Data on somatic and germline mutations; copy-number alterations; and clinicopathologic features, including age at diagnosis, platinum sensitivity, and overall survival (OS), were collected. Results: Following central pathology rereview, 119 patients with LGSC were identified for analysis. Of these, 110 (92%) had advanced-stage disease (stages III/IV). Somatic KRAS (33%), NRAS (11%), EIF1AX (10%), and BRAF (11%) alterations were the most common; MAPK pathway alterations were found in 60% (n = 71) of LGSCs. KRAS mutations were significantly associated with age at diagnosis more than 50 years (P = 0.02) and platinum-sensitive disease (P = 0.03). On multivariate analysis, MAPK pathway alterations (P = 0.02) and platinum sensitivity (P = 0.005) were significantly associated with improved OS. Seventy-nine patients (66%) underwent germline genetic testing; seven pathogenic germline mutations were identified: MUTYH (n = 2), BAP1 (n = 1), RB1 (n = 1), CHEK2 (n = 1), APC (n = 1), and FANCA (n = 1). There were no germline BRCA1/2 mutations. One germline MUTYH-associated LGSC harbored loss-of-heterozygosity at the MUTYH locus, and the patient with the germline BAP1 mutation also harbored a somatic BAP1 frameshift mutation. Conclusions: This study showed that MAPK pathway alterations in LGSC, including KRAS mutations, are independently associated with platinum sensitivity and prolonged survival. Germline data, which were limited, identified few pathogenic germline mutations in patients with LGSC. See related commentary by Veneziani and Oza, p. 4357

Aneuploidy Landscape in Precursors of Ovarian Cancer

Abstract Purpose: Serous tubal intraepithelial carcinoma (STIC) is now recognized as the main precursor of ovarian high-grade serous carcinoma (HGSC). Other potential tubal lesions include p53 signatures and tubal intraepithelial lesions. We aimed to investigate the extent and pattern of aneuploidy in these epithelial lesions and HGSC to define the features that characterize stages of tumor initiation and progression. Experimental Design: We applied RealSeqS to compare genome-wide aneuploidy patterns among the precursors, HGSC (cases, n = 85), and histologically unremarkable fallopian tube epithelium (HU-FTE; control, n = 65). On the basis of a discovery set (n = 67), we developed an aneuploidy-based algorithm, REAL-FAST (Repetitive Element AneupLoidy Sequencing Fallopian Tube Aneuploidy in STIC), to correlate the molecular data with pathology diagnoses. We validated the result in an independent validation set (n = 83) to determine its performance. We correlated the molecularly defined precursor subgroups with proliferative activity and histology. Results: We found that nearly all p53 signatures lost the entire Chr17, offering a “two-hit” mechanism involving both TP53 and BRCA1 in BRCA1 germline mutation carriers. Proliferatively active STICs harbor gains of 19q12 (CCNE1), 19q13.2, 8q24 (MYC), or 8q arm, whereas proliferatively dormant STICs show 22q loss. REAL-FAST classified HU-FTE and STICs into 5 clusters and identified a STIC subgroup harboring unique aneuploidy that is associated with increased proliferation and discohesive growth. On the basis of a validation set, REAL-FAST showed 95.8% sensitivity and 97.1% specificity in detecting STIC/HGSC. Conclusions: Morphologically similar STICs are molecularly distinct. The REAL-FAST assay identifies a potentially “aggressive” STIC subgroup harboring unique DNA aneuploidy that is associated with increased cellular proliferation and discohesive growth. REAL-FAST offers a highly reproducible adjunct technique to assist the diagnosis of STIC lesions.

Integrated Spatial Analysis Reveals the Molecular Landscape of Ovarian Precancerous Lesions

Abstract Studying precancerous lesions is essential for improving early detection and prevention, particularly in aggressive cancers such as ovarian carcinoma. In this study, we conducted integrated and spatial analyses of transcriptomes, aneuploidy, and clinicopathologic features in 166 ovarian precancerous lesions. Four precancerous transcriptomic subtypes were identified: proliferative, immunoreactive, dormant, and mixed. These subtypes varied in their frequency of germline BRCA1/2 mutations, aneuploidy, CCNE1/MYC amplification, proliferative activity, immunoregulatory gene expression, and histologic features. Notably, the immunoreactive subtype upregulated immunoregulatory genes, exhibited chronic inflammation, and was enriched in cases with germline BRCA1/2 mutations and deletions of chromosomes 17 (harboring TP53 and BRCA1) and 13 (harboring BRCA2), leading to a double “two-hit” involving TP53 and BRCA1/2. Tumor invasion was associated with the activation of interferon response pathways, epithelial–mesenchymal transition, and extracellular matrix remodeling. In summary, these results elucidate the earliest molecular landscape of ovarian precancerous lesions, serving as the foundation for future risk stratification to identify aggressive precancerous lesions. Significance: Integrated spatial multiomics analysis of ovarian precancerous lesions reveals molecular subtypes and mechanisms underlying tumor initiation, offering a foundation for future risk stratification and prevention. See related commentary by Soong et al. p. 1537