Ovarian RASoma With Mesonephric-like Adenocarcinoma and Mixed Mullerian Components: A Case Report With Molecular Analysis Demonstrating Multidirectional Mullerian Differentiation.

A 63-year-old patient was admitted to the Gynecology Department for a history of 3 months of pelvic pain. Ultrasound examination revealed a predominantly cystic mass of 120 mm diameter involving the left ovary. Total hysterectomy with bilateral salpingo-oophorectomy was performed. At macroscopic examination, the ovarian mass had a smooth and intact external surface while on cut surface, it demonstrated numerous cystic spaces with mucinous contents and smooth walls. However, one solid, white and hard area of 35 mm diameter was identified and contained numerous smaller cysts ( Figure 1A , B ). The right adnexa as well as the uterine cervix and corpus were normal in appearance. At microscopic examination, low power scanning demonstrated an admixture of micro- and macro cysts with most (approximately 80%) of the macrocystic and some of the microcystic spaces lined by a columnar mucin-rich non-stratified, hypoproliferative epithelium with small and uniform, basally located nuclei, morphologically consistent with a mucinous benign-looking component ( Figure 1C ). The mucinous epithelium had an endocervical-type appearance, lacking goblet cells, but this component was estrogen receptor (ER)/ progesterone receptor (PR) negative. The solid area was represented by a predominantly fibromatous stroma containing intimately admixed areas with various morphology: rounded tubulo-cystic structures of various sizes, lined by flat and low-cuboidal cells with flat-to-oval nuclei and scant cytoplasm, suggesting a clear cell carcinoma; some other glandular/tubulo-cystic structures had dense eosinophilic luminal secretions and were lined by cuboidal epithelium, suggesting mesonephric-like differentiation; while others areas were represented by glands, some of which were cystically dilated, surrounded by endometrioid stroma with stromal foam cells and lined by a columnar ciliated epithelium with rounded nuclei, suggesting endometrioid differentiation in association with endometriosis, and an endometrioid adenofibromatous component. Of interest, some of the cystic structures were partly lined by mixtures of mucinous, endometrioid and mesonephric-like epithelium ( Figure 1 D - J ). In a 1.6 cm focus, there was a more complex architecture with intracystic branching papillary structures, cribriform and solid architecture, lined by cuboidal and low columnar cells with minimal cytoplasm, increased mitotic activity and round nuclei with minimal nuclear atypia, sometimes with overlapping nuclei or nuclear grooves and cleared-out chromatin. This, together with tubulo-cystic structures lined by cuboidal epithelium, was thought to represent mesonephric-like adenocarcinoma ( Figure 1 K , L ). Immunohistochemically, the mucinous component expressed CK7 and was negative for CK20, ER, PR, CD10, GATA3 and TTF1. The endometrioid component (both epithelial and stromal) was positive for ER, PR and vimentin and lacked expression of mesonephric markers. The mesonephric-like (both cystadenomatous and malignant) components were diffusely positive for CD10 (luminal and apical), TTF1 (nuclear) and GATA3 (nuclear), and negative for all the other markers. Clear cell markers such as Napsin A and Racemase were negative in all components ( Figure 2 ). The different histologic components, mucinous, endometrioid and mesonephric-like, were microdissected separately and subjected to DNA extraction and next generation sequencing targeting 505 cancer-related genes (MSK-IMPACT at Memorial Sloan Kettering Cancer Center’s Integrated Genomics Operation) 3 , 11 . All three components were found to be diploid apart from a chromosome 1q gain ( Figure 3 ). Furthermore, all three histologic components harbored a clonal KRAS p.G12A hotspot mutation associated with loss of heterozygosity, a clonal ARHGAP35 p.L1295Cfs*20 frameshift mutation and a PPP2R1A p.R182W hotspot mutation. Analysis of the cancer cell fractions of the somatic mutations revealed that the PPP2R1A hotspot mutation was subclonal in the mucinous component and became clonal in the endometrioid and mesonephric-like components. Together with the finding of a subclonal IRS1 p.R410W missense mutation being present only in the endometrioid and mesonephric-like areas, and an increase of the chromosome 1q gain from 4 copies in the mucinous to 5 copies in the endometrioid and mesonephric-like components, provides evidence to suggest that the endometrioid and mesonephric-like components likely originated from the mucinous component. The morphology together with the immunohistochemical and molecular profile were in favor of a FIGO stage I ovarian MLA in a background of benign-appearing Mullerian proliferations in the form of endometriosis, endometrioid adenofibroma, focal endometrioid carcinoma and mucinous cystadenoma.

MLA has been only recently described by McCluggage et al as a peculiar and rare tumor resembling MA morphologically and immunohistochemically 2 . The WHO 2020 classification of ovarian neoplasms recognizes this entity but at present, we are still gaining experience with its morphology and clinical behaviour 1 . We present the case of an ovarian tumor harboring an MLA with mucinous and endometrioid components in which the molecular analysis demonstrated that all three components had a KRAS p.G12A hotspot mutation with 1q gains (i.e. “RASoma”), and that the mucinous component likely constituted the substrate for the endometrioid and mesonephric-like components. Ovarian MLA may be widely infiltrative and can present with a marked heterogeneity and an admixture of the following architectural patterns: tubular, solid, papillary, glomeruloid-like, sex-cord-like, retiform and spindle cells arranged in fascicles 1 . Tubular and glandular patterns often predominate, and a frequent finding is the presence of small round tubules containing luminal eosinophilic, colloid-like material (i.e. mesonephric-like tubules). The cytoplasm is usually minimal, while nuclei often have grooves and may show pseudoinclusions with nuclear overlap, resembling features classically used to describe papillary thyroid carcinoma. Squamous and mucinous differentiation is generally absent. In the present case however, an area of MLA was identified in a background of benign-appearing Mullerian components with mucinous and endometrioid morphology. The MLA component demonstrated mesonephric-like tubules, papillary and tubulo-cystic formations, with the latter two patterns resembling clear cell carcinoma. Since the diagnosis of ovarian MLA can be challenging, and the tumor may simulate endometrioid, clear cell and serous adenocarcinomas, immunohistochemical markers can refine the diagnosis. TTF1 and/or GATA3 with CD10 are usually positive, while ER and PR are negative 1 , 2 . Euscher et al suggested GATA3, TTF1, ER, and PR as first-line markers to help confirm an MLA, with CD10 and calretinin serving as supplemental immunostains 12 . In our case the mesonephric-like components were positive for CD10, GATA3 and TTF1 and negative for ER and PR. Several ovarian MLAs have been shown to coexist with endometriosis or Mullerian neoplasms, further supporting the idea that these tumors are not mesonephric in origin. In these particular cases, distinct areas of MLA have been reported to co-exist with areas of benign, borderline or malignant Mullerian ovarian tumors with their expected morphology and immunohistochemical profile, but also with shared various genetic mutations 5 , 6 . The present case harbors mucinous and endometrioid components with an adenofibromatous appearance that had molecular features more in common with malignancy, despite their “benign” appearance. Therefore, there is discordance between the morphology and molecular features, which is of uncertain significance given the context. Immunohistochemically, areas of Mullerian transdifferentiation preserved their expected immunohistochemical profile, which also helped to exclude clear cell carcinoma and confirm mesonephric-like differentiation in areas that resembled tubulo-cystic clear cell carcinoma and papillary clear cell carcinoma. Distinct molecular aberrations such as mutations in KRAS and NRAS as well as mutations in PIK3CA, ARID1A, TP53 (less common), 1q gains, lack of PTEN mutations and gain of chromosomes 10 and 12 have been identified in MLAs in a recent study 3 . This molecular profile seems to be similar to the one seen in MAs 13 . Analysis of mixed MLA has demonstrated that histologically distinct components generally harbor similar patterns of genetic alterations, providing evidence to support their clonal relatedness, but in addition demonstrating that each component harbored private mutations 1 , 3 , 13 . Based on the presence of common somatic genetic alterations in histologically distinct components of the mixed MLAs, the most concise explanation is that these do not constitute collision tumors, instead, these findings demonstrate that the histologically distinct components are clonally related. Clonal deconvolution analyses performed in one case reported in the literature showed that the mesonephric-like component may have been the substrate from which the mucinous component developed, since subclonal KRAS and PIK3CA mutations present in the mesonephric-like component became clonal in the mucinous component 3 . A recent small series of mixed MLA and mucinous ovarian tumors came to a similar conclusion 4 . In contrast, our molecular analysis revealed that the mucinous component was the likely substrate for the other 2 components, with a subclonal PPP2R1A hotspot mutation in the mucinous component that became clonal in the endometrioid and mesonephric-like components, and an IRS1 mutation acquired only in the endometrioid and mesonephric-like components but absent in the mucinous component. Based on this, one may suggest that the phenotypes observed are not solely the phenotypic manifestation of the underlying mutations but rather constitute the final product of the intricate interplay between genetic alterations, cell of origin and tumor microenvironment. MLAs are aggressive tumors although they frequently have a well differentiated appearance. There is a propensity for both lymph node metastases and spread beyond the pelvis, especially to lungs 3 . A recent study, including 44 uterine corpus MLAs and 25 ovarian MLAs, confirmed these tumours’ clinical aggressiveness 14 . In that particular study, 59% and 42% of patients with uterine corpus and ovarian MLAs, respectively, suffered recurrences, and many of them were distant recurrences. The 5-year disease-specific survival for MLA was approximately 70% irrespective of the organ in which the tumors arose 14 . Since clinically aggressive tumors can have a misleadingly low-grade histologic appearance, grading such tumors may not be relevant and, instead, the tumors should be regarded intrinsically high grade 14 . This will be endorsed in the upcoming update of the International Collaboration on Cancer Reporting (ICCR) guide for Ovarian, fallopian tube, and primary peritoneal carcinomas 15 . In conclusion, gynecologic carcinomas with RAS mutations may show a wide spectrum of histology, including mixed histotypes (i.e. “RASomas”), as seen in the present case. MLA is not diagnosed by its molecular features but by its morphology supported by immunohistochemistry. Pathologists should be aware of morphology mimicry that could lead to misdiagnosis. Since the mucinous component was the likely substrate of the two other components in the present case (MLA and endometrioid), we propose that RAS -mutated mucinous neoplasms can give rise to MLA.

Mesonephric adenocarcinoma (MA) of the cervix is a rare Human Papillomavirus (HPV)-independent tumor, reported to develop from mesonephric (Wolffian) remnants that are usually found in the lateral aspect of the cervical wall 1 . In contrast, mesonephric-like adenocarcinomas (MLA), an entity only recently described, can develop in the ovary or uterine corpus, despite the absence of native Wolffian structures 2 , 3 . Paradoxically, both ovarian and uterine MA and MLA present with similar morphology and immunohistochemical and molecular profiles. Ovarian MLAs are frequently pure (i.e. not mixed) and sometimes present with a sarcomatoid/spindle cell component. Recently published case reports and small series have demonstrated that ovarian MLAs may be found in association with a distinct Mullerian component (mucinous, serous and/or endometrioid) 3 - 9 . Moreover, ovarian endometriosis has been recently proposed as the origin of MLA on the basis of two cases in which there was evidence of endometriosis contiguous with the tumor and invasion from other sources was excluded 10 . Here we describe an ovarian MLA associated with endometriosis and mucinous and endometrioid components in which molecular study revealed an MLA-like genotype in all components.