β-catenin, PAX2, and PTEN Aberrancy Across the Spectrum of Endometrioid Ovarian Lesions

After approval from the UT Southwestern IRB, cases at the UT Southwestern Clements University and Parkland Hospitals were retrospectively identified through text searches. EBT was defined per WHO 2020 criteria: closely packed, crowded endometrioid glands, but falling short of the criteria for adenocarcinoma 26 . EA was defined as the presence of high-grade nuclear atypia visible at low magnification in endometrioma/endometriosis without overt architectural features of neoplasia (ie, gland crowding). Characteristic nuclear features included pleomorphism, angular shapes, hyperchromasia, and smudging. For EBT, seromucinous differentiation was allowed (ie, not an exclusion criterion), and 14 of the 19 cases of EBT exhibited features of seromucinous borderline tumors. Endometrioid adenocarcinomas were defined per standard histologic criteria, including back-to-back glands. The most representative tissue block for each case was selected for immunostaining. In 3 cases where analysis of a potential benign precursor (ie, endometriosis) required IHC of another block, IHC was performed on the additional block. PAX2, PTEN, and β-catenin staining protocols previously validated for clinical testing were performed on 4 μm sections in the clinical immunohistochemistry laboratory on a DAKO Autostainer Link 48 instrument. The following primary antibodies were used: β-catenin (prediluted, clone β-catenin-1, #IR70261-2, Agilent), PAX2 (prediluted, clone EP235, #BSB2567, Cancer Diagnostics, Durham, NC), and PTEN (prediluted, clone 6H2.1, #PM278AA, BioCare, Pacheco, CA) with antigen retrieval performed in low pH (6.0) for β-catenin and high pH (9.0) Tris/EDTA solution (Agilent) for the other markers at 97°C for 20 minutes. FLEX peroxidase block was performed for 10 minutes for β-catenin and 5 minutes for other markers. Primary antibody incubation time was 20 minutes for β-catenin, and 40 minutes for PAX2 and PTEN. Incubation with Mouse Linker (Agilent) for β-catenin and Rabbit Linker (Agilent) for PAX2 was performed for 10 minutes. Secondary antibody (Envision/HRP) incubation time was 20 minutes for PTEN, β-catenin, and 30 minutes for PAX2. For all antibodies, the enzymatic conversion of the 3,3′-diaminobenzidine tetrahydrochloride chromogen was performed for 10 minutes at room temperature. Aberrancy is manifested as nuclear localization versus its normal membranous/cytoplasmic localization, often associated with overexpression. Strong nuclear β-catenin is scored as aberrant, even if focal 20 , 21 . Nuclear staining is assessed only in glandular epithelium (not “squamous” morules), since true morules always exhibit nuclear β-catenin 27 . Low levels of nuclear β-catenin are normal; the criterion for strong nuclear expression is nuclear staining clearly greater than that of the lateral cell membranes 18 . PAX2 is a nuclear transcription factor expressed within the endometrial epithelium, and scoring aberrancy requires complete loss of expression within all the nuclei in an entire gland in a cross-section. Decreased expression is not scored as aberrant. In most specimens, residual normal glands that retain PAX2 serve as internal controls for PAX2 IHC. PTEN is ubiquitously expressed in endometrial glands, stroma, and leukocytes, and true loss within the endometrial glandular epithelium gives rise to a “punched-out” appearance of glands relative to the surrounding stroma. Leukocytes interspersed within endometrial glands, which can be abundant, retain PTEN expression even when true epithelial loss is present. Focal loss of PAX2 and PTEN in individual glands or small clusters of glands can occur in normal endometria, with >10% loss across the glands in the entire specimen used as the cutoff for aberrancy. However, most cases of EIN/AH exhibit PAX2 or PTEN loss in >25% of glands, making scoring more straightforward. In endometriotic cysts, the criterion for loss for PAX2 or PTEN was loss across >50% of the cyst epithelium.

PAX2, PTEN, and β-catenin immunostains were performed on endometriosis-associated endometrioid ovarian lesions from a total of 85 patients, including 32 usual-type (nonatypical) endometriosis/endometriomas, n=5 EA, n=19 EBT, and n=29 endometrioid adenocarcinomas (n=16 FIGO Grade 1, n=10 Grade 2, and n=3 Grade 3). Patient demographics are summarized in Table 1 . Scoring for each immunostain was performed per published criteria (see Methods) 12 , 19 , 23 . Patient Demographics The results are shown in Fig. 1 for the 4 diagnostic categories. Marker aberrance was rare in ovarian endometriosis without atypia, limited to a single case (1/32) aberrant for only PAX2 (Fig. 1 A). Marker aberrance occurred in 20% of EA, with 1 case being aberrant for both PAX2 and PTEN (Fig. 1 B). Marker aberrance was more frequent in EBT, with PAX2 having the highest rate of aberrance, followed by PTEN. One case of EBT exhibited β-catenin aberrance; this case also harbored morules, whereas no other EBT had morules or aberrant β-catenin (Fig. 1 C). Marker aberrance was most frequent in endometrioid adenocarcinomas of the ovary, with PAX2 aberrance in 93.1% of cases, followed by PTEN (27.6%) and β-catenin (48.3%). At least 1 of the 3 markers was aberrant in 96.6% of adenocarcinomas (Fig. 1 D); the one case with no marker aberrancy was FIGO 1. The 2/29 PAX2 nonaberrant adenocarcinomas (PAX2 expressors) had been clinically tested for mismatch repair (MMR) deficiency by IHC; both retained expression of all 4 MMR markers. Scoring for each case within the diagnostic categories is shown in a case matrix (Fig. 1 E). Marker aberrance across diagnostic categories. (A-D) Bar graphs for the 4 diagnostic entities. Percentages appear above each bar. (E) Heat map showing patterns of marker aberrancy in each of the 85 cases. Whereas most cases of endometriosis strongly expressed PAX2 and were thus nonaberrant, the one case aberrant for PAX2 exhibited broad loss of PAX2 expression across the entire epithelial lining of the endometriotic cyst (100%, 2 cm cyst). PAX2 is normally expressed in secretory tubal epithelial cells, and a portion of fallopian tube on the slide served as an incidental positive control for PAX2 expression, confirming true loss of expression (Fig. 2 A, B). Marker aberrancy in nonatypical endometriosis. Two different cases are shown. (A) Case #1. (B) Case #2. Inset = fallopian tube (FT) internal control. Cases of EA were characterized by flat nuclear atypia with nuclear hyperchromasia and pleomorphism. Cells often appeared dyscohesive, with an apparent detachment of some cells (Fig. 3 A, B). The single EA that exhibited marker aberrance resembled the other 4 cases with respect to histologic features and overall severity of nuclear atypia. This case exhibited nearly complete loss/aberrance (>90% of epithelial cells) for both PAX2 and PTEN (Fig. 3 C, D). The patient underwent hysterectomy 2 years later for failed medical management for endometriosis and persistent abnormal uterine bleeding; histopathologic findings included ovarian endometriosis with no evidence of malignancy. Marker aberrancy in a case of endometriosis with atypia. (A) H&E showing severe flat cytologic atypia. (B) H&E of different area from same case. (C). PAX2 immunostain, representative area. (D) PTEN immunostain, representative area. An example of EBT with seromucinous differentiation aberrant for PAX2 is shown in Fig. 4 A. This case (#1), as well as all other EBT, was nonaberrant for β-catenin, exhibiting the normal (wild-type) pattern of cytoplasmic/membranous localization and no significant nuclear localization. PTEN expression was low, especially in cells with more abundant mucin, but there was definitive cytoplasmic staining, indicating non-aberrance (Fig. 4 A). Only 1 EBT exhibited β-catenin aberrance, with distinct overexpression and nuclear localization in the nonmorular epithelium. This case also exhibited morules with nuclear β-catenin (Fig. 4 B). Whereas in most cases patterns of marker aberrancy were preserved with the adjacent endometriosis (see next paragraph), in this case, the adjacent endometriotic epithelium was nonaberrant for β-catenin, suggesting that a CTNNB1 mutation might have been the instigating molecular driver event within the endometriosis driving the formation of the EBT. Marker aberrancy in the endometrioid borderline tumor. Two different cases are shown. (A) Case #1. (B) Case #2. higher magnification of SM; small white arrow points to nuclear localization of β-catenin in the adjacent epithelium. SM indicate squamous morule. In 6/19 EBT and 4/29 adenocarcinomas, distinct areas of adjacent endometriosis and/or EBT (in the adenocarcinomas) were identifiable. Patterns of marker aberrancy were preserved in these areas (with the case above being an exception), arguing that the definitive neoplasms arose from precursors where initiation was driven by the aberrancy of the relevant markers. For example, 1 case of PAX2-aberrant adenocarcinoma was associated with nonatypical endometriosis similarly characterized by PAX2 loss (Fig. 5 A). In another adenocarcinoma that was characterized by squamous morules and β-catenin aberrancy, β-catenin was similarly aberrant in the normal-appearing adjacent endometriosis glands and the EBT areas (Fig. 5 B). PAX2 was similarly aberrant across all components (benign, EBT, adenocarcinoma) (not shown) strongly arguing for an origin in this adjacent endometriosis. In 8/10 of the EBT or adenocarcinoma cases with adjacent endometriosis, patterns of marker aberrancy were preserved in at least 1 marker, while 7/10 had identical marker patterns, supporting a mechanism in which benign-appearing precursors can harbor molecular alterations of the relevant factors promoting development into more malignant lesions. Marker aberrancy in 2 cases of endometrioid adenocarcinoma. (A) Case #1. Right-sided panels are of adjacent nonatypical endometriosis. The lower IHC panels correspond to the same areas shown in the H&E images. (B) Case #2. The single field in the H&E section shows distinct areas of adenocarcinoma, EBT, and endometriosis. β-catenin was aberrant across all areas.

Our study found that scoring criteria previously defined for the 3 markers in the endometrium are applicable to endometriosis and its neoplastic derivatives 12 , 18 , 23 , 25 . The overall results recall findings for the 3-marker panel in the endometrium, where the incidence of marker aberrance increases along the histologic spectrum of neoplastic progression (normal→disordered→nonatypical→EIN/AH→adenocarcinoma) 12 . Although significant differences in aberrancy rates for individual markers in ovarian versus endometrial lesions are noted (see below), the aberrancy rate of 97% in endometrioid adenocarcinomas of the ovary is comparable to the reported 93% rate in EIN 18 . The aberrancy rate for PTEN was lower across the spectrum in ovarian versus endometrial lesions (e.g., 27.6% in ovarian endometrioid adenocarcinomas [this study] relative to 59.5% in endometrioid adenocarcinomas 28 ). This finding is consistent with a lower reported incidence of PTEN mutations in ovarian endometrioid adenocarcinomas (17%) versus endometrial endometrioid adenocarcinomas (67%; P value of <0.0001) 29 . The β-catenin aberrancy rate we found in EBT (1/19 cases) is lower than reported in 1 small retrospective study of selected cases (n=8) 13 , a discrepancy that may be explained by different case selection criteria and a high proportion of seromucinous differentiation among our cases. The molecular basis of PAX2 loss in endometrial neoplasia remains unknown 23 . Nonetheless, our findings suggest that PAX2 loss is also an important driver of ovarian endometrioid neoplasia. PAX2 loss occurred in 1 case of nonatypical endometriosis and 1 case of EA. The loss of PAX2 across the entire benign lesion suggests that PAX2 loss might be an early or instigating event in ovarian endometrioid carcinogenesis, as is well-established in eutopic endometrium. In the case of EA with PAX2 loss, the biological significance of PAX2 loss is substantiated by concurrent PTEN loss/aberrancy, providing further evidence that a subset of EA may be significant precursor lesions (ie, intraepithelial carcinomas). Most cases of EA likely represent reactive changes, but this case suggests that a subset represents true intraepithelial carcinomas, a question that warrants further investigation. While the 3x panel could be useful in identifying EA at higher risk of neoplastic progression, important caveats are that there is no consensus if or how EA should be reported by pathologists, or how it should be clinically managed, other than it should prompt additional tissue sampling to exclude occult malignancy 5 . EA may also serve as a precursor for clear cell adenocarcinoma, a distinct endometriosis-associated malignancy 4 , 5 , although we did not observe histologic features suggestive of clear cell neoplasia among our EA cases. The finding of similar patterns of marker aberrancy in endometriosis adjacent to EBT and adenocarcinoma, or EBT adjacent to adenocarcinoma, further cements a link between aberrancy for β-catenin, PAX2, PTEN, and cancer initiation/progression in ovarian endometriosis-associated neoplasia. Significant limitations of our study are the small sample size and lack of systematic follow-up. Nonetheless, our findings provide support for the utility of the panel in endometriosis-associated neoplasms, such as difficult or ambiguous cases of EBT (analogous to EIN/AH), with the caveat that the sensitivity would be lower than for EIN/AH. In summary, our study adds to our understanding of the performance of the 3x biomarker panel in diverse diagnostic contexts.