Applied Immunohistochemistry & Molecular Morphology

Frequent CTNNB1 or PIK3CA Mutations Occurred in Endometrial Endometrioid Adenocarcinoma With High Levels of Microsatellite Instability and Loss of MSH2/MSH6 Expression

Background: DNA mismatch repair (MMR) proteins form 2 heterodimers—MutSα formed by MSH2 and MSH6, and MutLα by MLH1 and PMS2. In endometrial endometrioid adenocarcinomas, cases with MMR protein defect also usually harbor other recurrent genetic mutations of the neoplasm. However, it remains unknown whether defects of the 2 functionally different heterodimers are linked to mutations in different genes. We aimed to study the MMR protein expression, microsatellite instability (MSI), and other common genetic mutations of endometrial endometrioid adenocarcinoma. Materials and Methods: We investigated the MSI status of 107 endometrial endometrioid adenocarcinoma patients. MMR protein expression, and mutation of KRAS, CTNNB1, and PIK3CA were also evaluated by immunohistochemistry and sequencing. Results: An overall 34.6% (37/107) of endometrial endometrioid adenocarcinomas were MSI-H. All MSI-H tumors exhibited loss of MMR protein expression (loss of MLH1, PMS2, MSH6, and MSH2 was noted in 22, 25, 12, and 7 cases, respectively). CTNNB1, PIK3CA, and KRAS mutation were present in 9, 7, and 7 MSI-H tumors. Compared with patients with loss of PMS2 and/or MLH1 expression, patients with loss of MSH6 and/or MSH2 expression were associated with higher frequencies of CTNNB1 mutation (P=0.036) and PIK3CA mutation (P=0.025). Conclusions: In MSI-H endometrial endometrioid adenocarcinomas, different types of MMR protein deficiency indicate different molecular genetic alterations.

Role of Immunohistochemistry to Distinguish Grade 3 Endometrioid Carcinoma and Uterine Serous Carcinoma

Aim: The categorization of endometrial carcinomas into endometrioid and serous categories has prognostic implications but many-a-times, it is difficult to categorize based solely on morphology. The present study was conducted to determine an appropriate immunohistochemical panel to distinguish grade 3 endometrioid carcinoma from serous carcinoma. Experimental Design: This study was a retrospective and a prospective study including 63 cases of endometrial carcinoma diagnosed on morphology as either grade 3 endometrioid (n=29) or serous endometrial carcinomas (n=34). Immunohistochemistry (IHC) was performed using tissue microarrays for 8 immunomarkers on 60 cases. Results: The mean age of presentation was not significantly different for both types of carcinomas and the most common presentation was postmenopausal bleeding (93% of the total cases, P=0.66). Obesity (P=0.038), lymph nodal involvement (P=0.044), and stage at presentation (P=0.042) were found to be significantly different among the 2 types of carcinomas. Estrogen and progesterone receptor (ER, PR) positivity was more common (47.6% and 28.2%, respectively) in endometrioid carcinomas as compared with serous. Mutation type (diffuse or null) p53 staining was a powerful predictor of serous carcinomas. IMP3 and p16 were found to be positive in most cases of serous carcinoma (64.1% and 79.5%, respectively). Vimentin and β-catenin were found to be of limited utility. On the basis of IHC, 21 cases could be categorized as grade 3 endometrioid carcinomas and 39 as type 2 carcinomas (serous and clear cell carcinoma). Conclusions: The most appropriate IHC panel to differentiate endometrioid and serous endometrial carcinomas includes ER, PR, IMP3, p53, and p16.

Napsin-A and AMACR are Superior to HNF-1β in Distinguishing Between Mesonephric Carcinomas and Clear Cell Carcinomas of the Gynecologic Tract

Mesonephric carcinoma is a rare gynecologic neoplasm commonly mistaken for clear cell carcinoma, because of their overlapping morphologic features. Both tumors are negative for estrogen receptor and p16, magnifying this diagnostic dilemma. Recently, hepatocyte nuclear factor-1 beta (HNF-1β), a marker for clear cell carcinoma, has also been shown to be positive in mesonephric carcinomas. Other more recent markers for clear cell carcinoma, however, such as Napsin-A and alpha-methylacyl-CoA racemase (AMACR), have not yet been studied in mesonephric carcinomas. Here we examine HNF-1β, AMACR, and Napsin-A immunohistochemistry in 18 mesonephric and 55 endometrial/cervical clear cell carcinomas. HNF-1β was considered positive if nuclear staining was present in ≥70% of cells and at least moderate intensity; for Napsin-A and AMACR, any cytoplasmic staining was considered positive (≥1%). H-scores were determined by multiplying the intensity score by proportion score. HNF-1β was positive in a substantial portion of mesonephric carcinomas (9/18, 50%; H-score 98) and clear cell carcinomas (34/55, 62%; H-score 163) and did not distinguish between the 2 entities (specificity, 50%; P-value of H-score=0.08). Napsin-A and AMACR expression was significantly higher in clear cell [43/55 (78%) and 41/55 (75%), respectively] than mesonephric carcinomas [4/18 (22%) and 4/18 (22%) respectively], and helpful in this differential (specificity: 78% and 78%; P<0.05 for both). When Napsin-A and AMACR staining were seen in mesonephric carcinomas, staining was focal (≤5%), whereas staining in clear cell carcinomas was patchy/diffuse. In summary, Napsin-A and AMACR are helpful in distinguishing mesonephric carcinomas from clear cell carcinomas of the female genital tract, but HNF-1β is not.

The Immunohistochemical Pattern of Epithelial-Mesenchymal Transition Markers In Endometrial Carcinoma

The majority of endometrial carcinoma are diagnosed at an early stage and exhibit a favorable prognosis. However, 10% to 15% of ECs recur and the majority are type II tumors which are high-grade carcinomas. The epithelial-mesenchymal transition (EMT) has been considered as a fundamental step for the development of the invasive phenotype of cancer cells. During EMT, many of epithelial surface markers, primarily E-cadherin disappear, and mesenchymal markers including N-cadherin gain. This feature resides predominantly at the invasive front (IF) of the tumor. Therefore, we examined the immunohistochemical expression of E-cadherin and N-cadherin at the IF, in central areas of the tumor and lymphovascular space, in type I and type II endometrial carcinoma. The association of each protein with the clinicopathologic features was also evaluated. Our results confirmed a stronger E-cadherin immunostaining in type I tumors indicating that the loss of E-cadherin may be responsible for a more aggressive behavior of type II ECs. In both types, E-cadherin was strongly expressed in central areas and the reactivity decreased toward the IF. On contrary, N-cadherin was overexpressed at the IF confirming an inverse relationship between these markers. In addition, a decrease in E-cadherin expression was observed in cells within the lymphovascular space. Downregulation of E-cadherin was associated only with high-grade tumors while no correlations between both markers and other clinicopathologic features were found. Our results confirm that EMT occurs at the IF that represents a critical interface between the tumor and the host.

A Case Report of an Adenomatoid Tumor of the Uterus Mimicking an Endometrioid Adenocarcinoma on Endometrial Curetting: a Diagnostic Pitfall

Adenomatoid tumors (AT) arising in the female genital tract are usually incidental findings occurring most often in the fallopian tube and uterine serosa and rarely in the myometrium. In the myometrium, they appear grossly as deep seated, small, firm, ill circumscribed nodules mimicking leiomyoma. Histologically they show a glandular and invasive pattern making well-differentiated/low-grade endometrioid adenocarcinoma a major differential diagnosis. However, this differential is rarely encountered in practice because myometrial AT is usually seen on the hysterectomy specimen, because of their anatomic position in the deep myometrium, and only rarely in endometrial curettings. Our case is the first to report an AT, which presented as a polyp with associated fibroid on hysterescopic examination. Microscopically, the endometrial curetting and myomectomy showed irregular glands and cystic structures with occasional cytokeratin positive single signet-ring like cells invading into the myometrium, features consistent with low-grade endometrioid adenocarcinoma. On hysterectomy specimen, there was an ill-defined 5 cm mass in the myometrium with protrusion into the endometrium. The morphology was similar to that seen in the endometrial curetting. A larger panel of immunostains was done and the neoplastic cells were positive for AE1/3, CK7, CAM5.2, calretinin, and D2-40 and negative for CD34. A diagnosis of AT was rendered and no further treatment was required. Although AT is rarely seen in endometrial curetting, they should be in the differential diagnosis of glandular lesions to avoid pitfalls and unnecessary management especially in young patients desiring fertility.

Estrogen/Progesterone Receptor Loss, CTNNB1 and KRAS Mutations Are Associated With Local Recurrence or Distant Metastasis in Low-Grade Endometrial Endometrioid Carcinoma

A subset of endometrial endometrioid carcinomas (EECs) with low-grade histology recur with poor outcomes. Published evidence suggests that poor outcomes may be associated with loss of expression of ER-alpha (ER-α) as well as with β-Catenin-1 (CTNNB1) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. This study reports on institutional experience with the incidence of recurrence in low-grade EEC and their association with CTNNB1 and KRAS mutations as well as estrogen/progesterone receptor (ER/PR) expression. Forty-eight (8.5%) out of 568 cases of low-grade EEC with biopsy-proven recurrence were identified; and were analyzed by immunohistochemistry for ER, PR, p53, MMR protein, and mutation analysis for exon 3 of the CTNNB1 and exon 2 of KRAS in relation to recurrence type, local or distant metastasis/recurrence. Twenty-three patients (4%) developed local, and 25 patients (4.4%) developed distant metastases/recurrence. Decreased expression or loss of ER/PR was found in 17/44 (38.6%) patients with recurrence. Eighty-four percent of patients with low-grade EEC and local recurrence had CTNNB1 mutations. Seventy-three percent of patients with distant metastasis/recurrence had KRAS mutations. The association of these mutations with the type of recurrence was statistically significant for both. Five cases with the morphology of low-grade EEC were reclassified as mesonephric-like carcinoma and were universally characterized by distant metastasis/recurrence, loss of ER/PR expression, large tumor size, absence of CTNNB1 mutations, and the presence of KRAS mutations. In low-grade EEC, CTNNB1 and KRAS mutations are associated with local recurrence and distant metastasis/recurrence, respectively, suggesting that these 2 different progression types may be conditioned by tumor genotype. ER/PR immunohistochemistry may be helpful in identifying poor performers in low-grade EEC. Furthermore, identification of the decreased expression or loss of ER/PR in tumors with low-grade histology should prompt consideration of mesonephric-like carcinoma, which is a more aggressive tumor than the low-grade EEC. KRAS mutations were associated with distant metastasis/recurrence in tumors with and without mesonephric-like phenotype.

Coexistent Dedifferentiated Endometrioid Carcinoma of the Uterus and Adenocarcinoma of the Bladder in Lynch Syndrome: Case Report and Review of the Literature

Lynch syndrome is an autosomal dominant disorder, caused by an abnormality in DNA mismatch repair genes and characterized by the development of a variety of cancers. Upper urinary tract urothelial carcinoma is well characterized in Lynch syndrome; however, support for the inclusion of bladder urothelial carcinoma is limited, except for MSH2 mutation carriers. Urologic adenocarcinoma has not been documented in Lynch syndrome. Here we report, to the best of our knowledge, the first case of bladder adenocarcinoma, synchronous with uterine endometrioid dedifferentiated endometrioid adenocarcinoma in a patient with Lynch syndrome. We present a 47-year-old woman with an MLH1 gene mutation (G133X 397G>T) who presented with menorrhagia. Eleven family members have this mutation, 6 with carcinoma: 5 colorectal and 1 with a gynecologic primary of unknown type. Colonoscopy and endoscopy were unremarkable. Positron emission and computed tomography revealed a 3 cm anterior dome bladder mass without additional extrauterine disease or uterine connection. She underwent partial cystectomy, laparoscopic hysterectomy, bilateral salpingo-oophorectomy, and lymphadenectomy. The uterus demonstrated a dedifferentiated endometrioid adenocarcinoma, immunohistochemically positive for vimentin, ER, CK7, MSH2, MSH6, and p53 (focally) and negative for CEA, CDX2, CK20, β-catenin, MLH1, and PMS2. The bladder demonstrated a well-differentiated, enteric-type adenocarcinoma without muscularis propria invasion, positive for CEA, CDX2, CK20, p53, MSH2, and MSH6 and negative for vimentin, ER, CK7, MLH1, and PMS2. Eleven nodes were negative for carcinoma. The morphologic, immunohistochemical, and clinical findings support synchronous bladder adenocarcinoma, enteric type, and uterine dedifferentiated endometrioid adenocarcinoma, in a patient with Lynch syndrome.

Expression and Significance of MTA2 and CPNE1 in Cervical Squamous Cell Carcinoma

The aim of this study was to investigate the expression and clinical significance of MTA2 and CPNE1 proteins in cervical squamous cell carcinoma. In this study, high-risk human papillomavirus (HPV) typing was performed on cervical cancer tissues. Reverse transcription polymerase chain reaction and immunochemical EliVision method were used to examine the expressions of MTA2 and CPNE1 in the cervix, and their relationship with clinicopathologic features. We found that it is mainly distributed in these types, namely HPV-16 (23.8%), HPV-18 (20.9%), HPV-53 (17.1%), HPV-52 (15.5%), HPV-82 (11.7%), HPV-56 (10.8%). The expressions of MTA2 and CPNE1 in cervical squamous cell carcinoma tissues were significantly higher than those in normal tissues (P<0.01). The expressions of MTA2 and CPNE1 were correlated with FIGO stage, degree of differentiation, and lymph node metastasis of cervical cancer (P<0.05), but not with the patient’s age (P>0.05). The rank correlation coefficient of MTA2 and CPNE1 protein expression in cervical squamous cell carcinoma was 0.668 (P<0.01), and the 2 expressions were positively correlated. MTA2 and CPNE1 are closely related to the occurrence and development of cervical squamous cell carcinoma and may play a synergistic role in the evolution of cervical squamous cell carcinoma.

GPBAR1 Promotes Proliferation of Serous Ovarian Cancer by Inducing Smad4 Ubiquitination

Background: Ovarian cancer (OC) is the most lethal malignancy of all female cancers and lacks an effective prognostic biomarker. Serous ovarian cancer (SOC) is the most common OC histologic type. The expression and function of bile acid receptor, G-protein-coupled bile acid receptor-1 (GPBAR1), in tumor progression remains controversial, and its clinical significance in SOC is unclear. Materials and Methods: In our study, we detected the expression of GPBAR1 in SOCs and normal ovarian tissues with quantitative real-time polymerase chain reaction and immunohistochemistry to detect its expression pattern. Moreover, the prognostic significance of GPBAR1 was investigated with univariate and multivariate analyses. The function of GPBAR1 in regulating SOC proliferation was studied and the underlying mechanism was investigated with experiments in vitro. Results: GPBAR1 was overexpressed in SOCs compared with the normal ovarian tissues. In the 166 SOCs, subsets with low and high GPBAR1 accounted for 57.23% and 42.77%, respectively. Moreover, our results suggested that GPBAR1 expression was significantly associated with poor prognosis and can be considered as an independent prognostic biomarker. With experiments in vitro, we suggested that GPBAR1 promoted SOC proliferation by increasing Smad4 ubiquitination, which required the involvement of GPBAR1-induced ERK phosphorylation. Conclusions: GPBAR1 was overexpressed in SOC and predicted the poor prognosis of SOC. We showed that GPBAR1 promoted SOC proliferation by activating ERK and ubiquitining Smad4. Our results suggested that GPBAR1 was a supplement to better classify SOC on the basis of the molecular profile and that GPBAR1 may be a potential drug target of SOC.

mTOR Pathway Activation Assessed by Immunohistochemistry in Cervical Biopsies of HPV-associated Endocervical Adenocarcinomas (HPVA): Correlation With Silva Invasion Patterns

The Silva pattern of invasion, recently introduced to stratify patients at risk for lymph node metastases in human papillomavirus-associated endocervical adenocarcinomas (HPVAs), can only be assessed in cone and loop electrosurgical excision procedure excisions with negative margins or in a hysterectomy specimen. Previous studies found associations between destructive stromal invasion patterns (Silva patterns B and C) and mutations in genes involved in the MEK/PI3K pathways that activate the mammalian target of rapamycin (mTOR) pathway. The primary aim of this study was to use cervical biopsies to determine whether markers of mTOR pathway activation associate with aggressive invasion patterns in matched excision specimens. The status of the markers in small biopsy specimens should allow us to predict the final and biologically relevant pattern of invasion in a resection specimen. Being able to predict the final pattern of invasion is important, since prediction as Silva A, for example, might encourage conservative clinical management. If the pattern in the resection specimen is B with lymphovascular invasion or C, further surgery can be performed 34 HPVA biopsies were evaluated for expression of pS6, pERK, and HIF1α. Immunohistochemical stains were scored semiquantitatively, ranging from 0 to 4+ with scores 2 to 4+ considered positive, and Silva pattern was determined in follow-up excisional specimens. Silva patterns recognized in excisional specimens were distributed as follows: pattern A (n=8), pattern B (n=4), and pattern C (n=22). Statistically significant associations were found comparing pS6 and pERK immunohistochemistry with Silva pattern (P=0.034 and 0.05, respectively). Of the 3 markers tested, pERK was the most powerful for distinguishing between pattern A and patterns B and C (P=0.026; odds ratio: 6.75, 95% confidence interval: 1.111-41.001). Although the negative predictive values were disappointing, the positive predictive values were encouraging: 90% for pERK, 88% for pS6 and 100% for HIF1α. mTOR pathway activation assessed by immunohistochemistry in cervical biopsies of HPVA correlate with Silva invasion patterns.

Adenocarcinoma of the Uterine Cervix: Immunohistochemical Biomarker Expression and Diagnostic Performance

Immunohistochemistry (IHC) improves the diagnosis of cervical adenocarcinoma but is not adequately studied. The performance of 16 antibodies previously reported as potentially discriminating between some histotypes was investigated in 184 tumors comprised of 12 histotype groups collapsed into 3 categories [47 adenocarcinomas in situ (AIS), 121 probable human papillomavirus–dependent adenocarcinomas (HPVD), and 16 of probable independence (HPVI)]. IHC sections from 5 tissue microarrays were scanned, and 3 pathologists independently reviewed images to assess staining percentages and intensities. Biomarker expression was based on published positive and negative cutoffs and agreement between any 2 pathologists. Differences between the 3 categories in the hierarchical ranking of biomarker positivity were analyzed by Random Forest classification, and between select groups by Unsupervised Hierarchical Clustering. Important category discriminants were combined in logistic regression models and the area under the curve (AUC) computed. Potential group discriminants were terminal cluster biomarkers with a 50% or more difference in positivity. Strong associations occurred between the lower expression of carcinoembryonic antigen and stromal actin in AIS compared with HPVD [AUC=0.70, 95% confidence interval (CI), 0.59-0.80] and in the higher expression of p16 and estrogen receptor in comparison to HPVI (AUC=0.86, 95% CI, 0.73-0.98), and between the higher expression of p16, carcinoembryonic antigen and estrogen receptor in HPVD compared with HPVI (AUC=0.88, 95% CI, 0.77-0.99). Between select groups, 9 biomarkers emerged as potential discriminants. Select IHC biomarkers can discriminate AIS from invasive adenocarcinomas, and invasive adenocarcinomas stratified by human papillomavirus dependency from each other. Independent replication in larger studies is needed, and to confirm discriminants of histotype groups.

Evaluation of Sirtuin1 Overexpression by Immunohistochemistry in Cervical Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Cervical cancer is one of the most common genital cancers in the woman with approximately half a million new cases per year. Development of invasive squamous cell carcinoma (SCC) is the result of persistent and frequent human papilloma virus infection in premalignant lesions of cervix. Thereby identification of biomarkers that could predict progression of dysplastic mucosa to invasive cancer is of great clinical significance. Overexpression of SIRT1 has been reported to induce tumorogenesis in several organs. We evaluated SIRT1 expression in normal squamous epithelium of cervix, low-grade and high-grade cervical intraepithelial lesions and invasive SCC. A total of 104 cases were selected including 34 low-grade cervical intraepithelial lesions (CINs), 37 high-grade CINs, and 35 cases of invasive SCC. The normal cervical epithelium showed negative or weak SIRT1 positivity only in basal layers. SIRT1 cytoplasmic expression was found in 13 of 34 (38.2%) of low-grade CINs, 31 of 37 (83.8%) of high-grade CINs and all 35 (100%) cases of invasive SCC. Expression between 2 groups of CIN was statistically significant (P=0.001). Thus, SIRT1 appears to be a promising biomarker for predicting the progression of CIN to invasive SCC.

Testing Algorithms for the Diagnosis of Malignant Glandular Tumors of the Uterine Cervix Histotyped per the International Endocervical Adenocarcinoma Criteria and Classification (IECC) System

The International Endocervical adenocarcinoma Criteria and Classification (IECC) categorizes tumors into human papilloma virus (HPV) associated (HPVA), not associated (NHPV), and invasive adenocarcinoma not otherwise specified (IA NOS). HPVA and NHPV encompass 11 histotypes and an algorithm of mucin content, HPV ribonucleic acid (RNA), estrogen receptor and GATA3 is proposed for the diagnosis of most. In this study, the IECC algorithm’s diagnoses were compared with hematoxylin and eosin (H&E) based IECC histotyping. Kappa statistics measured performance agreement. With additional markers, hierarchical clustering by random forest (RF) classification identified the most discriminating between tumor types, and investigated other algorithms. Three pathologists independently reviewed digitized H&E images of n=152 primary cervical adenocarcinomas for IECC histotype and mucin content, and tissue microarrays for expression of HPV RNA by in situ hybridization and 16 antibodies by immunohistochemistry. Results were finalized by consensus. There were n=113 HPVA, n=22 NHPV, and n=17 IA NOS. Mucin was obvious in n=36 and limited in n=116. Among n=124 with satisfactory test results, HPV RNA was positive in n=96, estrogen receptor in n=72, and GATA3 in n=15. The IECC algorithm diagnosed n=99 which agreed with H&E histotyping in n=64 for a fair κ of 0.36 (95% confidence interval, 0.21-0.50): n=12 were undiagnosed and n=13 were IA NOS. Small sample sizes restricted RF to HPVA versus NHPV which were discriminated by p16, HPV RNA, and MUC6 with an area under the curve of 0.74 (95% confidence interval, 0.58-0.90). The IECC algorithm for histotyping under-performed. The RF algorithmin for categorization was favorable, but validation in larger studies and investigation of additional algorithms to discriminate between all IECC histotypes are needed.

P53 Puzzle: WWP1 and PARC Immunohistochemistry Illuminate New Pathways for Serous Ovarian Cancer

High-grade serous carcinoma is categorized based on p53 mutation status. A relationship is known to exist between p53 mutations and p53 immunoexpression patterns, including overexpression, complete absence, cytoplasmic, and wild-type patterns. The ubiquitin ligases WWP1 and PARC, known to regulate p53 activation, are hypothesized to influence the pathogenesis of serous ovarian tumors. This retrospective study examined 7 low-grade serous carcinomas, 38 high-grade serous carcinomas, and 15 serous cystadenomas, with immunohistochemical analyses performed for WWP1, PARC, and p53. High-grade serous carcinoma cases were classified into wild-type, cytoplasmic, complete absence, or overexpression categories based on p53 immunohistochemistry. PARC and WWP1 expressions were compared across p53 categories and diagnoses. Results showed a statistically significant reduction in WWP1 and PARC expression in serous carcinomas, with the most pronounced loss observed in high-grade cases. Among morphologically classified high-grade carcinomas, 17 overexpression, 11 complete absence, 6 wild-type, and 4 cytoplasmic p53 cases were identified. A statistically significant relationship was found between PARC, WWP1, and p53 status. Higher expression levels of PARC and WWP1 were detected in p53 wild-type cases, whereas lower expression levels were associated with cases exhibiting p53 overexpression and complete absence. This study suggests that PARC and WWP1 play a role in the pathogenesis of high-grade serous ovarian carcinoma, potentially mediated by p53, making them promising targets for treatment and prognostic markers in serous ovarian cancer.

Evaluation of Diagnostic Accuracy of Immunohistochemical Markers of SATB2 and Villin in Differential Diagnosis of Ovarian and Gastrointestinal Mucinous Carcinoma

Determining the origin of mucinous adenocarcinomas can be challenging due to overlapping histologic and immunohistochemical features. This study evaluates the utility of SATB2 and villin immunohistochemical markers for defining mucinous adenocarcinomas’ origin. We retrospectively analyzed 71 patients with mucinous adenocarcinomas—31 lower gastrointestinal (GI), 28 ovarian, and 12 cases of pseudomyxoma peritonei. Immunohistochemistry for SATB2 and villin was performed on ovarian and GI tumor samples. Sensitivity, specificity, and positive and negative predictive values were calculated to assess diagnostic performance.For identifying GI origin, SATB2 showed 87.1% sensitivity and 100% specificity, while villin exhibited 93.5% sensitivity but only 21.4% specificity. Dual SATB2/villin positivity demonstrated 80.6% sensitivity and 100% specificity for GI origin. For ovarian origin, dual SATB2/villin negativity provided 100% specificity. Logistic regression analysis on the adenocarcinoma cases showed 93.2% accuracy rate of dual staining with SATB2 and villin for predicting GI versus ovarian origin. SATB2 exhibits high specificity for GI mucinous adenocarcinomas, and villin is highly sensitive. Combining SATB2 and villin optimizes diagnostic performance for differentiating the primary origin site of mucinous adenocarcinomas and determining pseudomyxoma peritonei origin. Moreover, the presence of signet ring morphology prompted reconsideration of possible GI origin among our cases. A multimarker immunohistochemical panel, including SATB2 and villin can enhance the diagnostic accuracy of challenging mucinous adenocarcinoma cases.

The Role of Morphology in Predicting Fumarate Hydratase–deficient Uterine Leiomyomas in Young Women

Hereditary leiomyomatosis and renal cell carcinoma is caused by germline mutations in the fumarate hydratase (FH) gene and is associated with an increased incidence of leiomyomas and a potentially aggressive variant of renal cell carcinoma. Pathologic evaluation of uterine leiomyoma can provide an opportunity for early recognition of the syndrome. We reviewed all archived slides of the cases to identify the characteristic morphologic features described for FH-deficient leiomyomas. We performed immunohistochemistry on whole sections of patients with uterine leiomyoma to evaluate for both FH and 2-succinocysteine (2SC) expression. Of the 106 cases, 19 showed the characteristic eosinophilic nucleoli with perinuclear halos, and 24 revealed a characteristic eosinophilic cytoplasmic inclusion consisting of pink globules present within the cytoplasm. Both of these morphologic findings were present together in 15 cases, and hemangiopericytomatous vessels were detected in 23 cases. The loss of FH protein expression was detected in 14 out of 106 cases (13%), and 13 out of 106 cases (12%) were positive for 2SC. We detected 10 cases with both 2SC-positive and FH expression loss. The presence of eosinophilic nucleoli with perinuclear halos and eosinophilic cytoplasmic inclusion was associated with both loss of FH protein expression and 2SC positivity (P < 0.001). These findings underscore the importance of hematoxylin and eosin–based predictive morphology in FH-deficient uterine leiomyomas. Therefore, morphologic assessment of uterine leiomyomas for features of FH deficiency can serve as a screening tool for hereditary leiomyomatosis and renal cell carcinoma syndrome, allowing patients to be divided according to their hereditary risk assessment.

Tissue Factor Pathway Inhibitor-2 Expression in Uterine Cervical Clear Cell Carcinoma: A Potential Biomarker for Clinical Diagnosis

Cervical clear cell carcinoma (CCCC) is an extremely rare histologic type of uterine cancer. Tissue factor pathway inhibitor-2 (TFPI2) is a serine protease inhibitor that was recently shown to be expressed in ovarian clear cell carcinoma and endometrial clear cell carcinomas using immunohistological analyses. In this exploratory study, we conducted an immunohistochemical investigation to determine whether TFPI2 is expressed in cervical cancers, especially CCCC. Further, we examined the expression of hepatocyte nuclear factor 1 homeobox B (HNF-1β), a useful marker for immunohistological diagnosis of ovarian clear cell carcinoma. As a control group, we included 22 patients with cervical intraepithelial neoplasia grade 3 (CIN 3) and 40 patients with non-CCCC (21 with squamous cell carcinoma and 19 with adenocarcinoma). Immunohistochemical staining was positive for TFPI2 in all 3 CCCC cases (100%), whereas in non-CCCC, we observed only weak TFPI2 staining in 7 squamous cell carcinoma cases (33.3%), absence of staining in adenocarcinoma (0%), and staining in one CIN 3 case (4.5%). The histoscore for TFPI2 in CCCC was 166.7 ± 13.2 (mean ± SD), which was significantly higher than that in non-CCCC (3.3 ± 8.3) or CIN 3 (1.4 ± 6.4) (P<0.001). Similarly, HNF-1β staining was noted in all 3 CCCC cases and in 63.2% of the adenocarcinomas, whereas it was absent in CCCC and CIN 3. In conclusion, examination of TFPI2 expression, similar to that of HNF-1β, is useful for validating the immunohistological diagnosis of CCCC.

Use of SATB2 and CDX2 Immunohistochemistry to Characterize and Diagnose Colorectal Cancer

SATB2 has been reported to be highly specific for lower gastrointestinal tract tumors. On the basis of its ileum–colon conversion effects, which involve the activation of colonic genes in cooperation with CDX2 and HNF4A, we hypothesized that SATB2 and CDX2 might define the characteristics of colorectal cancers (CRCs). In the present study, the clinicopathologic and immunohistochemical characteristics of 269 CRCs were analyzed according to SATB2 and CDX2 expression. CRCs with SATB2− and/or CDX2− phenotypes showed associations with poorly differentiated histotypes (P<0.00001), mucus production (P=0.0019), and mismatch repair-deficient phenotypes (P<0.00001). SATB2−/CDX2− CRCs were significantly associated with CK20-negativity, with or without CK7 expression (P<0.00001), as well as with MUC5AC-positivity (P<0.00001), and CD10-negativity (P=0.00047). Negativity for SATB2 or CDX2 was associated with the expression of PD-L1 in both all CRC (P<0.00001) and mismatch repair-proficient CRC (P=0.000091). Multivariate Cox hazard regression analysis identified negativity for SATB2 and/or CDX2 as potential independent risk factors for patients with CRC. Regarding the diagnostic utility of SATB2, all of the 44 CRC metastases could be diagnosed as colorectal in origin if the immunohistochemical phenotypes (including CK7, CK20, and p53) of the primary lesions and patient history were considered. Among the other 684 tumors, we were unable to distinguish a case of CK7−/CK20+/CDX2+/SATB2+ ovarian mucinous cystadenocarcinoma from metastatic CRC without the patient history and clinical information.

Expression of the Receptor for Hyaluronic Acid–Mediated Motility (RHAMM) in Endometrial Cancer is Associated With Adverse Histologic Parameters and Tumor Progression

Endometrial cancer is one of the most common gynecologic malignancies worldwide. Only 2 agents have been approved by Food and Drug Administration for endometrial cancer since 1971. There is a need to identify molecular targets to treat advanced endometrial cancer. The receptor for hyaluronic acid–mediated motility (RHAMM) is upregulated in various types of cancer. Here, we aimed to determine the clinical significance of RHAMM expression in endometrial cancer. Two hundred twenty-five cases of endometrial cancer, including serous and endometrioid types, and 8 cases of normal endometrium were used for studying RHAMM protein levels. The Cancer Genome Atlas database was also queried for RHAMM mRNA expression in endometrial cancer. Increased expression of RHAMM protein was seen in endometrial cancer compared with no or weak expression in normal endometrium. RHAMM expression positively correlated with tumor grade. RHAMM expression was significantly increased in endometrial serous carcinomas, which are high-grade, aggressive types of endometrial cancer, compared with the relatively less aggressive endometrioid carcinomas. RHAMM expression also correlated with the presence of lymphovascular invasion. RHAMM mRNA expression correlated with decreased survival in The Cancer Genome Atlas cohort. Therefore, increased RHAMM expression in endometrial cancer is associated with high-grade tumors and is indicative of more aggressive behavior. These findings suggest RHAMM as a prognostic factor in endometrial cancer and as a potential therapeutic target in advanced endometrial cancer for future studies.

TTF-1-positive Metastatic Endometrioid Carcinoma: A Case Report and Review of Literature of a Potential Diagnostic Pitfall

A 75-year-old female patient, nonsmoker was addressed to our institution for a fracture of C5 vertebra with spinal cord compression by a tumor mass invading surrounding soft tissue. She had a previous history of breast ductal carcinoma and endometrioid carcinoma. Biopsy of the tumor mass showed a TTF-1-positive carcinoma. Molecular study showed a E545K mutation of PIK3CA. Lung imaging showed multiple nodules evocative of metastasis rather than a primitive tumor. Reviewing of slides of endometrioid carcinoma showed areas positive for TTF1, and the same E545K mutation was found in endometrial tumor. The final diagnosis was endometrioid metastatic carcinoma with aberrant TTF-1 expression. A subset of endometrial neoplasm expresses TTF-1, this situation might be confusing especially in case of metastatic disease.

High Homogeneity of Mesothelin Expression in Primary and Metastatic Ovarian Cancer

To study the extent of heterogeneity of mesothelin overexpression in primary ovarian cancers and their peritoneal and lymph node metastases, a tissue microarray (TMA) was constructed from multiple sites of 220 ovarian cancers and analyzed by immunohistochemistry. One tissue core each was taken from up to 18 different tumor blocks per cancer, resulting in a total of 2460 tissue spots from 423 tumor sites (188 primary cancers, 162 peritoneal carcinosis, and 73 lymph node metastases). Positive mesothelin expression was found in 2041 of the 2342 (87%) arrayed tissue spots and in 372 of the 392 (95%) tumor sites that were interpretable for mesothelin immunohistochemistry. Intratumoral heterogeneity was found in 23% of 168 primary cancer sites interpretable for mesothelin and decreased to 12% in 154 peritoneal carcinosis and to 6% in 71 lymph node metastases (P<0.0001). Heterogeneity between the primary tumor and matched peritoneal carcinosis was found in 16% of 102 cancers with interpretable mesothelin results. In these cancers, the mesothelin status switched from positive in the primary tumor to negative in the peritoneal carcinosis (3 cancers) in or vice versa (2 cancers), or a mixture of positive and negative peritoneal carcinoses was found (11 cancers). No such switch was seen between the mesothelin-interpretable primary tumors and their nodal metastases of 59 cancers, and only 1 mesothelin-positive tumor had a mixture of positive and negative lymph node metastases. In conclusion, mesothelin expression is frequent and highly homogeneous in ovarian cancer.

Prognostic Value of IMP3 Expression in Squamous Cervical Cancer

Cervical cancer remains one of the leading causes of death from malignant diseases in women worldwide. Primary and secondary prevention have led to better outcomes in developed countries, whereas in developing countries, cervical cancer continues to be responsible for an unjustifiably high number of fatalities. The discovery of new tumor biomarkers can lead to earlier diagnosis, better therapeutic decisions, and improved treatment methods. IMP3 is a protein responsible for invasiveness and other aggressive characteristics of tumor processes. Its highly specific expression has been proven in various malignant processes. The level of IMP3 expression in cervical cancer cells could be used as a prognostic factor for a worse disease course. In this study, IMP3 expression was examined in 80 patients who underwent surgery for squamous cell cervical cancer in the first FIGO stage of the disease, and its association with disease-free period and overall survival was investigated. Data analysis did not show a statistically significant association between IMP3 expression and the mentioned primary outcomes, despite its association with clinical-pathological indicators of advanced disease. In conclusion, the analysis of IMP3 protein expression in patients with early-stage cervical cancer is of limited utility.

TAGLN2-mediated Actin Cytoskeleton Stabilization Promotes Proliferation and Metastasis of Ovarian Carcinoma

Transgelin-2 (TAGLN2) is an actin-binding protein associated with tumor progression, particularly in gastric and brain tumors. However, its role in ovarian cancer metastasis is still not fully understood. This study investigated the role of TAGLN2 and its potential mechanism in ovarian cancer metastasis. TAGLN2 expression in ovarian cancer and normal tissues was assessed using Oncomine, Human Protein Atlas (HPA), and immunocytochemistry (IHC). Skov3 cells with TAGLN2 knockdown (transient knockdown of TAGLN2 of homo TAGLN2 was performed using specific small interfering RNAs), and Skov3 cells transfected with scrambled siRNAs (negative control group) were subjected to colony formation and wound healing assays to assess cell proliferation and migration in vitro. RT-PCR and western blot were used to assess TAGLN2 mRNA and protein expression; immunofluorescence staining was used to investigate the TAGLN2 impact on the cytoskeletal organization. Elevated TAGLN2 levels were observed in ovarian cancer compared with normal tissues, which was confirmed by IHC of a tissue microarray containing 65 ovarian tumor samples. TAGLN2 knockdown reduced cell proliferation and migration in vitro. Also, TAGLN2 was found to co-localize with F-actin; the knockdown of TAGLN2 impaired cytoskeletal organization, emphasizing its influence on cellular structures. In summary, TAGLN2, highly expressed in ovarian cancer, promotes migration and proliferation through cytoskeletal reorganization; thus, it may be a new therapeutic target for ovarian cancer.

HER2-positive Metastatic Melanoma: A Cautionary Tale!

A Retrospective Study and Literature Review of Cervical Villoglandular Adenocarcinoma: A Candidate Paradigm of Silva System Pattern A

The aim was to investigate the clinicopathologic characters of cervical villoglandular adenocarcinoma (VGA), the authors retrospectively reviewed 4 cases of VGA, including clinical characteristics, pathology, managements, together with outcome information. The median age of the patients was 42 (range: 37 to 58), with 3 of them presenting with stage IB disease and 1 presenting with IVB. Human papillomavirus infection was tested in 3 of the patients, with all positive with high-risk type. Three of the patients underwent a radical hysterectomy with bilateral salpingo-oophorectomy plus bilateral pelvic lymphadenectomy, and 2 of them underwent subsequent chemotherapy. One patient received a bilateral salpingo-oophorectomy plus pelvic and periaortic lymphadenectomy and postoperative radiochemotherapy. Lymph node metastasis was detected in 1 patient. The follow-up time ranged from 56 to 120 months (median: 70 mo). Except for 1 person who experienced recurrence, all patients are alive at present and no recurrence occurred. In conclusion, VGA is a rare subtype of adenocarcinoma of the uterine cervix with distinct exophytic, villous-papillary growth pattern and extremely excellent prognosis, which corresponds with pattern A in Silva system, while its underlying mechanism and genetic background is still far from well known.

Molecular Profiling Reveals Limited Targetable Biomarkers in Neuroendocrine Carcinoma of the Cervix

Neuroendocrine carcinoma of the cervix (NEC) is a rare and highly aggressive cervical malignancy. Given that no targeted therapy has been approved specifically to NEC, we investigated the presence of novel, potentially targetable biomarkers in a large cohort of NEC. Sixty-two NEC were molecularly profiled for biomarkers of targeted therapies including antibody-drug conjugates [delta-like canonical notch ligand 3 (DLL3), a trophoblast cell surface antigen 2 (TROP-2), and folate receptor 1 (FOLR1)], NTRK1-3 gene fusions, and immune checkpoint inhibitors [programmed death-ligand 1 (PD-L1), tumor mutational burden, and microsatellite instability] using immunohistochemistry and DNA/RNA next-generation sequencing assays. A cohort of squamous cell carcinomas of the cervix (n=599) was used for comparison for immune-oncology biomarkers. DLL3 expression was observed in 81% of the cases. DLL3 expression was inversely correlated with commonly observed pathogenic mutations in PIK3CA (17%) (P=0.018) and PTEN (10%) (P=0.006). Other more frequently seen pathogenic mutations (TP53 17%, KRAS 11%, and CTNNB1 5%) were not associated with DLL3 expression. TROP-2 expression was detected in only 1 case and no case expressed FOLR1. Although NTRK protein expression was observed in 21% of the cases, none of these had an NTRK gene fusion. PD-L1 expression (10%) and high tumor mutational burden (3%) were significantly less frequent in NEC compared with the squamous cell carcinoma cohort (79% and 11%, respectively). None of the NEC exhibited high microsatellite instability status. Despite frequent DLL3 expression in NEC, a potential therapeutic benefit of DLL3-targeted drugs remains uncertain given the recent failure of the Rova-T therapeutic trial in small cell lung carcinomas. Small cohorts of NEC enriched in PIK3CA/PTEN/AKT and programmed cell death protein 1/PD-L1 alterations indicate therapeutic roles for their respective inhibitors.

PD-L1 and MHC Class I Expression in High-grade Ovarian Cancers, Including Platinum-resistant Recurrences Treated With Checkpoint Inhibitor Therapy

Immune-modulating therapies targeting the programmed cell death-1/programmed cell death ligand-1 (PD-L1) immunosuppressive system have been used successfully in many solid tumor types. There is evidence that biomarkers such as PD-L1 and major histocompatibility complex (MHC) class I help identify candidates for anti-programmed cell death-1/PD-L1 checkpoint inhibition, though the evidence is limited in ovarian malignancies. PD-L1 and MHC Class I immunostaining was performed on pretreatment whole tissue sections in 30 cases of high-grade ovarian carcinoma. The PD-L1 combined positive score was calculated (a score of ≥1 is considered positive). MHC class I status was categorized as an intact or subclonal loss. In patients who received immunotherapy, drug response was assessed using RECIST criteria. PD-L1 was positive in 26 of 30 cases (87%; combined positive score: 1 to 100). Seven of 30 patients showed subclonal loss of MHC class I (23%), and this occurred in both PD-L1 negative (3/4; 75%) and PD-L1 positive (4/26; 15%) cases. Only 1 of 17 patients who received immunotherapy in the setting of a platinum-resistant recurrence responded to the addition of immunotherapy, and all 17 died of disease. In the setting of recurrent disease, patients did not respond to immunotherapy regardless of PD-L1/MHC class I status, suggesting that these immunostains may not be effective predictive biomarkers in this setting. Subclonal loss of expression of MHC class I occurs in ovarian carcinoma, including in PD-L1 positive cases, suggesting that the 2 pathways of immune evasion may not be mutually exclusive and that it may be important to interrogate MHC class I status in PD-L1 positive tumors to identify additional immune evasion mechanisms in these tumors.

Mismatch Repair Deficiency in Adult Granulosa Cell Tumors: an Immunohistochemistry-based Preliminary Study

Objective: Adult granulosa cell tumors (AGCTs) are rare ovarian malignant neoplasms; their etiopathogenetic mechanisms remain largely unelucidated. Lately, defects in mismatch repair (MMR) have been implicated in the pathogenesis of AGCTs. Demonstration of MMR deficiency in these tumors can help identify patients potentially eligible for immune checkpoint inhibition therapy. The present study was done to explore the role of MMR deficiency in the etiopathogenesis of AGCTs. Methods: This was a retrospective study conducted on histopathologically confirmed AGCT cases. MMR protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays using an antibody panel of MSH2, MSH6, MLH1, and PMS2. Results: Of a total of 40 ovarian AGCTs evaluated for MMR deficiency, none demonstrated loss of expression of any of the 4 MMR proteins. Conclusions: The results of our preliminary study show that there is no association between MMR deficiency with AGCT. Nevertheless, larger multicenter studies are needed to confirm or refute this observation.

Zinc Finger Protein 24 is a Prognostic Factor in Ovarian Serous Carcinoma

Objective: As a member of the zinc finger protein family, zinc finger protein 24 (ZNF24) contains a Cys2His2 zinc finger domain and acts as a transcription factor. ZNF24 has been reported to be downregulated in gastric cancer and breast cancer. However, little is known about its expression and function in ovarian serous carcinoma (OSC). Patients and Methods: We collected 117 OSC patients during 2011 to 2017 and retrospectively retrieved their clinicopathologic characteristics as well as their survival data. Protein level was analyzed by immunohistochemistry, mRNA level was evaluated by RT-qPCR assay, and transcriptional data was obtained from TCGA data sets. The correlations between ZNF24 expression and patients’ features were assessed using χ2 test. Univariate and multivariate analyses were used to identify the prognosis predicative potential of ZNF24 in OSC. The function of ZNF24 in the epithelial ovarian cancer cells was also verified by in vitro cellular experiments. Results: Among the 117 cases, ZNF24 was downregulated in 52 OSC samples (44.6%) and significantly correlated with tumor stages. According to univariate and multivariate analyses, ZNF24 can act as an independent prognostic indicator for the overall survival of OSC patients, whose lower expression was associated with poorer clinical outcomes. Ectopic overexpression and knockdown assays indicated that ZNF24 can negatively regulate the OSC cell viability. Conclusions: OSC patients with low level of ZNF24 have worse overall survival compared with those possess high-ZNF24 expression. Downregulated ZNF24 may be involved in the proliferation of OSC, and ZNF24 expression can serve as an independent survival predictor.

Assessment of HPV Risk Type in H&E-stained Biopsy Specimens of the Cervix by Microscopy Image Analysis

Objectives:The objective of this study was (a) to identify, by computer processing of digitized images of hematoxylin and eosin (H&E)-stained biopsy material of the cervix, differences in the structure of nuclei between high-risk (HR) and low-risk (LR) human papillomavirus virus (HPV) types and (b) to assess the HPV risk type by designing a decision-support system (DSS).Materials and Methods:Clinical material comprised H&E-stained biopsies from squamous intraepithelial lesions of 55 patients with polymerase chain reaction-verified HR-HPV (26 patients) or LR-HPV (29 patients) infection. From each patient’s biopsy specimen, we digitized 1 region of interest, guided by the expert physician. After the segmentation of nuclei, we quantified from each nucleus 77 textural and morphologic features. We represented each patient by a 77-feature vector, the feature means of all nuclei, and we created 2 classes for HR-HPV and LR-HPV types. We carried out (a) a statistical analysis to determine features with statistically significant differences between the 2 classes and (b) a discriminant analysis, by designing a DSS, to estimate the HPV risk type.Results:Statistical analysis revealed 40 features with between-classes statistically significant differences and discriminant analysis showed that the best DSS design achieved a high accuracy of about 93% in identifying the HPV risk type on data not used in the design of the DSS.Conclusions:Nuclei of HR-HPV types were of higher intensity, contained larger structures, had higher edges, were coarser, rougher, had higher contrast, were larger, and attained more irregular shapes. The proposed DSS indicates that discrimination of HPV risk type from images of H&E-stained biopsy material of the cervix is promising.

GATA3 Expression in HPV-associated and HPV-independent Vulvar Squamous Cell Carcinomas: Patterns of Expression and Prognostic Significance

Substantial diminution or loss of GATA3 expression is reportedly frequent in human papillomavirus–independent (HPVI), p53-mediated vulvar intraepithelial neoplasia. Herein, we study GATA3 expression in vulvar squamous cell carcinoma (VSCC) and assess its clinicopathologic significance. Eighty-six cases of VSCC diagnosed at a single institution were immunohistochemically assessed for their expression of GATA3, as well as any possible relationships with patient outcomes and other clinicopathologic parameters. Given that GATA3 expression pattern in the normal vulvar epidermis is typically strong basal staining with a uniform upward extension until at least the mid epidermal layers, VSCCs were scored using a previously reported tripattern system: pattern 0 (>75% tumor staining), pattern 1 (25% to 75% staining), and pattern 2 (<25% staining). Severe loss of GATA3 expression (pattern 2) was present in both human papillomavirus–associated (HPVA) and HPVI VSCC but was significantly more common in HPVI cases (P<0.001). Among 52 HPVA VSCCs, 16 (30.7%), 15 (28.8%), and 21 (40.3%) cases showed patterns 0, 1, 2 staining whereas among 34 HPVI VSCCs, the respective frequencies were 1 (2.9%), 5 (14.7%), and 28 (82.3%). None of the 30 p53 abnormal VSCCs showed pattern 0 staining (0%). Five (16.6%) and 25 (83.3%) showed patterns 1 and 2 staining, respectively. On univariate analysis, the pattern 2 cohort showed a significantly worse overall survival (OS) and disease-free survival (DFS) than the pattern 0 or 1 cohort (P=0.011 and 0.024, respectively), but this finding was not independent of stage on multivariate analysis (P=0.34; hazard ratio: 1.82; 95% CI: 0.55-6.06). Subgroup analysis of the p53 wild-type cases showed significantly worse OS for pattern 2 than the pattern 0 or 1 cohorts, independent of stage (P=0.04; hazard ratio: 6.5; 95% CI: 1.08-39.8). Subgroup analysis of p53 abnormal cases, however, showed no difference in OS and DFS among the 3-tiered GATA3 cohorts. In summary, loss of GATA3 may be seen in both HPVA and HPVI VSCCs but is significantly more common in HPVI SCCs. Loss or substantial diminution of GATA3 expression (pattern 2) is a negative prognostic factor in vulvar SCCs, but only in the p53 wild-type subset, where its negative prognostic significance appears to be independent of stage.

The Value of Intratumoral and Extratumoral Microvessel Density for the Tumor-dominated Area in the Endometrial Carcinoma

Objective: Microvessel density (MVD) measuring angiogenesis can influence clinicopathologic variables in endometrial carcinoma (EC). MVD is usually assessed in the densest vascular area the tumor, but the distinction between intratumoral and extratumoral MVD is not reported, and tumor-dominated area is not examined for MVD in the EC in the literature. Materials and Methods: A total of 151 cases with EC, which had hysterectomy from 2005 to 2020, were included. All histopathologic parameters were re-evaluated blindly. MVD was counted in the intratumoral (densest tumoral and vascular area) and extratumoral (periphery of the tumor, tumor invasion tip, densest tumoral, and vascular area) areas using immunohistochemical CD31 expression. Results: Large tumor size, deep myometrial invasion, high grade, nonendometrioid tumor type, cervix invasion, lymph node metastasis, human epidermal growth factor receptor 2 positivity, stage III to IV, substantial lymphovascular invasion, and overall survival had significant relations with intratumoral and extratumoral MVD (P<0.05). Age and lymphoplasmacytic inflammation showed marginal significance for extratumoral MVD. Extratumoral and intratumoral MVD had high (near-perfect) agreement (κ=0.870, P=0.001). Cervix invasion, stage III to IV, high grade, intratumoral, and extratumoral MVD were correlated with lower overall survival in the multivariate analyses. Conclusion: High MVD had an impact on the behavior and prognosis of EC and may be a potential indicator for antiangiogenic treatments for aggressive tumors. Although intratumoral and extratumoral MVD in the tumor-dominated area have mostly similar effects in our study, this situation can be cleared more by further investigations.

Human Epidermal Growth Factor Receptor 2 Overexpression/Amplification in Primary Ovarian Endometrioid Carcinoma

Human epidermal growth factor receptor 2 (HER2) expression has become increasingly helpful in predicting responses to anti-HER2 agents in gynecological cancers. This study retrospectively analyzed HER2 expression in 48 primary ovarian endometrioid carcinomas. HER2 immunohistochemistry was performed using the Ventana platform (Clone 4B5 monoclonal predilute) following the manufacturer’s protocol. HER2 expression was equivocal (score 2+) by image analysis in 2 cases (4.17%) based on the breast cancer criteria. Fluorescence in situ hybridization was negative for HER2 amplification in one case (International Federation of Gynecology and Obstetrics, grade 1) and positive in the other (International Federation of Gynecology and Obstetrics, grade 3). Our findings contribute to the growing evidence that HER2 is overexpressed in a small proportion of ovarian endometrioid carcinoma, and thus may serve as a potential therapeutic target in selected cases.

Applicability of the FDA-approved Immunohistochemical Panel for Identification of MMRd Phenotype in Uterine Endometrioid Carcinoma

Recently, the US Food and Drug Administration (FDA) approved the Ventana MMR RxDx Panel as the first immunohistochemical companion diagnostic test for identification of tumors with mismatch repair (MMR) status. The aim of this study was to investigate the accuracy of this test in comparison with polymerase chain reaction (PCR)-based microsatellite instability (MSI) analysis. We assessed the MMR/MSI concordance rate in 140 cases of endometrioid carcinoma. MMR status was evaluated by immunohistochemistry (MMR-IHC), and MSI status was evaluated by PCR-based analysis (MSI-PCR). Potential molecular mechanisms responsible for MSH6 staining variations were also analyzed. Immunohistochemistry showed that 34 tumors (24.3%) were MMRd; these included 26 with combined MLH1/PMS2 loss, 2 with combined MSH2/MSH6 loss, and 6 with isolated MSH6 loss. Heterogeneous MSH6 loss was found in 10 tumors and was recognized only in tumors with combined MLH1/PMS2 loss. Eight of 10 tumors with heterogeneous MSH6 loss harbored MSH6 C8 tract instability, suggesting a secondary somatic event after MLH1/PMS2 loss. MSI-PCR revealed that 102 tumors were MSS, 4 were MSI-low, and 34 were MSI-high. Consequently, MMR-IHC and MSI-PCR showed perfect concordance (kappa=0.080, P<0.0001). However, 10 of the 34 MSI-high tumors, including the 6 tumors with isolated MSH6 loss, showed only minimal microsatellite shift by MSI-PCR, which may have been erroneously interpreted as MSS or MSI-low. On the basis of these findings, we consider that the FDA-approved immunohistochemical panel can detect MMR variations consistently and is more accurate than MSI-PCR for determining the applicability of immune checkpoint inhibitors for treatment of endometrioid carcinomas.

Diagnostic Value of Combined BCOR, Cyclin D1, and CD10 in Differentiating Endometrial Stromal Sarcoma From Other Uterine Spindle Cell Lesions

Uterine spindle cell lesions share a dilemmatic overlapped features that needed to be addressed by the pathologist to reach a conclusive accurate diagnosis for its prognostic value and different management decisions. Usage of combined IHC panel can be an aiding guiding tool in this context. The aim of this study is to evaluate the diagnostic value of combined BCOR, Cyclin D1, and CD10 IHC panel in differentiating endometrial stromal sarcoma from other uterine spindle cell lesions. This study included 60 cases categorized into endometrial stromal sarcoma group (ESS) (12 cases high-grade endometrial stromal sarcoma [HGESS] and 18 cases low-grade endometrial stromal sarcoma [LGESS]), malignant uterine spindle cell lesions group (5 cases adenosarcoma [AS], 6 cases leiomyosarcoma [LS], 4 cases carcinosarcoma [CS]), and benign uterine lesions group (5 cases endometrial stromal nodule [ESN], 5 cases leiomyoma, and 5 cases adenomyosis). IHC staining procedure and evaluation for BCOR, Cyclin D1, and CD10 was performed on all studied cases. BCOR IHC staining was positive in all HGESS (12/12) of ESS group cases, with diffuse pattern in 75% of cases. BCOR-diffuse staining pattern was not recorded in any of LGESS (0/18), malignant mesenchymal lesions group (0/15), and also benign lesions group (0/15). Cyclin D1 positivity was observed only in HGESS cases, in parallel with positive-BCOR expression. On the contrary, CD10 was negatively expressed in all HGESS and positive in all LGESS, ESN, and adenomyosis cases. A specificity of 100% and sensitivity of 75% were recorded in differentiating HGESS from malignant mesenchymal lesions (including LMS, AS, and CS) and also HGESS from LGESS when using the combined panel BCOR+ve D/Cyclin D1+ve / CD10−ve, considering only the BCOR-diffuse staining pattern. In conclusion, BCOR+ve D/Cyclin D1+ve/CD10−ve as a combined panel is 100% specific and with lesser sensitivity in diagnosing HGESS as well as differentiating it from LGESS and other malignant uterine spindle cell lesions

p53, Pirh2, and L1CAM as Promising Prognostic Biomarkers of Endometrial Carcinoma: An Immunohistochemical and Genetic Study

Endometrial cancer (EC) is the most common gynecologic cancer and the current methods for the prediction of its prognosis and treatment response are unfortunately suboptimal. In this study, we evaluated the prognostic value of p53, Pirh2, and L1CAM in 60 cases of EC using immunohistochemistry (IHC) and polymerase chain reaction. TP53 missense mutations result in nuclear accumulation of p53 protein that can be detected as overexpression by IHC. This is in the form of diffuse strong nuclear positivity involving at least at least >50% of the tumor cells as a whole or if >50% of the tumor cells of a discrete geographical areas. Abnormal p53 IHC expression was expressed in 33.3% of the cases and significantly associated with the tumor grade, myometrial invasion (MI), lymphovascular invasion (LVSI), nodal metastasis, and FIGO stage, and the advanced European Society for Medical Oncology (ESMO) risk groups (P<0.001 for each). High IHC Pirh2 expression was noted in 58.3% of the cases, and significantly associated with MI, LVSI, nodal metastasis, FIGO stage, and high-risk group (P<0.001, P=0.011, P=0.010, P=0.024, P=0.005, respectively). There was a significant upregulation of Pirh2 mRNA expression in EC specimens as compared with the control adjacent tissues (P=0.001). Upregulated Pirh2 mRNA expression had a significant association with Pirh2 immunostaining, tumor grade, tumor stage, MI, lymph node involvement, LVSI, and relapse (P<0.001 for each). Positive L1CAM immunoexpression was noted in 26.7% and was significantly associated with grade, MI, LVSI, nodal metastasis, FIGO stage, and high-risk group (P=0.003, P=0.023, P=0.003, P<0.001, P<0.001, P=0.002, respectively). Analysis of follow-up period revealed that EC with abnormal p53 IHC expression, high pirh2 and positive L1CAM expression exhibited a potent relation with tumor relapse, shorter overall survival and disease-specific survival (P<0.001 for each). Mutant p53, high Pirh2, and L1CAM-positive EC are highly aggressive tumors with a shortened survival rate, dismal outcome, and high risk of relapse after the standard protocol of therapy.

Immunohistochemical Expression of LHRH Receptor in Different Compartments of Female Genital Tract in Patients With Endometrial Cancer

Luteinizing hormone–releasing hormone receptor (LHRHR) expression has been reported in various cancers, including endometrial neoplasms. Thus, LHRHR provides a potential point for therapeutic approach using LHRH analogs as carrier molecules for chemotherapeutic agents in this cancer population. However, clinical data did not prove any potential benefits for patients. We decided to assess LHRHR expression in patients with endometrial cancer to explain possible lack of efficacy in previous clinical reports. LHRHR expression was assessed immunohistochemically in different anatomic and histogenetic compartments of female genital tract of patients with endometrial cancer. The study sample consisted of paraffin tissue blocks obtained from patients who has undergone primary surgery owing to endometrial cancer. Strong LHRHR expression was found in endometrial cancer, fallopian tube, and concurrent atypical hyperplasia. Interestingly, LHRHR expression showed significant differences depending on the respective compartment of the ovary analyzed. Level of LHRHR expression in patients with primary advanced and unresectable disease, particularly in certain ovarian compartments may be substantially lower, which may influence the use of new targeted therapy regimens. The studies on secondary Müllerian system compartment and its hormonal receptor status may be crucial to understand mechanisms of lack of efficacy of LHRH hybrid molecules anti-cancer treatment.

Effects of Slide Storage on Detection of Molecular Markers by IHC and FISH in Endometrial Cancer Tissues From a Clinical Trial: An NRG Oncology/GOG Pilot Study

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P<0.01). Too few cases in the IHC cohort were FISH positive for any conclusions. HER2 amplification by FISH was unaffected by slide storage. We conclude that use of aged stored slides for proliferation markers TOP2A and Ki67 is feasible but may modestly underestimate true values in endometrial cancer. Pilot studies for particular storage conditions/durations/antigens to be used in translational studies are warranted.