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The purpose of this study is to assess the reproducibility among gynaecological pathologists in their diagnosis of mucinous alterations in endometrial sampling specimens. Twenty-six cases were independently reviewed by four experienced gynaecological pathologists from four academic medical centres. Pathologists were asked to classify each case into one of four groups, including three World Health Organization (WHO)-recognised categories: (1) mucinous metaplasia; (2) atypical mucinous glandular proliferation; (3) carcinoma; and (4) 'other' (absence of a true mucinous alteration and/or an alteration of non-endometrial origin). The overall reproducibility was 'fair' (κ = 0.39). In an analytical scenario that established three clinically significant groups ('benign/non-neoplastic', 'atypical', and 'carcinoma') by redistributing all group 4 responses, the resultant kappa improved to 0.51 (moderate reproducibility). In another analysis with only two categories-'benign/non-neoplastic' versus 'atypical/carcinoma'-reproducibility was similarly moderate (κ = 0.46). However, with one exception, all cases that were ultimately diagnosed as carcinoma in a follow-up hysterectomy specimen, were classified as atypical or carcinoma in the preceding sampling. For 11 cases that were classified as either 'carcinoma' or 'atypical' by all observers, there was moderate reproducibility (κ = 0.53) in making that distinction, and none of a wide array of morphological features were found to significantly distinguish between these two categories. For five cases that all observers classified as either mucinous metaplasia or benign endocervix, reproducibility was substantial (κ = 0.67). In summary, gynaecological pathologists show moderate reproducibility in categorising mucinous alterations in endometrial sampling specimens as benign, atypical, or carcinomatous. They accurately classify as at least 'atypical' those cases that are ultimately diagnosed as carcinoma in the subsequent resection. Our findings suggest that there are indeed some mucinous alterations which have features that do not allow for reproducible assignment by pathologists into the WHO-recognised categories. In this subset of cases, there may be a need for better-defined diagnostic criteria and/or extra-morphological diagnostic tools., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background and Objectives: To examine association of lympho-vascular space invasion (LVSI) with clinico-pathological factors and to evaluate survival of women with low-grade serous ovarian carcinoma containing areas of LVSI., Methods: This is a multicenter retrospective study examining consecutive cases of surgically treated stage I-IV low-grade serous ovarian carcinoma (n = 178). Archived histopathology slides for the ovarian tumors were reviewed, and LVSI was scored as present or absent. LVSI status was correlated to clinico-pathological findings and survival outcome., Results: LVSI was seen in 79 cases (44.4%, 95% confidence interval [CI] 37.1-51.7). LVSI was associated with increased risk of omental metastasis (87.0% vs 64.9%, odds ratio [OR] 3.62, P = 0.001), high pelvic lymph node ratio (median 12.9% vs 0%, P = 0.012), and malignant ascites (49.3% vs 32.6%, OR 2.01, P = 0.035). On multivariable analysis, controlling for age, stage, and cytoreductive status, presence of LVSI in the ovarian tumor remained an independent predictor for decreased progression-free survival (5-year rates 21.0% vs 35.7%, adjusted-hazard ratio 1.57, 95%CI 1.06-2.34, P = 0.026). LVSI was significantly associated with increased risk of recurrence in lymph nodes (OR 2.62, 95%CI 1.08-6.35, P = 0.047)., Conclusion: LVSI in the ovarian tumor is associated with adverse clinico-pathological characteristics and decreased progression-free survival in women with low-grade serous ovarian carcinoma., (© 2017 Wiley Periodicals, Inc.)

A 56-year-old G3P3 postmenopausal woman presented with a 5 month history of abnormal uterine bleeding and pelvic pain. A computed tomographic scan revealed a 5 cm right adnexal cystic mass in addition to a thickened, heterogenous endometrium and leiomyomatous uterus. A total laparascopic hysterectomy and bilateral salpingo-oophorectomy with omental and peritoneal biopsy were performed. Gross examination revealed a 12 week size uterus with small fibroids, normal bilateral atrophic ovaries, and a right paratubal cyst. A 4 cm vegetating mass was found in the right side of the uterine wall. Microscopically, the uterine mass was diagnosed as an endometrioid adenocarcinoma (EAC) FIGO 1 with 70% of myometrial invasion. The remaining endometrium showed a complex atypical hyperplasia. In addition, a 5 cm paratubal cystic mass was found that was separate from the uterus and the right adnexa. The cyst content was a chocolate brown fluid and the cyst wall was smooth with a single solid mass of 2 cm in size. The diagnosis of EAC, FIGO 1 was given. The remaining cyst lining showed endometriotic cyst and foci of endometriosis in the cyst wall. There was no lymphovascular invasion. The entire fallopian tube and ovaries were submitted and they were free of tumor. The patient was diagnosed with primary EAC of the paratubal cyst in addition to EAC of the uterine corpus (pT1b). A close follow-up was recommended. Because of our limited knowledge of carcinomas arising in the paratubal cyst, we will review the literature and discuss their clinical aspects, management, and behavior.

Objective: Evidence of potential prognostic and predictive value for nestin was investigated in well-annotated uterine cancers (UCs)., Methods: Nestin expression and previously-published biomarkers were evaluated by immunohistochemistry (IHC) in UC tissue microarrays. Biomarkers were categorized as low vs. high, and nestin was cut at 10% positive staining. Relationship between nestin and clinicopathologic factors, biomarkers and outcome were evaluated using exact/log-rank testing or logistic/Cox modeling., Results: There were 323 eligible cases, 34% had advanced stage disease, 37% had type II disease, and 5% were carcinosarcomas. High nestin, observed in 19% of cases, was more common in advanced vs. early stage disease, type II cancers or uterine carcinosarcoma vs. type I cancers, grade 3 disease, positive lymphovascular space invasion (LVSI) and tumors >6cm (p<0.05). Nestin was inversely correlated with ER, PR and TFF3, and correlated with p53 and IMP3. Women with high vs. low nestin had worse progression-free survival (PFS) and cancer-specific survival overall, and worse PFS in the subset who received no adjuvant therapy or radiation, or had early stage, type I disease or tumors with both low and high ER, PR, TFF3, PTEN, p53 or IMP3. The relationship between nestin and PFS was independent of stage, LVSI and risk categorization but not type of UC., Conclusions: High nestin was more common in UCs with aggressive features and poor outcome. Nestin may represent a predictive biomarker for treatment selection for patients previously considered to be lower risk and a candidate for no or radiation-based adjuvant therapy, and compliment ER/PR testing., (Published by Elsevier Inc.)

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three-dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early-stage EOC, generated by expressing CMYC and KRAS(G) (12) (V) in TERT-immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography-tandem mass spectrometry. Thirty-seven and 55 proteins were differentially expressed by CMYC and CMYC+KRAS(G) (12) (V) expressing cells respectively (p < 0.05; >2-fold). We evaluated expression of the top candidate biomarkers in ∼210 primary EOCs: CHI3L1 and FKBP4 are both expressed by >96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p = 0.042 and p = 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p = 0.0001) and suboptimal debulking (p = 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p = 3 × 10(-4) , p = 0.016, p = 0.010, respectively). We show in vitro cell biology models of early-stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease., (© 2014 UICC.)

Aims: To evaluate an immunohistochemical panel differentiating endometrial stromal sarcoma (ESS) from uterine leiomyosarcoma (ULMS) and leiomyoma (LM)., Methods: 94 cases (28 ESS, 41 ULMS, 25 LM) were retrieved and arrayed. 10 immunomarkers (estrogen receptor (ER), progesterone receptor (PR), CD10, smooth muscle actin, desmin, h-caldesmon, transgelin, GEM, ASC1, stathmin1) were used. A predictive model was constructed and examined by receiver operating characteristics curve analysis to determine area under the curve (AUC)., Results: The combination of ER(+)/PR(+)/CD10(+)/GEM(-)/h-caldesmon(-)/transgelin(-) can predict ESS versus ULMS with AUC predictive value of 0.872 (95% CI 0.784 to 0.961, p<0.0001). The combination of ER(+)/PR(+)/CD10(+)/h-caldesmon(-)/transgelin(-) can predict low grade (LG) ESS from 'LG' ULMS with AUC predictive value of 0.914 (95% CI 0.832 to 0.995, p<0.0001). Finally, ULMS and ESS, including the LGs, were more likely to be stathmin1(+) than LM., Conclusions: Due to the different clinical course and management, adding novel antibodies (GEM, transgelin) to the well established immunohistochemistry panel seemed to be useful in distinguishing ESS from ULMS and LG ESS from 'LG' ULMS. Finally, stathmin1 expression could be of value in differentiating LM from uterine sarcomas., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells, a considerable body of evidence suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in fallopian tube secretory epithelial cells, but there are conflicting reports about PAX8 expression in OSECs. The purposes of this study were to comprehensively characterize PAX8 expression in a large series of OSECs and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 messenger RNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44% to 71% of OSECs. Calretinin and E-cadherin were frequently coexpressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P = .028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P = .003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs, and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs, PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The microenvironment plays an important role in tumorigenesis. Fibroblast activation protein alpha (FAP) is overexpressed by fibroblasts present in the microenvironment of many tumors. High FAP expression is a negative prognostic factor in several malignancies, but this has not been investigated in epithelial ovarian cancer (EOC). The aim of this study is to define the value of FAP in EOC. Immunohistochemical staining using an anti-FAP antibody was performed on 338 EOC tissues. mRNA levels in cancer cell lines and FAP silencing using siRNA was also done. FAP immunoexpression by tumor stroma was a significant predictive factor for platinum resistance (p = 0.0154). In survival analysis of days to recurrence, FAP stoma (+) was associated with shorter recurrence than those with FAP (-) stroma (p = 0.0247). In 21.8 % of tumors, FAP protein was expressed by the tumor epithelium, and FAP mRNA was more highly expressed in tumors (n = 489) than in normal tissues (n = 8) (p = 3.88 × 10(-4)). In vitro, addition of FAP to EOC cells induced a 10-12 % increase in cell viability both in the presence and absence of cisplatin. Conversely, siRNA silencing of FAP resulted in ~10 % reduction in EOC cell proliferation. We have shown that FAP expression in EOC is associated with poorer clinical outcomes. FAP may have novel cell-autonomous effects suggesting that targeting FAP could have pleiotropic anti-tumor effects, and anti-FAP therapy could be a highly effective novel treatment for EOC, especially in cisplatinum-resistant cases.

Most solid tumors contain cancer-associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF-like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF-specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC., (© 2014 UICC.)

Cancer-associated fibroblasts (CAFs) play an important role in tumor initiation and progression. The aim of this study is to explore the role of 2 CAF markers, fibroblast activated protein (FAP) and α-smooth muscle actin (αSMA), in patients with epithelial ovarian cancer (EOC) post-neoadjuvant chemotherapy. Sixty-six patients with the diagnosis of EOC treated with debulking surgery after neoadjuvant therapy were retrieved from the archives. Immunohistochemistry for FAP and αSMA antibodies were performed on paraffin-embedded tissue. Fisher's exact test was performed to test the association between FAP and αSMA expression and disease status. Kaplan-Meier method with log-rank test was used to check the survival difference between different FAP tumor/stroma expressions. FAP stroma (pos) . expression was strongly associated with higher recurrences rate [OR: 15.95; 95 % CI: 1.521-835.206; p = 0.0072]. Cases with combined FAP stroma (pos) and FAP tumor (neg) had higher death rate [OR: 4.845; 95 % CI: 1.53-16.61; p = 0.0046] and higher recurrence rate [OR: 5.12; 95 % CI: 0.91-54.42; p = 0.0487] compared to all the others. Cases with combined FAP stroma (neg) and FAP tumor (neg) were more likely to have lower recurrence rates [OR: 0.086; 95 % CI: 0.001-0.997; p = 0.0248]. αSMA was expressed by tumor-associated stroma in 95 % of cases and by tumor cells in 9 % of cases. No statistical power was found for αSMA and disease status. Our data indicate that FAP plays an important role in predicting tumor aggressiveness in patients with EOC post-neoadjuvant therapy, and its frequent expression in this malignancy implicates that FAP targeted therapy could be a very attractive strategy.

Objective: The consequences of defective homologous recombination and other DNA repair pathways are important in disease outcomes of numerous tumor types. The objective of this study was to explore BRCA1, PARP, FANCD2, PTEN, H2AX, and ATM protein expression in endometrial cancer (EC)., Methods: PARP1, γH2AX, ATM, FANCD2, PTEN, BRCA1, and p53 proteins were evaluated in EC tissue microarray (TMA) and their expressions were correlated with clinical and pathological parameters in 357 patients., Results: In type I EC, PARP1(+), ATM(+), and FANCD2(+) were associated with high tumor grade (p 0.031, p 0.0045, p 0.0062 respectively); γH2AX(+) and FANCD2(+) with advanced tumor stage (p 0.0004, p 0.0085 respectively); γH2AX(+), FANCD2(+) and p53(+) with the presence of lympho-vascular invasion (p 0.0004, p 0.0042, p 0.0098 respectively); and γH2AX(+) and ATM(+) with tumor recurrence (p 0.0203, p 0.0465) respectively. In type II EC, only PARP1(+) was associated with tumor stage (p 0.0499). EC patients with p53(+) or FANCD2(+) were more likely to recur with 5year recurrence free survival (RFS) probability of 71.4% in comparison to 85.5% for the other patients and they were more likely to have shorter 5year overall survival (OS) of 66.46% in comparison to 78.5% of those other patients Finally, patients with ATM(+) and p53(+) or FANCD2(+) were more likely to recur with 5year RFS probability of 68% versus 80.3% for the other patients., Conclusion: DNA repair proteins seemed to play an important role in EC, and their expressions can forecast for poor outcomes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objectives: TFF3 has been found to be up-regulated at the gene and protein levels in endometrioid adenocarcinoma (EAC) when compared to uterine serous carcinoma (USC) and normal endometrium. In addition, TFF3 has been proven to be an estrogen-responsive gene and its expression level positively correlated to estrogen-receptor (ER) status in breast cancer cell culture. The aims of this study are to determine the expression and the prognostic value of TFF3 in a large series of human endometrial cancer and its relation with ER., Methods: We evaluated 328 endometrial carcinomas using TFF3 and ER antibody on paraffin-embedded tissue. 74% were type I (EAC), and 26% were type II (USC, CCC and carcinosarcoma)., Results: In type I carcinomas, TFF3(+) expression was associated with no lympho-vascular invasion (p=0.0131), disease status (p=0.0132), recurrence-free survival (p=0.0424) and overall survival (p=0.0018). There was a positive association between TFF3 and ER (p<.0001). The combination of TFF3(+)/ER(+) was associated with low FIGO grade (p=.0122), early FIGO stage (p=.0062), absence of recurrence (p=.0037), absence of LVI (p=.0011), no lymph node involvement (p=.0116) and disease status (p=.0107). TFF3 appeared to be an independent prognostic marker in predicting recurrences (p=.046). In type II carcinomas, TFF3 failed to have a prognostic value., Conclusion: 1-TFF3 seems to be a novel pathway in the pathogenesis of type I endometrial carcinomas. 2-The strong association of TFF3 and ER in the estrogen-dependent endometrioid carcinoma could explain the reason for its frequent expression by this tumor type. 3-TFF3(+) seems to forecast a good prognosis in type I endometrial carcinomas. Based on our data, TFF3 expression in endometrial cancer deserves further investigation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients., (Copyright © 2012 UICC.)

Aims: Differentiating endometrioid adenocarcinoma (EAC) from uterine serous carcinoma (USC) and clear cell carcinoma (CCC) of the endometrium can be challenging. We undertook an immunohistochemical study to address this issue., Methods and Results: We evaluated 401 endometrial carcinomas cases by using four immunomarkers - oestrogen receptor (ER), progesterone receptor (PR), insulin-like growth factor II mRNA - binding protein 3 (IMP3), and intestinal trefoil factor 3 (TFF3)-on a tissue microarray. The cases included 311 EACs (G1, 146; G2, 104; and G3, 61), 69 USCs, and 21 CCCs. ER, PR and TFF3 were most frequently expressed in EACs (P < 0.001), and IMP3 was more frequently expressed in USCs and CCCs (P < 0.001). ER(+) /PR(+) /TFF3(+) /IMP3(-) was the best marker combination associated with EAC [exact odds ratio (OR) 112; 95% confidence interval (CI) 19-∞; P < 0.0001]. This marker combination remained very reliable after adjustment for tumour grade (exact OR 19.2; 95% CI 3-∞; P = 0.0004). Because distinguishing EAC G3 from USC and CCC on the basis of morphology may be difficult, the use of immunomarkers to improve reproducibility is highly recommended. We found the ER(+) /PR(+) /TFF3(+) /IMP3(-) immunoprofile to be the best combination for confirming a diagnosis of endometrioid adenocarcinoma (exact OR 19.2; 95% CI 3-∞; P = 0.0004)., Conclusions: We recommend using an ER/PR/TFF3/IMP3 immunohistochemical panel in selected cases of endometrial carcinoma where the differential diagnosis is challenging., (© 2013 Blackwell Publishing Ltd.)

For many cancers, there is a real need for more effective therapies. Although many drugs show promising results in vitro, most fail to translate into an in vivo model system, and only ∼5% show anti-tumor activity in clinical trials. It remains a significant challenge to accurately replicate in vitro the complex in vivo microenvironment in which cancers thrive, but this will be key to increasing the success of translating novel therapies into clinical practice. Three-dimensional (3D) cell culture models may better mimic primary tumors in vivo than traditional two-dimensional (2D) cultures. Therefore, we established and characterized 3D in vitro models of 31 epithelial ovarian cancer (EOC) cell lines, compared their biological and molecular features with 2D cultures and primary tumors, and tested their efficacy as models for evaluating chemoresponse. When cultured in 3D using polyhydroxoethylamethacrylate-coated plastics, EOC lines formed multicellular aggregates that could be classified as 'large dense', 'large loose', and 'small', based on size, light permeability, and proportion of cells incorporated into the complex structures. Features of histological differentiation characteristic of primary tumors that were not present in 2D cultures were restored in 3D. For many cell lines, the transition from a 2D to 3D microenvironment induced changes in the expression of several biomarkers relevant to disease. Generally, EOC cell lines proliferated more slowly and were more chemoresistant in 3D compared with 2D culture. In summary, 3D models of EOCs better reflect the histological, biological, and molecular features of primary tumors than the same cells cultured using traditional 2D techniques; 3D in vitro models also exhibit different sensitivities to chemotherapeutic agents compared with 2D models, which may have a significant impact on the success of drug testing pipelines for EOC. These findings could also impact in vitro modeling approaches and drug development strategies for other solid tumor types.

Objectives: Pair-Box 8 (PAX8) is a transcription factor which has been found to be overexpressed in ovarian serous carcinoma (OSC). Silencing PAX8 by using shRNA led to a drop in cell viability in ovarian cancer cell lines, suggesting its use as a targeted therapeutic agent. The prognostic value of PAX8 in OSC is still widely unknown. The aim of this study was to evaluate PAX8 as a prognostic biomarker in patients with advanced stage OSC., Methods: PAX8 was evaluated using immunohistochemistry on a tissue microarray of 148 OSC and the expression was correlated to the following clinico-pathologic variables; age of diagnosis, tumor stage, optimal debulking, recurrence free survival (RFS) and overall survival (OS)., Results: We found that PAX8 was expressed in 61% of cases. There was no association between PAX8 and tumor stage, optimal debulking and disease recurrence. In addition, PAX8 failed to have a predictive value in disease outcome., Conclusion: Despite showing that PAX8 protein is not a useful predictive marker in patients with high grade, advanced stage OSC, its overexpression in a large number of these cases makes the inhibition of PAX8 a very attractive targeted therapy., Competing Interests: statement The authors declare that there are no conflicts of interest., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Differentiating uterine serous carcinoma (USC) from endometrioid adenocarcinoma (EAC) could be problematic, especially in high-grade EACs and tumors exhibiting architectural variations. To address this issue, we evaluated 103 endometrial carcinoma cases using 4 immunomarkers, beta-catenin, IMP3, PTEN, and p53. Cases included 31 USCs, 57 EACs, and 15 mixed EAC-USCs. Of 31 USCs and 57 EACs, 8 and 9, respectively, were considered diagnostically difficult and challenging. beta-catenin was more frequently expressed in EAC (P = .001); p53, PTEN, and IMP3 were more frequently found in USC (P < .001 for each). IMP3 was the best independent predictive marker for USCs. The best marker combination for predicting USCs was PTEN+/IMP3+ (exact odds ratio, 163.87; 95% confidence interval, 19.62 to infinity; P < .001). IMP3 was consistently negative in all 9 challenging EAC cases and consistently positive in all 8 challenging USC cases. None of the markers or their combinations demonstrated any value in making the diagnosis of serous component in mixed EAC-USC tumors. IMP3 immunoexpression and the IMP3+/PTEN+ pattern are the best independent and combination markers, respectively, to predict USCs. We strongly recommend using them in difficult and challenging cases.

NY-ESO-1 is a "cancer-testis" antigen frequently expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8(+) T cells derived from peripheral blood lymphocytes (PBLs), tumor-infiltrating lymphocytes (TILs), and tumor-associated lymphocytes (TALs) of EOC patients with NY-ESO-1-expressing tumors, with or without humoral immunity to NY-ESO-1. Whereas NY-ESO-1-specific CD8(+) T cells were readily detectable ex vivo with tetramers in TILs and TALs of seropositive patients, they were only detectable in PBLs following in vitro stimulation. Compared with PBLs, tumor-derived NY-ESO-1-specific CD8(+) T cells demonstrated impaired effector function, preferential usage of dominant T-cell receptor, and enriched coexpression of inhibitory molecules LAG-3 and PD-1. Expression of LAG-3 and PD-1 on CD8(+) T cells was up-regulated by IL-10, IL-6 (cytokines found in tumor ascites), and tumor-derived antigen-presenting cells. Functionally, CD8(+)LAG-3(+)PD-1(+) T cells were more impaired in IFN-gamma/TNF-alpha production compared with LAG-3(+)PD-1(-) or LAG-3(-)PD-1(-) subsets. Dual blockade of LAG-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8(+) T cells, indicating that antitumor function of NY-ESO-1-specific CD8(+) T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

Cancer-embryo antigens or developmentally restricted differentiation antigens (DRDAGs), such as PLAC1 (CT92) and developmental pluripotency associated-2 (DPPA2/CT100), are expressed in pluripotent embryonic cells. They are also recognized as cancer-testis antigens (CT) which are proteins normally expressed only in the human germ line but that are also present in a significant subset of malignant tumors. These antigens may prove to be markers of 'repopulating' cells with stem cell-like characteristics and could be critical targets for immunotherapy in epithelial ovarian cancer (EOC). Our objective was to define the frequency of expression and immunogenicity of PLAC1 and DPPA2 in EOC and correlate expression with clinical outcome. One-step reverse transcriptase PCR was performed on 101 EOC samples and a panel of normal tissues. Expression of PLAC1 and DPPA2 in the EOC specimens was 21/101 (21%) and 31/101 (31%) respectively. In normal tissues, PLAC1 expression was restricted to the placenta while DPPA2 expression was restricted to the placenta and testis. Immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were also performed on a subset of specimens. Humoral immunity was demonstrable in 2/12 serum samples from patients whose tumors expressed DPPA2. There was no demonstrable antibody response to PLAC1 in patients with PLAC1 positive tumors. The presence of PLAC1 and DPPA2 did not have a statistically significant effect on recurrence-free and overall survival. The tissue-restricted expression of PLAC1 and DPPA2, their expression in a significant proportion of EOC patients, and their potential to represent markers of stem cells make DRDAGs attractive targets for antigen-specific immunotherapy in EOC.

Background: Prognostic factors in predicting outcomes in patients with head and neck squamous-cell carcinoma (HNSCC) are limited to the clinical-pathological parameters, including lymph node metastasis, location, grade and stage of the disease., Aim: To determine whether the expression of these proteins has a value in predicting patient outcome., Methods: Ezrin, maspin and nm23-H1 immunohistochemistry in tissue samples of 120 patients with HNSCC were evaluated using the microarray technique., Results: In determining the association among each of the three proteins and the clinical-pathological parameters, low maspin expression was the only one found to be significantly associated with high tumour grade (p = 0.007); all others showed no significant associations. In univariate analysis, patients with tumours expressing high ezrin had a shorter disease-free survival (DFS) of 51% than those with low ezrin expression (DFS 84%; p = 0.08). In multivariate analysis, tumours with the combination of loss of maspin and low histological grade had longer DFS (83%) compared with those with high maspin and high histological grade (DFS 42%; p = 0.08)., Conclusion: Our study is the first to determine the value of ezrin and maspin in HNSCC in a large series of patients with long follow-up. Ezrin and maspin seem to have a potential prognostic value in patients with HNSCC but results should be confirmed with further studies.

Deficiency of glutathione S-transferase (GST) or NAD(P)H:quinone oxidoreductase 1 (NQO1) in humans is associated with increased risk of urothelial bladder cancer. Broccoli sprouts are a rich source of several isothiocyanates (ITCs), particularly sulforaphane (SF) which has shown promising chemopreventive activities. We report herein that a broccoli sprout ITC extract significantly induced both GST and NQO1 in cultured bladder cells in vitro and in rat bladder tissues in vivo. The inducer activity of the extract was comparable to that of pure SF on the basis of total ITC concentrations. The bladder was one of the most responsive organs to induction of the enzymes by the extract. Induction of the enzymes by the extract was largely mediated by Nrf2, a transcriptional factor that plays a critical role in the induction of many detoxification enzymes. Moreover, induction of GST and NQO1 in the rat bladder in vivo by the extract was associated with high levels of urinary ITC metabolites, but no toxic effects on the bladder mucosa were detected. In conclusion, broccoli sprout ITC extract is a potent inducer of GST and NQO1 in the bladder and is a promising agent for prevention of bladder cancer.