Guangming Han

TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma--evidence supporting the clonal relationship of the two lesions.

Serous tubal intraepithelial carcinomas (STICs) have been proposed to be the most likely precursor of ovarian, tubal and ‘primary peritoneal’ (pelvic) high‐grade serous carcinoma (HGSC). As somatic mutation of TP53 is the most common molecular genetic change of ovarian HGSC, occurring in more than 95% of cases, we undertook a mutational analysis of 29 pelvic HGSCs that had concurrent STICs to demonstrate the clonal relationship of STICs and HGSCs. In addition, we correlated the mutational data with p53 immunostaining to determine the role of p53 immunoreactivity as a surrogate for TP53 mutations in histological diagnosis. Somatic TP53 mutations were detected in all 29 HGSCs analysed and the identical mutations were detected in 27 of 29 pairs of STICs and concurrent HGSCs. Missense mutations were observed in 61% of STICs and frameshift/splicing junction/nonsense mutations in 39%. Interestingly, there were two HGSCs with two distinctly different TP53 mutations each, but only one of the mutations was detected in the concurrent STICs. Missense mutations were associated with intense and diffuse (≥ 60%) p53 nuclear immunoreactivity, while most of the null mutations were associated with complete loss of p53 staining (p < 0.0001). Overall, this p53 staining pattern yielded a sensitivity of 87% and a specificity of 100% in detecting TP53 missense mutations. In conclusion, the above findings support the clonal relationship of STIC and pelvic HGSC and demonstrate the utility of p53 immunostaining as a surrogate for TP53 mutation in the histological diagnosis of STIC. In this regard, it is important to appreciate the significance of different staining patterns. Specifically, strong diffuse staining correlates with a missense mutation, whereas complete absence of staining correlates with null mutations. Copyright

Intraepithelial T cells and prognosis in ovarian carcinoma: novel associations with stage, tumor type, and BRCA1 loss.

Intraepithelial tumor-infiltrating T cells have been correlated with improved outcomes in ovarian carcinoma, however, it is not known whether there is an association with disease stage, histological subtype, or BRCA mutation/expression. Two case series of ovarian carcinomas were included in the study; a retrospective series of 500 patients, and 40 prospectively collected cases fully characterized for BRCA1 mutation status and expression. Intraepithelial immune cells were assessed as present or absent by immunohistochemical staining of tissue microarrays. In the retrospective case series, the presence of intraepithelial CD8+ T-cells correlated with improved disease-specific survival (P=0.027), whereas intraepithelial CD3+ T cells did not (P=0.49). For serous ovarian carcinomas, the presence of intraepithelial CD3+ and CD8+ T-cells correlated with improved disease-specific survival (P=0.0016 and P≤0.0001, respectively). The presence of intraepithelial CD8+ T cells was not associated with improved survival in endometrioid or clear cell carcinomas. On multivariate analysis, disease stage and CD8+ T cells were found to be independently predictive of improved disease-specific survival, whereas grade, age at surgery, and type of adjuvant treatment were not. In the prospective patient cohort, intraepithelial CD8+ T-cells correlated with the presence of mutation or loss of expression of BRCA1 through promoter methylation (P=0.019). Intraepithelial CD8+ tumor-infiltrating T-cells correlate with improved clinical outcomes for all stages of ovarian cancer; this association is restricted to the serous ovarian cancer subtype, and is an independent prognostic factor on multivariate analysis. The presence of intraepithelial CD8+ T cells also significantly correlates with loss of BRCA1.

Reproducibility of histological cell type in high-grade endometrial carcinoma

Subclassification of endometrial carcinoma according to histological type shows variable interobserver agreement. The aim of this study was to assess specifically the interobserver agreement of histological type in high-grade endometrial carcinomas, recorded by gynecological pathologists from five academic centers across Canada. In a secondary aim, the agreement of consensus diagnosis with immunohistochemical marker combinations was assessed including six routine (TP53, CDKN2A (p16), ER, PGR, Ki67, and VIM) and six experimental immunohistochemical markers (PTEN, ARID1A, CTNNB1, IGF2BP3, HNF1B, and TFF3). The paired interobserver agreement ranged from κ 0.50 to 0.63 (median 0.58) and the intraobserver agreement from κ 0.49 to 0.67 (median 0.61). Consensus about histological type based on morphological assessment was reached in 72% of high-grade endometrial carcinomas. A seven-marker immunohistochemical panel differentiated FIGO grade 3 endometrioid from serous carcinoma with a 100% concordance rate compared with the consensus diagnosis. More practically, a three-marker panel including TP53, ER, and CDKN2A (p16) can aid in the differential diagnosis of FIGO grade 3 endometrioid from endometrial serous carcinoma. Our study demonstrates that the inter- and intraobserver reproducibility of histological type based on morphology alone are mostly moderate. Ancillary techniques such as immunohistochemical marker panels are likely needed to improve diagnostic reproducibility of histological types within high-grade endometrial carcinomas.

Mixed Ovarian Epithelial Carcinomas With Clear Cell and Serous Components are Variants of High-grade Serous Carcinoma : An Interobserver Correlative and Immunohistochemical Study of 32 Cases

There are conflicting data about chemoresistance and prognosis in ovarian clear cell carcinoma (CCC). This could be due to significant interobserver variation in the diagnosis of CCC and other ovarian surface epithelial tumors containing clear cells. Thirty-two cases previously diagnosed as CCC, high-grade ovarian serous carcinoma (SC), and mixed surface epithelial carcinoma (SEC) with clear cell and serous components were reviewed by 4 gynecologic pathologists blinded to the original diagnoses. Interobserver reproducibility was evaluated. Each case was also assessed using immunohistochemical markers Wilm tumor 1, estrogen receptor, and p53. The interobserver reproducibility was greatest for pure CCC (κ of 0.82), and lowest for the mixed SEC (κ of 0.32). Moderate agreement was seen in the pure SC category (κ of 0.59). All pure SC and most mixed SEC presented as stage III or IV diseases. Most pure CCC presented as stage I or II diseases. Immunoreactivities of the mixed SECs were similar to those of pure SC, but significantly different from those of pure CCC for Wilm tumor 1 (P=0.0011 for both components), estrogen receptor (P=0.0003 for clear cell component, P=0.0001 for serous component), and p53 (P=0.0062 for both components). The serous and clear cell components of mixed SEC showed higher mitotic rates than pure CCC (P=0.004 and P=0.023, respectively), but the mitotic rate of pure SC was similar to the mixed SEC. We conclude that (1) pure CCC is reproducibly diagnosed. (2) The diagnosis of mixed ovarian SEC with clear cell component is not reproducible. (3) Mixed SEC with clear cell and serous components show similar stage, mitotic activities, and immunoreactivities to those of pure SC, and likely represent SC with clear cell changes.

Histotype-genotype correlation in 36 high-grade endometrial carcinomas.

Endometrioid, serous, and clear cell carcinomas are the major types of endometrial carcinoma. Histologic distinction between these different tumor types can be difficult in high-grade cases, in which significant interobserver diagnostic disagreement exists. Endometrioid and clear cell carcinomas frequently harbor ARID1A and/or PTEN mutations. Serous carcinoma acquires TP53 mutations/inactivation at onset, with a significant subset harboring an additional mutation in PPP2R1A. This study examines the correlation between tumor histotype and genotype in 36 previously genotyped high-grade endometrial carcinomas. This included 23 endometrioid/clear cell genotype and 13 serous genotype tumors. Eight subspecialty pathologists reviewed representative online slides and rendered diagnoses before and after receiving p53, p16, and estrogen receptor immunostaining results. &kgr; statistics for histotype-genotype concordance were calculated. The average &kgr; values for histotype-genotype concordance was 0.55 (range, 0.30 to 0.67) on the basis of morphologic evaluation alone and it improved to 0.68 (range, 0.54 to 0.81) after immunophenotype consideration (P<0.001). Genotype-incompatible diagnoses were rendered by at least 2 pathologists in 12 of 36 cases (33%) (3 cases by 2/8 pathologists, 2 by 3/8, 2 by 4/8, 3 by 6/8, 1 by 7/8, and 1 case by 8/8 pathologists). Six of the 12 were endometrioid/clear cell genotype tumors, and the other 6 were serous genotype tumors. The histopathologic features associated with histotype-genotype–discordant cases were reviewed, and specific diagnostic recommendations were made to improve concordance. This study found that although the majority of morphologic diagnoses are genotype concordant, genotype-incompatible diagnoses are made in a significant subset of cases. Judicious use and interpretation of p53 immunohistochemistry in selected scenarios can improve histotype-genotype concordance.

Immunohistochemical characterization of prototypical endometrial clear cell carcinoma—diagnostic utility of HNF-1β and oestrogen receptor

The great majority of ovarian clear cell carcinomas have a hepatocyte nuclear factor 1 homeobox B (HNF‐1β)‐positive and oestrogen receptor (ER)‐negative immunoprofile. However, the pattern of HNF‐1β and ER immunostaining in clear cell carcinomas of the endometrium and the usefulness of this panel in distinguishing clear cell carcinoma from other histological types of endometrial carcinoma have yet to be well defined.

Complex atypical hyperplasia of the uterus: characteristics and prediction of underlying carcinoma risk

OBJECTIVE The objective of the study was to identify additional factors that may improve the ability to predict underlying carcinoma risk in patients with complex atypical hyperplasia (CAH) of the uterus. STUDY DESIGN All subjects diagnosed with CAH of the uterus on endometrial sampling from March 1994 to May 2008 were identified. CAH was classified as CAH suspicious, CAH polypoid, CAH focal, or CAH not otherwise specified (NOS). Subjects were then exclusively assigned to 1 of 3 categories: CAH suspicious, CAH NOS, or CAH focal and/or polypoid. RESULTS We identified 197 cases of CAH diagnosed on preoperative endometrial sampling. Carcinoma was subsequently diagnosed in the hysterectomy specimen in 67 subjects (34%). The risk of underlying carcinoma if assigning subjects as CAH suspicious, CAH NOS, or CAH polypoid and/or focal was 56%, 36%, and 20%, respectively (P < .001). CONCLUSION The risk of underlying carcinoma in patients with CAH on preoperative endometrial sampling is associated with the method of sampling and age and can be significantly modified by the nature of pathologic assessment.

MMR deficiency is common in high-grade endometrioid carcinomas and is associated with an unfavorable outcome

OBJECTIVE To assess the prevalence of MMR deficiency (dMMR) in contemporary reclassified high-grade endometrial carcinomas and correlate dMMR with molecular alterations and patient outcome. METHODS In this study we evaluated the expression of MLH1, MSH2, PMS2 and MSH6 assessed by two different methods in a series of 102 high-grade endometrial carcinomas. The series was comprised of 64 high-grade endometrioid carcinomas (HGEC), 27 serous (ESC), and 11 clear cell (CCC) carcinomas. Absence of expression in any of the proteins was considered dMMR. dMMR was correlated with clinicopathological parameters using a Chi-square test. Univariate and multivariate survival analysis was performed using Kaplan-Meier and Cox regression analyses. RESULTS The overall prevalence of dMMR was 28% (29/102) and was seen in 29/64 (45%) HGEC but not detected in any of the ESC and CCC. Within HGEC, dMMR was associated with loss of ARID1A (p=0.0099), loss of PTEN (p=0.044) and wild-type TP53 (p=0.024) expression. dMMR was associated with increased risk for disease specific death by univariate analysis (p=0.013) among stage III/IV HGEC but not in multivariate analysis (p=0.12). CONCLUSIONS Among high-grade endometrial carcinomas, dMMR is restricted to HGEC and could be used as an adjunct diagnostic tool to refute a diagnosis of ESC. The association with dMMR in HGEC with ARID1A/PTEN alterations, TP53 wild type expression pattern and unfavorable outcome suggests that different oncogenetic pathways within HGEC are present.

The diagnostic and biological implications of laminin expression in serous tubal intraepithelial carcinoma.

There is compelling evidence to suggest that serous tubal intraepithelial carcinoma (STIC) is the likely primary site for the development of many pelvic high-grade serous carcinomas (HGSCs). Identifying molecules that are upregulated in STIC is important not only to provide biomarkers to assist in the diagnosis of STIC but also to elucidate our understanding of the pathogenesis of HGSC. In this study, we performed RNA sequencing to compare transcriptomes between HGSC and normal fallopian tube epithelium (FTE), and we identified LAMC1 encoding laminin &ggr;1 as one of the preferentially upregulated genes associated with HGSC. Reverse transcription polymerase chain reaction further validated LAMC1 upregulation in HGSC as compared with normal FTE. Immunohistochemical analysis was performed on 32 cases of concurrent HGSC and STIC. The latter was diagnosed on the basis of morphology, TP53 mutations, and p53 and Ki-67 immunohistochemical patterns. Laminin &ggr;1 immunostaining intensity was found to be significantly higher in STIC and HGSC compared with adjacent FTE in all cases (P<0.001). In normal FTE, laminin &ggr;1 immunoreactivity was predominantly localized in the basement membrane or on the apical surface of ciliated cells, whereas in STIC and HGSC cells, laminin &ggr;1 staining was diffuse and intense throughout the cytoplasm. More importantly, strong laminin &ggr;1 staining was detected in all 13 STICs, which lacked p53 immunoreactivity because of null mutations. These findings suggest that the overexpression of laminin &ggr;1 immunoreactivity and alteration of its staining pattern in STICs can serve as a useful tissue biomarker, especially for those STICs that are negative for p53 and have a low Ki-67 labeling index.

The diagnostic utility of TP53 and CDKN2A to distinguish ovarian high-grade serous carcinoma from low-grade serous ovarian tumors

Low-grade serous carcinomas and serous borderline tumors, combined herein and referred to as low-grade serous tumors, show distinct molecular alterations and clinical behaviors compared with high-grade serous carcinomas. The discrimination between low-grade serous tumors and high-grade serous carcinomas can be challenging on small tissue samples, such as cell blocks of paracentesis fluid or biopsies from omental disease. The purpose of this study was to test the ability of TP53 and CDKN2A immunohistochemistry to distinguish between high-grade serous carcinomas and low-grade serous tumors on small tissue samples. Tissue microarrays containing 582 high-grade serous carcinomas, 45 low-grade serous carcinomas, and 49 serous borderline tumors, confirmed by contemporary histopathological review, were stained for TP53 and CDKN2A (DO7 and E6H4 antibody clones, respectively). TP53 was scored as completely absent, wild-type pattern or overexpressed (>60%), and CDKN2A was scored as either negative/patchy (<90%) or block expression (>90%). The combination of the two markers, ie, the TP53 wild-type pattern and CDKN2A patchy expression, had sensitivity for low-grade serous tumors of 89%, a specificity of 93%, a positive predictive value of 68%, and a negative predictive value of 98%. These markers can, therefore, be used on small biopsies/cell blocks to refute a diagnosis of low-grade serous tumors. These findings may inform emerging neoadjuvant therapeutic strategies in advanced ovarian cancers and may be crucial for future clinical trials on molecular-based therapies.