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Peroxisome proliferator-activated receptor-delta (PPAR-[delta]) is overexpressed in human colon cancer, but its contribution to colonic tumorigenesis is controversial. We generated a mouse model in which PPAR-[delta] was genetically disrupted in colonic epithelial cells by targeted deletion of exon 4. Elimination of colon-specific PPAR-[delta] expression was confirmed by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), immunoblotting, and activity assays. Mice with and without targeted PPAR-[delta] genetic disruption (10-11 mice per group) were tested for incidence of azoxymethane-induced colon tumors. The effects of targeted PPAR-[delta] deletion on vascular endothelial growth factor expression were determined by realtime RT-PCR. Targeted PPAR-[delta] genetic disruption inhibited colonic carcinogenesis: Mice with PPAR-[[delta].sup.-/-]) colons developed 98.5% fewer tumors than wild-type mice (PPAR-[[delta].sup.-/-]) vs wild-type, mean = 0.1 tumors per mouse vs 6.6 tumors per mouse, difference = 6.5 tumors per mouse, 95% confidence interval = 4.9 to 8.0 tumors per mouse, P < .001, two-sided test). Increased expression of vascular endothelial growth factor in colon tumors vs normal colon was suppressed by loss of PPAR-[delta] expression. These findings indicate that PPAR-[delta] has a crucial role in promoting colonic tumorigenesis.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ajog.2003.09.048 Byline: Russell R. Broaddus, Susu Xie, Ching-Ju Hsu, Jian Wang, Sui Zhang, Changping Zou Keywords: Leiomyoma; 4-(N-hydroxyphenyl)retinamide; [alpha]-Difluoromethylornithine; Apoptosis Abstract: This study examined the effects of the chemopreventive agents 4-(N-hydroxyphenyl)retinamide (4-HPR) and [alpha]-difluoromethylornithine (DFMO) on leiomyoma growth. Article History: Received 10 April 2003; Revised 25 June 2003; Accepted 26 September 2003 Article Note: (footnote) [star] Supported in part by a grant from the Women's Fund for Health, Education, and Research (C. Z.) and an Institutional Research Grant from the Physician's Referral Service at the University of Texas M.D. Anderson Cancer Center (R. R. B.).

Immunohistochemistry for mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 is an effective screen to detect individuals at risk for Lynch syndrome. College of American Pathologists guidelines stipulate that protein expression should be reported as present versus absent, as most patients with germline mutations in a mismatch repair gene have complete loss of protein expression in tumor cells. A similar approach is employed to screen for cancer patients eligible for immune checkpoint blockade. This “all or none” interpretive approach ignores substantial evidence that mismatch repair may be more finely regulated by other mechanisms. We have observed clinically that MSH6 expression is variable, even in carcinomas that are overall considered positive for MSH6 expression. A proof-of-principle study was therefore designed to more rigorously quantify the protein expression of MSH6 and its binding partner, MSH2, using image analysis applied to age-matched endometrioid grade 2 subsets that were either mismatch repair intact or MLH1-deficient due to MLH1gene methylation. In both endometrioid groups, MSH6 expression was significantly lower than MSH2 expression. MSH6 expression increased in higher grade, mismatch repair intact serous carcinomas, but it was still significantly lower than that for MSH2. MSH2 expression was consistently high across the 3 different tumor groups. These results suggest that MSH6 expression is subject to wide fluctuations in expression, even when overall its expression is considered intact. While such fluctuations are likely not relevant for Lynch syndrome screening, they may be more impactful when considering patients eligible for immune checkpoint blockade.

Endometrial cancer remains the most common gynecological malignancy in the United States. While the loss of the tumor suppressor, PTEN(phosphatase and tensin homolog), is well studied in endometrial cancer, recent studies suggest that DICER1, the endoribonuclease responsible for miRNA genesis, also plays a significant role in endometrial adenocarcinoma. Conditional uterine deletion of Dicer1and Ptenin mice resulted in poorly differentiated endometrial adenocarcinomas, which expressed Napsin A and HNF1B (hepatocyte nuclear factor 1 homeobox B), markers of clear-cell adenocarcinoma. Adenocarcinomas were hormone-independent. Treatment with progesterone did not mitigate poorly differentiated adenocarcinoma, nor did it affect adnexal metastasis. Transcriptomic analyses of DICER1deleted uteri or Ishikawa cells revealed unique transcriptomic profiles and global miRNA downregulation. Computational integration of miRNA with mRNA targets revealed deregulated let-7 and miR-16 target genes, similar to published human DICER1-mutant endometrial cancers from TCGA (The Cancer Genome Atlas). Similar to human endometrial cancers, tumors exhibited dysregulation of ephrin-receptor signaling and transforming growth factor-beta signaling pathways. LIM kinase 2 (LIMK2), an essential molecule in p21 signal transduction, was significantly upregulated and represents a novel mechanism for hormone-independent pathogenesis of endometrial adenocarcinoma. This preclinical mouse model represents the first genetically engineered mouse model of poorly differentiated endometrial adenocarcinoma.

There is currently no blood-based marker in routine use for endometrial cancer patients. Such a marker could potentially be used for early detection, but it could also help to track tumor recurrence following hysterectomy. This is important, as extra-vaginal recurrence of endometrial endometrioid adenocarcinoma is usually incurable. This proof-of-principle study was designed to determine if tumor-associated mutations could be detected in cell-free DNA from the peripheral blood of early and late stage endometrial endometrioid carcinoma patients. Approximately 90% of endometrioid carcinomas have at least one mutation in the genes CTNNB1, KRAS, PTEN, or PIK3CA. Using a custom panel targeting 30 hotspot amplicons in these four genes, next-generation sequencing was performed on cell-free DNA extracted from plasma obtained from a peripheral blood draw at the time of hysterectomy and the matching tumor DNA from 48 patients with endometrioid endometrial carcinomas. At least one mutation in the tumor was detected in 45/48 (94%) of patients. Fifteen of 45 patients (33%) had a mutation in the plasma that matched a mutation in the tumor. These same mutations were not detected in the matched negative control buffy coat. Presence of a plasma mutation was significantly associated with advanced stage at hysterectomy, deep myometrial invasion, lymphatic/vascular invasion, and primary tumor size. Detecting a plasma-based mutation was independent of the amount of cell-free DNA isolated from the plasma. Overall, 18% of early stage patients had a mutation detected in the plasma. These results demonstrate that mutations in genes relevant to endometrial cancer can be identified in the peripheral blood of patients at the time of surgery. Future studies can help to determine the post-operative time course of mutation clearance from the peripheral blood and if mutation re-emergence is predictive of recurrence.

Lynch syndrome (LS) is a hereditary cancer syndrome caused by a germline mutation in a DNA mismatch repair gene, usually MLH1, MSH2, MSH6, or PMS2. The most common cancers associated with LS are colorectal adenocarcinoma and endometrial carcinoma. Identification of women with LS-associated endometrial cancer is important, as these women and their affected siblings and children are at-risk of developing these same cancers. Germline testing of all endometrial cancer patients is not cost effective, and screening using young age of cancer diagnosis and/or presence of family history of syndrome-associated is underutilized and ineffective. Therefore, most groups now advocate for tumor tissue testing to screen for LS, with germline testing targeted to women with abnormal tissue testing results. Immunohistochemistry for MLH1, MSH2, MSH6, and PMS2 is used in many clinical laboratories for this tumor screening step, as immunohistochemistry is relatively inexpensive and is technically more accessible for smaller clinical labs. PCR-based tissue testing, whereas technically more challenging, does play an important role in the identification of these patients. MLH1methylation analysis identifies women with tumor MLH1 loss who likely have sporadic endometrial cancer and do not need heightened cancer prevention surveillance. High levels of microsatellite instability have been identified in tumors with retained positive expression of mismatch repair proteins. Somatic sequencing of mismatch repair genes from tumor DNA, whereas not currently available in most clinical laboratories, is helpful in resolution of cases in which germline sequencing fails to identify a mutation in a mismatch repair gene. The tumor tissue testing approach can help to identify most women at-risk for germline mutations in a LS gene, but not all patients will be captured using this approach. Clinical suspicion can still play a pivotal role in accurately identifying a subset of these patients.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer–associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP–CUL3 substrates that are preferentially degraded by endometrial cancer–associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer–specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

Although the majority of low grade, early stage endometrial cancer patients will have good survival outcomes with surgery alone, those patients who do recur tend to do poorly. Optimal identification of the subset of patients who are at high risk of recurrence and would benefit from adjuvant treatment has been difficult. The purpose of this study was to evaluate the impact of somatic tumor mutation on survival outcomes in this patient population. For this study, low grade was defined as endometrioid FIGO grades 1 or 2, while early stage was defined as endometrioid stages I or II (disease confined to the uterus). Next-generation sequencing was performed using panels comprised of 46–200 genes. Recurrence-free and overall survival was compared across gene mutational status in both univariate and multivariate analyses. In all, 342 patients were identified, 245 of which had endometrioid histology. For grades 1–2, stages I–II endometrioid endometrial cancer patients, age (HR 1.07, 95% CI 1.03–1.10), CTNNB1 mutation (HR 5.97, 95% CI 2.69–13.21), and TP53 mutation (HR 4.07, 95% CI 1.57–10.54) were associated with worse recurrence-free survival on multivariate analysis. When considering endometrioid tumors of all grades and stages, CTNNB1 mutant tumors were associated with significantly higher rates of grades 1–2 disease, lower rates of deep myometrial invasion, and lower rates of lymphatic/vascular space invasion. When both TP53 and CTNNB1 mutations were considered, presence of either TP53 mutation or CTNNB1 mutation remained a statistically significant predictor of recurrence-free survival on multivariate analysis and was associated with a more precise confidence interval (HR 4.69, 95% CI 2.38–9.24). Thus, mutational analysis of a 2 gene panel of CTNNB1 and TP53 can help to identify a subset of low grade, early stage endometrial cancer patients who are at high risk of recurrence.

Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. cfDNA from 31 patients with diverse advanced cancers and known tumor mutation status was deep sequenced using targeted hotspot panels. Forty-five of 52 expected mutations and two additional mutations (KRASp.Q61H and EZH2p.Y646F) were detected in plasma through a custom bioinformatics pipeline. We observed comparable cfDNA concentration/integrity between collection tubes within 16 hours of plasma separation and equal analytical performance among NGS platforms, with 1% detection sensitivity for cfDNA genotyping.

Although the majority of low grade, early stage endometrial cancer patients will have good survival outcomes with surgery alone, those patients who do recur tend to do poorly. Optimal identification of the subset of patients who are at high risk of recurrence and would benefit from adjuvant treatment has been difficult. The purpose of this study was to evaluate the impact of somatic tumor mutation on survival outcomes in this patient population. For this study, low grade was defined as endometrioid FIGO grades 1 or 2, while early stage was defined as endometrioid stages I or II (disease confined to the uterus). Next-generation sequencing was performed using panels comprised of 46–200 genes. Recurrence-free and overall survival was compared across gene mutational status in both univariate and multivariate analyses. In all, 342 patients were identified, 245 of which had endometrioid histology. For grades 1–2, stages I–II endometrioid endometrial cancer patients, age (HR 1.07, 95% CI 1.03–1.10), CTNNB1mutation (HR 5.97, 95% CI 2.69–13.21), and TP53mutation (HR 4.07, 95% CI 1.57–10.54) were associated with worse recurrence-free survival on multivariate analysis. When considering endometrioid tumors of all grades and stages, CTNNB1mutant tumors were associated with significantly higher rates of grades 1–2 disease, lower rates of deep myometrial invasion, and lower rates of lymphatic/vascular space invasion. When both TP53and CTNNB1mutations were considered, presence of either TP53mutation or CTNNB1mutation remained a statistically significant predictor of recurrence-free survival on multivariate analysis and was associated with a more precise confidence interval (HR 4.69, 95% CI 2.38–9.24). Thus, mutational analysis of a 2 gene panel of CTNNB1and TP53can help to identify a subset of low grade, early stage endometrial cancer patients who are at high risk of recurrence.

Minimally invasive procedures, such as fine needle aspiration and core needle biopsy, are commonly used for the diagnosis in solid organ malignancies. In the era of targeted therapy, it is crucial for molecular testing to be performed on these limited volume specimens. Although several recent studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to whether core needle biopsy specimens are more reliable than fine needle aspiration for molecular studies. In this study, we reviewed concurrently acquired fine needle aspiration and core needle biopsy samples (n=24), and compared overall cellularity, tumor fraction, and the results of next-generation sequencing. All somatic mutations detected in core needle biopsy samples were also detected in fine needle aspiration samples. The estimated tumor fraction was significantly higher in fine needle aspiration smears than core needle biopsy samples (P=0.003), whereas the overall DNA yield from smears was significantly lower than that obtained from the core needle biopsy specimens (P=0.01). The normalized average amplicon coverage for the genes analyzed was significantly higher in cytology smears than paired core needle biopsy samples, with lower numbers of failed amplicons and higher overall mutation allelic frequencies seen in the former. We further evaluated 100 malignant fine needle aspiration and core needle biopsy samples, acquired concurrently, for overall cellularity and tumor fraction. Overall cellularity and tumor fraction of fine needle aspiration samples was significantly higher than concurrently acquired core needle biopsy samples (P<0.001). In conclusion, we show that fine needle aspiration samples frequently provide better cellularity, higher tumor fraction, and superior sequencing metrics than concurrently acquired core needle biopsy samples. Cytologic specimens, therefore, should be better integrated into routine molecular diagnostics workflow to maximize limited tissues for clinically relevant genomic testing.

Clinical next-generation sequencing (NGS) assay choice requires careful consideration of panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, compatibility with low quality and quantity of nucleic acids, and work flow feasibility. Herein, in a high-volume clinical molecular diagnostic laboratory, we have validated a targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay) coupled with high-throughput sequencing using Ion Proton sequencer for routine screening of solid tumors. The panel screens 143 genes using low amounts of formalin-fixed, paraffin-embedded DNA (20 ng) and RNA (10 ng). A large cohort of 121 tumor samples representing 13 tumor types and 6 cancer cell lines was used to assess the capability of the panel to detect 148 single-nucleotide variants, 49 insertions or deletions, 40 copy number aberrations, and a subset of gene fusions. High levels of analytic sensitivity and reproducibility and robust detection sensitivity were observed. Furthermore, we demonstrated the critical utility of sequencing paired normal tissues to improve the accuracy of detecting somatic mutations in a background of germline variants. We also validated use of the Ion Chef automated bead templating and chip loading system, which represents a major work flow improvement. In summary, we present data establishing the OncoMine Comprehensive Assay–Ion Proton platform to be well suited for implementation as a routine clinical NGS test for solid tumors.

Hereditary endometrial carcinoma is associated with germline mutations in Lynch syndrome genes. The role of other cancer predisposition genes is unclear. We aimed to determine the prevalence of cancer predisposition gene mutations in an unselected endometrial carcinoma patient cohort. Mutations in 25 genes were identified using a next-generation sequencing-based panel applied in 381 endometrial carcinoma patients who had undergone tumor testing to screen for Lynch syndrome. Thirty-five patients (9.2%) had a deleterious mutation: 22 (5.8%) in Lynch syndrome genes (three MLH1, five MSH2, two EPCAM-MSH2, six MSH6, and six PMS2) and 13 (3.4%) in 10 non-Lynch syndrome genes (four CHEK2, one each in APC, ATM, BARD1, BRCA1, BRCA2, BRIP1, NBN, PTEN, and RAD51C). Of 21 patients with deleterious mutations in Lynch syndrome genes with tumor testing, 2 (9.5%) had tumor testing results suggestive of sporadic cancer. Of 12 patients with deleterious mutations in MSH6 and PMS2, 10 were diagnosed at age >50 and 8 did not have a family history of Lynch syndrome-associated cancers. Patients with deleterious mutations in non-Lynch syndrome genes were more likely to have serous tumor histology (23.1 vs 6.4%, P=0.02). The three patients with non-Lynch syndrome deleterious mutations and serous histology had mutations in BRCA2, BRIP1, and RAD51C. Current clinical criteria fail to identify a portion of actionable mutations in Lynch syndrome and other hereditary cancer syndromes. Performance characteristics of tumor testing are sufficiently robust to implement universal tumor testing to identify patients with Lynch syndrome. Germline multi-gene panel testing is feasible and informative, leading to the identification of additional actionable mutations.

This article reviews the main tissue testing modalities for Lynch Syndrome in the pathology laboratory, such as immunohistochemistry and PCR based analyses, and discusses their routine application, interpretation pitfalls, and troubleshooting of common technical performance issues. Discrepancies between laboratory and genetic testing may arise, and are examined in the context of the complexity of molecular abnormalities associated with Lynch Syndrome. The merits of targeted versus universal screening in a changing healthcare climate are addressed. In the absence of comprehensive screening programs, specific tumor topography and histological features that may prompt pathologist-initiated molecular tumor testing are outlined.

Endometrial endometrioid carcinomas are related to estrogen excess and express estrogen and progesterone receptors. However, hormone receptor expression can be variable from tumor to tumor, and this variability is not always explained by differences in tumor grade. Variable expression of other biomarkers that may be used in the diagnostic work-up of endometrial cancer has also been noted. We hypothesized that mismatch repair (MMR) defects may contribute to this variability. A total of 411 unselected endometrial carcinomas were evaluated for immunohistochemical expression of DNA MMR proteins and MLH1 methylation. Loss of immunohistochemical expression of MLH1, MSH2, MSH6, or PMS2 was defined as MMR deficient; positive expression was defined as MMR intact. A case-control cohort of 80 Grade 2 endometrioid carcinomas was selected from this set (40 MMR deficient, 40 MMR intact). Cases were matched for histotype, grade, and age. Estrogen receptor, progesterone receptor, CK7, CK20, and Pax-8 immunohistochemistry was evaluated. The median percentage of CK7+tumor cells was significantly lower in the MMR deficient group compared with the MMR intact group. The mean percentage of tumor cells exhibiting estrogen receptor expression was similar in both the MMR-deficient and MMR intact groups. However, there was greater variability in the MMR-deficient group. Our study shows that MMR defects influence the expression of clinically important biomarkers for endometrioid-type endometrial carcinoma as decreased cytokeratin 7 expression is more commonly associated with MMR deficiency.

Rhabdoid histology in clear-cell renal cell carcinoma is associated with a poor prognosis. The prognosis of patients with clear-cell renal cell carcinoma may also be influenced by molecular alterations. The aim of this study was to evaluate the association between histologic features and salient molecular changes in rhabdoid clear-cell renal cell carcinoma. We macrodissected the rhabdoid and clear-cell epithelioid components from 12 cases of rhabdoid clear-cell renal cell carcinoma. We assessed cancer-related mutations from eight cases using a clinical next-generation exome-sequencing platform. The transcriptome of rhabdoid clear-cell renal cell carcinoma (n=8) and non-rhabdoid clear-cell renal cell carcinoma (n=37) was assessed by RNA-seq and gene expression microarray. VHL(63%) showed identical mutations in all regions from the same tumor. BAP1(38%) and PBRM1(13%) mutations were identified in the rhabdoid but not in the epithelioid component and were mutually exclusive in 3/3 cases and 1 case, respectively. SETD2(63%) mutations were discordant between different histologic regions in 2/5 cases, with mutations called only in the epithelioid and rhabdoid components, respectively. The transcriptome of rhabdoid clear-cell renal cell carcinoma was distinct from advanced-stage and high-grade clear-cell renal cell carcinoma. The diverse histologic components of rhabdoid clear-cell renal cell carcinoma, however, showed a similar transcriptomic program, including a similar prognostic gene expression signature. Rhabdoid clear-cell renal cell carcinoma is transcriptomically distinct and shows a high rate of SETD2and BAP1mutations and a low rate of PBRM1mutations. Driver mutations in clear-cell renal cell carcinoma are often discordant across different morphologic regions, whereas the gene expression program is relatively stable. Molecular profiling of clear-cell renal cell carcinoma may improve by assessing for gene expression and sampling tumor foci from different histologic regions.

IMPORTANCE: Chemotherapy response in the majority of patients with ovarian cancer remains unpredictable. OBJECTIVE: To identify novel molecular markers for predicting chemotherapy response in patients with ovarian cancer. DESIGN, SETTING, AND PARTICIPANTS: Observational study of genomics and clinical data of high-grade serous ovarian cancer cases with genomic and clinical data made public between 2009 and 2014 via the Cancer Genome Atlas project. MAIN OUTCOMES AND MEASURES: Chemotherapy response (primary outcome) and overall survival (OS), progression-free survival (PFS), and platinum-free duration (secondary outcome). RESULTS: In 512 patients with ovarian cancer with available whole-exome sequencing data, mutations from 8 members of the ADAMTS family (ADAMTS mutations) with an overall mutation rate of approximately 10.4% were associated with a significantly higher chemotherapy sensitivity (100% for ADAMTS-mutated vs 64% for ADAMTS wild-type cases; P &lt; .001) and longer platinum-free duration (median platinum-free duration, 21.7 months for ADAMTS-mutated vs 10.1 months for ADAMTS wild-type cases; P = .001). Moreover, ADAMTS mutations were associated with significantly better OS (hazard ratio [HR], 0.54 [95% CI, 0.42-0.89]; P = .01 and median OS, 58.0 months for ADAMTS-mutated vs 41.3 months for ADAMTS wild-type cases) and PFS (HR, 0.42 [95% CI, 0.38-0.70]; P &lt; .001 and median PFS, 31.8 for ADAMTS-mutated vs 15.3 months for ADAMTS wild-type cases). After adjustment by BRCA1 or BRCA2 mutation, surgical stage, residual tumor, and patient age, ADAMTS mutations were significantly associated with better OS (HR, 0.53 [95% CI, 0.32-0.87]; P = .01), PFS (HR, 0.40 [95% CI, 0.25-0.62]; P &lt; .001), and platinum-free survival (HR, 0.45 [95% CI, 0.28-0.73]; P = .001). ADAMTS-mutated cases exhibited a distinct mutation spectrum and were significantly associated with tumors with a higher genome-wide mutation rate than ADAMTS wild-type cases across the whole exome (median mutation number per sample, 121 for ADAMTS-mutated vs 69 for ADAMTS wild-type cases; P &lt; .001). CONCLUSIONS AND RELEVANCE: ADAMTS mutations may contribute to outcomes in ovarian cancer cases without BRCA1 or BRCA2 mutations and may have important clinical implications.

The indolent nature and expression of progesterone receptor (PR), a well-known estrogen-induced gene, in a subset of pancreatic neuroendocrine tumors (PanNETs), raise the possibility of hormonal regulation in these tumors.

For the first time, we report a case of a primary high-grade serous carcinoma of the fallopian tube in a 69-yr-old woman with intraepithelial involvement of endocervical glands. The patient had a remote history of tubal ligation and no known personal or family history of breast or gynecologic cancer. She initially presented with an abnormal Pap smear, and, on a subsequent endometrial curettage, detached fragments of high-grade adenocarcinoma were identified. Upon hysterectomy and bilateral salpingoophorectomy, invasive high-grade serous carcinoma was found in the proximal stump of a ligated fallopian tube. Morphologically identical tumor extensively involved the endocervical glands. A single focus of malignant cells was also found at the surface of the lower uterine segment endometrium. By immunohistochemistry, the tumors in the fallopian tube and in the cervix showed the same immunopheonotype. This included diffuse staining for WT-1, PAX-8, p16, and p53, and focal staining for the monoclonal carcinoembryonic antigen. The estrogen receptor was negative. Human papillomavirus-in situhybridization was also negative. The focus of intramucosal tumor within the endometrium was similarly positive for WT-1 and p53 and negative for the estrogen receptor. The patient was subsequently treated with 6 cycles of carboplatinum and taxol. She remains well with no evidence of recurrent disease. The phenomenon of intramucosal tumor spread to the cervix mimicking primary invasive or in situcervical adenocarcinoma may present a diagnostic challenge for the pathologist, requiring consideration of an appropriately wide differential diagnosis when interpreting cervical cytology and endocervical and endometrial biopsies.

Molecular diagnostic testing of endometrial carcinomas in the pathology laboratory has recently emerged as a key component of the clinical evaluation of Lynch syndrome in many centers. Testing modalities involve immunohistochemical and PCR-based analyses. This article outlines the routine application of these analyses, provides a practical guide for troubleshooting some of the common technical issues related to their performance, and reviews common pitfalls in their interpretation. Discrepancies between tissue testing and genetic testing results are discussed in the context of the current understanding of endometrial cancer biology. The merits of universal versus targeted tissue testing based on clinical patient history and histological tumor appearance are also addressed.

Despite a wealth of preclinical studies, it is unclear whether PIK3CAor phosphatase and tensin homolog (PTEN) gene aberrations are actionable in the clinical setting. Of 1,656 patients with advanced, refractory cancers tested for PIK3CAor PTEN abnormalities, PIK3CAmutations were found in 9% (146/1,589), and PTEN loss and/or mutation was found in 13% (149/1,157). In multicovariable analysis, treatment with a phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitor was the only independent factor predicting response to therapy in individuals harboring a PIK3CAor PTENaberration. The rate of stable disease ≥6 months/partial response reached 45% in a subgroup of individuals with H1047R PIK3CAmutations. Aberrations in the PI3K/AKT/mTOR pathway are common and potentially actionable in patients with diverse advanced cancers. This work provides further important clinical validation for continued and accelerated use of biomarker-driven trials incorporating rational drug combinations.

Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTENsequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. MLH1methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared with non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTENwild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial carcinomas for Lynch Syndrome.

PTEN(phosphatase and tensin homolog) is a tumor suppressor that negatively regulates the PI3K–AKT signaling pathway, which is implicated in the pathogenesis of endometrial carcinoma. Sanger sequencing has been considered to be the gold standard for detection of PTENsequence abnormalities. However, this approach fails to address the epigenetic mechanisms that contribute to functional PTEN loss. Using a study cohort of 154 endometrioid and non-endometrioid endometrial carcinomas, we performed full-length PTENsequencing and PTEN immunohistochemistry on each tumor. PTENsequence abnormalities were detected in a significantly lower proportion of cases (43%) than PTEN protein loss (64%, P=0.0004). Endometrioid tumors had a significantly higher proportion of PTENsequence abnormalities and PTEN protein loss than non-endometrioid tumors. Within the latter group, PTENsequence abnormalities and PTEN protein loss were most frequent in undifferentiated carcinomas, followed by mixed carcinomas; they were least frequent in carcinosarcomas. Overall, at least one PTENsequence abnormality was detected in each exon, and the greatest number of sequence abnormalities was detected in exon 8. Pure-endometrioid tumors had a significantly higher frequency of sequence abnormalities in exon 7 than did the non-endometrioid tumors (P=0.0199). Importantly, no mutational hotspots were identified. While PTEN protein loss by immunohistochemistry was identified in 89% of cases with a PTENsequence abnormality, PTEN protein loss was detected by immunohistochemistry in 44% of cases classified as PTENwild type by sequencing. For the first time, we demonstrate that PTEN immunohistochemistry is able to identify the majority of cases with functional PTEN loss. However, PTEN immunohistochemistry also detects additional cases with PTEN protein loss that would otherwise be undetected by gene sequencing. Therefore, for clinical purposes, immunohistochemistry appears to be a preferable technique for identifying endometrial tumors with loss of PTEN function.

Early stage endometrial cancer is generally curable. However, progress in the treatment of advanced and recurrent endometrial cancer has been limited. This has led to a shift from the use of traditional chemotherapeutic agents and radiotherapy regimens to the promising area of targeted therapy, given the large number of druggable molecular alterations found in endometrial cancer. To maximize the effects of directed targeted therapy, careful molecular characterization of the endometrial tumor is necessary. This represents an important difference in the use of targeted therapy vs. traditional chemotherapy or radiation treatment. This review will discuss relevant pathways to target in endometrial cancer as well as the challenges that arise during development of a personalized oncology approach.

Endometrial carcinomas show frequent PTEN-PI3Kpathway abnormalities, and there are currently multiple trials focused on PI3K pathway inhibitors in patients with endometrial carcinoma. PTEN immunohistochemistry may help to select patients with potential for response to targeted therapy, making it important to develop and validate this stain in formalin-fixed, paraffin-embedded tissue. Immunohistochemistry for PTEN was performed and scored independently on 118 cases of endometrial carcinomas from 2 cancer centers using monoclonal DAKO 6H2.1 antibody. Cases were scored as positive, negative, or heterogeneous; reproducibility of PTEN staining and interpretation was assessed. Overall interobserver agreement was good (weighted =0.80), with 82 concordance, similar for nonendometrioid (81) and endometrioid carcinomas (85). Twenty-one of 118 cases showed discrepant results (17) that resulted from differences in interpretation and not staining. Our study shows that evaluation of PTEN loss by immunohistochemistry is highly reproducible with the application of standard immunohistochemical techniques and simple scoring criteria.

Assessment of estrogen receptor (ER) expression by immunohistochemistry has yielded inconsistent results as a prognostic indicator in ovarian carcinoma. In breast and endometrial carcinomas, panels of estrogen-induced genes have shown improved prognostic capability over the use of ER immunohistochemistry alone. For both breast and endometrial cancers, overexpression of estrogen-induced genes is associated with better prognosis. We hypothesized that analysis of a panel of estrogen-induced genes can predict the outcome in ovarian carcinoma and potentially differentiate between tumors of varying hormonal responsiveness. From a cohort of 219 women undergoing ovarian cancer surgery from 2004 to 2007, 83 patients were selected for inclusion. All patients had advanced stage ovarian/primary peritoneal high-grade serous carcinoma and underwent primary surgical debulking, followed by adjuvant treatment with platinum and taxane agents. The expression of ERα and six genes known to be induced by estrogen in the female reproductive tract (namely EIG121, sFRP1, sFRP4, RALDH2, PR, and IGF-1) was measured using quantitative RT-PCR. Unsupervised cluster analyses were used to categorize patients as high or low gene expressors. Gene expression results were then compared with those for ER immunohistochemistry. Clusters were compared using χ2analyses, and Cox proportional hazards models were used to evaluate survival outcomes. The median follow-up time was 38.7 months (range: 1–68). A cluster defined by EIG121 and ERα segregated tumors into distinct groups of high and low gene expressors. Shorter overall survival (OS) was associated with high gene expression (HR 2.84 (1.11–7.30), P=0.03), even after adjustment for other covariates. No difference in ER immunohistochemistry expression was noted between gene clusters. In contrast to other hormonally driven cancers, high expression of ERα and the estrogen-induced gene EIG121 predicts shorter OS in patients with high-grade serous ovarian carcinoma. Such a biomarker panel may potentially be used to guide management with estrogen antagonists in this patient population.

Assessment of estrogen receptor (ER) expression by immunohistochemistry has yielded inconsistent results as a prognostic indicator in ovarian carcinoma. In breast and endometrial carcinomas, panels of estrogen-induced genes have shown improved prognostic capability over the use of ER immunohistochemistry alone. For both breast and endometrial cancers, overexpression of estrogen-induced genes is associated with better prognosis. We hypothesized that analysis of a panel of estrogen-induced genes can predict the outcome in ovarian carcinoma and potentially differentiate between tumors of varying hormonal responsiveness. From a cohort of 219 women undergoing ovarian cancer surgery from 2004 to 2007, 83 patients were selected for inclusion. All patients had advanced stage ovarian/primary peritoneal high-grade serous carcinoma and underwent primary surgical debulking, followed by adjuvant treatment with platinum and taxane agents. The expression of ERαand six genes known to be induced by estrogen in the female reproductive tract (namely EIG121, sFRP1, sFRP4, RALDH2, PR, and IGF-1) was measured using quantitative RT-PCR. Unsupervised cluster analyses were used to categorize patients as high or low gene expressors. Gene expression results were then compared with those for ER immunohistochemistry. Clusters were compared using χ2analyses, and Cox proportional hazards models were used to evaluate survival outcomes. The median follow-up time was 38.7 months (range: 1–68). A cluster defined by EIG121and ERαsegregated tumors into distinct groups of high and low gene expressors. Shorter overall survival (OS) was associated with high gene expression (HR 2.84 (1.11–7.30), P=0.03), even after adjustment for other covariates. No difference in ER immunohistochemistry expression was noted between gene clusters. In contrast to other hormonally driven cancers, high expression of ERαand the estrogen-induced gene EIG121predicts shorter OS in patients with high-grade serous ovarian carcinoma. Such a biomarker panel may potentially be used to guide management with estrogen antagonists in this patient population.

OBJECTIVE. EphA2 overexpression predicts poor prognosis in endometrial cancer. To explore mechanisms for this association and assess its potential as therapeutic target, the relationship of EphA2 expression to markers of angiogenesis was examined using patient samples and an orthotopic mouse model of uterine cancer.EXPERIMENTAL DESIGN. Expression of EphA2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, vascular endothelial growth factor (VEGF) and microvessel density (MVD) was evaluated using immunohistochemistry in 85 endometrioid endometrial adenocarcinomas (EEC) by two independent investigators. Results were correlated with clinicopathological characteristics. The effect of EphA2- agonist monoclonal antibody EA5, alone or in combination with docetaxel was studied in vitro and in vivo. Samples were analyzed for markers of angiogenesis, proliferation and apoptosis.RESULTS. Of 85 EEC samples, EphA2 was overexpressed in 47% of tumors and was significantly associated with high VEGF expression (p=0.001) and high MVD counts (p=0.02). High EphA2 expression, high VEGF expression and high MVD counts were significantly associated with shorter disease-specific survival. EA5 led to decrease in EphA2 expression and phosphorylation in vitro. In the murine model, while EA5 (33-88%) and docetaxel (23-55%) individually led to tumor inhibition over controls, combination therapy had the greatest efficacy (78-92%, p&lt;0.001). In treated tumors, combination therapy resulted in significant reduction in MVD counts, percent proliferation and apoptosis over controls. CONCLUSIONS. EphA2 overexpression is associated with markers of angiogenesis and is predictive of poor clinical outcome. EphA2 targeted therapy reduces angiogenesis and tumor growth in orthotopic uterine cancer models and should be considered for future clinical trials.

Previous work has identified Indian hedgehog (Ihh) as a major mediator of progesterone signaling during embryo implantation. Ihhacts through its downstream effector smoothened (Smo) to activate the GLI family of transcription factors. In order to gain a better understanding of Ihhaction during embryo implantation, we expressed a Cre-recombinase-dependent constitutively activated SMO in the murine uterus using the Pgrtm2(cre)Lyd(PRcre) mouse model [Pgrtm2(cre)Lyd+Gt(ROSA)26Sortm1(Smo/EYFP)Amc+(PRcre/+SmoM2+)]. Female PRcre/+SmoM2+mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but their ova failed to be fertilized in vivo and their uterus failed to undergo the artificially induced decidual response. Examination of the PRcre/+SmoM2+uteri revealed numerous features such as uterine hypertrophy, the presence of a stratified luminal epithelial cell layer, a reduced number of uterine glands, and an endometrial stroma that had lost its normal morphologic characteristics. Microarray analysis of 3-mo-old PRcre/+SmoM2+uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of PRcre/+SmoM2+increased extracellular matrix production. Thus, constitutive activation of Smoin the mouse uterus alters postnatal uterine differentiation which interferes with early pregnancy. These results provide new insight into the role of Hedgehog signaling during embryo implantation.

Conditional ablation of Indian hedgehog (Ihh) in the murine uterus results in mice that are sterile because of defects in embryo implantation. We performed microarray analysis on these mice at the time point at which the Ihhtarget genes are induced by the administration of exogenous hormone to mimic Day 3.5 of pregnancy. This analysis identified 863 genes altered by the conditional ablation of Ihh. Of these, genes that regulated the cell cycle were overrepresented. In addition, genes involved in epidermal growth factor (EGF) and estrogen (E2) signaling were found to be deregulated upon Ihhablation. Furthermore, upon conditional ablation of Ihh, 15-mo-old mice exhibited hallmarks of estrogenized uteri, such as cystically dilated glands and hyalinized stroma. Thus, Ihhregulates embryo implantation by having an impact on the cell cycle, EGF signaling, and E2 signaling.

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making.  Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers.  cDNA microarray and qRT- PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium.  These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR.  Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics.  Time to recurrence by cluster was analyzed using the Kaplan-Meier method.  A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis.  Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas.  Unsupervised cluster analysis revealed two distinct groups based on gene expression.  The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology.   The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster.  ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors.  We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk.  This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Endometrial cancer has been generally categorized into two broad groups of tumors, type I (TI) and type II (TII), with distinct epidemiological/clinical features and genetic alterations. Because telomere attrition appears to trigger genomic instability in certain cancers, we explored the role of telomere dysfunction in endometrial cancer by analyzing telomeres and other markers of telomere status in both tumor types. We describe a new method, telomere chromogenic in situhybridization, which permitted us to detect cells with short telomeres relative to control (stromal) cells within the same tissue section. Using this method, we found that both types of tumor cells had short telomeres. However, only TII tumors were significantly associated with critical telomere shortening in adjacent, morphologically normal epithelium, suggesting that telomere shortening contributes to the initiation of TII but not TI tumors. To explore this hypothesis, we analyzed mice with critically short telomeres and documented distinctive endometrial lesions that histologically resembled the in situprecursor of TII serous carcinomas; these lesions have not been observed previously in TI mouse models of endometrial cancer. Based on this and previous studies, we propose a model in which telomere attrition contributes to the initiation of TII and progression of TI endometrial cancers.

Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.Modern Pathology (2008) 21, 912–923; doi:10.1038/modpathol.2008.82; published online 16 May 2008

Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.

Expression of various S100 genes has been associated with clinically aggressive subtypes in a variety of different cancers. We hypothesized that S100A4 would be overexpressed in endometrial carcinoma compared to benign endometrium. Quantitative real-time RT-PCR (qRT-PCR) was used to quantify the mRNA level of S100A4 in benign endometrium (n=19), endometrioid adenocarcinoma (n=87), and non-endometrioid tumors (n=21). Immunohistochemistry was used to verify the results of qRT-PCR and to assess protein localization. Possible mechanisms of S100A4 gene regulation were also examined. S100A4 was overexpressed in the grade 3 endometrioid tumors, uterine papillary serous carcinoma, and uterine malignant mixed müllerian tumor. Expression in grade 1 and grade 2 endometrioid tumors was comparable to that of normal endometrium, which was quite low. Expression was significantly higher in stage III and IV tumors compared with stage I. By immunohistochemistry, S100A4 was expressed in the tumor cell cytoplasm of poorly differentiated tumors, but was not detected in normal endometrial glandular epithelium. In benign endometrium, S100A4 expression was confined to stromal cells. S100A4 was not regulated by estrogen or progesterone, and its expression in tumors was not significantly correlated to estrogen receptor or progesterone receptor content. However, methylation of the S100A4 gene was detected in benign endometrium and grade 1 tumors with low S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 mRNA and protein expression showed no methylation of the gene. These methylation results were verified in endometrial cancer cell lines with differential baseline levels of S100A4 protein. These results suggest that hypomethylation is an important mechanism of regulating the expression of the S100A4 gene. These results support the emerging concept that hypomethylation may play a role in the upregulation of genes during later stages of tumorigenesis.Modern Pathology (2007) 20, 1045–1054; doi:10.1038/modpathol.3800940; published online 3 August 2007

Expression of various S100genes has been associated with clinically aggressive subtypes in a variety of different cancers. We hypothesized that S100A4would be overexpressed in endometrial carcinoma compared to benign endometrium. Quantitative real-time RT-PCR (qRT-PCR) was used to quantify the mRNA level of S100A4in benign endometrium (n=19), endometrioid adenocarcinoma (n=87), and non-endometrioid tumors (n=21). Immunohistochemistry was used to verify the results of qRT-PCR and to assess protein localization. Possible mechanisms of S100A4gene regulation were also examined. S100A4was overexpressed in the grade 3 endometrioid tumors, uterine papillary serous carcinoma, and uterine malignant mixed müllerian tumor. Expression in grade 1 and grade 2 endometrioid tumors was comparable to that of normal endometrium, which was quite low. Expression was significantly higher in stage III and IV tumors compared with stage I. By immunohistochemistry, S100A4 was expressed in the tumor cell cytoplasm of poorly differentiated tumors, but was not detected in normal endometrial glandular epithelium. In benign endometrium, S100A4 expression was confined to stromal cells. S100A4was not regulated by estrogen or progesterone, and its expression in tumors was not significantly correlated to estrogen receptoror progesterone receptorcontent. However, methylation of the S100A4gene was detected in benign endometrium and grade 1 tumors with low S100A4expression. In contrast, grade 3 endometrioid tumors with high S100A4mRNA and protein expression showed no methylation of the gene. These methylation results were verified in endometrial cancer cell lines with differential baseline levels of S100A4 protein. These results suggest that hypomethylation is an important mechanism of regulating the expression of the S100A4gene. These results support the emerging concept that hypomethylation may play a role in the upregulation of genes during later stages of tumorigenesis.

Background: We identified the BRK tyrosine kinase in a PCR-based screen of tyrosine kinases expressed by ovarian tumors. BRK expression is restricted to normal differentiating epithelial cells and its overexpression may play a role in processes related to tumor development and growth. Its expression in normal ovary and ovarian tumors has not previously been described, and is the focus of this study.Methods: BRK expression levels were determined in 14 normal ovaries and 138 high-grade, late stage serous carcinomas of the ovary by immunohistochemical analysis, and in 19 ovarian cancer cell lines and immortalized ovarian surface epithelium by western blot analysis. Furthermore, BRK/PTK6 gene copy number was determined in 7 primary serous carcinomas by fluorescence in situ hybridization.Results: Immunohistochemical studies indicate that BRK is highly expressed in 97/138 (70%) of high-grade, serous carcinomas of the ovary, but is absent in normal ovarian surface epithelia. BRK is also expressed by 9/19 of ovarian cancer cell lines, but is undetectable in immortalized ovarian surface epithelium. Interestingly, the BRK gene has been mapped to chromosome 20q13.3, a site frequently amplified in ovarian cancers, and associated with poor prognosis. We have determined by fluorescence in situ hybridization (FISH) that BRK is specifically amplified at low levels in 6/7 primary ovarian carcinomas.Conclusions: The amplification of the BRK gene and over-expression of BRK protein in the majority of high-grade serous carcinomas and ovarian cancer cell lines suggest that BRK may play a role in the development and growth of ovarian tumors.

Abstract Recent studies have estimated that the lifetime risk of endometrial cancer in women with Lynch syndrome/hereditary non-polyposis colorectal cancer syndrome (Lynch/HNPCC) is 40–60%. This risk equals or exceeds their risk for colon cancer. While much research has been done to define the natural history and molecular features of Lynch/HNPCC associated colon cancer, there has been considerably less research defining Lynch/HNPCC associated endometrial cancer. This article will review current information regarding the clinico-pathologic features of Lynch/HNPCC associated endometrial cancer. In addition, current consensus guidelines for endometrial cancer screening and prevention for women with Lynch/HNPCC will be discussed. Given the increased risk of multiple cancers, changing the name of this syndrome from hereditary non-polyposis colorectal cancer syndrome to Lynch Syndrome may benefit both patients and clinicians. Clinicians caring for women with Lynch/HNPCC may stress colon cancer screening and prevention without reviewing endometrial cancer risks and symptoms or screening and prevention options. Perhaps more importantly, women with Lynch/HNPCC may focus on colon cancer risks and lack understanding of endometrial cancer risks. With increasing evidence that women with Lynch/HNPCC have significant risks for both colon and endometrial cancers, we believe a multi-disciplinary approach to the management of these individuals is crucial.

Patients with hyperplastic polyposis have multiple hyperplastic polyps (HPs) and increased risk of colorectal carcinomas. Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesions in colorectal carcinogenesis. We evaluated BRAFmutations by DNA sequencing in 53 ACF from patients with sporadic colorectal carcinomas and familial adenomatous polyposis, in 18 sporadic HPs from patients with resected colorectal cancer, and in 70 HPs, 4 serrated adenomas, 3 admixed hyperplastic-adenomatous polyps, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis. BRAFmutation status was compared with clinicopathological features and other genetic alterations by marginal logistic regression. BRAFmutation was present in only 2% of ACF and 6% of sporadic HPs. In contrast, BRAFmutation was present in 43% of HPs (P= 0.01 versus sporadic HPs), 75% of serrated adenomas, 33% of admixed hyperplastic-adenomatous polyps, 30% of tubular adenomas, and 33% of carcinomas from patients with multiple/large HPs and/or hyperplastic polyposis. BRAFmutation status in patients with multiple/large HPs and/or hyperplastic polyposis correlated with HPs from the same patient (odds ratio, 5.8; P= 0.0002) but associated with younger age (odds ratio, 0.83; P= 0.006 compared to older age), with a large HP (odds ratio, 22.5; P= 0.01 compared with patients with multiple HPs), with location of HPs in the right colon (odds ratio, 3.0; P= 0.03), and with methylation of the p16gene and the MINT31 locus [odds ratio, 12.2 (P= 0.0001) and 4.4 (P= 0.02), respectively]. Our study shows that BRAFmutation status is heterogeneous among patients with multiple/large HPs and/or hyperplastic polyposis, suggesting differences in pathogenesis of HPs that indicate subsets within this phenotype.

Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesion in colorectal carcinogenesis, and CpG island methylation has been described as an important molecular pathway. We therefore studied methylation in ACF from patients with familial adenomatous polyposis (FAP) or sporadic colorectal cancer. We assessed methylation status of the p16tumor suppressor gene, MINT1 (methylated in tumor 1), MINT2, MINT31, O6-methylguanine-DNA methyltransferasegene, and hMLH1mismatch repair gene. We compared methylation to ACF histopathology, K-rasproto-oncogene mutation, loss of heterozygosity at chromosome 1p, and microsatellite instability. Methylation was present in 34% (21 of 61) of ACF, including both FAP and sporadic types, but was more frequent in sporadic ACF [53% (18 of 34) versus11% (3 of 27), P= 0.002], especially dysplastic sporadic ACF [75% (3 of 4) versus8% (2 of 24), P= 0.004]. MINT31 was more frequently methylated in heteroplastic ACF than dysplastic ACF [35% (11 of 31) versus7% (2 of 30), P= 0.01]. Strong associations of ACF methylation with K-ras mutation (P= 0.007) and with loss of chromosome 1p (P= 0.04) were observed, but methylation was the only molecular abnormality identified in 16% (10 of 61) of ACF. Our findings suggest that methylation in ACF is an early event in the pathogenesis of a subset of colorectal carcinomas, and that ACF from FAP patients and patients with sporadic colorectal cancer have distinct epigenetic changes that reflect differences in molecular pathogenesis.

Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2). Women with this syndrome are at high risk for developing endometrial cancer and several other types of cancer. The lifetime risk for affected women of developing endometrial cancer is 40. Between 1 and 5 of endometrial cancers can be attributed to LS. Women with the syndrome are at high risk of developing a second cancer, and their first-degree relatives with and without cancer are also at risk for having the syndrome. Diagnosing LS leads to heightened surveillance and risk-reducing strategies that facilitate prevention of endometrial cancer andor early detection and also allows for cancer prevention strategies in first-degree relatives.

ABSTRACTThe prevailing view of CD73 in cancer is that it is overexpressed in tumors and promotes cancer progression by dampening local T cell-mediated immune responses. We recently found that CD73 is down-regulated in poorly-differentiated and advanced stage endometrial carcinoma compared to normal endometrium and well-differentiated, early stage tumors. We revealed that CD73-generated adenosine induces a physiological response to protect epithelial integrity in well-differentiated, early stage endometrial carcinoma. The ability of CD73-generated adenosine to protect the barrier is not so different from its ability to induce immunosuppression and other physiological responses in cancerous tissues. In this commentary we examine the complexity of CD73 in cancer and suggest that a “one size fits all” approach to the role of CD73/adenosine in cancer is no longer warranted. Given that tumors often hijack normal cellular responses, we also provide consideration on how CD73s known role to protect barrier function may have implications in promoting tumor progression.

Endometrial cancer is the most common gynecological cancer. Estrogen-dependent endometrioid carcinoma is the most common type of endometrial cancer and alterations in the expression of PTEN have been associated with this disease. Ablation of Mig-6in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. To investigate the impact of overexpression of Mig-6in endometrial cancer development and progression, we have generated mice with conditionally overexpression of Mig-6in the uterus. The elevated expression of Mig-6is accomplished by placing the coding region of Mig-6under the control of a ubiquitously expressed promoter, the chicken beta actin-cytomegalovirus hybrid (CAGGS) promoter. The construction also contained the "Stop Cassette" flanked by LoxP sites (LSL). In order to ensure uniform expression in mice, the CAGGS-LSL-Mig-6transgene is inserted into the ROSA26 locus. In order to investigate the effects of the Mig-6and the PTEN/PI3K/AKT signaling pathways on uterine tumorigenesis, mice with Ptenfloxed (Ptenf/f) and LoxP-STOP-LoxP Mig-6 (R26Mig-6LSL) were bred to the PRCremouse model to generate ablation of Ptenand overexpression of Mig-6in the uterus (PRcre+R26Mig-6LSLPtenf/f). PRcre+R26Mig-6LSLPtenf/fmice showed significantly decreased uterine weight compared to PRcre/+Ptenf/fat 3 months of age. Gross morphology at 3 months of age showed that double mutant mice dramatically retarded the development of endometrial cancer compared to ablation of Pten gene. Histological analysis showed that endometrial adenocarcinoma with invasion into the myometrium was observed in the PRcre/+Ptenf/fmice at 3 months of age. However, double mutant mice did not develop endometrial cancer. Therefore, Mig-6exerts a tumor suppressor function in endometrial cancer. These results will contribute to the understanding of the molecular mechanism of tumorigenesis and to the development of therapeutic approaches for endometrial cancer.(platform)