Russell Broaddus

High Frequency of PIK3R1 and PIK3R2 Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability

We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function, including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.

Patterns of Gene Expression in Different Histotypes of Epithelial Ovarian Cancer Correlate with Those in Normal Fallopian Tube, Endometrium, and Colon

Purpose: Epithelial ovarian cancers are thought to arise from flattened epithelial cells that cover the ovarian surface or that line inclusion cysts. During malignant transformation, different histotypes arise that resemble epithelial cells from normal fallopian tube, endometrium, and intestine. This study compares gene expression in serous, endometrioid, clear cell, and mucinous ovarian cancers with that in the normal tissues that they resemble. Experimental Design: Expression of 63,000 probe sets was measured in 50 ovarian cancers, in 5 pools of normal ovarian epithelial brushings, and in mucosal scrapings from 4 normal fallopian tube, 5 endometrium, and 4 colon specimens. Using rank-sum analysis, genes whose expressions best differentiated the ovarian cancer histotypes and normal ovarian epithelium were used to determine whether a correlation based on gene expression existed between ovarian cancer histotypes and the normal tissues they resemble. Results: When compared with normal ovarian epithelial brushings, alterations in serous tumors correlated with those in normal fallopian tube (P = 0.0042) but not in other normal tissues. Similarly, mucinous cancers correlated with those in normal colonic mucosa (P = 0.0003), and both endometrioid and clear cell histotypes correlated with changes in normal endometrium (P = 0.0172 and 0.0002, respectively). Mucinous cancers displayed the greatest number of alterations in gene expression when compared with normal ovarian epithelial cells. Conclusion: Studies at a molecular level show distinct expression profiles of different histologies of ovarian cancer and support the long-held belief that histotypes of ovarian cancers come to resemble normal fallopian tube, endometrial, and colonic epithelium. Several potential molecular markers for mucinous ovarian cancers have been identified.

Gynecologic cancer as a sentinel cancer for women with hereditary nonpolyposis colorectal cancer syndrome

OBJECTIVE: Women with hereditary nonpolyposis colorectal cancer syndrome have a 40–60% lifetime risk for colon cancer, a 40–60% lifetime risk for endometrial cancer, and a 12% lifetime risk for ovarian cancer. A number of women with hereditary nonpolyposis colorectal cancer syndrome will have more than one cancer in their lifetime. The purpose of this study was to estimate whether women with hereditary nonpolyposis colorectal cancer syndrome who develop 2 primary cancers present with gynecologic or colon cancer as their “sentinel cancer.” METHODS: Women whose families fulfilled Amsterdam criteria for hereditary nonpolyposis colorectal cancer syndrome and who developed 2 primary colorectal/gynecologic cancers in their lifetime were identified from 5 large hereditary nonpolyposis colorectal cancer syndrome registries. Information on age at cancer diagnoses and which cancer (colon cancer or endometrial cancer/ovarian cancer) developed first was obtained. RESULTS: A total of 117 women with dual primary cancers from 223 Amsterdam families were identified. In 16 women, colon cancer and endometrial cancer/ovarian cancer were diagnosed simultaneously. Of the remaining 101 women, 52 (51%) women had an endometrial or ovarian cancer diagnosed first. Forty-nine (49%) women had a colon cancer diagnosed first. For women who developed endometrial cancer/ovarian cancer first, mean age at diagnosis of endometrial cancer/ovarian cancer was 44. For women who developed colon cancer first, the mean age at diagnosis of colon cancer was 40. CONCLUSION: In this large series of women with hereditary nonpolyposis colorectal cancer syndrome who developed 2 primary colorectal/gynecologic cancers, endometrial cancer/ovarian cancer was the “sentinel cancer,” preceding the development of colon cancer, in half of the cases. Therefore, gynecologists and gynecologic oncologists play a pivotal role in the identification of women with hereditary nonpolyposis colorectal cancer syndrome. LEVEL OF EVIDENCE: II-3

Integrated Analyses Identify a Master MicroRNA Regulatory Network for the Mesenchymal Subtype in Serous Ovarian Cancer

Integrated genomic analyses revealed a miRNA-regulatory network that further defined a robust integrated mesenchymal subtype associated with poor overall survival in 459 cases of serous ovarian cancer (OvCa) from The Cancer Genome Atlas and 560 cases from independent cohorts. Eight key miRNAs, including miR-506, miR-141, and miR-200a, were predicted to regulate 89% of the targets in this network. Follow-up functional experiments illustrate that miR-506 augmented E-cadherin expression, inhibited cell migration and invasion, and prevented TGFβ-induced epithelial-mesenchymal transition by targeting SNAI2, a transcriptional repressor of E-cadherin. In human OvCa, miR-506 expression was correlated with decreased SNAI2 and VIM, elevated E-cadherin, and beneficial prognosis. Nanoparticle delivery of miR-506 in orthotopic OvCa mouse models led to E-cadherin induction and reduced tumor growth.

Somatic Mutations in BRCA1 and BRCA2 Could Expand the Number of Patients That Benefit From Poly (ADP Ribose) Polymerase Inhibitors in Ovarian Cancer

PURPOSE The prevalence of BRCA(1/2) mutations in germline DNA from unselected ovarian cancer patients is 11% to 15.3%. It is important to determine the frequency of somatic BRCA(1/2) changes, given the sensitivity of BRCA-mutated cancers to poly (ADP ribose) polymerase-1 (PARP1) inhibitors and platinum analogs. PATIENTS AND METHODS In 235 unselected ovarian cancers, BRCA(1/2) was sequenced in 235, assessed by copy number analysis in 95, and tiling arrays in 65. 113 tumors were sequenced for TP53. BRCA(1/2) transcript levels were assessed by quantitative polymerase chain reaction in 220. When available for tumors with BRCA(1/2) mutations, germline DNA was sequenced. RESULTS Forty-four mutations (19%) in BRCA1 (n = 31)/BRCA2 (n = 13) were detected, including one homozygous BRCA1 intragenic deletion. BRCA(1/2) mutations were particularly common (23%) in high-grade serous cancers. In 28 patients with available germline DNA, nine (42.9%) of 21 and two (28.6%) of seven BRCA1 and BRCA2 mutations were demonstrated to be somatic, respectively. Five mutations not previously identified in germline DNA were more commonly somatic than germline (four of 11 v one of 17; P = .062). There was a positive association between BRCA1 and TP53 mutations (P = .012). BRCA(1/2) mutations were associated with improved progression-free survival (PFS) after platinum-based chemotherapy in univariate (P = .032; hazard ratio [HR] = 0.65; 95% CI, 0.43 to 0.98) and multivariate (P = .019) analyses. BRCA(1/2) deficiency, defined as BRCA(1/2) mutations or expression loss (in 24 [13.3%] BRCA(1/2)-wild-type cancers), was present in 67 ovarian cancers (30%) and was also significantly associated with PFS in univariate (P = .026; HR = 0.67; 95% CI, 0.47 to 0.96) and multivariate (P = .008) analyses. CONCLUSION BRCA(1/2) somatic and germline mutations and expression loss are sufficiently common in ovarian cancer to warrant assessment for prediction of benefit in clinical trials of PARP1 inhibitors.

CpG Island Methylation in Aberrant Crypt Foci of the Colorectum

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 p16 tumor suppressor gene, MINT1 (methylated in tumor 1), MINT2, MINT31, O(6)-methylguanine-DNA methyltransferase gene, and hMLH1 mismatch repair gene. We compared methylation to ACF histopathology, K-ras proto-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) versus 11% (3 of 27), P = 0.002], especially dysplastic sporadic ACF [75% (3 of 4) versus 8% (2 of 24), P = 0.004]. MINT31 was more frequently methylated in heteroplastic ACF than dysplastic ACF [35% (11 of 31) versus 7% (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.

Pathologic features of endometrial carcinoma associated with HNPCC: A comparison with sporadic endometrial carcinoma

Endometrial carcinoma is a common malignancy in hereditary nonpolyposis colorectal carcinoma (HNPCC). Like colon carcinoma, endometrial carcinoma is diagnosed at an earlier age in women with HNPCC. In contrast to colon carcinoma, the pathologic features of endometrial carcinoma in HNPCC have not been studied in detail. It was the purpose of this study to pathologically characterize a series of HNPCC associated endometrial carcinomas.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer

Background:Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH).Methods:Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines.Results:Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10−11). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10−5 and 10−29), and identified breast and pancreatic cell lines with BRCA defects.Conclusion:The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearsons correlation −0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearsons correlation −0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3′-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

Prospective Determination of Prevalence of Lynch Syndrome in Young Women With Endometrial Cancer

PURPOSE Age younger than 50 years at the time of colon cancer diagnosis is often used as a screening criterion for Lynch syndrome (hereditary nonpolyposis colorectal cancer syndrome). The purpose of this study was to determine the prevalence of MLH1, MSH2, and MSH6 mutations in an unselected cohort of women diagnosed with endometrial cancer at age younger than 50 years. METHODS A prospective, multicenter study was performed at three institutions. After written consent was obtained, germline mutation testing by full sequencing and large deletion analysis of the MLH1, MSH2, and MSH6 genes was performed. Tumor studies included immunohistochemistry of MLH1, MSH2, and MSH6; microsatellite instability analysis; and hypermethylation of the MLH1 promoter. RESULTS Of the 100 women, nine (9%; 95% CI, 4.2 to 16.4) carried a deleterious germline mutation: seven women with mutations in MSH2, one woman with a mutation in MLH1, and one woman with a mutation in MSH6. Two additional women had molecular studies consistent with the diagnosis of Lynch syndrome. The mean body mass index (BMI) for the entire cohort was 34.4, which is significantly higher than 29.2, the mean BMI for the mutation carriers. Predictors of finding a germline mutation included having a first-degree relative with a Lynch syndrome-associated cancer, endometrial tumor with loss of MSH2 expression, tumors with high microsatellite instability, and lower BMI. CONCLUSION In this prospective study of endometrial cancer patients younger than age 50 years, 9% were found to carry germline Lynch syndrome-associated mutations. In addition to young age of onset, family history, BMI, and molecular tumor studies can improve the likelihood of identifying a Lynch syndrome-associated germline mutation in MLH1, MSH2, and MSH6.