Andrew K. Godwin

Expression of Epiregulin and Amphiregulin and K-ras Mutation Status Predict Disease Control in Metastatic Colorectal Cancer Patients Treated With Cetuximab

PURPOSE The antiepidermal growth factor receptor (EGFR) antibody cetuximab shows activity in multiple epithelial tumor types; however, responses are seen in only a subset of patients. This study was conducted to identify markers that are associated with disease control in patients treated with cetuximab. PATIENTS AND METHODS One hundred ten patients with metastatic colorectal cancer were enrolled onto a cetuximab monotherapy trial. Transcriptional profiling was conducted on RNA from mandatory pretreatment metastatic biopsies to identify genes whose expression correlates with best clinical responses. EGFR and K-ras mutation analyses and EGFR gene copy number analyses were performed on DNA from pretreatment biopsies. RESULTS Gene expression profiles showed that patients with tumors that express high levels of the EGFR ligands epiregulin and amphiregulin are more likely to have disease control with cetuximab (EREG, P = .000015; AREG, P = .000025). Additionally, patients whose tumors do not have K-ras mutations have a significantly higher disease control rate than patients with K-ras mutations (P = .0003). Furthermore, patients with tumors that have high expression of EREG or AREG also have significantly longer progression-free survival (PFS) than patients with low expression (EREG: P = .0002, hazard ratio [HR] = 0.47, and median PFS, 103.5 v 57 days, respectively; AREG: P < .0001, HR = 0.44, and median PFS, 115.5 v 57 days, respectively). CONCLUSION Patients with tumors that have high gene expression levels of epiregulin and amphiregulin and patients with wild-type K-ras are more likely to have disease control on cetuximab treatment. The identified markers could be developed further to select patients for cetuximab therapy.

MicroRNA-34b and MicroRNA-34c Are Targets of p53 and Cooperate in Control of Cell Proliferation and Adhesion-Independent Growth

MicroRNAs (miRNA) are a recently discovered class of noncoding RNAs that negatively regulate gene expression. Recent evidence indicates that miRNAs may play an important role in cancer. However, the mechanism of their deregulation in neoplastic transformation has only begun to be understood. To elucidate the role of tumor suppressor p53 in regulation of miRNAs, we have analyzed changes in miRNA microarray expression profile immediately after conditional inactivation of p53 in primary mouse ovarian surface epithelium cells. Among the most significantly affected miRNAs were miR-34b and miR-34c, which were down-regulated 12-fold according to quantitative reverse transcription-PCR analysis. Computational promoter analysis of the mir-34b/mir-34c locus identified the presence of evolutionarily conserved p53 binding sites approximately 3 kb upstream of the miRNA coding sequence. Consistent with evolutionary conservation, mir-34b/mir-34c were also down-regulated in p53-null human ovarian carcinoma cells. Furthermore, as expected from p53 binding to the mir-34b/c promoter, doxorubicin treatment of wild-type, but not p53-deficient, cells resulted in an increase of mir-34b/mir-34c expression. Importantly, miR-34b and miR-34c cooperate in suppressing proliferation and soft-agar colony formation of neoplastic epithelial ovarian cells, in agreement with the partially overlapping spectrum of their predicted targets. Taken together, these results show the existence of a novel mechanism by which p53 suppresses such critical components of neoplastic growth as cell proliferation and adhesion-independent colony formation.

AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth

Activation of the PI3K/AKT pathway may contribute to tumorigenesis. AKT mediates survival signals that protect cells from apoptosis and, thus, is a potentially important therapeutic target. To determine the frequency of AKT activation in human ovarian cancer, we screened a tumor tissue microarray with a phospho-specific pan-AKT (Ser473) antibody, which revealed elevated staining in 21 of 31 (68%) ovarian carcinomas. Phospho-AKT staining was associated with that of phospho (active)-mTOR in 27 of 31 (87%) ovarian tumors, with 17 (55%) tumors showing elevated phospho-mTOR positivity. We tested the effects of AKT/mTOR activation on the therapeutic sensitivity of ovarian cancer cells. Pretreatment of SKOV3 cells, which exhibit constitutive AKT activity under low serum conditions, with the PI3K inhibitor LY294002 augmented cisplatin-induced apoptosis. In contrast, ovarian cancer cell lines OVCAR4 and OVCAR5, which have low basal levels of AKT activity, did not show increased cisplatin-induced apoptosis when pretreated with LY294002. In addition, inhibition of mTOR activity with rapamycin resulted in G1 arrest in SKOV3 cells, but not in OVCAR4 or OVCAR5 cells. Collectively, these findings indicate that active AKT and downstream mTOR represent potentially important therapeutic and/or chemopreventive targets in ovarian cancer.

Association between BRCA1 and BRCA2 mutations and survival in women with invasive epithelial ovarian cancer

CONTEXT Approximately 10% of women with invasive epithelial ovarian cancer (EOC) carry deleterious germline mutations in BRCA1 or BRCA2. A recent article suggested that BRCA2-related EOC was associated with an improved prognosis, but the effect of BRCA1 remains unclear. OBJECTIVE To characterize the survival of BRCA carriers with EOC compared with noncarriers and to determine whether BRCA1 and BRCA2 carriers show similar survival patterns. DESIGN, SETTING, AND PARTICIPANTS A pooled analysis of 26 observational studies on the survival of women with ovarian cancer, which included data from 1213 EOC cases with pathogenic germline mutations in BRCA1 (n = 909) or BRCA2 (n = 304) and from 2666 noncarriers recruited and followed up at variable times between 1987 and 2010 (the median year of diagnosis was 1998). MAIN OUTCOME MEASURE Five-year overall mortality. RESULTS The 5-year overall survival was 36% (95% CI, 34%-38%) for noncarriers, 44% (95% CI, 40%-48%) for BRCA1 carriers, and 52% (95% CI, 46%-58%) for BRCA2 carriers. After adjusting for study and year of diagnosis, BRCA1 and BRCA2 mutation carriers showed a more favorable survival than noncarriers (for BRCA1: hazard ratio [HR], 0.78; 95% CI, 0.68-0.89; P < .001; and for BRCA2: HR, 0.61; 95% CI, 0.50-0.76; P < .001). These survival differences remained after additional adjustment for stage, grade, histology, and age at diagnosis (for BRCA1: HR, 0.73; 95% CI, 0.64-0.84; P < .001; and for BRCA2: HR, 0.49; 95% CI, 0.39-0.61; P < .001). The BRCA1 HR estimate was significantly different from the HR estimated in the adjusted model (P for heterogeneity = .003). CONCLUSION Among patients with invasive EOC, having a germline mutation in BRCA1 or BRCA2 was associated with improved 5-year overall survival. BRCA2 carriers had the best prognosis.

Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer

Testicular germ cell tumors (TGCT) have been expected to have a strong underlying genetic component. We conducted a genome-wide scan among 277 TGCT cases and 919 controls and found that seven markers at 12p22 within KITLG (c-KIT ligand) reached genome-wide significance (P < 5.0 × 10−8 in discovery). In independent replication, TGCT risk was increased threefold per copy of the major allele at rs3782179 and rs4474514 (OR = 3.08, 95% CI = 2.29–4.13; OR = 3.07, 95% CI = 2.29–4.13, respectively). We found associations with rs4324715 and rs6897876 at 5q31.3 near SPRY4 (sprouty 4; P < 5.0 × 10−6 in discovery). In independent replication, risk of TGCT was increased nearly 40% per copy of the major allele (OR = 1.37, 95% CI = 1.14–1.64; OR = 1.39, 95% CI = 1.16–1.66, respectively). All of the genotypes were associated with both seminoma and nonseminoma TGCT subtypes. These results demonstrate that common genetic variants affect TGCT risk and implicate KITLG and SPRY4 as genes involved in TGCT susceptibility.

Focus on epithelial ovarian cancer

Epithelial ovarian cancer (EOC) will be diagnosed in 24,400women in the United States in 2004, with an estimated 14,300deaths (Jemal et al., 2003).Neoplasms from the surface epithe-lium of the ovary exhibit a variety of Mullerian-type cells, includ-ing serous, mucinous, endometrioid, and clear cell, reflecting acommon pathway in embryological development.In the westernworld, EOC is the most lethal gynecologic cancer, accountingfor more deaths than endometrial and cervical cancer com-bined. Spread of the disease via the lymphatics and by peri-toneal implantation is not associated with any specific signs orsymptoms, and the vast majority of women are diagnosed withdisseminated intraperitoneal carcinomatosis (FIGO Stage III).Also contributing to the high mortality is the advanced age atdiagnosis (median 63 years), with an increase aftermenopause. While ultrasound and computerized tomogramsare useful in definition of sites of bulk disease, surgical evalua-tion is necessary for accurate staging and to remove largemetastases (cytoreduction). Due to the propensity for diffusesmall-volume disease, surgery is rarely able to render patientsdisease free, and postoperative chemotherapy is usuallyrequired. While overall mortality rates have remained relativelyconstant for the past two to three decades, five-year survivalrates have increased from 30% in the 1960s to over 50% in thepast several years.

Common Breast Cancer-Predisposition Alleles Are Associated with Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Phase II Study of Gefitinib in Patients with Relapsed or Persistent Ovarian or Primary Peritoneal Carcinoma and Evaluation of Epidermal Growth Factor Receptor Mutations and Immunohistochemical Expression: A Gynecologic Oncology Group Study

Purpose: This phase II trial assessed the activity and tolerability of a daily oral dose of 500 mg gefitinib (ZD1839, Iressa) in patients with recurrent or persistent epithelial ovarian or primary peritoneal carcinoma, and explored the clinical value of determining the status of the epidermal growth factor receptor (EGFR). Experimental Design: Primary measure of efficacy was progression-free survival at 6 months. Mutations in exons 18 to 21 of EGFR and/or immunohistochemical expression of EGFR were evaluated in tumor specimens from patients enrolled in this trial as well as from patients not treated with gefitinib. Results: Twenty-seven of 30 (90%) patients were eligible and evaluable for analysis of gefitinib efficacy and toxicity. Of these, four survived progression-free >6 months with one objective response (4%). The most commonly observed grade 3 toxicities were dermatologic (15%, 4 of 27) and diarrhea (30%, 8 of 27). Specimens from 26 of 26 or 25 of 26 patients were evaluable for immunohistochemical or mutation analysis, respectively. The response rate for patients with EGFR-positive tumors was 9% (1 of 11). EGFR expression was associated with longer progression-free survival (P = 0.008) and possibly longer survival (P = 0.082). The patient with the only objective response had a mutation in the catalytic domain of the tumors EGFR (P = 0.04). Among 32 invasive tumors from patients not treated with gefitinib, one exhibited a catalytic domain mutation. Conclusions: Gefitinib was well tolerated but had minimal activity in unscreened patients with recurrent ovarian or primary peritoneal carcinoma. Prescreening patients for activating mutations in EGFR may improve response rate to gefitinib. This report is the first to document activating mutations in catalytic domain of EGFR in 3.5% (2 of 57) of ovarian cancers.

Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes

Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ∼74,000 deaths annually. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology. We used whole-exome sequencing to comprehensively search for somatic mutations within ∼22,000 protein-encoding genes in 13 primary serous endometrial tumors. We subsequently resequenced 18 genes, which were mutated in more than 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors. We identified high frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%). Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

Modification of BRCA1-Associated Breast Cancer Risk by the Polymorphic Androgen-Receptor CAG Repeat

Compared with the general population, women who have inherited a germline mutation in the BRCA1 gene have a greatly increased risk of developing breast cancer. However, there is also substantial interindividual variability in the occurrence of breast cancer among BRCA1 mutation carriers. We hypothesize that other genes, particularly those involved in endocrine signaling, may modify the BRCA1-associated age-specific breast cancer risk. We studied the effect of the CAG repeat-length polymorphism found in exon 1 of the androgen-receptor (AR) gene (AR-CAG). AR alleles containing longer CAG repeat lengths are associated with a decreased ability to activate androgen-responsive genes. Using a sample of women who inherited germline BRCA1 mutations, we compared AR-CAG repeat length in 165 women with and 139 women without breast cancer. We found that women were at significantly increased risk of breast cancer if they carried at least one AR allele with >/=28 CAG repeats. Women who carried an AR-CAG allele of >/=28, >/=29, or >/=30 repeats were given a diagnosis 0.8, 1.8, or 6.3 years earlier than women who did not carry at least one such allele. All 11 women in our sample who carried at least one AR-CAG allele with >/=29 repeats had breast cancer. Our results support the hypothesis that age at breast cancer diagnosis is earlier among BRCA1 mutation carriers who carry very long AR-CAG repeats. These results suggest that pathways involving androgen signaling may affect the risk of BRCA1-associated breast cancer.