Arto Leminen

A phase 3 trial of bevacizumab in ovarian cancer.

BACKGROUND Angiogenesis plays a role in the biology of ovarian cancer. We examined the effect of bevacizumab, the vascular endothelial growth factor inhibitor, on survival in women with this disease. METHODS We randomly assigned women with ovarian cancer to carboplatin (area under the curve, 5 or 6) and paclitaxel (175 mg per square meter of body-surface area), given every 3 weeks for 6 cycles, or to this regimen plus bevacizumab (7.5 mg per kilogram of body weight), given concurrently every 3 weeks for 5 or 6 cycles and continued for 12 additional cycles or until progression of disease. Outcome measures included progression-free survival, first analyzed per protocol and then updated, and interim overall survival. RESULTS A total of 1528 women from 11 countries were randomly assigned to one of the two treatment regimens. Their median age was 57 years; 90% had epithelial ovarian cancer, 69% had a serous histologic type, 9% had high-risk early-stage disease, 30% were at high risk for progression, and 70% had stage IIIC or IV ovarian cancer. Progression-free survival (restricted mean) at 36 months was 20.3 months with standard therapy, as compared with 21.8 months with standard therapy plus bevacizumab (hazard ratio for progression or death with bevacizumab added, 0.81; 95% confidence interval, 0.70 to 0.94; P=0.004 by the log-rank test). Nonproportional hazards were detected (i.e., the treatment effect was not consistent over time on the hazard function scale) (P<0.001), with a maximum effect at 12 months, coinciding with the end of planned bevacizumab treatment and diminishing by 24 months. Bevacizumab was associated with more toxic effects (most often hypertension of grade 2 or higher) (18%, vs. 2% with chemotherapy alone). In the updated analyses, progression-free survival (restricted mean) at 42 months was 22.4 months without bevacizumab versus 24.1 months with bevacizumab (P=0.04 by log-rank test); in patients at high risk for progression, the benefit was greater with bevacizumab than without it, with progression-free survival (restricted mean) at 42 months of 14.5 months with standard therapy alone and 18.1 months with bevacizumab added, with respective median overall survival of 28.8 and 36.6 months. CONCLUSIONS Bevacizumab improved progression-free survival in women with ovarian cancer. The benefits with respect to both progression-free and overall survival were greater among those at high risk for disease progression. (Funded by Roche and others; ICON7 Controlled-Trials.com number, ISRCTN91273375.).

Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer

MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of ≈15% and at least ≈36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.

MicroRNA Microarray Identifies Let-7i as a Novel Biomarker and Therapeutic Target in Human Epithelial Ovarian Cancer

MicroRNAs (miRNA) are approximately 22-nucleotide noncoding RNAs that negatively regulate protein-coding gene expression in a sequence-specific manner via translational inhibition or mRNA degradation. Our recent studies showed that miRNAs exhibit genomic alterations at a high frequency and their expression is remarkably deregulated in ovarian cancer, strongly suggesting that miRNAs are involved in the initiation and progression of this disease. In the present study, we performed miRNA microarray to identify the miRNAs associated with chemotherapy response in ovarian cancer and found that let-7i expression was significantly reduced in chemotherapy-resistant patients (n = 69, P = 0.003). This result was further validated by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.015). Both loss-of-function (by synthetic let-7i inhibitor) and gain-of-function (by retroviral overexpression of let-7i) studies showed that reduced let-7i expression significantly increased the resistance of ovarian and breast cancer cells to the chemotherapy drug, cis-platinum. Finally, using miRNA microarray, we found that decreased let-7i expression was significantly associated with the shorter progression-free survival of patients with late-stage ovarian cancer (n = 72, P = 0.042). This finding was further validated in the same sample set by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.001) and in an independent sample set by in situ hybridization (n = 53, P = 0.049). Taken together, our results strongly suggest that let-7i might be used as a therapeutic target to modulate platinum-based chemotherapy and as a biomarker to predict chemotherapy response and survival in patients with ovarian cancer.

Surveillance for endometrial cancer in hereditary nonpolyposis colorectal cancer syndrome

The estimated lifetime risk for endometrial carcinoma (EC) in hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is 32–60%, thus supporting surveillance. The survival rate of EC patients is, however, favourable questioning the need for surveillance. Yet, the effectiveness of gynecological surveillance remains to be shown. The 2 previously published studies were based on transvaginal ultrasound (TVUS) alone. Intrauterine biopsy has not been tested in surveillance for EC in HNPCC families. The effect of gynecological surveillance was evaluated among 175 Finnish mutation carriers. During 759 person years at risk, there were 503 surveillance visits including TVUS and intrauterine biopsy of endometrium at 94% and 74% of the visits, respectively. EC occurred in 14 cases, 11 of which were diagnosed by surveillance, 8 by intrauterine biopsies. TVUS indicated only 4 EC patients but missed 6 other cases. Intrauterine sampling detected 14 additional cases of potentially premalignant hyperplasia. The stage distribution and survival tended to be more favorable in the 14 EC cases of the surveilled group (no deaths) than in the group of 83 symptomatic mutation carriers of whom 6 died of EC, but with no statistical significance. Four cases of ovarian cancer occurred but none was detected by surveillance in TVUS examinations. In conclusion, EC surveillance in HNPCC seems more effective with endometrial biopsies than with TVUS alone. A definite improvement in survival remains to be shown. The detection of early cancer stages and premalignant lesions offers the opportunity to avoid extensive adjuvant treatment.

Gene amplification, mutation, and protein expression of EGFR and mutations of ERBB2 in serous ovarian carcinoma

EGFR and erbB-2 are targets for specific cancer therapy. The purpose of this study was to examine the frequency and clinicopathological correlations of gene amplification, protein expression, and mutations of EGFR and ERBB2 in serous carcinoma, the most common and aggressive type of ovarian cancer. Tissue microarray constructed of 398 carcinomas was examined by chromogenic in situ hybridization (CISH) and by immunohistochemistry. Cases with amplification of EGFR by CISH were further analyzed by fluorescence in situ hybridization. One hundred ninety-eight samples were analyzed for mutations in exons 18, 19, or 21 of EGFR and in exon 20 of ERBB2 using denaturating high-performance liquid chromatography and direct sequencing. Amplification of EGFR was present in 12% (41/333), low-level gain in 43% (144/333), and protein overexpression in 17% (66/379) of the tumors. Both increased copy number and overexpression of EGFR were associated with high tumor grade, greater patient age, large residual tumor size, high proliferation index, aberrant p53, and poor patient outcome. Furthermore, increased copy number of EGFR was associated with increased copy number of ERBB2. No mutations were identified in EGFR, whereas one tumor had an insertion mutation in exon 20 of ERBB2. Both amplification and protein overexpression of EGFR occur in serous ovarian carcinoma, but EGFR copy number has a stronger prognostic value. This makes EGFR amplification a potentially useful criterion for selecting patients in clinical trials testing the effect of EGFR inhibitors in serous ovarian carcinoma.

Sociodemographic determinants of incidence of primary fallopian tube carcinoma, Finland 1953–97

Primary fallopian tube carcinoma is very rare. In Western countries, it accounts for about 1% of all female genital malignant tumors. Its etiology remains poorly known, but high parity is considered to be protective. We studied determinants of incidence of primary fallopian tube carcinoma in Finland. Incidence rates for primary fallopian tube carcinoma, according to the population based Finnish Cancer Registry, from 1953– 97 were assessed by age, year of diagnosis, and type of residential area. Standardized incidence ratios (SIRs) for the years 1971– 95 were calculated by occupation and social class variables taken from the 1970 Population Census. There were 485 cases of primary fallopian tube carcinoma registered during 45 years. The age‐adjusted incidence rate increased from 1.2/1,000,000 in 1953–57 to 5.4/1,000,000 in 1993–97. This 4.5‐fold increase in incidence rate corresponds to a 7‐fold increase in the number of new cases. The increase is attributable to the age group beyond 55 years, the peak incidence occurring between 60–64 years. Although the relative increase in incidence rate has been larger in rural areas than in cities, the rate in the latter remains 2‐fold. Women in the 2 highest social classes had a 1.8‐fold incidence (95% CI = 1.2–2.6) as compared to the lowest social class. Women in agriculture and those not working outside the home had only half the cancer incidence of those in academic or clerical occupations. The incidence of primary fallopian tube carcinoma increases in Finland. Evidently, the incidence has increased simultaneously with the affluence of urban life. Part of the variation in incidence correlates with variation in parity.

Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients

OBJECTIVE Ezrin is a membrane-cytoskeleton linker protein, which regulates cell polarity and signaling. Increased ezrin expression in astrocytomas and uveal melanomas is correlated with unfavorable prognostic factors and reduced patient survival. We investigated ezrin IR in normal ovarian surface epithelium and serous ovarian carcinomas, and its relation with clinical parameters and patient outcome. METHODS Tissue microarray blocks were constructed of all serous ovarian carcinoma tissue samples removed at a primary operation in Helsinki University Central Hospital between 1964 and 1999 and of healthy ovarian tissue samples. Ezrin expression was assessed by indirect immunohistochemistry using a monoclonal 3C12 ezrin antibody. Tissue samples (n = 440) were scored for the intensity of ezrin immunoreactivity, and the scores were compared with patient age, the stage and grade of disease, and disease outcome. RESULTS Healthy ovarian epithelium showed strong polarized ezrin immunoreactivity. In serous ovarian carcinoma, the reactivity varied from strong (15.0% of samples) to moderate (57.3%) or weak/negative (27.7%) and the subcellular distribution was typically diffuse. Weak or negative expression of ezrin was associated with shorter survival (P = 0.027) but also with an advanced age of the patients (P = 0.0001), and a higher histological grade of the disease (P = 0.032). In Cox multivariate survival analysis, ezrin immunoreactivity had no independent effect on survival, when controlling for the stage and grade of the disease, and patient age. CONCLUSIONS In contrast with astrocytomas and uveal melanomas, negative or weak ezrin immunoreactivity in serous ovarian carcinoma correlates with poor patient outcome.

Adenocarcinoma of the uterine cervix

In Finland, the incidence of cervical cancer has shown a decreasing tendency since the 1960s. The same trend, however, has not been noticed in the incidence of cervical adenocarcinoma. The reason for this is not known, although many studies have shown differences in the cause, epidemiology, and biology of the epidermoid and adenocarcinoma of the uterine cervix. A total of 106 new patients with cervical adenocarcinoma were treated at our institution from 1976 to 1980, which represents 20.4% of all cervical carcinomas treated. The mean age of the patients was 58.1 years (range, 29 to 82 years) and the peak incidence was in the group 60 to 69 years of age. Most of the patients were postmenopausal (71.7%) and the main symptom was abnormal vaginal bleeding (78.3%). The proportion of Stage I was 61.3%. Combined operative and radiation therapy was used in 74.5% of the patients. The overall 5‐year survival rate was 65.1% (corrected 74.5%), which did not differ from that of patients with squamous cell carcinoma. The most significant prognostic factors were the size of the tumor, presence of pelvic lymph node metastases, and the stage of the disease.

Elevated Cyclooxygenase-2 Expression Is Associated with Altered Expression of p53 and SMAD4, Amplification of HER-2/neu, and Poor Outcome in Serous Ovarian Carcinoma

Purpose and Experimental Design: Cyclooxygenase-2 (COX-2) is frequently expressed in human adenocarcinomas and inhibition of COX-2 suppresses tumor formation in various animal models of carcinogenesis. We analyzed expression of COX-2 protein in human serous ovarian carcinomas by immunohistochemistry (n = 442) and by Western blotting (n = 12) and COX-2 mRNA by reverse transcriptase PCR (n = 12). COX-2 immunoreactivity was correlated to clinicopathological variables and to expression of p53 and SMAD4 as detected by immunohistochemistry and to amplification of HER-2/neu as detected by in situ hybridization. Results: COX-2 mRNA expression was detected in 75% (9 of 12) and COX-2 protein in 42% (5 of 12) of the serous ovarian adenocarcinoma specimens as detected by reverse transcriptase-PCR and Western blot analysis, respectively. Moderate to strong (elevated) immunoreactivity for COX-2 was detected in 70% (310 of 442) of the tumors. Elevated COX-2 expression associated with reduced disease-specific survival (P = 0.0011), high histological grade (P < 0.0001), residual tumor size > 1 cm (P = 0.0111), and age > 57 years (P = 0.0099). Tumors with altered immunostaining pattern for p53 or SMAD4 expressed more frequently elevated levels of COX-2 when compared with the tumors with normal staining pattern of these tumor suppressor genes (P < 0.0001 and P = 0.0004, respectively). In addition, elevated COX-2 expression associated with amplification of HER-2/neu oncogene (P = 0.0479). Conclusions: Our results suggest that elevated expression of COX-2 associates with reduced survival in serous ovarian carcinomas and that expression of COX-2 may be induced in these tumors by loss of tumor suppressor genes such as p53and SMAD4and by amplification of HER-2/neuoncogene.

Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer

Significance The major portion of hereditary breast cancer still remains unexplained, and many susceptibility loci are yet to be found. Exome sequencing of 24 high-risk familial BRCA1/2-negative breast cancer patients and further genotyping of a large sample set of breast/ovarian cancer cases and controls was used to discover previously unidentified susceptibility alleles and genes. A significant association of a FANCM nonsense mutation with breast cancer, especially triple-negative breast cancer, identifies FANCM as a breast cancer susceptibility gene. Identification of such risk alleles is expected to improve cancer risk assessment for breast cancer patients and families, and may lead to improvements in the prevention, early diagnosis, and treatment of cancer. Inherited predisposition to breast cancer is known to be caused by loss-of-function mutations in BRCA1, BRCA2, PALB2, CHEK2, and other genes involved in DNA repair. However, most families severely affected by breast cancer do not harbor mutations in any of these genes. In Finland, founder mutations have been observed in each of these genes, suggesting that the Finnish population may be an excellent resource for the identification of other such genes. To this end, we carried out exome sequencing of constitutional genomic DNA from 24 breast cancer patients from 11 Finnish breast cancer families. From all rare damaging variants, 22 variants in 21 DNA repair genes were genotyped in 3,166 breast cancer patients, 569 ovarian cancer patients, and 2,090 controls, all from the Helsinki or Tampere regions of Finland. In Fanconi anemia complementation gene M (FANCM), nonsense mutation c.5101C>T (p.Q1701X) was significantly more frequent among breast cancer patients than among controls [odds ratio (OR) = 1.86, 95% CI = 1.26–2.75; P = 0.0018], with particular enrichment among patients with triple-negative breast cancer (TNBC; OR = 3.56, 95% CI = 1.81–6.98, P = 0.0002). In the Helsinki and Tampere regions, respectively, carrier frequencies of FANCM p.Q1701X were 2.9% and 4.0% of breast cancer patients, 5.6% and 6.6% of TNBC patients, 2.2% of ovarian cancer patients (from Helsinki), and 1.4% and 2.5% of controls. These findings identify FANCM as a breast cancer susceptibility gene, mutations in which confer a particularly strong predisposition for TNBC.