Ralf Bützow

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.

Pathology of Ovarian Cancers in BRCA1 and BRCA2 Carriers

Purpose: Germline mutations in the BRCA1 and BRCA2 genes confer increased susceptibility to ovarian cancer. There is evidence that tumors in carriers may exhibit a distinct distribution of pathological features, but previous studies on the pathology of such tumors have been small. Our aim was to evaluate the morphologies and immunophenotypes in a large cohort of patients with familial ovarian cancer. Experimental Design: We performed a systematic review of ovarian tumors from 178 BRCA1 mutation carriers, 29 BRCA2 mutation carriers, and 235 controls with a similar age distribution. Tumors were evaluated by four pathologists blinded to mutation status. Both morphological features and immunochemical staining for p53 and HER2 were evaluated. Results: Tumors in BRCA1 mutation carriers were more likely than tumors in age-matched controls to be invasive serous adenocarcinomas (odds ratio, 1.84; 95% confidence interval, 1.21–2.79) and unlikely to be borderline or mucinous tumors. Tumors in BRCA1 carriers were of higher grade (P < 0.0001), had a higher percentage solid component (P = 0.001), and were more likely to stain strongly for p53 (P = 0.018). The distribution of pathological features in BRCA2 carriers was similar to that in BRCA1 carriers. Conclusions: Use of pathological features can substantially improve the targeting of predictive genetic testing. Results also suggest that BRCA1 and BRCA2 tumors are relatively aggressive and may be expected to have poor prognosis, although this may be treatment dependent.

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.

Characterization of Uterine Leiomyomas by Whole-Genome Sequencing

BACKGROUND Uterine leiomyomas are benign but affect the health of millions of women. A better understanding of the molecular mechanisms involved may provide clues to the prevention and treatment of these lesions. METHODS We performed whole-genome sequencing and gene-expression profiling of 38 uterine leiomyomas and the corresponding myometrium from 30 women. RESULTS Identical variants observed in some separate tumor nodules suggested that these nodules have a common origin. Complex chromosomal rearrangements resembling chromothripsis were a common feature of leiomyomas. These rearrangements are best explained by a single event of multiple chromosomal breaks and random reassembly. The rearrangements created tissue-specific changes consistent with a role in the initiation of leiomyoma, such as translocations of the HMGA2 and RAD51B loci and aberrations at the COL4A5-COL4A6 locus, and occurred in the presence of normal TP53 alleles. In some cases, separate events had occurred more than once in single tumor-cell lineages. CONCLUSIONS Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).

Biallelic Inactivation of Fumarate Hydratase (FH) Occurs in Nonsyndromic Uterine Leiomyomas but Is Rare in Other Tumors

Germline mutations in the fumarate hydratase (FH) gene at 1q43 predispose to dominantly inherited cutaneous and uterine leiomyomas, uterine leiomyosarcoma, and papillary renal cell cancer (HLRCC syndrome). To evaluate the role of FH inactivation in sporadic tumorigenesis, we analyzed a series of 299 malignant tumors representing 10 different malignant tumor types for FH mutations. Additionally, 153 uterine leiomyomas from 46 unselected individuals were subjected to and informative in loss of heterozygosity analysis at the FH locus, and the five (3.3%) tumors displaying loss of heterozygosity were subjected to FH mutation analysis. Although mutation search in the 299 malignant tumors was negative, somatic FH mutations were found in two nonsyndromic leiomyomas; a splice site change IVS4 + 3A>G, leading to deletion of exon four, and a missense mutation Ala196Thr. The occurrence of somatic mutations strongly suggests that FH is a true target of the 1q43 deletions. Although uterine leiomyomas are the most common tumors of women, specific inactivating somatic mutations contributing to the formation of nonsyndromic leiomyomas have not been reported previously. Taking into account the apparent risk of uterine leiomyosarcoma associated with FH germline mutations, the finding raises the possibility that also some nonsyndromic leiomyomas may have a genetic profile that is more prone to malignant degeneration. Our data also indicate that somatic FH mutations appear to be limited to tumor types observed in hereditary leiomyomatosis and renal cell cancer.

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary.

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95–97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

LKB1 Somatic Mutations in Sporadic Tumors

Germline mutations of LKB1/Peutz-Jeghers syndrome gene predispose carriers to hamartomatous polyposis of the gastrointestinal tract as well as to cancer of different organ systems. Although Peutz-Jeghers syndrome patients frequently present with neoplasms of the colon, stomach, small intestine, pancreas, breast, ovaries, and cervix, somatic mutations appear to be rare in the sporadic tumor types thus far studied (colorectal, gastric, testicular, and breast cancers). To evaluate whether somatic mutations of LKB1 contribute to the tumorigenesis of yet unstudied tumor types, we screened 14 cell lines and 129 tumor specimens from different cancers for a genetic defect in LKB1. Six melanoma and eight myeloma cell lines were scrutinized for LKB1 somatic mutations by genomic sequencing. No changes were found in the coding LKB1 sequence and exon/intron boundaries. Next, we analyzed 12 pancreatic, 8 gastric, 12 ovarian granulosa cell, 26 cervical, 28 lung, 24 soft tissue, and 19 renal tumors by single-strand conformational polymorphism analysis. Three changes in LKB1 coding nucleotide sequence were identified. One base pair deletion at A957 and G958 substitution by T occurred in a cervical adenocarcinoma sample, resulting in a frameshift and premature stop codon at position 335. Substitution of A581 by T occurred in a lung adenocarcinoma sample, resulting in the change of aspartic acid at position 194 to valine. A loss of another allele was detected in this sample. One silent change, C1257T, was found in a pancreatic carcinoma sample. The changes were not present in the matched normal tissue DNA samples. Our results suggest that mutational inactivation of LKB1 is a rare event in most sporadic tumor types.

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.

Integrative Genomic Analysis of Protein Kinase C (PKC) Family Identifies PKCι as a Biomarker and Potential Oncogene in Ovarian Carcinoma

The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCiota (43.8%), PKCbeta1 (37.1%), PKCgamma (27.6%), PKCzeta (22.5%), and PKCtheta (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCiota and PKCtheta, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCiota expression correlated with tumor stage or grade, and PKCiota overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCiota protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCiota did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCiota expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCiota contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCiota may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer.