Ronald P. Zweemer

Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer

The aim of this study was to investigate the occurrence of (pre)neoplastic lesions in overtly normal Fallopian tubes from women predisposed to developing ovarian carcinoma. The presence of (pre)neoplastic lesions was scored in histological specimens from 12 women with a genetically determined predisposition for ovarian cancer, of whom seven tested positive for a germline BRCA1 mutation. A control group included 13 women. Immunohistochemistry was used to determine the expression of p21, p27, p53, cyclin A, cyclin D1, bcl‐2, Ki67, HER‐2/neu, and the oestrogen and progesterone receptors. Loss of heterozygosity (LOH) analysis on the BRCA1 locus was also assessed on dysplastic tissue by PCR studies. Of the 12 women with a predisposition for ovarian cancer, six showed dysplasia, including one case of severe dysplasia. Five harboured hyperplastic lesions and in one woman no histological aberrations were found in the Fallopian tube. No hyperplastic, dysplastic or neoplastic lesions were detected in the Fallopian tubes of control subjects. In the cases studied, morphologically normal tubal epithelium contained a higher proportion of Ki67‐expressing cells (p=0.005) and lower fractions of cells expressing p21 (p<0.0001) and p27 (p=0.006) than in the control group. Even higher fractions of proliferating cells were found in dysplastic areas (p=0.07) and accumulation of p53 was observed in the severely dysplastic lesion. Expression patterns of other proteins studied, including the hormone receptors, were similar in cases and controls. One subject, a germline BRCA1 mutation carrier, showed loss of the wild‐type BRCA1 allele in the severely dysplastic lesion. In conclusion, the Fallopian tubes of women predisposed to developing ovarian cancer frequently harbour dysplastic changes, accompanied by changes in cell‐cycle and apoptosis‐related proteins, indicating an increased risk of developing tubal cancer. Copyright

Histopathologic features of genetically determined ovarian cancer.

Inheritance of germline mutations of BRCA1 or BRCA2 genes account for approximately 10% of ovarian carcinomas, but the characterization of these genetically determined cancers is incomplete. The objective of our study was to characterize the histologic features of ovarian carcinomas associated with germline mutations of BRCA1 and BRCA2. Thirty-two ovarian carcinomas associated with germline BRCA1 or BRCA2 mutations and 40 ovarian carcinomas from patients screened as negative for germline mutations were obtained from three centers. A gynecologic pathologist, blinded to mutation status, reviewed each case, with documentation of the histologic type, Gynecologic Oncology Group (GOG) grade, architectural and nuclear grade, Silverberg grade, and mitotic activity. All BRCA1 and BRCA2 mutation-associated cases were invasive serous carcinomas, and of these 50% were GOG grade 3, 41% had an architectural grade of 3 (predominant solid architecture), 84% a nuclear grade of 3, 72% a mitotic score of 3 (>25 mitoses per 10 HPF), and 75% a Silverberg grade of 3. The differences in histologic type (p=0.001) and Silverberg grade (p=0.002) between these tumors and the control group were statistically significant and remained so when comparisons between BRCA carriers and noncarriers were restricted to carcinomas of serous histology alone. Ovarian carcinomas associated with germline mutations of BRCA1/BRCA2 are, in this study, invasive serous carcinomas, with a statistically significant higher histologic grade than ovarian carcinomas without BRCA mutations when using the recently proposed Silverberg grading system.

Prognostic significance of low volume sentinel lymph node disease in early-stage cervical cancer

OBJECTIVE Evaluate prognostic significance of low volume disease detected in sentinel nodes (SN) of patients with early stages cervical cancer. Although pathologic ultrastaging of SN allows for identification of low volume disease, including micro-metastasis and isolated tumor cells (ITC), in up to 15% of cases, prognostic significance of these findings is unknown. METHODS A total of 645 records from 8 centers were retrospectively reviewed. Enrolled in our study were patients with early-stage cervical cancer who had undergone surgical treatment including SN biopsy followed by pelvic lymphadenectomy and pathologic ultrastaging of SN. RESULTS Macrometastasis, micrometastasis, and ITC were detected by SN ultrastaging in 14.7%, 10.1%, and 4.5% patients respectively. False negativity of SN ultrastaging reached 2.8%. The presence of ITC was not associated with significant risk, both for recurrence free survival and overall survival. Overall survival was significantly reduced in patients with macrometastasis and micrometastasis; hazard ratio for overall survival reached 6.85 (95% CI, 2.59-18.05) and 6.86 (95% CI, 2.09-22.61) respectively. Presence of micrometastasis was an independent prognostic factor for overall survival in a multivariable model. CONCLUSION Presence of micrometastasis in SN in patients with early stage cervical cancer was associated with significant reduction of overall survival, which was equivalent to patients with macrometastasis. No prognostic significance was found for ITC. These data highlight the importance of SN biopsy and pathologic ultrastaging for the management of cervical cancer.

Bilateral ultrastaging of sentinel lymph node in cervical cancer: Lowering the false-negative rate and improving the detection of micrometastasis

OBJECTIVE To evaluate the sensitivity of sentinel node (SN) ultrastaging and to define parameters that may reduce the overall false-negative rate in women with early-stage cervical cancer. METHODS We analyzed data from a large retrospective multicenter cohort group with FIGO stages IA-IIB cervical cancer in whom at least one SN was identified and systematic pelvic lymphadenectomy was uniformly performed. All who were SN negative by initial evaluation were subjected to ultrastaging. RESULTS In all, 645 patients were evaluable. SN were detected bilaterally in 72% of cases and unilaterally in 28%. Patients with optimal bilateral SN detection were significantly more likely to have any metastasis detected (33.3% vs. 19.2%; P<0.001) as well as micrometastasis detected in their SN (39.6% vs. 11.4%). SN ultrastaging resulted in a low overall false-negative rate of 2.8% (whole group) and an even lower false-negative rate of 1.3% for patients with optimal bilateral mapping. Patients with false-negative SN after ultrastaging had a higher prevalence of LVSI and more frequent unilateral SN detection. Sensitivity of SN ultrastaging was 91% (95% CI: 86%-95%) for the whole group and 97% (95% CI: 91%-99%) in the subgroup with bilateral SN detection. CONCLUSION These data confirm previous observations that optimal bilateral SN detection substantially decreases the false negative rate of SN ultrastaging and increases detection of micrometastasis. In patients with bilateral SN detection, the sensitivity of SN ultrastaging is not reduced in more advanced stages of the disease. SN mapping and ultrastaging should become standard practice in the surgical management of early-stage cervical cancer.

Clinical and genetic evaluation of thirty ovarian cancer families

OBJECTIVES Our purpose was to determine the prevalence of BRCA1 and BRCA2 germline mutations among patients from ovarian cancer families and to evaluate age at diagnosis, histologic diagnosis, and International Federation of Gynecology and Obstetrics stage in this group. STUDY DESIGN We reviewed 50 ovarian cancer patients from 30 ovarian cancer families and compared relevant clinical characteristics with those of a cancer registry reference group. BRCA1 (exons 2 to 24) and BRCA2 (exon 11) germline mutations were detected by a protein truncation test and sequencing of BRCA1 exon 2 (185delAG mutation) in 25 of the 30 families. RESULTS Ten (40%) of 25 families tested revealed a germline BRCA1 or BRCA2 mutation. Patients with ovarian cancer from the study group were young with an advanced International Federation of Gynecology and Obstetrics stage at diagnosis and had a relatively high frequency of serous adenocarcinoma. CONCLUSION Direct mutation analysis of BRCA1 and BRCA2 revealed a high frequency of germline mutations in ovarian cancer families. Some clinical characteristics of hereditary ovarian cancer may differ from those of sporadic disease.

The influence of the b-value combination on apparent diffusion coefficient based differentiation between malignant and benign tissue in cervical cancer

To analyze the influence of different b‐value combinations on apparent diffusion coefficient (ADC)‐based differentiation of known malignant and benign tissue in cervical cancer patients.

Molecular evidence for putative tumour suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer

Background/Aims: Loss of heterozygosity (LOH) on chromosome 13q has been reported to occur frequently in human ovarian cancer, and indications have been found that chromosome 13 may also play a specific role in the inherited form of ovarian cancer. The aim of this study was to define regions on chromosome 13 that may harbour additional tumour suppressor genes involved in the tumorigenesis of BRCA1 related ovarian and fallopian tube cancer. Materials/methods: DNA extracted from paraffin wax blocks of 36 BRCA1 associated ovarian and fallopian tube carcinomas was analysed by LOH polymerase chain reaction using seven highly polymorphic microsatellite markers spanning chromosome 13q. Results: High LOH frequencies were found on loci 13q11, 13q14, 13q21, 13q22–31, 13q32, and 13q32–4, suggesting the presence of putative tumour suppressor genes on the long arm of chromosome 13 that may play a role in the pathogenesis of BRCA1 related ovarian and fallopian tube cancer. LOH patterns appeared to be independent of the type of BRCA1 mutation, stage, and grade. Although in some cases there were indications for loss of larger parts of chromosome 13, in most cases losses were fairly randomly distributed over chromosome 13 with retained parts in between lost parts. Microsatellite instability was found in six cases. Conclusion: Several loci on chromosome 13q show high frequencies of LOH in BRCA1 related ovarian and fallopian tube cancer, and may therefore harbour putative tumour suppressor genes involved in the carcinogenesis of this particular type of hereditary cancer.

Genomic and transcriptomic plasticity in treatment-naïve ovarian cancer

Intra-tumor heterogeneity is a hallmark of many cancers and may lead to therapy resistance or interfere with personalized treatment strategies. Here, we combined topographic mapping of somatic breakpoints and transcriptional profiling to probe intra-tumor heterogeneity of treatment-naïve stage IIIC/IV epithelial ovarian cancer. We observed that most substantial differences in genomic rearrangement landscapes occurred between metastases in the omentum and peritoneum versus tumor sites in the ovaries. Several cancer genes such as NF1, CDKN2A, and FANCD2 were affected by lesion-specific breakpoints. Furthermore, the intra-tumor variability involved different mutational hallmarks including lesion-specific kataegis (local mutation shower coinciding with genomic breakpoints), rearrangement classes, and coding mutations. In one extreme case, we identified two independent TP53 mutations in ovary tumors and omentum/peritoneum metastases, respectively. Examination of gene expression dynamics revealed up-regulation of key cancer pathways including WNT, integrin, chemokine, and Hedgehog signaling in only subsets of tumor samples from the same patient. Finally, we took advantage of the multilevel tumor analysis to understand the effects of genomic breakpoints on qualitative and quantitative gene expression changes. We show that intra-tumor gene expression differences are caused by site-specific genomic alterations, including formation of in-frame fusion genes. These data highlight the plasticity of ovarian cancer genomes, which may contribute to their strong capacity to adapt to changing environmental conditions and give rise to the high rate of recurrent disease following standard treatment regimes.

Comparative genomic hybridization of microdissected familial ovarian carcinoma: Two deleted regions on chromosome 15q not previously identified in sporadic ovarian carcinoma

The vast majority of familial ovarian cancers harbor a germline mutation in either the breast cancer gene BRCA1 or BRCA2 tumor suppressor genes. However, mutations of these genes in sporadic ovarian cancer are rare. This suggests that in contrast to hereditary disease, BRCA1 and BRCA2 are not commonly involved in sporadic ovarian cancer and may indicate that there are two distinct pathways for the development of ovarian cancer. To characterize further differences between hereditary and sporadic cancers, the comparative genomic hybridization technique was employed to analyze changes in copy number of genetic material in a panel of 36 microdissected hereditary ovarian cancers. Gains at 8q23-qter (18 of 36, 5 cases with high-level amplifications), 3q26.3-qter (18 of 36, 2 cases with high-level amplifications), 11q22 (11 of 36) and 2q31–32 (8 of 36) were most frequent. Losses most frequently occurred (in decreasing order of frequency) on 8p21-pter (23 of 36), 16q22-pter (19 of 36), 22q13 (19 of 36), 9q31–33 (16 of 36), 12q24 (16 of 36), 15q11–15 (16 of 36), 17p12–13 (14 of 36), Xp21–22 (14 of 36), 20q13 (13 of 36), 15q24–25 (12 of 36), and 18q21 (12 of 36). Comparison with the literature revealed that the majority of these genetic alterations are also common in sporadic ovarian cancer. Deletions of 15q11–15, 15q24–25, 8p21-ter, 22q13, 12q24 and gains at 11q22, 13q22, and 17q23–25, however, appear to be specific to hereditary ovarian cancer. Aberrations at 15q11–15 and 15q24–25 have not yet been described in familial ovarian cancer. In these regions, important tumor suppressor genes, including the hRAD51 gene, are located. These and other yet unknown suppressor genes may be involved in a specific carcinogenic pathway for familial ovarian cancer and may explain the distinct clinical presentation and behavior of familial ovarian cancer.

Methylation profiles of endometrioid and serous endometrial cancers

Promoter methylation is a gene- and cancer type-specific epigenetic event that plays an important role in tumour development. As endometrioid (endometrioid endometrial carcinoma, EEC) and serous endometrial cancers (uterine papillary serous carcinoma, UPSC) exhibit different clinical, histological and molecular genetic characteristics, we hypothesized that these differences may be reflected in epigenetic phenomena as well. Identification of a panel of methylation biomarkers could be helpful in a correct histological classification of these two subtypes, which solely on the basis of morphology is not always easy. Methylation-specific multiplex ligation-dependent probe amplification was used to assess the extent of promoter methylation of different tumour suppressor genes in EEC and UPSC. Methylation results were correlated with histology and survival. The median cumulative methylation index of all genes was significantly higher in EEC (124) than in UPSC (93) (P<0.001). Promoter methylation of CDH13 and MLH1 was more frequently present in EEC, while CDKN2B and TP73 were more frequently methylated in UPSC. Almost 90% of EEC and 70% of UPSC could be predicted by CDH13 and TP73. In EEC, methylation of MLH1 was associated with a shorter disease-free survival (DFS; P<0.0001) and overall survival (OS; P=0.005). In a multivariate model, MLH1 methylation emerged as an additional prognostic factor to stage for DFS (P=0.002). In conclusion, promoter methylation is more common in EEC than UPSC. A panel of methylation biomarkers could be useful to distinguish between the two histological subtypes of endometrial cancer. Furthermore, methylation of MLH1 may have prognostic value in EEC.