M. Zikan

The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's Cancer

Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.

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.

Diagnosis, Treatment, and Follow-Up of Borderline Ovarian Tumors

Borderline ovarian tumors represent a heterogeneous group of noninvasive tumors of uncertain malignant potential with characteristic histology. They occur in younger women, are present at an early stage, and have a favorable prognosis, but symptomatic recurrence and death may be found as long as 20 years after therapy in some patients. The molecular changes in borderline ovarian tumors indicate linkage of this disease to type I ovarian tumors (low-grade ovarian carcinomas). The pathological stage of disease and subclassification of extraovarian disease into invasive and noninvasive implants, together with the presence of postoperative macroscopic residual disease, appear to be the major predictor of recurrence and survival. However, it should be emphasized that the most important negative prognostic factor for recurrence is just the use of conservative surgery, but without any impact on patient survival because most recurrent diseases are of the borderline type-easily curable and with an excellent prognosis. Borderline tumors are difficult masses to correctly preoperatively diagnose using imaging methods because their macroscopic features may overlap with invasive and benign ovarian tumors. Over the past several decades, surgical therapy has shifted from a radical approach to more conservative treatment; however, oncologic safety must always be balanced. Follow-up is essential using routine ultrasound imaging, with special attention paid to the remaining ovary in conservatively treated patients. Current literature on this topic leads to a number of controversies that will be discussed thoroughly in this article, with the aim to provide recommendations for the clinical management of these patients.

Early-stage cervical cancer: Tumor delineation by magnetic resonance imaging and ultrasound - A European multicenter trial

OBJECTIVE To compare the diagnostic accuracy of ultrasound (US) and magnetic resonance imaging (MRI) in the preoperative assessment of early-stage cervical cancer using pathologic findings as the reference standard. PATIENTS AND METHODS Prospective multi-center trial enrolling 209 consecutive women with early-stage cervical cancer (FIGO IA2-IIA) scheduled for surgery. The following parameters were assessed on US and MRI and compared to pathology: remaining tumor, size, tumor stromal invasion<2/3 (superficial) or ≥2/3 (deep), and parametrial invasion. RESULTS Complete data were available for 182 patients. The agreement between US and pathology was excellent for detecting tumors, correctly classifying bulky tumors (>4cm), and detecting deep stromal invasion (kappa values 0.84, 0.82, and 0.81 respectively); and good for classifying small tumors (<2cm) and detecting parametrial invasion (kappa values 0.78 and 0.75, respectively). The agreement between MRI and histology was good for classifying tumors as <2cm, or >4cm, and detecting deep stromal invasion (kappa values 0.71, 0.76, and 0.77, respectively). It was moderately accurate in tumor detection, and in assessing parametrial invasion (kappa values 0.52 and 0.45, respectively). The agreement between histology and US was significantly better in assessing residual tumor (p<0.001) and parametrial invasion (p<0.001) than the results obtained by MRI. Imaging methods were not significantly influenced by previous cone biopsy. CONCLUSION US and MRI are highly accurate for the preoperative assessment of women with early-stage cervical cancer, although US may be more accurate in detecting residual tumors and assessing parametrial invasion.

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.

Common variants associated with breast cancer in genome-wide association studies are modifiers of breast cancer risk in BRCA1 and BRCA2 mutation carriers.

Recent studies have identified single nucleotide polymorphisms (SNPs) that significantly modify breast cancer risk in BRCA1 and BRCA2 mutation carriers. Since these risk modifiers were originally identified as genetic risk factors for breast cancer in genome-wide association studies (GWASs), additional risk modifiers for BRCA1 and BRCA2 may be identified from promising signals discovered in breast cancer GWAS. A total of 350 SNPs identified as candidate breast cancer risk factors (P < 1 x 10(-3)) in two breast cancer GWAS studies were genotyped in 3451 BRCA1 and 2006 BRCA2 mutation carriers from nine centers. Associations with breast cancer risk were assessed using Cox models weighted for penetrance. Eight SNPs in BRCA1 carriers and 12 SNPs in BRCA2 carriers, representing an enrichment over the number expected, were significantly associated with breast cancer risk (P(trend) < 0.01). The minor alleles of rs6138178 in SNRPB and rs6602595 in CAMK1D displayed the strongest associations in BRCA1 carriers (HR = 0.78, 95% CI: 0.69-0.90, P(trend) = 3.6 x 10(-4) and HR = 1.25, 95% CI: 1.10-1.41, P(trend) = 4.2 x 10(-4)), whereas rs9393597 in LOC134997 and rs12652447 in FBXL7 showed the strongest associations in BRCA2 carriers (HR = 1.55, 95% CI: 1.25-1.92, P(trend) = 6 x 10(-5) and HR = 1.37, 95% CI: 1.16-1.62, P(trend) = 1.7 x 10(-4)). The magnitude and direction of the associations were consistent with the original GWAS. In subsequent risk assessment studies, the loci appeared to interact multiplicatively for breast cancer risk in BRCA1 and BRCA2 carriers. Promising candidate SNPs from GWAS were identified as modifiers of breast cancer risk in BRCA1 and BRCA2 carriers. Upon further validation, these SNPs together with other genetic and environmental factors may improve breast cancer risk assessment in these populations.

Oral contraceptives and risk of ovarian and breast cancers in BRCA mutation carriers: a meta-analysis

Prophylactic salpingo-oophorectomy is currently the only effective strategy available for decreasing ovarian cancer risk in BRCA1/2 mutation carriers. Significantly decreased risk of ovarian cancer associated with the use of combined oral contraceptives (COCs) was shown in the general population, which could be an alternative approach for those who do not accept risk-reducing surgery. Cohort, case–control and case–case studies published in English up to December 2009 reporting the association of ovarian or breast cancer risk with the use of COCs and presenting BRCA status were selected for meta-analysis. Meta-analysis of three case–control studies showed a significant risk reduction of ovarian cancer in BRCA1/2 mutation carriers who were associated with any past COC use (odds ratio [OR]: 0.57; 95% CI: 0.47–0.70; p < 0.001) and significant trend by duration of COC use (OR: 0.95; 95% CI: 0.93–0.97; p < 0.001). No significant increase in breast cancer risk associated with COC use has been found in case–control studies in BRCA1 (OR: 1.08; p = 0.250), in BRCA2 (OR: 1.03; p = 0.788) mutation carriers or in case–case studies in BRCA1/2 carriers (OR: 0.80; p = 0.147). Significantly increased risk of breast cancer was only shown on a subset of cohort studies in BRCA1 mutation carriers (OR: 1.48; 95% CI: 1.14–1.92). In conclusion, meta-analysis confirmed significantly decreased ovarian cancer risk in BRCA1/2 mutation carriers associated with the use of COCs comparable to the relative extent shown in the general population. Data on the risk of breast cancer associated with COC use in BRCA mutation carriers are heterogeneous and results are inconsistent. COCs can be considered as an alternative strategy in the chemoprevention of ovarian cancer in BRCA1 mutation carriers who do not accept prophylactic salpingo-oophorectomy above the age of 30 years.

Abdominal radical trachelectomy in fertility-sparing treatment of early-stage cervical cancer.

Background: Abdominal radical trachelectomy (ART) is one of the fertility-sparing procedures in women with early-stage cervical cancer. In comparison with vaginal radical trachelectomy, the published results of ART are so far limited. Methods: Enrolled were women referred for ART either by laparoscopy or laparotomy. The main inclusion criterion was stage IA2 or IB1 with a cranial extent that allows for preservation of at least 1 cm of the endocervical canal. Results: A total of 24 women were referred for the procedure, but fertility could not be preserved in 7 (29%) of them. Four women underwent immediate completion of radical hysterectomy because of a positive cranial surgical margin (n = 2) or sentinel node macrometastasis (n = 2) on frozen section. We found no correlation between tumor volume and inability to preserve fertility. A positive sentinel node was identified in 4 patients (17%); there were no false-negative results. Of the 9 women (53%) who have tried to conceive so far, 6 (67%) have conceived and 5 given birth, 2 of which were premature deliveries. Conclusions: Fertility cannot be preserved because of positive cranial margins or involved lymph nodes in almost one third of patients originally referred for radical trachelectomy. The main criterion for the selection of suitable patients should be the cranial extent of the tumor. Abdominal radical trachelectomy allows for achievement of satisfactory obstetrical outcomes.

High proportion of recurrent germline mutations in the BRCA1 gene in breast and ovarian cancer patients from the Prague area.

BackgroundGermline mutations in the BRCA1 and BRCA2 genes have been shown to account for the majority of hereditary breast and ovarian cancers. The purpose of our study was to estimate the incidence and spectrum of pathogenic mutations in BRCA1/2 genes in high-risk Czech families.MethodsA total of 96 Czech families with recurrent breast and/or ovarian cancer and 55 patients considered to be at high-risk but with no reported family history of cancer were screened for mutations in the BRCA1/2 genes. The entire coding sequence of each gene was analyzed using a combination of the protein truncation test and direct DNA sequencing.ResultsA total of 35 mutations in the BRCA1/2 genes were identified in high-risk families (36.5%). Pathogenic mutations were found in 23.3% of breast cancer families and in 59.4% of families with the occurrence of both breast and ovarian cancer. In addition, four mutations were detected in 31 (12.9%) women with early onset breast cancer. One mutation was detected in seven (14.3%) patients affected with both a primary breast and ovarian cancer and another in three (33.3%) patients with a bilateral breast cancer. A total of 3 mutations in BRCA1 were identified among 14 (21.4%) women with a medullary breast carcinoma. Of 151 analyzed individuals, 35 (23.2%) carried a BRCA1 mutation and 9 (6.0%) a BRCA2 mutation. One novel truncating mutation was found in BRCA1 (c.1747A>T) and two in BRCA2 (c.3939delC and c.5763dupT). The 35 identified BRCA1 mutations comprised 13 different alterations. Three recurrent mutations accounted for 71.4% of unrelated individuals with detected gene alterations. The BRCA1 c.5266dupC (5382insC) was detected in 51.4% of mutation positive women. The mutations c.3700_3704del5 and c.181T>G (300T>G) contributed to 11.4% and 8.6% of pathogenic mutations, respectively. A total of eight different mutations were identified in BRCA2. The novel c.5763dupT mutation, which appeared in two unrelated families, was the only recurrent alteration of the BRCA2 gene identified in this study.ConclusionMutational analysis of BRCA1/2 genes in 151 high-risk patients characterized the spectrum of gene alterations and demonstrated the dominant role of the BRCA1 c.5266dupC allele in hereditary breast and ovarian cancer.

HOXA methylation in normal endometrium from premenopausal women is associated with the presence of ovarian cancer: a proof of principle study.

DNA methylation of polycomb group target (PCGT) genes is an early step in carcinogenesis and could potentially be assayed to determine cancer risk prediction. To assess whether methylation changes in PCGT genes in normal tissue is able to predict the presence of cancer, we studied HOXA gene methylation in normal endometrium from premenopausal ovarian cancer patients and age‐matched healthy controls without ovarian cancer. DNA methylation of HOXA9 and HOXA11 genes in normal endometrium was associated with ovarian cancer in an initial test set and this was subsequently confirmed in independent validation sample sets. The overall risk of ovarian cancer was increased 12.3‐fold by high HOXA9 methylation for all stages, and 14.8‐fold for early stage ovarian cancers, independent of age, phase of the menstrual cycle and histology of the cancer. The results of this proof of principle study demonstrate the potential to detect ovarian cancer via analysis of normal endometrial cells and provide insight into the possible contribution of this novel approach in ovarian cancer risk prediction and prevention.