Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing.

BACKGROUND: Circulating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via non invasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample. METHODS: Serum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qPCR targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing. RESULTS: In control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples. CONCLUSION: MBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics.

Association between family history, mutation locations, and prevalence of BRCA1 or 2 mutations in ovarian cancer patients.

We investigated the prevalence of germline BRCA mutations in a population-based cohort of Austrian women diagnosed with ovarian cancer and its association with family history of cancer. We prospectively collected family pedigrees of 443 Austrian ovarian cancer patients who had been tested for the presence of a germline BRCA or 2 mutations and correlated the familial breast and ovarian cancer burden with the prevalence of BRCA mutations and disease onset. The probability of carrying a gBRCA mutation in patients without family history of cancer is 14% (95% CI 9%-22%), as opposed to 45% (95% CI 31%-59%) of patients with at least one family member with ovarian cancer, and 47% (95% CI 40%-54%) if other relatives have developed breast cancer. If both breast and ovarian cancer are diagnosed in the family, the probability of carrying a germline BRCA1 or 2 mutations is 60% (95% CI 50%-68%). germline BRCA1 or mutations in families with ovarian cancer only are commonly located in the Ovarian Cancer Cluster Regions when compared to families with both breast and ovarian cancer (P = 0.001, and P = 0.020, respectively). While gBRCA mutation carriers with ovarian cancer do not have a significantly different age at onset than patients with a family history of cancer, gBRCA1 carriers in general have an earlier onset than gBRCA2 carriers (P = 0.002) and patients without a mutation (P = 0.006). The rate of germline BRCA1 or 2 mutations in ovarian cancer patients without a family history or breast or ovarian cancer is low. However, in women with additional family members affected, the prevalence is considerably higher than previously reported.

Identification and management of familial breast cancer in Austria.

Aim The aim of this study is to review the legal implications, the technology, the indications and the management of women with a familial background of breast and/or ovarian cancer. Methods We have reviewed the literature and national Austrian guidelines to describe the uptake of genetic counseling and the management options offered in Austria. Results Genetic testing for the BRCA1 and 2 mutations is free and readily available through a nation-wide program. Increased awareness and the availability of screening programs and prophylactic surgery have resulted in a profound increase in genetic counseling and testing in women with a familial background of breast and ovarian cancer in Austria. Conclusion While readily available country-wide counseling has led to an increase in counseling and testing, Austrian legislation mandates "non-directional counseling" resulting in a comparatively low uptake of prophylactic surgery.

The pre-analytical processing of blood samples for detecting biomarkers on protein microarrays.

UNLABELLED: Specimen collection method and quality insurance are pivotal in biomarker discovery. Pre-analytical variables concerning blood collection and sample handling might affect analytical results and should be standardised prior application. In this study, we examine pre-analytical characteristics of blood samples using protein microarray. The influences of 1) standby times until centrifugation (1 h, 4 h, 24 h and 48 h), 2) four blood collection methods, and 3) IgG purified from those samples on differentially reactive antigens between samples ("DIRAGs") were investigated. Spearman correlation analyses of intra-individual arrays demonstrated remarkable differences (0.75-0.98 vs. 0.5-0.75) of antibody reactivities within and between serum and plasma samples. Class comparison showed that reactive antigen profiles were best preserved using IgG purified samples of serum tubes without separation gel as after 24h 83% of the 1h baseline DIRAGs were re-found. Testing dilution series with protein microarrays and Luminex xMap® Technology, we found linear correlations (R(2) = 0.94-0.99) between IgG concentration and read-out when using purified IgG instead of serum. Therefore, we highly recommend standardising pre-analytics and proposing the use of purified IgG for autoantibody immune-profiling with protein microarrays to reduce potential unspecific binding of matrix proteins abundant in serum and plasma samples. SIGNIFICANCE: Although purified IgG cannot completely compensate the influence of pre-analytics, in highly parallel immune-profiling IgG enables reduction of unspecific effects, which occur when using serum or plasma for analysis on protein microarrays. Reproducibility problems due to pre-analytical processing of blood samples might explain discrepant results reported in various biomarker studies.

Central European BRCA2 mutation carriers: birth cohort status correlates with onset of breast cancer.

BACKGROUND: Mutations in brca1 and 2 genes lead to a significant increase in the lifetime risk of developing breast (BC) and ovarian cancer (OC). There are indications that birth cohort can influence the cancer risk in brca1 mutation carriers. Therefore, we investigated the risks for BC and OC associated with brca2 mutations in a cohort of female mutation carriers of a genetically heterogeneous Central European population. PATIENTS AND METHODS: This study included 246 women in whom a functional mutation in the brca2 gene had been identified at our institution. At the time of analysis, 153 women had developed cancer (142 BC, 9 OC, 2 BC and OC). Risks were estimated using the product limit method. The log rank test was used to compare different strata. RESULTS: After correction for risk-reducing surgeries, the cumulative risk of developing cancer to age 70 was found to be 88% for BC (95% CI 81-95%) and 31% for OC (95% CI 17-45%). Female brca2 mutation carriers born in 1958 or later were at a significantly higher risk of developing BC at a younger age (p<0.001), while no such age cohort-dependent correlation was found for OC. CONCLUSION: The age cohort-dependent early onset in BC in women born after 1958 strongly suggests the importance of exogenous factors such as lifestyle modification while this does not seem to be the case for OC. Female brca2 mutation carriers should be counseled about their age cohort-dependent breast cancer risk.

[Guideline for the prevention and early detection of breast and ovarian cancer in high risk patients, particularly in women from HBOC (hereditary breast and ovarian cancer) families]. / Leitlinie zur Prävention und Früherkennung von Brust- und Eierstockkrebs bei Hochrisikopatientinnen, insbesondere bei Frauen aus HBOC (Hereditary Breast and Ovarian Cancer) Familien.

The Austrian guideline for prevention and early detection of breast and ovarian cancer in high risk patients--particularly in women from hereditary breast and ovarian cancer families--were established with particular consideration of the most recent position paper of the European Society of Breast Cancer Specialists (EUSOMA) by the authors mentioned above. The guideline is aimed at facilitating and standardizing the care and early detection strategies in women with an elevated life time risk for breast and ovarian cancer.

Impact of lifestyle factors on preneoplastic changes in prophylactic oophorectomies of BRCA mutation carriers.

BRCA mutation carriers are at high risk of developing ovarian cancer. Ovarian malignancies are usually identified at an advanced stage with poor prognosis, attributed to inadequate options of early detection. Because of its risk-reducing effect of nearly 96%, prophylactic salpingo-oophorectomy is still the leading option for risk-reduction in women with a positive BRCA mutation status. The presence of ovarian cancer precursor lesions, such as epithelial inclusion cysts (EICs) or cortical invaginations (CIs), has previously been discussed in several studies with diverse conclusions. We retrospectively investigated a large and consistent population (n=94) of BRCA mutation carriers for the presence of potential preneoplastic and neoplastic changes. We also examined the role of specific lifestyle factors. Ninety-four women with disease-associated germline BRCA1 or BRCA2 mutations were included in this retrospective study. All women had undergone genetic counseling and prophylactic salpingo-oophorectomy, which was performed at a mean age of 43.33 years (range 27-66). Histological slides of both ovaries were reviewed by an independent pathologist. Data concerning lifestyle factors were collected from medical files and questionnaires. Two malignant lesions (2.1%), one bilateral serous papillary adenocarcinoma of the epithelial surface and one adenocarcinoma of the peritoneum with involvement of the left adnexa, and one lesion (1.1%) with obvious malignant potential, one mucinous borderline tumor of the right ovary, have been identified. We registered a high prevalence of CIs (30; 31.9%) and EICs (44; 46.8%) in prophylactically removed ovaries of BRCA mutation carriers. A significant correlation (P=0.002) was found with regard to the presence of EICs in women with increased BMI. Concerning the regular consumption of alcohol as a risk factor for premalignant lesions, in particular CIs, a statistically insignificant trend (P=0.083) was noted. Overweight women seem to be at risk of developing more cortical invaginations than women of normal weight. To improve the final outcome of the disease, women at increased risk of ovarian cancer should be appropriately informed of potential increased risk factors.