Dominic Varga

Does Guideline-Adherent Therapy Improve the Outcome for Early-Onset Breast Cancer Patients?

Background and Objective: Guidelines for the treatment of early-onset breast cancer have been proposed in several countries, but to date, their impact on outcomes is unverified. The objective of this study was to evaluate the association between guideline-adherent versus nonadherent treatment and recurrence-free survival (RFS) and overall survival (OAS) in early-onset breast cancer patients. Methods: A total of 1,778 patients were included in the study, of whom 111 were 35 years or younger and 1,667 were between 36 and 55 years. RFS and OAS were compared between the two groups, with respect to multiple parameters. All survival data were adjusted for tumor characteristics and analyzed with respect to guideline adherence according to the German Step 3 guidelines. Results: Statistically significant differences between the two groups (<35 years, 36–55 years) were observed with regard to breast surgery (p = 0.002) and hormone therapy (p = 0.006). Both groups were treated identically in terms of guideline adherence concerning axillary dissection (p = 0.9), radiation therapy (p = 0.7) and chemotherapy (p = 0.556). Young breast cancer patients whose treatment adhered to guideline recommendations had increased RFS and OAS [RFS: p = 0.030, hazard ratio (HR) 2.95, 95% confidence interval (CI) 1.11–7.83; OAS: p ≤ 0.001, HR 2.92, 95% CI 2.01–4.23]. Conclusion: Guideline-adherent treatment for early-onset breast cancer patients significantly improves OAS and RFS and should therefore be demanded for all patients.

Heritability of baseline and induced micronucleus frequencies

The scoring of micronuclei (MN) is widely used in biomonitoring and mutagenicity testing as a surrogate marker of chromosomal damage inflicted by clastogenic agents or by aneugens. Individual differences in the response to a mutagenic challenge are known from studies on cancer patients and carriers of mutations in DNA repair genes. However, it has not been studied to which extent genetic factors contribute to the observed variability of individual MN frequencies. Our aim was to quantify this heritable genetic component of both baseline and radiation-induced MN frequencies. We performed a twin study comprising 39 monozygotic (MZ) and 10 dizygotic (DZ) twin pairs. Due to the small number of DZ pairs, we had to recruit controls from which 38 age- and gender-matched random control pairs (CPs) were generated. For heritability estimates, we used biometrical modelling of additive genetic, common environmental, and unique environmental components (ACE model) of variance and direct comparison of variance between the sample groups. While heritability estimates from MZ to DZ comparisons produced inconclusive results, both estimation methods revealed a high degree of heritability (h(2)) for baseline MN frequency (h(2) = 0.68 and h(2) = 0.72) as well as for the induced frequency (h(2) = 0.68 and h(2) = 0.57) when MZ were compared to CP. The result was supported by the different intraclass correlation coefficients of MZ, DZ and CP for baseline (r = 0.63, r = 0.31 and r = 0.0, respectively) as well as for induced MN frequencies (r = 0.79, r = 0.74 and r = 0.0, respectively). This study clearly demonstrates that MN frequencies are determined by genetic factors to a major part. The strong reflection of the genetic background supports the idea that MN frequencies represent an intermediate phenotype between molecular DNA repair mechanisms and the cancer phenotype and affirms the approaches that are made to utilise them as predictors of, for example, cancer risk.

The power of DNA double-strand break (DSB) repair testing to predict breast cancer susceptibility

Most presently known breast cancer susceptibility genes have been linked to DSB repair. To identify novel markers that may serve as indicators for breast cancer risk, we performed DSB repair analyses using a case‐control design. Thus, we examined 35 women with defined familial history of breast and/or ovarian cancer (first case group), 175 patients with breast cancer (second case group), and 245 healthy women without previous cancer or family history of breast cancer (control group). We analyzed DSB repair in peripheral blood lymphocytes (PBLs) by a GFP‐based test system using 3 pathway‐specific substrates. We found increases of microhomology‐mediated nonhomologous end joining (mmNHEJ) and nonconservative single‐strand annealing (SSA) in women with familial risk vs. controls (P=0.0001‐0.0022) and patients with breast cancer vs. controls (P=0.0004‐0.0042). Young age (<50) at initial diagnosis of breast cancer, which could be indicative of genetic predisposition, was associated with elevated SSA using two different substrates, amounting to similar odds ratios (ORs=2.54‐4.46, P= 0.0059‐0.0095) as for familial risk (ORs=2.61‐4.05, P=0.0007‐0.0045). These findings and supporting validation data underscore the great potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility beyond limitations of genotyping and to predict responsiveness to therapeutics targeting DSB repair‐dysfunctional tumors.—Keimling, M., Deniz, M., Varga, D., Stahl, A., Schrezenmeier, H., Kreienberg, R., Hoffmann, I., König, J., Wiesmüller, L. The power of DNA double‐strand break (DSB) repair testing to predict breast cancer susceptibility. FASEB J. 26, 2094‐2104 (2012). www.fasebj.org

Impact of Guideline Conformity on Breast Cancer Therapy: Results of a 13-Year Retrospective Cohort Study

Background: To date, few studies have investigated whether the implementation of national breast cancer guidelines fulfills the goal to optimize the national standard of care. Therefore, we aimed to evaluate retrospectively the guideline-related 13-year data on breast cancer patients treated at our institution. Patients and Methods: In a retrospective cohort study, the records of a total of 2,231 patients with primary breast cancer treated during the period of 1992–2005 at the Department of Obstetrics and Gynecology, University of Ulm, Germany, were analyzed. Based on the German national Step 3 (S3) guideline, a model was created to classify groups according to therapy ‘conforming’ and ‘non-conforming’ to guideline recommendations. Results: In 2005, 70.2% of all patients included received both surgical and systemic adjuvant therapies conforming to the guideline. Guideline-conforming treatment was accompanied with significant advantages in terms of recurrence-free survival (RFS) and overall survival (OAS) rates. Conclusions: It has to be demanded that breast cancer patients are treated in con-formity with the S3 guidelines. The reasons for a treatment not conforming to the guidelines should be analyzed for the detection of barrier factors, in order to optimize adherence to the guidelines and therefore to prolong RFS and OAS.

Validation of the Manchester scoring system for predicting BRCA1/2 mutations in 9,390 families suspected of having hereditary breast and ovarian cancer

The Manchester scoring system (MSS) allows the calculation of the probability for the presence of mutations in BRCA1 or BRCA2 genes in families suspected of having hereditary breast and ovarian cancer. In 9,390 families, we determined the predictive performance of the MSS without (MSS‐2004) and with (MSS‐2009) consideration of pathology parameters. Moreover, we validated a recalibrated version of the MSS‐2009 (MSS‐recal). Families were included in the registry of the German Consortium for Hereditary Breast and Ovarian Cancer, using defined clinical criteria. Receiver operating characteristics (ROC) analysis was used to determine the predictive performance. The recalibrated model was developed using logistic regression analysis and tested using an independent random validation sample. The area under the ROC curves regarding a mutation in any of the two BRCA genes was 0.77 (95%CI 0.75–0.79) for MSS‐2004, 0.80 (95%CI 0.78–0.82) for MSS‐2009, and 0.82 (95%CI 0.80–0.83) for MSS‐recal. Sensitivity at the 10% mutation probability cutoff was similar for all three models (MSS‐2004 92.2%, MSS‐2009 92.2%, and MSS‐recal 90.3%), but specificity of MSS‐recal (46.0%) was considerably higher than that of MSS‐2004 (25.4%) and MSS‐2009 (32.3%). In the MSS‐recal model, almost all predictors of the original MSS were significantly predictive. However, the score values of some predictors, for example, high grade triple negative breast cancers, differed considerably from the originally proposed score values. The original MSS performed well in our sample of high risk families. The use of pathological parameters increased the predictive performance significantly. Recalibration improved the specificity considerably without losing much sensitivity.

Radiosensitivity detected by the micronucleus test is not generally increased in sporadic prostate cancer patients

The micronucleus test (MNT) has shown increased micronuclei (MN) frequencies in BRCA associated and sporadic breast cancer patients, Ataxia telangiectasia and Nijmegen Breakage Syndrome patients, demonstrating a common cellular phenotype of increased radiosensitivity. Some genes, causative of these diseases, have also recently been associated with prostate cancer. In order to investigate if prostate cancer exhibits the cellular phenotype of increased radiosensitivity, we performed MNT analysis on 22 sporadic prostate cancer patients and 43 male controls. We determined the baseline MN frequency, in order to see in vivo chromosomal damage without radiation, and induced (after irradiation with 2 Gy) frequency of MN, both in binucleated cells (BNC) obtained from cultured peripheral blood lymphocytes. An automated image analysis system was used to score the MN employing two different classifiers (Classifier A and B) for detection of BNC. The mean baseline frequencies were 48/43 MN/1000 BNC (A/B) for the controls and 42/50 (A/B) for prostate cancer patients. The induced MN frequencies amounted to 107/111 MN/1000 BNC (A/B) for controls and 111/114 MN/1000 BNC (A/B) for prostate cancer patients. The obtained MN frequencies did not result in a statistically significant difference between unselected cases and controls. However, restricting the analysis to young patients (50–60 years, N = 7) and age-matched controls (N = 7) revealed marginally significant higher MN frequencies in patients. We conclude that increased radiosensitivity is not a property of prostate cancer patients in general.

Ovarian cancer: in search of better marker systems based on DNA repair defects.

Ovarian cancer is the fifth most common female cancer in the Western world, and the deadliest gynecological malignancy. The overall poor prognosis for ovarian cancer patients is a consequence of aggressive biological behavior and a lack of adequate diagnostic tools for early detection. In fact, approximately 70% of all patients with epithelial ovarian cancer are diagnosed at advanced tumor stages. These facts highlight a significant clinical need for reliable and accurate detection methods for ovarian cancer, especially for patients at high risk. Because CA125 has not achieved satisfactory sensitivity and specificity in detecting ovarian cancer, numerous efforts, including those based on single and combined molecule detection and “omics” approaches, have been made to identify new biomarkers. Intriguingly, more than 10% of all ovarian cancer cases are of familial origin. BRCA1 and BRCA2 germline mutations are the most common genetic defects underlying hereditary ovarian cancer, which is why ovarian cancer risk assessment in developed countries, aside from pedigree analysis, relies on genetic testing of BRCA1 and BRCA2. Because not only BRCA1 and BRCA2 but also other susceptibility genes are tightly linked with ovarian cancer-specific DNA repair defects, another possible approach for defining susceptibility might be patient cell-based functional testing, a concept for which support came from a recent case-control study. This principle would be applicable to risk assessment and the prediction of responsiveness to conventional regimens involving platinum-based drugs and targeted therapies involving poly (ADP-ribose) polymerase (PARP) inhibitors.

The role of preoperative ultrasound evaluation of inguinal lymph nodes in patients with vulvar malignancy

OBJECTIVES Inguinal lymphadenectomy in vulvar malignancies is associated with significant morbidity, especially in patients over 70 years old. Under certain conditions, surgical guidelines recommend biopsy and evaluation of the sentinel node in early vulvar cancer. The purpose of our study is to evaluate ultrasonography as a predictor of inguinal lymph node involvement. METHODS A retrospective study was performed with 60 patients who had vulvar malignancies (92% of which were squamous cell carcinomas) and who were treated at our hospital between 2002 and 2012. The patients ranged in age from 35 to 89 years, with a median age of 76 years. In total, 118 groin scans were retrospectively evaluated for sonographic evidence of lymph node involvement (i.e., absence of fatty hilum, irregular shape, cortical region diameter and vascularization pattern). The results were then compared with histopathologically confirmed lymph node status. RESULTS Histopathologically confirmed lymph node status was available for 107 of the inguinal nodes examined by ultrasound, and lymph node metastases were found in 38 (35.5%) cases. The presence or absence of inguinal lymph node metastases was correctly identified by sonography in 92 (86.0%) of the scanned areas. Sensitivity was 76.3%, specificity was 91.3%, and positive and negative predictive values were 82.9% and 87.5%, respectively. CONCLUSIONS Ultrasonography of the inguinal lymph nodes showed a relatively high sensitivity and specificity for predicting inguinal tumor metastases. However, our results indicate that surgical lymph node staging is still needed to precisely determine inguinal lymph node status in vulvar cancer, especially because a missed lymph node-metastasis is often fatal.

Increased Radiosensitivity as an Indicator of Genes Conferring Breast Cancer Susceptibility

Purpose:This paper briefly summarizes the research on increased radiosensitivity in breast cancer patients measured by the micronucleus test (MNT) and its association to genetic variants in DNA repair genes. More preliminary data are presented on the distribution of chromosomes and chromosome fragments in micronuclei (MN) in order to gain more information on clastogenic and aneugenic effects and better understand the phenotype of increased radiosensitivity.Material and Methods:Reports of relevant studies obtained from a search of PubMed and studies referenced in those reports were reviewed. In four patients with high MN frequency (three cancer patients, one control) and four probands with low MN frequency, the presence of chromosome fragments or whole chromosomes in MN was determined by fluorescence in situ hybridization analysis for chromosomes 1, 7, and 17.Results:An increased MN frequency in breast cancer patients compared to controls has consistently been reported with high significance. Higher MN frequencies were observed in 20–50% of breast cancer patients. Chromosomal fragments of chromosome 17, but not of chromosomes 1 and 7 were more frequent in the probands with high MN frequency than in those with low frequency (p = 0.045).Conclusion:The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness.Ziel:Diese Arbeit fasst die bisherige Forschung zur erhöhten Radiosensitivität bei Brustkrebspatientinnen zusammen, wie sie mit Hilfe des Mikronukleustests (MNT) bestimmt werden kann. Des Weiteren zeigen vorläufige Fluoreszenz-in-situ-Hybridisierungs-(FISH-)Analysen die Verteilung einzelner Chromosomen oder Chromosomenbruchstücke in Mikrokernen (MN), um aneugene oder klastogene Effekte zu detektieren und mehr Informationen über den Phänotyp der erhöhten Radiosensitivität zu erhalten.Material und Methodik:Als Grundlage für diese Übersicht dienten in PubMed veröffentlichte Untersuchungen sowie darin zitierte Studien. Des Weiteren wurden bei vier Probandinnen mit hohen MN-Raten (drei Krebspatientinnen, eine Kontrolle) und vier Probandinnen mit niedrigen MN-Raten MN mittels FISH auf die Frage hin untersucht, ob sie Fragmente oder die ganzen Chromosomen 1, 7 und 17 enthalten.Ergebnisse:Brustkrebspatientinnen und Kontrollen zeigen in der Literatur statistisch signifikante Unterschiede bezüglich der MN-Rate. Höhere MN-Raten wurden bei 20–50% der Brustkrebspatientinnen beobachtet. Die Chromosomenfragmente von Chromosom 17 kamen häufiger bei Probandinnen mit hoher MN-Rate als bei Probandinnen mit niedriger MN-Rate vor (p = 0,045).Schlussfolgerung:Der MNT detektiert einen Phänotyp, welcher einen Anteil der sporadischen Brustkrebspatientinnen erfasst. Dieser Phänotyp ist wahrscheinlich genetisch determiniert. Für genetische Untersuchungen zur Brustkrebssuszeptibilität könnte sich dieser intermediäre Phänotyp als geeigneter erweisen als das Auftreten der Erkrankung Brustkrebs selbst. Zusätzliche Parameter, die zusammen mit der MN-Rate bestimmt werden können, könnten seine Anwendbarkeit verbessern.

Medium-sized deletion in the BRCA1 gene: limitations of Sanger sequencing and MLPA analyses

We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions. Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.