Tobias Anzeneder

Promoter hypermethylation of the tumor- suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening

IntroductionFor early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.MethodsWe examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.ResultsBased on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.ConclusionsTumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.

Reprogramming of the ERRα and ERα Target Gene Landscape Triggers Tamoxifen Resistance in Breast Cancer

Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer.

AGR3 in breast cancer: prognostic impact and suitable serum-based biomarker for early cancer detection.

Blood-based early detection of breast cancer has recently gained novel momentum, as liquid biopsy diagnostics is a fast emerging field. In this study, we aimed to identify secreted proteins which are up-regulated both in tumour tissue and serum samples of breast cancer patients compared to normal tissue and sera. Based on two independent tissue cohorts (n = 75 and n = 229) and one serum cohort (n = 80) of human breast cancer and healthy serum samples, we characterised AGR3 as a novel potential biomarker both for breast cancer prognosis and early breast cancer detection from blood. AGR3 expression in breast tumours is significantly associated with oestrogen receptor α (P<0.001) and lower tumour grade (P<0.01). Interestingly, AGR3 protein expression correlates with unfavourable outcome in low (G1) and intermediate (G2) grade breast tumours (multivariate hazard ratio: 2.186, 95% CI: 1.008-4.740, P<0.05) indicating an independent prognostic impact. In sera analysed by ELISA technique, AGR3 protein concentration was significantly (P<0.001) elevated in samples from breast cancer patients (n = 40, mainly low stage tumours) compared to healthy controls (n = 40). To develop a suitable biomarker panel for early breast cancer detection, we measured AGR2 protein in human serum samples in parallel. The combined AGR3/AGR2 biomarker panel achieved a sensitivity of 64.5% and a specificity of 89.5% as shown by receiver operating characteristic (ROC) curve statistics. Thus our data clearly show the potential usability of AGR3 and AGR2 as biomarkers for blood-based early detection of human breast cancer.

Abstract 398: Real-life example of biobanking: results of the PATH Biobank

Introduction: Since 2002, PATH Foundation has kept a biobank collecting high-quality fresh frozen breast cancer specimens adhering to uniform SOPs at seven certified breast cancer centres in Germany. Research groups from academia and industry can obtain samples after application and review. PATH Biobank is a not-for-profit organisation and asks for a cost recovery fee in exchange for sample allocation to sustainably finance the expenses it incurs. Material and methods: The PATH Biobank consists of a centralized database and a decentralized bio repository. The samples are collected and stored at seven institutes for pathology at certified German breast cancer centres. Tumour tissue, along with normal (benign) adjacent tissue and blood serum aliquots are processed, labelled and stored according to uniform SOPs. Informed consent to biobanking and the use of the samples and data for research is obtained from the donors individually during pre-operation discussions. Results: Since 2004, more than 8,600 breast cancer patients have given their informed consent to the storage and analysis of their tissue and blood serum for research purposes. Breast cancer tissue samples from 59% of donors could be stored due to size of the surgically removed tissue specimen. In addition, normal adjacent tissue is available from 62% of donors and blood serum aliquots can be derived from 92% of patients. Using the annotating data, 96% of donors can be classified into the intrinsic molecular breast cancer subtypes in accordance with the St. Gallen Criteria. In 2008, PATH Biobank started to support research groups by providing breast cancer samples and data as well as informing the public about the projects on-line. In 2015 three co operations resulted in scientific publications. One landmark collaborations started in 2012 and has been studying the frequency and background of defined mutations in breast cancer. A number of 701 tissue samples have been used for this study. All samples were subject to quality testing, only 11% failed due to less than 5% tumour content. Tumour content depended on clinical conditions and staging (ranging from 34% failure in samples derived from neo adjuvantly treated patients to 2% in cases with staging UICC IV). Additional results concerning the quality of samples from PATH Biobank will be presented. Conclusions: For breast cancer research and biomarker studies, PATH Biobank can be a valuable resource. Citation Format: Tobias Anzeneder, Ulla Ohlms, Heinz Bodenmuller. Real-life example of biobanking: results of the PATH Biobank. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 398.

Abstract 569: AKT1 (E17K) mutation: coexistence with oncogenic alterations, prevalence, and correlation to clinical parameter in a large series of breast cancer patients

The AKT1 (E17K) mutation is rare and occurs in colon, ovarian, lung, and especially breast cancer where its frequency ranges between 1.4% and 8.2%. It9s precise role in cancer development and progression in clinical context is still unknown. To increase our understanding of the AKT1 (E17K) mutation in breast cancer we analyzed more than 600 tumor samples from breast cancer patients (UICC I - IV, including untreated and neoadjuvantly treated patients) which were provided by the non-profit organization PATH (Patients’ Tumor Bank of Hope, Germany). Extensive clinical data with a median follow-up time of 4.8 years to record disease progression were available for 95% of the patients included in this study. The AKT1 (E17K) mutation was detected in ∼6% of samples in the analyzed cohort using the BEAMing technology. Correlation with clinical parameters showed that the prevalence of the AKT1 (E17K) mutation was statistically independent of age or post-/pre-menopausal stage and was comparable between HER-2 positive and negative patients. In addition, FOUNDATION ONE ® targeted exome Next Generation Sequencing (NGS) analysis of some of the tumor samples was done to demonstrate the fingerprint of individual tumors in correlation with the AKT1 (E17K) mutation. NGS and BEAMing technology had a ∼98% concordance for AKT1 (E17K) mutated and non-mutated samples. In 12 out of 36 AKT1 (E17K) mutated samples no additional somatic mutations (SNVs, indels) described to drive cancer development were detected. Moreover, neither amplification nor deletion of tested genes known to be recurrently amplified or deleted in cancer were found in 10 out of these 12 samples. This supports the hypothesis that AKT1 (E17K) can be a driver mutation. However, in all of these samples mutations with yet unannotated function in additional oncogenes were detected. It remains open whether these aberrations impact the role of AKT1 (E17K) as a driver mutation in tumor growth. Analyses of patient cohort data from large databases, as demonstrated here, holds promise for discovering the role of rare somatic mutations in known oncogenes (such as AKT1 (E17K)) in the development of breast cancer. Citation Format: Marion Rudolph, Tobias Anzeneder, Matthias Ocker, Eleni Lagkadinou, Oliver Politz, Martin Michels, Anke Schulz, Georg Beckmann, Michael Teufel, Henrik Seidel, Richie Soong, Heinz Bodenmuller, Ulla Ohlms, Khusru Asadullah, Joachim Reischl. AKT1 (E17K) mutation: coexistence with oncogenic alterations, prevalence, and correlation to clinical parameter in a large series of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 569. doi:10.1158/1538-7445.AM2014-569

Abstract 3835: The PATH biobank - procedures and results of a breast cancer biospecimen research resource

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: The purpose of PATH is to provide high quality fresh frozen breast cancer specimen for research, annotated with highly differentiated datasets. To this end, we have developed and established standardized methods for collection, processing, labeling, longterm storage, retrieval and distribution of the specimen. PATH is a non-profit, patient-driven biobank in Germany. Procedures: The PATH biobank consists of a centralized database and a decentralized biorepository. The samples are collected and stored at seven institutes for pathology being part of certified German breast cancer centers. Breast cancer patients can join PATH by informed consent; all PATH procedures include ethical approval. Using unique SOPs, tumor tissue, normal adjacent tissue and blood serum aliquots are being processed, labeled and stored long-term under fresh frozen conditions using vapor phase of liquid nitrogen. Serum aliquots have a minimum volume of 1ml; tissue samples at least 3mm edge length. All data is stored in an Oracle® database, containing an in-house LIMS. Follow up data is collected by directly questioning the breast cancer patients who donated their tissue and blood to PATH. Follow up events, such as local or distant recurrence are validated by physicians in charge or regional German cancer registries. Specimen and datasets can be obtained upon request. The application process follows particular terms and conditions and goes through a peer review. The PATH Foundation charges a fee for specimen allocation to enable it to continue its work. Results: Since 2004, more than 7,200 breast cancer patients have given informed consent. From 59% of all donors a tumor tissue specimen could be stored. The same applies to 62% of all cases in accordance to adjacent normal tissue specimen and in 92% with blood serum samples. Tumor stages T1 or T2 are present in 93% of the women. Overall, 81% are ER+, 71% PR+ and 13% HER2-neu+. The triple negative subgroup includes 11% of all patients. E.g. PATH stores 1,142 fresh frozen tumor aliquots of 453 triple negative cases. In a sub-cohort, comprising the years of diagnosis 2006 - 2009, 76% of all individuals could be reached through follow-up inquiries by PATH. Subsequently, in a test-collective (108 patients, median follow-up time 4.8 years) only 5% were “lost to follow up”. Research groups having used PATHs biobank applied different techniques successfully for their investigations, among them were laser capture microdissection, next generation sequencing, whole genome sequencing, proteomics technologies and immuno histochemical procedures. Several publications and presentations demonstrate the quality of PATHs biobank. Conclusion: The biobank and annotating datasets containing detailed clinical, biological and follow up information are a valuable resource for breast cancer research. Sample quality enables the application of modern analytical methods without prior patient recruitment or sample collection. Citation Format: Tobias Anzeneder, Cathrin Mayer, Ulla Ohlms, Doris C. Schmitt, Carmen Waldner, Reinhard Buttner, Heinz Bodenmueller. The PATH biobank - procedures and results of a breast cancer biospecimen research resource. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2014-3835

Abstract LB-382: DKK3, ITIH5 and RASSF1A gene methylation as novel biomarkers for blood-based breast cancer screening: Towards improving early detection of breast cancer

Aims: For early detection of breast cancer the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory and thus a growing field of research. The most common alterations in human cancers including breast cancer are changes in the status of DNA methylation, which are therefore quickly emerging as a new pool of potential biomarkers. Thus, we investigated the feasibility of detecting aberrant tumor suppressor gene methylation in cancer cell-derived free circulating DNA in the bloodstream of breast cancer patients. Methods: Using qualitative MSP, we examined the methylation status of seven biologically significant putative tumor suppressor genes, i.e. ITIH5, DKK3, WIF1, RASSF1A, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum. Free circulating DNA was isolated with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Clinical performance was determined in a training study on 240 serum samples (120 breast cancers, 120 age-matched healthy controls). 20 benign gynaecological disease and 30 colon cancer serum samples were included for additional specificity testing. Results: Based on the training study we could evaluate the top candidate biomarkers with the best values for sensitivity and specificity. A marker panel with DKK3 and ITIH5 detected breast cancer with a sensitivity of 42% (50/120). Specificity of the panel was sufficient with 83%, 100% and 93% in colon cancer samples, benign and healthy control samples, respectively. Control samples revealed unacceptable high methylation rates of SFRP1 and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2 and WIF1 showed a considerable methylation frequency in sera from healthy controls. The well-established potential biomarker RASSF1A was included as a reference gene. RASSF1A was highly specific in the analysed benign and healthy control samples (100%) why we integrated RASSF1A in our independent test study to improve sensitivity and specificity of the marker panel with DKK3 and ITIH5. Conclusions: The current study suggests that cancer-specific methylation of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be valuable biomarkers for early detection of breast cancer. In the second phase of this project we are currently validating with quantitative MSP ITIH5, DKK3 and RASSF1A as reliable methylation biomarkers in an independent test set consisting of 140 breast cancer serum samples, 140 age-matched healthy controls, 40 benign gynaecological disease and 30 colon cancer serum samples for additional specificity testing. To date, this is the first study investigating the potential of ITIH5 and DKK3 in combination with the well-established biomarker RASSF1A as reliable blood-based methylation biomarkers in a large-scale serum collective of breast cancer and matched control samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-382. doi:1538-7445.AM2012-LB-382

Abstract P4-13-01: Follow-Up in Biobanking — Strategy and Outcomes of Patients’ Tumor Bank of Hope (PATH)

Purpose: Breast Cancer Patients established PATH in 2002 to collect tumor sam-ples, blood samples and data at high ethical standards and under uniform SOPs. Since 2004 more then 4500 women and men in Germany gave their informed consent for the collection. In addition, PATH has successfully started to collect follow-up data from all patients, covering both disease and therapy process. Design: Specimen (tumor, normal tissue, blood serum) are stored in liquid nitrogen tanks (fresh frozen quality) operated by pathologists at seven certified breast cancer centers in Germany. To provide a benefit for the donors, the first aliquot is stored exclusively for the patient. The rest of the material is donated to research purposes. Processing, handling and labelling of the samples is defined in rigid SOPs, accompanied by monitoring. Data regarding clinical findings, tumor-biology and sample processing are collected and centrally managed. In order to annotate the samples follow up started in early 2009. As a patient driven, non-profit organization PATH has a special reliability. Thus, the Foundation is given the right to establish direct contact with any patient that has given informed con-sent. This approach was confirmed by ethics committee, the Bavarian Commission of Data Protection and a university professor of medical law. In order to get the follow-up data, PATH contacts the patient by letter. A structured phone call follows, carried out by female medical students, who are specially trained. The patient is asked to provide details on their individual course of disease. Additionally they are asked about their compliance with therapy. Procedure, data volume and data quality are specified and standardized. If patients could not be reached by call they are asked to complete an additionally mailed questionnaire. As the last source for data PATH will try to get data from tumor registers. In case of re-currence the data obtained from the patients is validated by check-ups with medical reports from the practitioners. Results: By May 2010 more than 4500 patients gave informed consent. 4042 cases have been documented in their entirety in the data base. 75% have stored a patient9s tumor specimen and 61% a research tumor sample in the bio bank. As many patients have multiple samples there is a total of 4080 tumor specimen, 9029 blood serum and 4075 normal tissue aliquots for research purposes. 2253 cases were contacted for follow up, 1491 (66,2%) patients were interviewed by phone and 142 (6,3%) women returned the questionnaire. In 72,5% of the con-tacted cases follow-up data is available. The mean age of all patients who do-nated their tumor specimen is 60,3 years. 78,9% of the tumors are ER-positive, 11,4% triple negative. The data includes 20 cases of recurrence and 61 cases of metastases. Conclusion: Within 16 months PATH contacted 40% of all patients included in the bio bank and gained follow up data. Thus PATH will provide a great variety and quantity of fresh frozen tissue and blood samples with excellent quality and a mean follow up of 5 years in less than 12 months. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-13-01.

5 Years Experience in Collecting Tumor Specimen at Patient's Tumor Bank of Hope (PATH) – A Decentralized, Independent Biobank in Germany Controlled by Patients.

Purpose: PATH was established by a German breast cancer advocacy group in 2002. While there are other tumor banks around the world, the immediate benefit for the donating patient is not always clear. With PATH, we set out to prove that patients, physicians and researchers can join forces to operate a tumor bank at the highest ethical standards while collecting tumor specimen at highest and standardized quality. In addition, PATH has developed data acquisition and management methods for disease and therapy process and follow-up.Design: PATH Foundation is a non-profit organization.Seven certified German „breast centers“ are currently our partners. PATH is presented to patients in pre-OP discussion in these centers and they are given the opportunity to take part, by agreeing to an informed consent. The informational documents and consent form we give to patients were developed in collaboration with Prof. Taupitz of Germany9s National Board of Ethics. The whole PATH procedure causes no additional cost for the patient.PATH tissuebank contains tumor-, normal tissue- and blood serum-specimen in fresh frozen quality stored in -152°C freezers or liquid nitrogen tanks. The first aliquot of each specimen is stored exclusively for the patient, the rest of the material is donated to PATH for research purposes. All cooperation partners (OB/Gyns and pathologists) commit to working strictly according to PATH9s SOPs. There are 4 SOPs (extraction of blood serum und tissue aliquots, labeling of the specimen and quality-check with RIN assay).Cooperation partners9 compliance and the implementation of the SOPs is monitored in biannual visits and meetings.All important data regarding tumor biology and therapy as well as sample processing are collected in PATH9s own centrally managed MySQL database. The process was approved by the Bavarian Commission of Data Protection.Furthermore, PATH collects necessary follow-up data directly from the patient as well as from local tumor registries.Results: We began the collection of specimen in 2004.Data acquisition and implementation of the data base started in 2005. Follow-up began in late 2008. As of 04/2009, we have 3255 sufficiently documented patient data sets correlating with patients taking part in the PATH procedure (median age is 56). The available PATH collection contains 2732 tumor research specimen from 1251 patients (according to the last analysis of a collective of 2020 patients in 12/2008). Other quantities of collected specimen incl. patients9 aliquot (ps9) tumor = 1578; ps9 normal tissue = 1591; ps9 serum = 1876; resarch aliquots (ra) normal tissue = 2667; ra serum = 6068. This means that in 78,1% it was possible to collect a patient9s tumor aliquot and in 61,9% at least one research specimen, varying with tumor stage.Conclusion: Over the last 5 years, PATH has gained broad experience in tumorbanking that is allowing it to operate a biobank at the highest ethical and qualitative standards. With its follow-up efforts, PATH will be able to provide a great variety of research specimen with at least a mean follow-up time of 5 years in 2011! Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3074.