Marianna Alunni-Fabbroni

DNA methylation markers for early detection of women's cancer: promise and challenges

Breast, ovarian and endometrial cancers cause significant morbidity and mortality. Despite the presence of existing screening, diagnostic and treatment modalities, they continue to pose considerable unsolved challenges. Overdiagnosis is a growing problem in breast cancer screening and neither screening nor early diagnosis of ovarian or endometrial cancer is currently possible. Moreover, treatment of the diversity of these cancers presenting in the clinic is not sufficiently personalized at present. Recent technological advances, including reduced representation bisulfite sequencing, methylation arrays, digital PCR, next-generation sequencing and advanced statistical data analysis, enable the analysis of methylation patterns in cell-free tumor DNA in serum/plasma. Ongoing work is bringing these methods together for the analysis of samples from large clinical trials, which have been collected well in advance of cancer diagnosis. These efforts pave the way for the development of a noninvasive method that would enable us to overcome existing challenges to personalized medicine.

Therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer: concept of the DETECT study program

AbstractPurpose The aim of the ongoing DETECT study program is to evaluate therapeutic intervention based on phenotypes of circulating tumor cells (CTC) in patients with metastatic breast cancer (MBC). Currently (as of July 2015) more than half of the projected about 2000 patients with MBC have already been screened for CTC.MethodsWomen with HER2-negative primary tumor and presence of CTC are recruited into different DETECT trials according to the HER2-phenotype of CTC. Patients with HER2-positive CTC are randomized to treatment with physicians’ choice therapy (standard chemo- or endocrine therapy) with or without additional HER2-targeted therapy with lapatinib in the DETECT III trial. In DETECT IVa, postmenopausal patients with hormone-receptor positive primary cancer and HER2-negative CTC receive everolimus and standard endocrine therapy. For women with HER2-negative CTC and triple negative MBC or hormone-receptor positive tumor and indication for chemotherapy, a treatment with eribulin is offered (DETECT IVb). The clinical efficacy is investigated by CTC-Clearance and progression-free survival (PFS). The DETECT V/CHEVENDO trial extends the DETECT study program for women with HER2-positive and hormone-receptor positive MBC. The primary objective of this trial is to compare safety and quality of life (QoL) as assessed by the occurrence of adverse events in patients treated with dual (trastuzumab plus pertuzumab) HER2-targeted therapy plus either endocrine or chemotherapy. The translational research projects of the DETECT study program focus on further molecular characterization of CTC and evaluation of markers for their suitability to predict treatment response and to facilitate the development of more personalized treatment options.

Monitoring in Metastatic Breast Cancer: Is Imaging Outdated in the Era of Circulating Tumor Cells?

In clinical practice imaging technologies such as computed tomography (CT), positron emission tomography (PET)/CT and magnetic resonance imaging (MRI) are well-established methods for monitoring metastatic breast cancer (MBC) patients and for assessing therapeutic efficacy. However, several weeks of treatment are required before these technologies can offer any reliable information on effective disease regression, and, in the meanwhile, the patients are exposed to potentially unnecessary therapy. Circulating tumor cells (CTCs) have been shown to be powerful prognostic and predictive markers and provide clinicians with valuable information. However, in one clinical trial, an early change of chemotherapy based on CTC detection did not result in improved survival. Currently, CTC detection outside clinical trials should be limited to selected clinical situations, i.e. increased treatment toxicity or as risk estimation.

Quantifying HER-2 expression on Circulating Tumor Cells by ACCEPT

Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch® and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC’s HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators’ scoring results for HER-2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches.

Methylation patterns in serum DNA for early identification of disseminated breast cancer

BackgroundMonitoring treatment and early detection of fatal breast cancer (BC) remains a major unmet need. Aberrant circulating DNA methylation (DNAme) patterns are likely to provide a highly specific cancer signal. We hypothesized that cell-free DNAme markers could indicate disseminated breast cancer, even in the presence of substantial quantities of background DNA.MethodsWe used reduced representation bisulfite sequencing (RRBS) of 31 tissues and established serum assays based on ultra-high coverage bisulfite sequencing in two independent prospective serum sets (n = 110). The clinical use of one specific region, EFC#93, was validated in 419 patients (in both pre- and post-adjuvant chemotherapy samples) from SUCCESS (Simultaneous Study of Gemcitabine-Docetaxel Combination adjuvant treatment, as well as Extended Bisphosphonate and Surveillance-Trial) and 925 women (pre-diagnosis) from the UKCTOCS (UK Collaborative Trial of Ovarian Cancer Screening) population cohort, with overall survival and occurrence of incident breast cancer (which will or will not lead to death), respectively, as primary endpoints.ResultsA total of 18 BC specific DNAme patterns were discovered in tissue, of which the top six were further tested in serum. The best candidate, EFC#93, was validated for clinical use. EFC#93 was an independent poor prognostic marker in pre-chemotherapy samples (hazard ratio [HR] for death = 7.689) and superior to circulating tumor cells (CTCs) (HR for death = 5.681). More than 70% of patients with both CTCs and EFC#93 serum DNAme positivity in their pre-chemotherapy samples relapsed within five years. EFC#93-positive disseminated disease in post-chemotherapy samples seems to respond to anti-hormonal treatment. The presence of EFC#93 serum DNAme identified 42.9% and 25% of women who were diagnosed with a fatal BC within 3–6 and 6–12 months of sample donation, respectively, with a specificity of 88%. The sensitivity with respect to detecting fatal BC was ~ 4-fold higher compared to non-fatal BC.ConclusionsDetection of EFC#93 serum DNAme patterns offers a new tool for early diagnosis and management of disseminated breast cancers. Clinical trials are required to assess whether EFC#93-positive women in the absence of radiological detectable breast cancers will benefit from anti-hormonal treatment before the breast lesions become clinically apparent.

Comparison of HER2 Expression in Primary Tumor and Disseminated Tumor Cells in the Bone Marrow of Breast Cancer Patients.

Objective: The aim of this study was to measure the human epidermal growth factor receptor 2 (HER2) status of disseminated tumor cells (DTCs) from bone marrow (BM) aspirates and to assess correspondence or discrepancy with the primary tumor. Methods: DTCs were isolated from the BM of 156 breast cancer patients. Cytokeratin-positive DTCs were further analyzed by the chromogenic in situ hybridization method to detect HER2 gene amplification. Results: A significant correlation (p = 0.021) was found between the HER2 status of DTCs and the primary tumors. Sixty-one (68.5%) patients had a corresponding status. However, a shift of phenotype between primary tumor and DTCs was found in the remaining patients. Conclusion: This study showed a significant grade of discordance of the HER2 status between primary tumors and DTCs in the BM of a relevant subgroup of patients. Detection of HER2 amplification on DTCs could therefore help to better stratify patients for a more tailored therapy, since they would benefit from a HER2-targeted therapy.

Abstract P4-01-16: Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment

Background: Metastasis are thought to be induced by occult spreading of tumor cells already during the early phases of the disease. Circulating Tumor Cells (CTCs) are regarded as precursors of distant metastasis, while detaching from the primary tumor and originating micrometastases in distant organs. Recent evidences pointed to CTC heterogeneity, showing that CTCs can present different phenotypes. Goal of this study was to identify in metastatic breast cancer (mBC) patients CTCs with EMT features and to further characterize them with respect to cellular heterogeneity. Methods: This prospective ongoing study included mBC patients (n=12) with a median age of 58,5 years (range: 35-78 years), enrolled in a time frame of 7 months while undergoing therapy. The majority of patients were estrogen and/or progesterone receptor positive (11/12) and HER2 non-amplified (10/12). Patients had metastatic lesions in viscera as well as bone (6/12), only bone (2/12), only viscera (2/12), only viscera in combination with locoregional recurrence (1/12), or visceral and bone metastases in combination with locoregional recurrence (1/12). Current therapy was endocrine therapy (4/12), chemotherapy (3/12), chemotherapy and HER2-targeted therapy (2/12), HER2-targeted monotherapy (1/12), antiangiogenic therapy (1/12), or surgical therapy only (1/12). Blood samples, withdrawn at any time point during treatment, were depleted of EpCAM+ cells and CD45+ white blood cells (EpCAM/CD45 depleted fraction) (Mego et al., Int J Cancer 2012;130(4):808-816) and expression of epithelial markers (EpCAM, E-Cadherin, Cytokeratin 8,18,19), mesenchymal markers (n-Cadherin, Vimentin), EMT-inducing factors (Twist1, Snail1, SLUG, Zeb1 and FoxC2), anoikis markers (TrkB1, Bcl2) and stem cell markers (CD24, CD44, CD133) were analyzed by qRT-PCR. CTC counting with the CellSearchTM system (Veridex, Raritan NJ) was run in parallel. Healthy donors (n=10) were included in the study as negative controls. Results: The data collected so far showed that 50% of the patients were positive for CTCs in the EpCAM+ fraction as detected with the CellSearchTM system, while 33% were still CTC positive in the EpCAM/CD45 depleted fraction. 50% of the patients, found CTC negative with CellSearchTM, were nevertheless positive for the epithelial and EMT markers in the EpCAM/CD45 depleted fraction (EpCAM 25%, E-Cadherin 25%, CK8 16,6%, CK18 16,6%, CK19 16,6%, SLUG 8,3%, Zeb1 25%, Twist1 8,3%, Vimentin 58,3%). N-cadherin, FoxC2, Snail1, TrkB1, CD24, CD44 and CD133 were never detectable. Conclusions: These preliminary results suggest that mBC patients undergoing different lines of therapy present heterogeneous CTCs. 50% of the patients with undetectable EpCAM+ CTCs, were found CTC positive in the EpCAM/CD45 depleted fraction. mBC patients might be insensitive to treatment due to a selection of resistant CTCs subpopulations with different phenotypes. Additional patients with the same clinical characteristics will be analyzed in the next 6 coming months and updated results will be included. Citation Format: Elisabeth K Trapp, Brigitte Rack, Leonie Majunke, Julian Koch, Simone Hofmann, Thomas WP Friedl, Julia Neugebauer, Julia Juckstock, Bernadette Jager, Jens Huober, Wolfgang Janni, Marianna Alunni-Fabbroni. Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-16.

Endocrine Treatment with 2 Years of Tamoxifen versus 2 Years of Exemestane in Postmenopausal Patients with High-Risk Early Breast Cancer and Persisting Circulating Tumor Cells - First Results of the SUCCESS C Endocrine Treatment Sub-Study

Background: Optimal choice and sequence of endocrine treatment following adjuvant chemotherapy in postmenopausal early breast cancer patients are still under discussion and treatment stratification factors are missing. Patients and Methods: Postmenopausal women with HER2-negative, hormone receptor-positive tumors and persisting circulating tumor cells (CTCs; assessed using the FDA-approved CellSearch® System, Janssen Diagnostics, LLC) after chemotherapy were randomized to 2 years of tamoxifen followed by 3 years of exemestane (tamoxifen-exemestane group, n = 54) or 5 years of exemestane (exemestane-only group, n = 54). CTCs were again assessed after the first 2 years of endocrine treatment. In addition, safety data were compared between the 2 groups. Results: The 2 groups were well-balanced with regard to baseline characteristics. The CTC clearance rate after 2 years was 89% in the exemestane-only group and 97% in the tamoxifen-exemestane group (exact Fisher test, p = 0.36). The safety profile showed good tolerability with few grade 3 or 4 adverse events in both groups. Conclusion: The similar CTC clearance rate after 2 years of endocrine therapy with exemestane or tamoxifen, and the safety profiles obtained may indicate comparable efficacy and tolerability of both endocrine treatment regimens. However, these results have to be confirmed by final survival and safety analysis.

Discordance in Human Epidermal Growth Factor Receptor 2 (HER2) Phenotype Between Primary Tumor and Circulating Tumor Cells in Women With HER2-Negative Metastatic Breast Cancer

PurposeDiscordance in human epidermal growth factor receptor 2 (HER2) status between primary tumor and metastases might have important implications for treatment response and therapy decisions. Here, we evaluate both the frequency of circulating tumor cells (CTCs) and the factors predicting HER2 discordance between primary tumor and CTCs as a potential surrogate for tumor biology and tumor heterogeneity in patients with metastatic breast cancer.Patients and MethodsThe number of CTCs in 7.5 mL of peripheral blood and HER2 status were evaluated in 1,123 women with HER2-negative metastatic breast cancer. HER2 discordance was defined as the presence of at least one CTC with a strong immunocytochemical HER2 staining intensity. Factors predicting discordance in HER2 phenotype were assessed using multivariable logistic regression.ResultsOverall, 711 (63.3%) of 1,123 screened patients were positive for CTCs (≥ one CTC). Discordance in HER2 phenotype between primary tumor and CTCs was observed in 134 patients (18....

Prevalence of Circulating Tumor Cells After Adjuvant Chemotherapy With or Without Anthracyclines in Patients With HER2-negative, Hormone Receptor-positive Early Breast Cancer

Background Use of anthracycline‐based chemotherapy in patients with early breast cancer (EBC) has been well‐established but is often associated with cardiotoxicity. Based on data suggesting a limited benefit of anthracyclines in human epidermal growth factor receptor 2 (HER2)‐negative patients, the Simultaneous Study of Docetaxel Based Anthracycline Free Adjuvant Treatment Evaluation, as well as Life Style Intervention Strategies (SUCCESS) C study randomized patients to either anthracycline‐containing or anthracycline‐free chemotherapy. Given the proven prognostic value of circulating tumor cells (CTCs) in EBC, we compared the prevalence of CTCs after chemotherapy between both treatment arms for a preliminary efficacy assessment. Methods The SUCCESS C trial (NCT00847444) is an open‐label, phase III study randomizing 3547 patients with HER2‐negative EBC to either 3 cycles of epirubicin, 5‐fluorouracil, and cyclophosphamide followed by 3 cycles of docetaxel (FEC‐DOC) or 6 cycles of docetaxel and cyclophosphamide (DOC‐C). CTC status was prospectively evaluated in hormone receptor‐positive patients at the time of last chemotherapy cycle using the US Food and Drug Administration‐approved CellSearch System (Janssen Diagnostics). Results Data on CTC status were available for 1766 patients. Overall, CTCs were found in 221 (12.5%) patients. Univariate analyses revealed that presence of CTCs at time of last chemotherapy cycle was not significantly associated with tumor or patient characteristics (all P > .1). There was no significant difference with respect to presence of CTCs between patients randomized to FEC‐DOC or DOC‐C (11.5% vs. 13.6%; P = .18). Conclusions The comparable prevalence of CTCs at the time of last chemotherapy cycle may indicate that anthracycline‐free chemotherapy is equally effective to anthracycline‐containing chemotherapy in HER2‐negative, hormone receptor‐positive EBC. However, efficacy data from the final survival analysis of SUCCESS C have to be awaited to confirm these preliminary findings. Micro‐Abstract The prevalence of circulating tumor cells (CTCs) in patients with human epidermal growth factor receptor 2‐negative, hormone receptor‐positive early breast cancer after adjuvant chemotherapy was compared between anthracycline‐free and anthracycline‐containing treatment cohorts within the Simultaneous Study of Docetaxel Based Anthracycline Free Adjuvant Treatment Evaluation, as well as Life Style Intervention Strategies (SUCCESS) C trial and showed no significant difference. Though CTCs have been established as an early treatment monitoring tool, comparison of CTC prevalence after therapy may have clinical implications with respect to equal effectiveness of both treatment regimens.