Mitra Tewes

Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients.

IntroductionThe persistence of circulating tumor cells (CTC) in breast cancer patients might be associated with stem cell like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells also may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Here we evaluated 226 blood samples of 39 metastatic breast cancer patients during a follow-up of palliative chemo-, antibody – or hormonal therapy for the expression of the stem cell marker ALDH1 and markers for EMT and correlated these findings with the presence of CTC and response to therapy.Methods2 × 5 ml blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1 and HER2 transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist1, Akt2, PI3Kα] and separately for the tumor stem-cell markers ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample.Results97% of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts. CTC were detected in 69/226 (31%) cancer samples. In the CTC (+) group, 62% were positive for at least one of the EMT markers and 69% for ALDH1, respectively. In the CTC (-) group the percentages were 7% and 14%, respectively. In non-responders, EMT and ALDH1 expression was found in 62% and 44% of patients, in responders the rates were 10% and 5%, respectively.ConclusionsOur data indicate that a major proportion of CTC of metastatic breast cancer patients shows EMT and tumor stem cell characteristics. Further studies are needed to prove whether these markers might serve as an indicator for therapy resistant tumor cell populations and, therefore, an inferior prognosis.

Comparison of estrogen and progesterone receptor status of circulating tumor cells and the primary tumor in metastatic breast cancer patients

OBJECTIVES The expression of predictive markers including the estrogen (ER) and progesterone receptor (PR) expression can change during the course of the disease. Therefore, reassessment of these markers at the time of disease progression might help to optimize treatment decisions. Metastatic tissue may be difficult to obtain for repeated analysis. In this context, characterization of circulating tumor cells (CTCs) could be of relevance. It was the purpose of the present study (1) to reevaluate the ER/PR expression by CTCs and (2) to compare the hormone receptor status expression profile of CTCs with the primary tumor. METHODS We evaluated 193 blood samples from metastatic breast cancer patients at the time of first diagnosis of metastatic disease or disease progression. All samples underwent immunomagnetic enrichment using the AdnaTest BreastCancerSelect (AdnaGen AG, Germany) within 4h after blood withdrawal followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR in separated tumor cells using the AdnaTest BreastCancerDetect. CTCs were analyzed for the three breast cancer-associated markers: EpCAM, Muc-1, Her-2 and actin as an internal PCR control. Expression of the ER and PR was assessed in an additional RT-PCR. The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. RESULTS The overall detection rate for CTCs was 45% (87/193 patients) with the expression rates of 71% for EpCAM (62/87 patients), 73% for MUC1 (64/87 patients), 48% for HER2 (42/87 patients), 19% for ER (17/87 patients) and 10% for PR (9/87 patients), respectively. Comparisons with the primary tumor were only performed in CTC+ patients (n=87). In 48/62 (77%) patients with ER+ tumors, CTCs were ER- and 46/53 (87%) patients with PR+ tumors did not express PR on CTCs. Primary tumors and CTCs displayed a concordant ER and PR status in only 41% (p=0.260) and 45% (p=0.274) of cases, respectively. CONCLUSION Most of the CTCs were ER/PR-negative despite the presence of an ER/PR- positive primary tumor. The predictive value of hormone receptor status expression profile of CTCs for palliative endocrine therapy has to be prospectively evaluated. STATEMENT: We recently demonstrated in more than 400 primary breast cancer patients that the expression profile between CTCs and the primary tumor with regard to ER/PR/HER2 positivity differs. The concordance rate between ER, PR and HER2 status of CTCs and the primary tumor was 29%, 25% and 53%, respectively (Fehm T et al., Breast Cancer Res Aug 10 2009, 11(4) pR59). Based on these results we studied blood samples of 193 metastatic breast cancer patients participating in the German DETECT study (1) to reevaluate the ER/PR expression by CTCs and (2) to compare the hormone receptor status expression profile of CTCs with the primary. As already shown for primary breast cancer, most of the CTCs were ER/PR-negative despite the presence of an ER/PR- positive primary tumor. In the metastatic setting the phenotype of CTC reflects the phenotype of metastatic disease. Therefore palliative treatment selected based on the expression profile may not be effective since the phenotype has changed during disease progression. To our knowledge, this study is one of the biggest to compare hormonal receptor expression on CTC and the primary tumor. We hope that our manuscript is suitable for publication in Gynecologic Oncology.

Establishment of a multimarker qPCR panel for the molecular characterization of circulating tumor cells in blood samples of metastatic breast cancer patients during the course of palliative treatment

Background Circulating tumor cells (CTC) are discussed to be an ideal surrogate marker for individualized treatment in metastatic breast cancer (MBC) since metastatic tissue is often difficult to obtain for repeated analysis. We established a nine gene qPCR panel to characterize the heterogeneous CTC population in MBC patients including epithelial CTC, their receptors (EPCAM, ERBB2, ERBB3, EGFR) CTC in Epithelial-Mesenchymal-Transition [(EMT); PIK3CA, AKT2), stem cell-like CTC (ALDH1) as well as resistant CTC (ERCC1, AURKA] to identify individual therapeutic targets. Results At TP0, at least one marker was detected in 84%, at TP1 in 74% and at TP2 in 79% of the patients, respectively. The expression of ERBB2, ERBB3 and ERCC1 alone or in combination with AURKA was significantly associated with therapy failure. ERBB2 + CTC were only detected in patients not receiving ERBB2 targeted therapies which correlated with no response. Furthermore, patients responding at TP2 had a significantly prolonged overall-survival than patients never responding (p = 0.0090). Patients and Methods 2 × 5 ml blood of 62 MBC patients was collected at the time of disease progression (TP0) and at two clinical staging time points (TP1 and TP2) after 8–12 weeks of chemo-, hormone or antibody therapy for the detection of CTC (AdnaTest EMT-2/StemCell Select™, QIAGEN Hannover GmbH, Germany). After pre-amplification, multiplex qPCR was performed. Establishment was performed using various cancer cell lines. PTPRC (Protein tyrosine phosphatase receptor type C) and GAPDH served as controls. Conclusions Monitoring MBC patients using a multimarker qPCR panel for the characterization of CTC might help to treat patients accordingly in the future.

Validity of bone marker measurements for monitoring response to bisphosphonate therapy with zoledronic acid in metastatic breast cancer

Bone is the most common site of metastasis in breast cancer. Detection relies on imaging technology which is costly and can only be performed to a certain degree. Bone markers are non-invasive, inexpensive and may potentially serve as predictive and prognostic surrogate endpoints in detecting bone metastases and response to bisphosphonates. This study analyzed the value of the serum bone turnover markers PINP and ICTP for bone metastases in metastatic breast cancer patients receiving zoledronic acid. The results were compared with the serum levels of CEA and CA 15-3, and analyzed with respect to the number of bone metastases as well as clinical response. Forty patients with confirmed bone metastases who received chemotherapy and/or hormonal therapy and zoledronic acid i.v. q4 weeks participated in the present study. blood (5 ml) was collected at the start of the study and q3 months for a period of one year for the analysis of PINP, ICTP, CEA and CA 15-3 using radioimmunoassays and ELISA, respectively. Imaging of bone metastases was performed at the same time points. In 29 out of 40 patients, more than 3 bone metastases were confirmed by imaging and 11 out of 40 patients presented with 3 or less. At the start of the study, the median value for ICTP was 6 µg/l and for PINP 58.7 µg/l. At the end of the study the median values were 4.5 µg/l for ICTP and 21 µg/l for PINP. When patients were stratified into responders and non-responders, a decrease in both PINP (P<0.0001) and ICTP (P=0.048) was observed for the responders, while the level of ICTP (P=0.02) increased for the non-responders. Serum PINP and ICTP concentrations were significantly different when patients were stratified into groups of those having more than 3 bone metastases and 3 or less, respectively (P<0.05). CEA and CA 15-3 levels did not differ with respect to the number of bone metastases, while the tumor marker levels determined at the end of the study significantly distinguished responders from non-responders (P=0.002 and P=0.004). In conclusion, in contrast to serum tumor markers, the determination of PINP and ICTP allows inferences to the number of bone metastases and appears to be a useful tool for prediction and monitoring metastatic breast cancer patients undergoing bisphosphonate therapy with zoledronic acid for the treatment of bone metastases.

Gene Expression Signatures in Circulating Tumor Cells Correlate with Response to Therapy in Metastatic Breast Cancer

BACKGROUND Circulating tumor cells (CTCs) are thought to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). We investigated the prediction of treatment response in CTCs of MBC patients on the basis of the expression of 46 genes. METHODS From 45 MBC patients and 20 healthy donors (HD), 2 × 5 mL of blood was collected at the time of disease progression (TP0) and at 2 consecutive clinical staging time points (TP1 and TP2) to proceed with the AdnaTest EMT-2/StemCellSelectTM (QIAGEN). Patients were grouped into (a) responder (R) and non-responder (NR) at TP1 and (b) overall responder (OR) and overall non-responder (ONR) at TP2. A 46-gene PCR assay was used for preamplification and high-throughput gene expression profiling. Data were analyzed by use of GenEx (MultiD) and SAS. RESULTS The CTC positivity was defined by the four-gene signature (EPCAM, KRT19, MUC1, ERBB2 positivity). Fourteen genes were identified as significantly differentially expressed between CTC+ and CTC- patients (KRT19, FLT1, EGFR, EPCAM, GZMM, PGR, CD24, KIT, PLAU, ALDH1A1, CTSD, MKI67, TWIST1, and ERBB2). KRT19 was highly expressed in CTC+ patients and ADAM17 in the NR at TP1. A significant differential expression of 4 genes (KRT19, EPCAM, CDH1, and SCGB2A2) was observed between OR and ONR when stratifying the samples into CTC+ or CTC-. CONCLUSIONS ADAM17 could be a key marker in distinguishing R from NR, and KRT19 was powerful in identifying CTCs.

Comparison of the PI3KCA pathway in circulating tumor cells and corresponding tumor tissue of patients with metastatic breast cancer

The aim of the present study was to compare the phosphatidylinositol 3-kinase (PI3KCA)-AKT serine/threonine kinase (AKT) pathway in circulating tumor cells (CTCs) and corresponding cancerous tissues. Stemness-like circulating tumor cells (slCTCs) and CTCs in epithelial-mesenchymal transition (EMT) have been implicated as the active source of metastatic spread in breast cancer (BC). In this regard, the PI3KCA-AKT signaling pathway was demonstrated to be implicated in and to be frequently mutated in BC. The present study compared this pathway in slCTCs/CTCs in EMT and the corresponding tumor tissues of 90 metastatic BC patients (pts). slCTCs and CTCs in EMT were isolated using the AdnaTest EMT-1/StemCell for the detection of aldehyde dehydrogenase 1 family member A1 (ALDH1) (singleplex PCR) and PI3KCA, AKT2 and twist family bHLH transcription factor 1 (multiplex PCR). Tumor tissue was investigated for PI3KCA hotspot mutations using Sanger sequencing of genomic DNA from micro-dissected formalin-fixed paraffin-embedded tissue, and for the expression of ALDH1 and phosphorylated AKT (pAKT), and phosphatase and tensin homolog (PTEN) loss, by immunohistochemistry. slCTCs were identified in 23% of pts (21/90 pts) and CTCs in EMT in 56% (50/90 pts) of pts. pAKT and ALDH1 positivity in tumor tissue was identified in 47 and 9% of cases, respectively, and a PTEN loss was observed in 18% of pts. A significant association was detected between pAKT expression in cancerous tissue and AKT2 expression in CTCs (P=0.037). PI3KCA mutations were detected in 32% of pts, most frequently on exons 21 (55%) and 10 (45%). Pts with PI3KCA mutations in tumor tissue had a significantly longer overall survival than pts with wild-type PI3KCA expression (P=0.007). Similar results were obtained for pts with aberrant PI3KCA signaling in CTCs and/or aberrant signaling in cancerous tissue (P=0.009). Therapy-resistant CTCs, potentially derived from the primary tumor or metastatic tissue, may be eliminated with specific PI3K pathway inhibitors, alone or in combination, to improve the prognosis of metastatic BC pts.

Stem cell and epithelial-mesenchymal transition markers on circulating tumor cells in patients with metastatic breast cancer.

Abstract #107 Background: Stem cell like tumor cells have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Assuming that metastasis requires a dissemination of tumor stem cells or tumor cells showing EMT, it was the purpose of this study to identify these cells among circulating tumor cells (CTC) in blood samples of 28 metastatic breast cancer patients.
 Materials and Methods: 5 ml blood was analyzed for EpCAM, MUC-1 and HER-2 transcripts with the AdnaTest BreastCancer (AdnaGen AG). The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist, Akt2, PI3K (TAP)] and separately for the tumor stem-cell marker ALDH1. The analytical sensitivity was determined by the detection of a low number of target cells (5 IGROV cells spiked into 5 ml blood of healthy donors) using the AdnaTest BreastCancer procedure. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Healthy donor samples without spiked tumor cells were used to determine the specificity of the test. The resulting PCR fragments were separated and quantified using the Agilent Bioanalyzer 2100.
 Results: Applying an amplicon cut-off value of 0.2 ng/μl for Akt2, 0.15 ng/μl for Twist, 0.25 ng/μl for PI3K and 0.15 ng/μl for ALDH1, 97% of 30 healthy donor samples investigated were negative for TAP and 95% for ALDH1 transcripts. The spiking experiments revealed 80% recovery of the IGROV cells. CTC were detected in 12/28 (43%) breast cancer samples. All samples were further examined for tumor stem cell or TAP markers. In the CTC (+) group 50% were positive for at least one of the TAP markers and 42% for ALDH1. In the CTC (-) group the percentages were 19% and 12%, respectively. The expression of TAP and/or stem cell markers in CTC was compared with the clinical follow up results. In CTC (+) patients diagnosed as non-responders the expression of TAP and ALDH1 was found in 5/12 (40%) samples. In the CTC (+) responder group only one sample expressed TAP and no positive case was found for ALDH1. In the CTC (-) non-responder group 1/14 samples was positive for TAP and ALDH1. 2/14 (14%) of CTC (-) responders were positive for TAP and 1/14 for ALDH1, respectively.
 Conclusion: A major proportion of CTC found in the blood of metastatic breast cancer patients shows EMT and tumor stem cell characteristics supporting the hypothesis that these markers are an indicator for therapy resistant tumor cell populations and, therefore, for an inferior prognosis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 107.

Predictors of outpatients’ request for palliative care service at a medical oncology clinic of a German comprehensive cancer center

PurposeEarly integration of palliative care (PC) is recommended. The determination of predictors for patients’ request for PC may guide implementation in clinical practice. Toward this end, we analyzed the symptom burden and distress of cancer patients in outpatient care and examined their need and request for PC.MethodsBetween October 2013 and March 2016, 705 patients receiving outpatient cancer treatment took part in the survey. We used the new MInimal DOcumentation System to detect symptom clusters. Additionally, patients’ request for palliative and psychosocial support was assessed. Groups of patients with PC request were compared to patients without PC request regarding their symptom clusters. Logistic regression analysis was applied to discover significant predictors for the requested inclusion of PC.ResultsA total of 159 patients (25.5%) requested additional support by PC. Moderate and severe tiredness (40.3%), weakness (37.9%), pain (25.0%), loss of appetite (22.3%), and dyspnea (19.1%) were the most frequent symptoms. The group of patients requesting PC differed significantly in terms of pain, nausea, dyspnea, constipation, weakness, loss of appetite, tiredness, depression, and anxiety from patients without request for PC (p < .01). The perceived need for PC was identified by the significant predictors “depression,” “anxiety,” and “weakness” with an explained variance of 22%.ConclusionCombining a standardized screening questionnaire and the assessment of patients’ request for PC allows systematic monitoring for patients’ need for PC in a large Medical Oncology clinic. Depression, anxiety, and weakness are predictors of requesting PC service by patients receiving outpatient cancer treatment.

Patient-Reported-Outcome-Messung (PROM) psychosozialer Belastung und Symptome für ambulante Patienten unter kurativer oder palliativer Tumortherapie

ZusammenfassungHintergrundPalliative Unterstützung sollte früh in die Tumortherapie eingebunden werden, doch der Bedarf der Patienten bleibt oft unerkannt und unbehandelt. Die Autoren analysierten die Rücklaufquote einer quartalsweisen Patientenbefragung mit validierten Fragebögen und erfassten die Bedarfe der Tumorpatienten, die sich in kurativer oder palliativer Behandlung befanden.MethodenBei der Entwicklung des Fragebogens wurden die 3 validierten Instrumente Hornheider Fragebogen (HSI), PHQ4 Fragebogen (Patient Health Questionnaire) und der MIDOS Fragebogen (MInimal DOcumentation System) kombiniert. 96 (13,6 %) kurativ und 609 (81,4 %) palliativ behandelte Patienten füllten einen Fragebogen aus.ErgebnisseZwischen Oktober 2013 und März 2016 wurden 75 % der ausgeteilten Fragebögen ausgefüllt. Unter den kurativ und palliativ behandelten Patienten hatten 34 (35,8 %) und 272 (45,0 %) Patienten einen pathologischen HSI-Score (p = 0,094), bei 7 (11,3 %) und 51 (13,7 %) Patienten ergab der PHQ4 einen mittleren oder schlechten Score (p = 0,845) und 6 (6,5 %) und 70 (12,1 %) Patienten gaben ein schlechtes oder sehr schlechtes Allgemeinbefinden an (p = 0,388). 340 von 669 (50,8 %) Patienten wiesen auf die zusätzliche Belastung ihrer Angehörigen hin.SchlussfolgerungDie Analyse zeigt die Durchführbarkeit einer quartalsweise durchgeführten Patientenbefragung zur Erfassung des palliativen Bedarfs im Routinebetrieb einer onkologischen Ambulanz. Hauptsymptome beider Patientengruppen waren Müdigkeit und Schwäche. Weiterhin gab ein Großteil der Patienten eine Belastung der Angehörigen an.AbstractBackgroundPalliative care should be integrated at an early stage in the cancer treatment trajectory, but distress and symptom burden are often underdetected or undertreated. We analyzed the response rate of validated questionnaires to detect symptom burden and distress of patients with curative and palliative treatment concepts.MethodsIn the development of the questionnaire we combined validated instruments, e. g. the Hornheide screening instrument (HSI), patient health questionnaire (PHQ-4) and the minimal documentation system (MIDOS). In this study 96 (13.6%) and 609 (81.4%) patients treated with curative and palliative intent, respectively, completed a questionnaire.ResultsBetween October 2013 and March 2016, 75% of the questionnaires were completed. In a curative and palliative setting 34 (35.8%) and 272 patients (45.0%), respectively, had pathological scores for HSI (p = 0.094), 7 (11.3%) and 51 (13.7%) patients had moderate and severe PHQ-4 scores (p = 0.845) and 6 (6.5%) and 70 (12.1%) patients had poor and very poor general condition (p = 0.388). Out of 669 patients 340 (50.8%) indicated a burden on their relatives.ConclusionOur analysis showed the feasibility of a quarter yearly patient survey to assess palliative needs in the routine operation of an oncology outpatient clinic. The main symptoms of both patient groups were fatigue and weakness. Furthermore, a large proportion of the patients reported a burden on their relatives.

Integration von Palliativmedizin in onkologische Spitzenzentren Deutschlands – ambulante Sprechstunden und Rotationsprogramme der spezialisierten Palliativmedizin

BACKGROUND  Comprehensive Cancer Centers are characterized by interdisciplinary exchange for meeting increasingly complex care needs during the course of the disease. Tumor consultations hours and fellowship rotations build hereby bridges between the subjects and disciplines. OBJECTIVES  In order to be able to provide support in the Comprehensive Cancer Center Network for the further integration of specialized palliative medicine, it was highlighted to what extent consultation hours and fellowship rotations for the palliative care are integrated into the centers. METHODS  Information about the spS and fellowship rotation of the Comprehensive Cancer Center (n = 16), which had previously been funded by the Cancer Aid, was paper-based collected with a survey questionnaire. For this purpose, the heads of the palliative care departments of the centers were interviewed from July to August 2017. The evaluation was performed by SPSS (frequency, mean value, median, range). RESULTS  15 from 16 centers responded to the survey (93.75 %). Nine centers (60 %) have a consultation hour for palliative care. Four from nine centers can submit this offer for ≥ 4 hours (44.4 %). Fellowship rotations in the palliative care occur primarily in all centers from oncology / hematology (n = 11, 73.3 %) and anaesthesia (n = 6) and often for a twelve months (n = 11) period of time. CONCLUSION  Outpatient structures of the palliative care have been insufficiently implemented to a consultation hour within Comprehensive Cancer Centers. The existing effort to integrate palliative care into the oncological course of disease requires further structuring in order to increase the visibility of palliative care services. Fellowship rotations in the palliative care department are regularly implemented in the network even if only for some of the subjects in order to raise awareness of the possibilities of the palliative care.