The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

The impact of HER2 phenotype of circulating tumor cells in metastatic breast cancer: a retrospective study in 107 patients.

BACKGROUND: In metastatic breast cancer (MBC), antigen profiles of metastatic tissue and primary tumor differ in up to 20 % of patients. Reassessment of predictive markers, including human epidermal growth factor receptor 2 (HER2) expression, might help to optimize MBC treatment. While tissue sampling is invasive and often difficult to repeat, circulating tumor cell (CTC) analysis requires only a blood sample and might provide an easy-to-repeat, real-time "liquid biopsy" approach. The present retrospective study was conducted to compare HER2 expression in primary tumors, metastatic tissue, and circulating tumor cells (CTCs) from MBC patients and to analyze the potential impact of HER2 overexpression by CTCs on progression-free (PFS) and overall survival (OS) in MBC. METHODS: CTC-positive (five or more CTCs/7.5 mL blood; CellSearch®, Janssen Diagnostics) MBC patients starting a new line of systemic treatment were eligible for the study. HER2 status of CTCs was determined by immunofluorescence (CellSearch®). HER2 status of primary (PRIM) and metastatic (MET) tumor tissue was determined by immunohistochemistry. Data were analyzed using descriptive statistics and Kaplan-Meier plots. RESULTS: One hundred seven patients (median age (range) 57 (33-81) years) were included. 100/107 (93%) patients were followed-up for a median [95% confidence interval (CI)] of 28.5 [25.1-40.1] months. Of 37/107 (35%) CTC-HER2-positive patients only 10 (27%) were PRIM-HER2-positive. 6/46 (13%) patients were MET-HER2-positive; only 2/10 (20%) CTC-HER2-positive patients were MET-HER2-positive. Overall accuracy between CTC-HER2 expression and PRIM-HER2 and MET-HER2 status was 69% and 74%, respectively. Kaplan-Meier plots of PFS and OS by CTC-HER2 status revealed significantly longer median [95% CI] PFS of CTC-HER2-positive versus CTC-HER2-negative patients (7.4 [4.7-13.7] versus 4.34 [3.5-5.9] months; p = 0.035). CTC-HER2-positive status showed no significant difference for OS (13.7 [7.7-30.0] versus 8.7 [5.9-15.3] months; p = 0.287). CONCLUSIONS: HER2 status can change during the course of breast cancer. CTC phenotyping may serve as an easy-to-perform "liquid biopsy" to reevaluate HER2 status and potentially guide treatment decisions. Further, prospective studies are needed.

Serial enumeration of circulating tumor cells predicts treatment response and prognosis in metastatic breast cancer: a prospective study in 393 patients.

BACKGROUND: To prospectively assess circulating tumor cell (CTC) status at baseline (CTCBL) and after one cycle of a new line of systemic therapy (CTC1C), and changes from CTCBL to CTC1C (CTC kinetics, CTCKIN) for their utility in predicting response, progression-free (PFS) and overall survival (OS) in metastatic breast cancer (MBC). METHODS: CTCBL and CTC1C status was determined as negative (-) or positive (+) for < 5 or ≥ 5 CTCs/7.5 ml blood using CellSearch™ (Veridex). CTCKIN was categorized as favorable (CTC1C-) or unfavorable (CTC1C+). Tumor response was to be assessed every 2-3 months using the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Statistical analysis focused on the relation between CTC status and CTCKIN, and response, PFS, and OS. RESULTS: 133/393 (34%) patients enrolled were CTCBL+. CTC1C status after one cycle and radiological tumor response were assessed after median (range) periods of 1.2 (0.5-3.2) and 2.9 (0.5-4.8) months, respectively. 57/201 (28%) were CTC1C+. Median [95% confidence interval] PFS and OS (months) were significantly reduced in CTCBL+ vs. CTCBL- patients (PFS 4.7 [3.7-6.1] vs. 7.8 [6.4-9.2]; OS 10.4 [7.9-15.0] vs. 27.2 [22.3-29.9]), and for CTC1C+ vs. CTC1C- patients (PFS 4.3 [3.6-6.0] vs. 8.5 [6.6-10.4]; OS 7.7 [6.4-13.9] vs. 30.6 [22.6-not available]). Unfavorable CTCKIN was significantly associated with progressive disease. Multivariate Cox regression analysis revealed prognostic factors for shorter PFS (CTCBL+, persistent CTCs after one cycle, ≥ 3rd-line therapy, and triple-negative receptor status) and shorter OS (CTCBL+, persistent CTCs after one cycle, bone-and-visceral/local metastases, ≥ 3rd-line therapy, and triple-negative receptor status). CONCLUSIONS: CTCBL, CTC1C, and CTCKIN are predictive of outcome in MBC. Serial CTC enumeration is useful in tailoring systemic treatment of MBC. TRIAL REGISTRATION: Not applicable.

Protocol of the IntenSify-Trial: An open-label phase I trial of the CYP3A inhibitor cobicistat and the cytostatics gemcitabine and nab-paclitaxel in patients with advanced stage or metastatic pancreatic ductal adenocarcinoma to evaluate the combination's pharmacokinetics, safety, and efficacy.

Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2 ) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2 ) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.