Jan Dominik Kuhlmann

MicroRNA-148a is down-regulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B

MicroRNAs (miRNAs: short non-coding RNAs) are emerging as a class of potential novel tumor markers, as their dysregulation is being increasingly reported in various types of cancers. In the present study, we investigated the transcription status of miRNA-148a (miR-148a) in human pancreatic ductal adenocarcinoma (PDAC) and its role in the regulation of the dual specificity protein phosphatase CDC25B. We observed that miR-148a exhibited a significant 4-fold down-regulation in PDAC as opposed to normal pancreatic ductal cells. In addition, we observed that stable lentiviral-mediated overexpression of miR-148a in the pancreatic cancer cell line IMIM-PC2, inhibited tumor cell growth and colony formation. Furthermore, CDC25B was identified as a potential target of miR-148a by in silico analysis using PicTar, Targetscan and miRanda in conjunction with gene ontology analysis. The proposed interaction between miR-148a and the 3′ untranslated region (UTR) of CDC25B was verified by in-vitro luciferase assays. We demonstrate that the activity of a luciferase reporter containing the 3′UTR of CDC25B was repressed in the presence of miR-148a mimics, confirming that miR-148a targets the 3′UTR of CDC25B. Finally, CDC25B was down-regulated at the protein level in miR-148a overexpressing IMIM-PC2-cells, and in transiently transfected pancreatic cell lines (as detected by Western blot analysis), as well as in patient tumor samples (as detected by immunohistochemistry). In summary, we identified CDC25B as a novel miR-148a target which may confer a proliferative advantage in PDAC.

Regorafenib in combination with FOLFOX or FOLFIRI as first- or second-line treatment of colorectal cancer: results of a multicenter, phase Ib study

Background Metastatic colorectal cancer (mCRC) is commonly treated with 5-fluorouracil, folinic acid, and oxaliplatin or irinotecan. The multitargeted kinase inhibitor, regorafenib, was combined with chemotherapy as first- or second-line treatment of mCRC to assess safety and pharmacokinetics (primary objectives) and tumor response (secondary objective). Patients and methods Forty-five patients were treated every 2 weeks with 5-fluorouracil 400 mg/m2 bolus then 2400 mg/m2 over 46 h, folinic acid 400 mg/m2, and either oxaliplatin 85 mg/m2 or irinotecan 180 mg/m2. On days 4–10, patients received regorafenib 160 mg orally once daily. Results The median duration of treatment was 108 (range 2–345 days). Treatment was stopped for adverse events or death (17 patients), disease progression (11 patients), and consent withdrawal or investigator decision (11 patients). Six patients remained on regorafenib at data cutoff (two without chemotherapy). Drug-related adverse events occurred in 44 patients [grade ≥3 in 32 patients: mostly neutropenia (17 patients) and leukopenia, hand–foot skin reaction, and hypophosphatemia (four patients each)]. Thirty-three patients achieved disease control (partial response or stable disease) for a median of 126 (range 42–281 days). Conclusion Regorafenib had acceptable tolerability in combination with chemotherapy, with increased exposure of irinotecan and SN-38 but no significant effect on 5-fluorouracil or oxaliplatin pharmacokinetics.

ERCC1-Positive Circulating Tumor Cells in the Blood of Ovarian Cancer Patients as a Predictive Biomarker for Platinum Resistance

BACKGROUND Platinum resistance constitutes one of the most recognized clinical challenges for ovarian cancer. Notably, the detection of the primary tumor-based excision repair cross-complementation group 1 (ERCC1) protein by immunohistochemistry was recently shown to be inaccurate for the prediction of platinum resistance. On the basis of the previous finding that circulating tumor cells (CTC) in the blood of ovarian cancer patients are prognostically significant, and given our hypothesis that the negative prognostic impact of CTC may arise from a cellular phenotype associated with platinum resistance, we asked whether expression of the excision repair cross-complementation group 1 (ERCC1) gene in the form of the ERCC1 transcript in CTC may be a suitable blood-based biomarker for platinum resistance. METHODS The presence of CTC was analyzed by immunomagnetic CTC enrichment (n = 143 patients) targeting the epithelial epitopes epithelial cell adhesion molecule (EPCAM) (also known as GA733-2) and mucin 1, cell surface associated (MUC1), followed by multiplex reverse-transcription PCR to detect the transcripts EPCAM, MUC1, and mucin 16, cell surface associated (MUC16) (also known as CA125), including ERCC1 transcripts in a separate approach. ERCC1 expression in primary tumors was comparatively assessed by immunohistochemistry, using the antibody 8F1. RESULTS At primary diagnosis, the presence of CTC was observed in 14% of patients and constituted an independent predictor of overall survival (OS) (P = 0.041). ERCC1-positive CTC (ERCC1(+)CTC) were observed in 8% of patients and constituted an independent predictor, not only for OS but also for progression-free survival (PFS) (P = 0.026 and P = 0.009, respectively). More interestingly, we discovered the presence of ERCC1(+)CTC at primary diagnosis to be likewise an independent predictor of platinum resistance (P = 0.010), whereas ERCC1 expression in corresponding primary tumor tissue predicted neither platinum resistance nor prognosis. CONCLUSIONS The presence of ERCC1(+)CTC can serve as a blood-based diagnostic biomarker for predicting platinum resistance at primary diagnosis of ovarian cancer.

microRNA and the pathogenesis of ovarian cancer--a new horizon for molecular diagnostics and treatment?

Abstract Ovarian cancer is the leading cause of death among gynecologic malignancies and despite advances in treatment, more than 50% of all patients will experience recurrence, resulting in worse overall prognosis. Therefore, identification of novel biomarkers for ovarian cancer is of significant interest. microRNA (miRNA) constitute a class of small gene regulatory RNA molecules (18–24 nt) and by sequence complementarity, they negatively regulate messenger RNA (mRNA) translation of target genes. Rising data are available that miRNA are functionally involved in the pathogenesis of ovarian cancer. In this regard, recent advances in profiling studies revealed a variety of miRNA candidates, differently expressed in ovarian carcinomas and in disease-specific conditions like hypoxia or chemo-resistance. This review abstracts recent efforts on establishing miRNA as novel molecular biomarkers for ovarian cancer and depicts the existing preliminary framework for defining peripheral-blood derived miRNA as novel circulating biomarkers. Beside these clinical implications, we highlight the current functional understanding of miRNA alteration and discuss major challenges in miRNA profiling approaches. Finally, we briefly outline methodologies to therapeutically modulate miRNA expression in cancer and try to assess how miRNA can improve our conceptual understanding and the clinical management of ovarian cancer.

Circulating U2 Small Nuclear RNA Fragments as a Novel Diagnostic Tool for Patients with Epithelial Ovarian Cancer

BACKGROUND Ovarian cancer is the leading cause of death among malignancies in women. Despite advances in treatment, >50% of patients relapse. For disease monitoring, the identification of a blood-based biomarker would be of prime interest. In this regard, noncoding RNAs, such as microRNA (miRNA) or small nuclear RNA (snRNA), have been suggested as biomarkers for noninvasive cancer diagnosis. In the present study, we sought to identify differentially expressed miRNA/snRNA in sera of ovarian cancer patients and investigate their potential to aid in therapy monitoring. METHODS miRNA/snRNA abundance was investigated in serum (n = 10) by microarray analysis and validated in an extended serum set (n = 119) by reverse-transcription quantitative PCR. RESULTS Abundance of U2-1 snRNA fragment (RNU2-1f) was significantly increased in sera of ovarian cancer patients (P < 0.0001) and paralleled International Federation of Gynecology and Obstetrics stage as well as residual tumor burden after surgery (P < 0.0001 and P = 0.011, respectively). Moreover, for patients with suboptimal debulking, preoperative RNU2-1f concentration was associated with radiographic response after chemotherapy and with platinum resistance (P = 0.0088 and P = 0.0015, respectively). Interestingly, according to the RNU2-1f abundance dynamics, persistent RNU2-1f positivity before surgery and after chemotherapy identified a subgroup of patients with high risk of recurrence and poor prognosis. CONCLUSIONS This is the first report to suggest that a circulating snRNA can serve as an auxiliary diagnostic tool for monitoring tumor dynamics in ovarian cancer. Our results provide a rationale to further investigate whether this high-risk patient group may benefit from additional therapies that are directly applied after chemotherapy.

Gene expression profiling of single circulating tumor cells in ovarian cancer - Establishment of a multi-marker gene panel.

The presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was shown to correlate with decreased overall survival, whereby CTCs with epithelial–mesenchymal‐transition (EMT) or stem‐like traits are supposed to be involved in metastatic progression and recurrence. Thus, investigating the transcriptional profiles of CTCs might help to identify therapy resistant tumor cells and to overcome treatment failure. For this purpose, we established a multi‐marker panel for the molecular characterization of single CTCs, detecting epithelial (EpCAM, Muc‐1, CK5/7), EMT (N‐cadherin, Vimentin, Snai1/2, CD117, CD146, CD49f) and stem cell (CD44, ALDH1A1, Nanog, SOX2, Notch1/4, Oct4, Lin28) associated transcripts.

LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival

BackgroundWe recently showed that LOH proximal to M6P/IGF2R locus (D6S1581) in primary ovarian tumors is predictive for the presence of disseminated tumor cells (DTC) in the bone marrow (BM). For therapy-monitoring, it would be highly desirable to establish a blood-based biomarker. Therefore, we quantified circulating DNA (cirDNA) in sera of 63 ovarian cancer patients before surgery and after chemotherapy, measured incidence of LOH at four cancer-relevant chromosomal loci, correlated LOH with tumor cell spread to the BM and evaluated prognostic significance of LOH.MethodscirDNA was fractionated into high- and low molecular-weight fraction (HMWF, LMWF) for LOH-profiling, utilizing PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.ResultscirDNA levels in the HMWF before surgery were predictive for residual tumor load (p = 0.017). After chemotherapy, we observed a significant decline of cirDNA in the LMWF (p = 0.0001) but not in the HMWF. LOH was prevalently detected in the LMWF with an overall frequency of 67%, only moderately ablating after chemotherapy (45%). Before surgery, LOH in the LMWF at marker D10S1765 and D13S218 significantly correlated with tumor grading and FIGO stage (p = 0.033, p = 0.004, respectively). In both combined fractions, LOH at D6S1581 additionally associated with overall survival (OS) (p = 0.030). Moreover, solely LOH at D10S1765 in LMWF after therapy correlated with DTC in BM after therapy (p = 0.017).ConclusionWe demonstrate the applicability and necessity of DNA-fractionation prior to analyzing circulating LOH and identify LOH at D10S1765 and D6S1581 as novel blood-based biomarkers for ovarian cancer, being relevant for therapy-monitoring.

Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance.

OBJECTIVES The identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patients survival. METHODS A total of 73 patients with primary ovarian cancer were enrolled into this study. Primary tumor tissue was analyzed for the expression of VEGF-R1, VEGF-R2 and VEGF-R3 by immunohistochemistry. The presence of disseminated tumor cells (DTC) in the BM was analyzed by immunocytochemistry using the pancytokeratin antibody A45B/B3 and subsequent automatic detection based on staining and cytomorphology. RESULTS In primary ovarian cancer tissue, VEGF-receptor expression, detected with an overall frequency of 44%, was mostly located in the vascular wall and across the stroma; positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 were 34%, 18% and 26%, respectively. Total VEGF-receptor expression correlated with residual tumor after primary debulking surgery and the presence of DTC at primary diagnosis (p=0.035, p=0.023, respectively). Interestingly, VEGF-R1 positivity significantly correlated with decreased progression-free survival (p=0.026). CONCLUSIONS This is the first report, suggesting total VEGF-receptor status as a molecular biomarker for monitoring tumor cell spread to the BM and, particularly, revealing prognostic significance of VEGF-R1.

Targeting Cancer Stem Cells: Promises and Challenges.

Recent discoveries have provided the compelling evidence that stem cell populations within each individual tumor are key contributors of therapy failure regardless of whether these populations are transient or stable. Thus, it is becoming increasingly clear that efficient tumor treatment requires eradication of the entire CSC population. The potential role of CSCs in tumor initiation and relapse has motivated an investigation of the CSCspecific treatments. However, development of the therapeutic strategies targeting CSCs might be challenged by a high diversity and plasticity of CSC features. Moreover, taking in account that an origin of CSC remains controversial and accumulating experimental evidence suggests a possibility of tumor cell reprogramming, efficient anti-cancer treatment should eradicate both CSCs and tumor bulk as well as prevent tumor dedifferentiation. In this article we discuss new insights into the stem cell concept of tumor development, review the treatment strategies eradicating CSCs that have been evaluated in preclinical and clinical studies and summarize the strategies to identify new CSC-targeted therapy.

Increased level of circulating U2 small nuclear RNA fragments indicates metastasis in melanoma patients.

Abstract Background: Melanoma is the most aggressive skin cancer and, despite recent advances in therapy, about 20% of the patients die of their disease. Early relapse detection and monitoring of therapy response are crucial for efficient treatment of advanced melanoma. Thus, there is a need for blood-based biomarkers in melanoma management. Serum-derived U2 small nuclear RNA fragments (RNU2-1f) were previously shown to be blood-based biomarkers for gastrointestinal and gynecologic malignancies. Here we examined whether RNU2-1f may also serve as diagnostic biomarker in advanced melanoma. Methods: Circulating RNU2-1f levels were quantified by comparative reverse transcription PCR in a training cohort of patients with metastatic melanoma (n=33, thereof regionally metastasized to skin and lymph nodes, n=23, and distantly metastasized, n=10) vs. patients with benign naevi (n=16) vs. healthy controls (n=39). Results were validated in an independent patient cohort with distant metastasis (n=16) vs. controls (n=18). Results: Circulating RNU2-1f levels in the training cohort were significantly increased in serum of regionally and distantly metastatic patients, compared with patients with benign naevi or healthy controls (p<0.0001) and allowed accurate detection of regional (AUC 0.80) as well as distant (AUC 0.84) metastasis. In the validation cohort, increased RNU2-1f levels were confirmed and enabled highly specific detection of distant metastasis (sensitivity 81%, specificity 100%, AUC 0.94). Conclusions: This is the first report to suggest a blood-based snRNA serving as a diagnostic biomarker for melanoma metastasis. Our data provide a rationale for further defining clinical utility of circulating RNU2-1f in metastasis detection in the management of melanoma patients at risk of relapse and/or with advanced disease.