Detection of Recurrence through microRNA-371a-3p Serum Levels in a Follow-up of Stage I Testicular Germ Cell Tumors in the DRKS-00019223 Study.

PURPOSE: Surveillance of clinical stage I (CSI) testicular germ cell tumors (GCT) is hampered by low sensitivity and specificity of current biomarkers for detecting relapses. This study evaluated if serum levels of microRNA371a-3p (M371 test) can: (i) Accurately detect relapses, (ii) detect relapses earlier than conventional technology, and (iii) if elevated postoperative M371 levels may predict relapse. EXPERIMENTAL DESIGN: In a multicentric setting, 258 patients with testicular CSI GCT were prospectively followed by surveillance for a median time of 18 months with serial measurements of serum M371 levels, in addition to standard diagnostic techniques. Diagnostic characteristics of M371 for detecting relapses were calculated using ROC curve analysis. RESULTS: Thirty-nine patients recurred (15.1%), all with elevated M371 levels; eight without relapse had elevations, too. The test revealed the following characteristics: area under the ROC curve of 0.993, sensitivity 100%, specificity 96.3%, positive predictive value 83%, negative predictive value 100%. Earlier relapse detection with the test was found in 28%, with non-significant median time gain to diagnosis. Postoperative M371 levels did not predict future relapse. CONCLUSIONS: The sensitivity and specificity of the M371 test for detecting relapses in CSI GCTs are much superior to those of conventional diagnostics. However, post-orchiectomy M371 levels are not predictive of relapse, and there is no significant earlier relapse detection with the test. In all, there is clear evidence for the utility of the M371 test for relapse detection suggesting it may soon be ready for implementation into routine follow-up schedules for patients with testicular GCT.

Associations of serum levels of microRNA-371a-3p (M371) with risk factors for progression in nonseminomatous testicular germ cell tumours clinical stage 1.

PURPOSE: Lymphovascular invasion (LV1) and presence of > 50% embryonal carcinoma (> 50% EC) represent risk factors for progression in patients with clinical stage 1 (CS1) nonseminomatous (NS) testicular germ cell tumours. As serum levels of microRNA-371a-3p (M371) are capable of detecting small amounts of GCT, we evaluated if LV1 and > 50% EC are associated with M371 levels. METHODS: M371 serum levels were measured postoperatively in 153 NS CS1 patients and both pre- and postoperatively in 131 patients. We registered the following factors: age, tumour size, LV status, > 50% EC, teratoma in primary, preoperative elevation of classical tumour markers. M371 expression was compared among subgroups. The ability of M371 to predict LV1 was calculated by receiver operating characteristics (ROC) curves. Multiple regression analysis was used to look for associations of M371 levels with other factors. RESULTS: Postoperatively elevated M371 levels were found in 29.4% of the patients, but were neither associated with LV status nor with > 50% EC. Likewise, relative decrease of M371 was not associated. ROC analysis of postoperative M371 levels revealed an AUC of 0.5 for the ability to predict LV1 while preoperative M371 had an AUC of 0.732. Multiple regression analysis revealed significant associations of preoperative M371 levels with LV status (p = 0.003), tumour size (p = 0.001), > 50% EC (p = 0.004), and teratoma component (p = 0.045). CONCLUSION: Postoperatively elevated M371 levels are not associated with risk factors for progression in NS CS1 patients. However, the significant association of preoperative M371 expression with LV1 deserves further evaluation.

Serum Levels of MicroRNA-371a-3p (M371 Test) as a New Biomarker of Testicular Germ Cell Tumors: Results of a Prospective Multicentric Study.

PURPOSE: Previous studies suggested that serum levels of microRNA (miR)-371a-3p (so-called M371 test) have a much higher sensitivity and specificity than the classic markers of testicular germ cell tumors (GCTs) and are applicable toward both seminoma and nonseminoma. We sought to confirm the usefulness of this test as a novel biomarker for GCT. PATIENTS AND METHODS: In a prospective, multicentric study, serum samples of 616 patients with testicular GCTs and 258 male controls were examined for serum levels of miRNA-371a-3p (miR levels) by quantitative polymerase chain reaction. The GCT population encompassed 359 patients with seminoma and 257 with nonseminoma; 371 had clinical stage I disease, 201 had systemic disease, and 46 had relapses. Paired measurements before and after orchiectomy were performed in 424 patients; 118 with systemic disease had serial measurements during treatment. miR levels were compared with those of ß-human chorionic gonadotropin, α-fetoprotein, and lactate dehydrogenase. RESULTS: For the primary diagnosis of GCT, the M371 test showed a sensitivity of 90.1%, a specificity of 94.0%, an area under the curve of 0.966 upon receiver operating characteristic analysis, and a positive predictive value of 97.2%. α-Fetoprotein, ß-human chorionic gonadotropin, and lactate dehydrogenase had sensitivities of less than 50% in seminoma and slightly higher sensitivities in nonseminomas. miR levels were significantly associated with clinical stage, primary tumor size, and response to treatment. Relapses had elevated miR levels that subsequently dropped to normal upon remission. Teratoma did not express miR-371a-3p. CONCLUSION: The M371 test outperforms the classic markers of GCT with both a sensitivity and a specificity greater than 90%. All histologic subgroups, except teratoma, express this marker. The test could be considered for clinical implementation after further validation.

Seminoma Clinical Stage 1 - Patterns of Care in Germany.

INTRODUCTION: International guidelines are ambivalent regarding the recommendations for the management of clinical stage 1 (CS1) seminoma. PATIENTS AND METHODS: During 2008-2013, 1,050 patients with seminoma CS1 were prospectively registered with regard to assessing management modality (radiotherapy, carboplatin, surveillance). Associations with tumor size, rete testis invasion, age, year of diagnosis, type of institution, and geographic location were assessed. RESULTS: Of the total number of patients, 49.3% received carboplatin 1 course, 8.5% carboplatin 2 courses, 35.9% surveillance, and 6.3% radiotherapy. In 2013, surveillance increased significantly to 57.9%. Treatment decisions were significantly associated with rete testis invasion and tumor size. Carboplatin was applied significantly more in office clinics than elsewhere. There is some regional variation regarding treatment preferences. CONCLUSIONS: The rising acceptance of surveillance mirrors international trends. The associations with prognostic factors demonstrate care givers to be compliant with contemporary guidelines. The association with the type of institution suggests non-oncological factors to be also relevant in decision making.

Targeting mTOR and AREG with everolimus, sunitinib and sorafenib in HPV-positive and -negative SCC.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm appearing in the upper aerodigestive tract and the sixth most common cancer worldwide. The five-year survival rate remains poor despite advances in surgery, radiation and chemotherapy. Furthermore, the incidence of human papillomavirus (HPV)-associated oropharyngeal cancer is rising. Thus, innovative therapy approaches are imperative in order to improve the situation. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) and sorafenib and sunitinib, multityrosine kinase inhibitors, have been notably effective in the therapy of different tumor entities. The modest side-effects and oral application of the drugs might improve patient compliance. Expression levels of mTOR and Amphiregulin (AREG) in p16-positive and -negative SCC (squamous cell carcinoma) and the effect of everolimus, sorafenib or sunitinib on the expression levels of these target proteins were assessed. As far as we are aware of, this is one of the first in vitro studies to evaluate the effect of these small-molecule drugs with regard to the p16 status of SCC cells. MATERIALS AND METHODS: p16-negative HNSCC 11A and 14C cells and p16-positive CERV196 cells were exposed to different concentrations of everolimus, sorafenib and sunitinib for 2-8 days. Expression levels of mTOR and AREG were determined by enzyme-linked immunosorbent assay (ELISA) and compared against a chemonaïve control. RESULTS: AREG and mTOR were expressed in all tested cell lines. CERV196 displayed a remarkable increase of mTOR expression compared to p16-negative HNSCC. On the contrary, AREG levels were reduced by 50% in CERV196. Everolimus, sorafenib and sunitinib significantly reduced mTOR expression. Everolimus significantly decreased AREG expression independently of the HPV status. Sunitinib and sorafenib increased AREG expression in HNSCC 11A and 14C but not in CERV196. CONCLUSION: The applied drugs showed remarkable suppression of mTOR expression, which might delay tumor progression. Interestingly, sorafenib and sunitinib increased AREG in HNSCC 11A and 14C, which could be a possible evasive mechanism following incubation with these drugs. On the contrary, p16-positive CERV196 showed increased susceptibility to sorafenib and sunitinib concerning suppression of AREG expression. Further studies are required to evaluate the HPV-dependent differences of therapy response and the possible consequences for treatment options.

Small molecules alter VEGFR and PTEN expression in HPV-positive and -negative SCC: new hope for targeted-therapy.

BACKGROUND/AIM: Prognosis for patients with head and neck squamous cell carcinoma (HNSCC) is poor in most cases and has not improved despite advances in therapy. Novel therapeutic approaches are mandatory in order to improve the situation. Everolimus, an inhibitor of mammalian target of rapamycin, as well as the multi-tyrosine kinase inhibitors sorafenib and sunitinib, has demonstrated a substantial therapeutic effect in various types of human cancer with moderate side-effects. Expression of vascular endothelial growth factor receptor (VEGFR) 1 and 2, and of the tumor-suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were evaluated in chemonaïve human papillomavirus (HPV)-positive and -negative squamous cell carcinoma (SCC) and after exposure to everolimus, sorafenib or sunitinib. MATERIALS AND METHODS: p16-positive CERV196 and p16-negative HNSCC 11A and 14C cells were incubated with different drug concentrations for 48-192 h. Expression of VEGFR1 and -2 as well as PTEN were determined by enzyme-linked immunosorbent assay and was compared to a chemonaïve control. RESULTS: VEGFR1 and -2, as well as PTEN, were expressed in all three cell lines. Sunitinib, sorafenib and everolimus significantly reduced the expression of VEGFR1 and -2, especially in p16-positive CERV196 cells. Sunitinib appeared to be more effective in reducing VEGFR1 and -2 expression than sorafenib and everolimus. PTEN levels were remarkably lower in HPV-positive CERV196 cells. PTEN expression increased significantly under sunitinib and sorafenib in HNSCC 11A and CERV196 cells. Everolimus, on the other hand, led to a significant decrease of PTEN expression in these cell lines. CONCLUSION: The tested drugs displayed a remarkable anti-angiogenic effect by inhibition of VEGFR1 and -2 expression. Sunitinib and sorafenib were able to increase PTEN expression, which might induce apoptosis of cancer cells. HPV-positive CERV196 cells were characterized by an increased susceptibility to these small-molecule drugs. Further studies are imperative to scrutinize HPV status-dependent differences in drug response and possible implications for future treatment options.

Matrix metalloproteinase-2 and -14 in p16-positive and -negative HNSCC after exposure To 5-FU and docetaxel In Vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world. While the incidence of HNSCC associated with tobacco and alcohol abuse is falling, the incidence of HNSCC associated with human papilloma virus (HPV) is rising. Proliferation, cell migration and formation of metastases are dependent on interactions between the tumor cells, tumor stromal cells and the extracellular matrix (ECM). Degradation of the ECM is a crucial step in the process of local tumor infiltration and formation of locoregional and distant metastases. Matrix metalloproteinases (MMPs) are a family of enzymes that are able to degrade the ECM. Locally advanced HNSCC with cervical node metastases are treated with docetaxel in induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU) as standard clinical anti-neoplastic regimens. This study evaluated the expression of MMP-14 and MMP-2 in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the alteration of expression levels after exposure to either docetaxel or 5-FU. MATERIALS AND METHODS: Tumor cells were exposed to 5-FU or docetaxel in concentrations of 1.0 and 5.0 µmol/ml. MMP-protein expression was evaluated by enzyme-linked immunosorbent assay (ELISA) after 2, 3, 5, 8 and 10 days of incubation. RESULTS: Docetaxel exposure significantly decreased MMP-14 expression in HNSCC 11A and especially 14C but not in CERV196 apart from an apoptotic process. 5-FU had no significant effect on MMP-14 expression independent of the HPV-status. Significant alterations of MMP-2 could be detected in HNSCC 11A only. CONCLUSION: Although neither of the applied drugs were selective inhibitors of MMP-expression, surprisingly docetaxel significantly decreased MMP-14 in HNSCC 14C and 11A in this study. Interestingly, HPV-positive CERV196 was not sensitive to decreased MMP-14 or -2 expression following incubation with 5-FU or docetaxel.

Unexpected alteration of ß-catenin and c-KIT expression by 5-FU and docetaxel in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. In several tumour entities, the tyrosine kinase receptor c-KIT is associated with tumour transformation in the epithelial tissue in cases of aberrant expression. Furthermore, tumour development and dissemination are a result of dysregulated cellular pathways such as the WNT/ß-catenin pathway. ß-Catenin is a multifunctional protein within the canonical WNT signalling pathway and a pivotal factor for the stabilization of cell-cell interactions. In malignant tissues, ß-catenin triggers tumour proliferation and progression. The aim of this study is to investigate the expression patterns of c-KIT and ß-catenin in human papillomavirus-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutical agents docetaxel and 5-fluorouracil (5-FU). MATERIALS AND METHODS: We incubated the tumour cell lines with docetaxel (5 µmol/ml) and 5-FU (1 µmol/ml) and detected ß-catenin and c-KIT by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. RESULTS: We found a reliable trend towards decreased ß-catenin expression levels in p16-positive and p16-negative tumour cell lines when incubated with docetaxel, in addition to induced apoptotic effect. At best, 5-FU had a slight influence on the alteration of the expression of ß-catenin. Dose escalation of docetaxel and 5-FU had no statistically significant effect on the expression of ß-catenin or c-KIT. In HPV-negative HNSCC, a reduced expression level of ß-catenin and c-KIT was detected in an incubation period-dependent manner. p16-transformed SCC (CERV196) cells were characterized by a reduced susceptibility to docetaxel induced alteration of ß-catenin expression. CONCLUSION: We were unable to confirm the clinically-substantiated increased chemosensitivity of p16-positive tumour cells in vitro. Extended studies and clinical trials are needed to investigate these findings further in HPV-associated HNSCC.

Chemotherapeutic alteration of VEGF, PDGF and PDGFRα/ß expression under 5-FU vs. docetaxel in HPV-transformed squamous cell carcinoma compared to HPV-negative HNSCC in vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the most common malignant epithelial tumor in the upper aerodigestive tract. The incidence of HNSCC induced by the oncogenic human papilloma virus (HPV) is rising, indicating a growing importance of the viral etiology. Cell proliferation, migration and tumor vascularization are regulated by a set of angiogenic peptides such as PDGF (platelet-derived growth factor), PDGFRα/ß (platelet-derived growth factor receptor α/ß) and VEGF (vascular endothelial growth factor). In locally advanced HNSCC docetaxel is used for induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU). This study sought to evaluate the expression of angiogenic factors (VEGF, PDGF and PDGFRα/ß) in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the efficacy of chemotherapy with docetaxel as a potential treatment modality, compared to 5-FU as a single-drug application. MATERIALS AND METHODS: Tumor cell lines were incubated with 5-FU or docetaxel at a concentration of 1.0 and 5.0 µmol/ml. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical analyses were carried out after 48, 72, 120, 192 and 240 hours, in order to identify changes in protein expression of VEGF, PDGF and PDGFRα/ß. RESULTS: We demonstrated a significant reduction of VEGF and PDGFRß expression after incubation with docetaxel by ELISA and of PDGF by immunohistochemistry, irrespective of the HPV status, whereas the application of 5-FU had a significantly weaker impact on the expression of angiogenic peptides. HPV-positive CERV196 cells were characterized by a reduced susceptibility to a docetaxel-altered expression. CONCLUSION: Although neither of the applied drugs are selective anti-angiogenic agents, docetaxel surprisingly was demonstrated to cause a significant decrease of angiogenic factors in this study.