Serum Levels of MicroRNA-371a-3p for Predicting the Histology of Postchemotherapy Residual Masses of Germ Cell Tumours.

BACKGROUND AND OBJECTIVE: Serum levels of microRNA-371a-3p (M371) represent a novel and sensitive biomarker of germ cell tumours (GCTs). This study analysed the utility of M371 to identify viable cancer (VC) in postchemotherapy (pc) residual masses with the underlying goal of avoiding overtreatment. METHODS: A multicentric, prospective diagnostic study was conducted in 180 GCT patients undergoing pc resection of residual masses. A correlation of M371 measurement results with the histological presence of VC in masses was found. A receiver operating characteristic analysis was performed for exploring the performance characteristics of the test. KEY FINDINGS AND LIMITATIONS: The sensitivity was found to be 68.9%, specificity 99.3%, area under the curve 0.813, positive predictive value 0.969, and negative predictive value 0.905; sensitivity is significantly associated with the percentage of VC in the mass. In specimens with ≤10% VC, there were 33.3% elevated M371 levels as opposed to 85.7% in specimens with >50% VC. Teratoma and somatic-type malignancy do not express M371. A lack of a central pathological review is a limitation. CONCLUSIONS AND CLINICAL IMPLICATIONS: The M371 test can identify 68.9% of patients with VC in pc masses. However, cases with <10% VC in the mass may escape detection. Teratoma does not express M371. The test alone cannot correctly identify patients requiring pc surgery, but it may be a tool for scheduling the extent of surgery. PATIENT SUMMARY: The microRNA-371a-3p (M371) test can identify about two-thirds of patients with viable cancer in residual metastatic masses following chemotherapy for germ cell tumours. Only masses with high percentages of viable cancer cells can be identified, and the histological subtype teratoma remains undetected with the test.

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.

MicroRNA-371a-3p-The Novel Serum Biomarker in Testicular Germ Cell Tumors.

INTRODUCTION: Testicular germ cell tumors (TGCTs) are a paradigm for the use of serum tumor markers in clinical management. However, conventional markers such as alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (hCG), and lactate dehydrogenase (LDH) have quite limited sensitivities and specificities. Within the last decade, the microRNA-371a-3p (miR371) emerged as a possible new biomarker with promising features. AREAS COVERED: This review covers the typical features as well as possible clinical applications of miR371 in TGCT patients, such as initial diagnosis, therapy monitoring, and follow-up. Additionally, technical issues are discussed. EXPERT OPINION: With a sensitivity of around 90% and specificity >90%, miR371 clearly outperforms the classical serum tumor markers in TGCTs. The unique features of the test involve the potential of modifying recent standards of care in TGCT. In particular, miR371 is expected to aid clinical decision-making in scenarios such as discriminating small testicular TGCT masses from benign ones prior to surgery, assessing equivocal lymphadenopathies, and monitoring chemotherapy results. Likewise, it is expected to make follow-up easier by reducing the intensity of examinations and by sparing imaging procedures. Overall, the data presently available are promising, but further prospective studies are required before the test can be implemented in standard clinical care.

Testicular neoplasms: the interrelationships of serum levels of microRNA-371a-3p (M371) and classical tumor markers with histology, clinical staging, and age-a statistical analysis.

PURPOSE: In testicular neoplasms, the interrelationship of elevations of the novel serum tumor marker microRNA-371a-3p (M371) and traditional markers with other clinical features is still incompletely understood. The present study evaluated marker expression rates in relation to various other clinical parameters. METHODS: The following data were retrospectively registered from 641 consecutive patients with testicular neoplasms: histology, such as seminoma (n = 365), nonseminoma (n = 179), benign tumor (n = 79), other malignant tumor (n = 18); patients age (years); clinical stage (CS1, CS2a/b, CS2c, CS3); and preoperative elevation of beta HCG, AFP, LDH, M371 (yes/no). Descriptive statistical methods were employed with comparisons of various subgroups to disclose associations of marker expression rates with age, histology and CS, and of age with histology. RESULTS: The histologic subgroups revealed significantly different expression rates of tumor markers. M371 performed best with expression rates of 82.69% and 93.58% in seminoma and in nonseminoma, respectively. In germ cell tumors, all markers had significantly higher expression rates in metastasized stages than in localized disease. All markers except LDH have significantly higher expression rates in younger than in older patients. Nonseminoma is most prevalent in the youngest age category, seminoma predominates in patients > 40 years, other malignancies were restricted to patients > 50 years. CONCLUSION: The study documented significant associations of serum marker expression rates with histology, age and clinical staging, with highest rates in nonseminomas, young age and advanced clinical stages. M371 showed significantly higher expression rates than other markers suggesting its superior clinical usefulness.

Testicular Neoplasms: Primary Tumour Size Is Closely Interrelated with Histology, Clinical Staging, and Tumour Marker Expression Rates-A Comprehensive Statistical Analysis.

The role of primary tumour size (TS) in the clinical course of testicular tumours is incompletely understood. We retrospectively evaluated 641 consecutive patients with testicular neoplasms with regard to TS, histology, clinical stage (CS), serum tumour marker (STM) expression and patient age using descriptive statistical methods. TS ≤ 10 mm was encountered in 13.6% of cases. Median TS of 10 mm, 30 mm, 35 mm, and 53 mm were found in benign tumours, seminomas, nonseminomas, and other malignant tumours, respectively. In cases with TS ≤ 10 mm, 50.6% had benign tumours. Upon receiver operating characteristics analysis, TS of > 16 mm revealed 81.5% sensitivity and 81.0% specificity for detecting malignancy. In subcentimeter germ cell tumours (GCTs), 97.7% of cases had CS1, and CS1 frequency dropped with increasing TS. Expression rates of all STMs significantly increased with TS. MicroRNA-371a-3p (M371) serum levels had higher expression rates than classical STMs, with a rate of 44.1% in subcentimeter GCTs. In all, TS is a biologically relevant factor owing to its significant associations with CS, STM expression rates and histology. Importantly, 50% of subcentimeter testicular neoplasms are of benign nature, and M371 outperforms the classical markers even in subcentimeter tumours.

Serum Levels of MicroRNA-371a-3p (M371) Can Predict Absence or Presence of Vital Disease in Residual Masses After Chemotherapy of Metastatic Seminoma.

Background: Radiological evaluation of postchemotherapy residual masses of metastatic seminoma is characterized by poor diagnostic accuracy. Serum levels of microRNA-371a-3p (M371) involve high specificity and sensitivity for the primary diagnosis of seminoma. We evaluated if M371 levels can indicate the presence of vital disease in postchemotherapy residual masses in patients with metastatic seminoma. Methods: Twenty-three seminoma patients (median age 52 years) with residual masses had posttreatment measurements of serum M371 levels (group A), fourteen of whom had measurements also beforehand. The posttreatment results were compared with the clinical outcome during follow-up. Eleven patients with complete remission after treatment of metastatic seminoma (group B) and 33 men with non-malignant testicular diseases (group C) served as controls. M371 serum levels were measured by quantitative real-time PCR using miR-30b-5p as endogenous control. An evaluation was performed with descriptive statistical methods. Results: Twenty-two patients of Group A had uneventful follow-up so far, twenty-one of whom had M371 level <5, and one other had a mildly elevated level below relative quantity (RQ) = 10. One patient with a level of RQ = 26.2 rapidly progressed. The median posttreatment M371 level of the non-progressing patients of group A is not significantly different from the median level of the control group with complete remission (B). Before treatment, the median M371 levels in groups A and B were 507.6 and 143.9, respectively. In both groups, significant drops in M371 levels resulted from treatment. Conclusion: Normal M371 serum levels at the time of completion of treatment of metastatic seminoma indicate the absence of vital seminoma in residual masses, while elevated levels >RQ = 10 predict the presence of disease. The optimal timing of M371 measurement after chemotherapy and the appropriate cutoff level still need to be determined. Based on the present results, measuring serum M371 levels involves the potential of a novel tool for assessing postchemotherapy residual masses of metastatic seminoma.

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.

A Multi-institutional Pooled Analysis Demonstrates That Circulating miR-371a-3p Alone is Sufficient for Testicular Malignant Germ Cell Tumor Diagnosis.

BACKGROUND: Circulating microRNAs have clear potential for improving malignant germ-cell-tumor (MGCT) diagnosis. Here, we address the central issue of whether measurement of a single microRNA is sufficient for detecting testicular MGCTs, or whether there is added benefit in quantifying other members of the 4-microRNA panel previously identified (miR-371a-3p/miR-372-3p/miR-373-3p and miR-367-3p). PATIENTS AND METHODS: We performed a pooled analysis of available published raw data where all 4 panel miRNAs had been assessed using pre-amplification PCR technology (4 studies; total 329 patients). Two studies using identical methodology (and identical normalization using endogenous miR-30b-5p) were used in the discovery phase (n = 51 patients: 17 MGCT, 34 controls). The 2 other studies (n = 278 patients: 140 MGCT, 138 controls), which assessed the same test panel but with different normalization approaches (endogenous miR-93-5p, exogenous cel-miR-39-3p), were used for the validation phase. We derived sensitivity, specificity, positive- and negative-predictive-values (PPV/NPV) for the detection thresholds that maximised the Youden Index (YI). RESULTS: In the discovery-phase, the YI was 0.97 for miR-371a-3p (sensitivity = 1, specificity = 0.97), 0.71 (miR-367-3p), 0.68 (miR-372-3p), and 0.50 (miR-373-3p). These findings were confirmed in the validation-phase, with YI of 0.75 for miR-371a-3p (sensitivity = 0.90, specificity 0.85), 0.55 (miR-367-3p), 0.47 (miR-372-3p), and 0.51 (miR-373-3p). Importantly, no combination of markers added additional diagnostic benefit to miR-371a-3p alone, in either the discovery or the validation phase. CONCLUSION: Quantifying circulating miR-371a-3p alone is sufficient for testicular MGCT diagnosis. PCR measurement of this single miRNA marker will be more cost-effective and easier to interpret, facilitating future incorporation into routine clinical practice.

Circulating MicroRNAs, the Next-Generation Serum Biomarkers in Testicular Germ Cell Tumours: A Systematic Review.

CONTEXT: Clinical management of testicular germ cell tumours (GCTs) is hampered by low sensitivity and specificity of the biomarkers currently in use. Circulating microRNAs (miRs) might offer the potential to address areas of unmet clinical need. OBJECTIVE: To systematically evaluate the evidence for clinical applications of serum levels of miR302/367 and miR371-3 in adult testicular GCTs in terms of primary diagnosis, various clinical scenarios, and the costs of clinical implementation. EVIDENCE ACQUISITION: We performed a critical review of PubMed/Medline, Embase and the Cochrane Library in January 2021 in accordance with Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement. EVIDENCE SYNTHESIS: Thirty-one manuscripts addressed miR performance and potential clinical use in testicular GCT. Of these, 23 evaluated the utility in primary diagnosis, seven in early-stage disease, and 13 in metastatic disease, and two addressed the costs of clinical implementation. Of the various miRs studied, miR-371a-3p appears the most useful and potentially the only one that needs to be assayed, with an area under the receiver operating characteristic curve >0.90, sensitivity of 89-96%, and specificity of >90% for both seminoma and nonseminoma, surpassing the classic serum tumour markers. The miRs studied to date are not elevated in cases with teratoma only. Levels of miR-371a-3p correlate with primary tumour mass, clinical stage, and International Germ Cell Cancer Collaborative Group risk groups. Serial measurements mirror treatment efficacy in all clinical stages. CONCLUSIONS: Circulating miRNA levels, particularly of miR-371a-3p, have potential for incorporation in clinical practice and may aid in clinical decision-making in various clinical scenarios in GCT. PATIENT SUMMARY: We analysed the current evidence on the usefulness of blood levels of molecules called microRNAs in the management of testicular tumours. The microRNA-371a-3p molecule has better sensitivity and specificity than the markers currently being measured. This new biomarker may soon have a place in clinical practice.

Serum levels of microRNA-371a-3p are not elevated in testicular tumours of non-germ cell origin.

PURPOSE: Serum levels of microRNA-371a-3p (M371) have been shown to be a highly sensitive and specific biomarker for testicular germ cell tumours (TGCT). Little information exists on the expression of this marker in testicular neoplasms deriving from the gonadal stroma or other structures of the gonad. This study presents an expression analysis of the novel TGCT-biomarker M371 in a large cohort of testicular non-germ cell tumours. METHODS: The M371 expression was measured by quantitative real time PCR in serum of 99 patients with testicular tumours of non-germ cell origin, thereof 30 patients with malignant testicular lymphomas and 61 patients with gonadal stroma tumours such as Leydig cell tumours, Sertoli cell tumours and 8 cases with miscellaneous benign testicular tumours. Their M371 levels were compared to those of 20 patients with TGCT and to 37 tumour-free male controls. RESULTS: The median expression levels of benign testicular tumours and testicular lymphoma are close to zero, thus, identical with those of controls and significantly lower than those of TGCT. In summary, this study provides further evidence for the notion that M371 is exclusively expressed by germ cell tumours and not by testicular neoplasms of the non-germ cell subtypes. CONCLUSION: Clinically, the test might be of value in preoperative characterization of benign testicular tumours eligible for conservative surgery.

High Expression of microRNA-371a-3p in Cystic Fluid of Post-Chemotherapy Teratoma with Concurrent Normal Serum Levels in Patients with Non-Seminomatous Testicular Germ Cell Tumours.

BACKGROUND: MicroRNA-371a-3p (miR-371), the novel serum biomarker of testicular germ cell tumours (GCTs), is produced by undifferentiated subtypes of GCTs but not by teratoma. Cystic teratoma developing from retroperitoneal metastases of GCT subsequent to chemotherapy had been shown to contain high levels of classical serum tumour markers of GCT in the presence of normal marker levels in serum. To date, no information is available regarding the presence of miR-371 in the cystic fluid of residual teratoma after chemotherapy. METHODS: Four patients (age 18-26 years) undergoing retroperitoneal lymph node dissection (RPLND) for cystic residual masses resulting from chemotherapy of bulky retroperitoneal GCT had measurements of miR-371 in both serum and cystic fluid aspirated from surgical specimens. Measurement of the miR was performed with quantitative real-time PCR using miR-30b-5p as reference. Results were tabulated and analysed in a descriptive manner. RESULTS: Histologically, all of the surgical specimens involved teratoma only with no evidence of vital undifferentiated GCT tissue. All patients were cured. Prior to RPLND, miR-371 serum levels were not measurable or close to zero in all of the patients. Cystic fluid revealed elevated levels of miR-371 in 3 patients and traces of miR in one. CONCLUSIONS: The detection of miR-371 in the cystic fluid of teratoma is somewhat enigmatic since this GCT subtype usually does not express the miR. Two hypotheses may explain the finding: First, miR-371 molecules were released into the cystic fluid by active GCT tissue prior to chemotherapy. High levels were kept after regression of vital GCT tissue because the cystic lumen is without a specific drainage system. Second, teratoma cells lining the interior cyst wall may shed small amounts of miR-371 into the lumen. Because of the lacking drainage system, even small levels may accumulate. The present finding adds to the understanding of the biology of the novel biomarker of GCT.

Graded expression of microRNA-371a-3p in tumor tissues, contralateral testes, and in serum of patients with testicular germ cell tumor.

Background: Serum levels of microRNA-371a-3p represent a specific tumor marker of testicular germ cell tumors (GCTs) but the origin of circulating miR-371a-3p is not finally resolved. The correlation between miR-levels in tissue and serum is unclear. Results: MiR-levels in GCT tissue are 399-fold higher than in contralateral testicular tissue and 5843-fold higher than in non-testicular tissue. MiR tissue levels correlate with corresponding serum levels (r 2 = 0.181). ISH detected miR-371a-3p intracellularly in GCT cells except teratoma. A low expression was also detected in normal testicular germ cells. Conclusions: Circulating miR-371a-3p is specifically derived from GCT tissue. The miR is present in GCT cells except teratoma. A low expression is also found in normal testicular tissue but not in non-testicular tissue. MiR-371a-3p levels in tissue and serum correlate significantly. This study underscores the usefulness of serum miR-371a-3p as tumor marker of GCT. Patients and methods: Expression levels of miR-371a-3p were concurrently measured in tissues of GCT, contralateral testes ( n = 38), and in serum ( n = 36) with real time PCR. For control, 5 healthy testicles and 4 non-testicular tissue samples were examined. MiR-levels were compared using descriptive statistical methods. We also performed in situ hybridization (ISH) of GCT tissue with a probe specific for miR-371a-3p.

Non-Coding microRNAs as Novel Potential Tumor Markers in Testicular Cancer.

Testicular cancer is an important disease with increasing incidence and a high burden of morbidity and mortality in young men worldwide. Histological examination of the testicular tissue after orchiectomy plays an important role alongside patient history, imaging, clinical presentation and laboratory parameters. Surgical procedures and chemotherapeutic treatment provide a high chance of cure in early stages, though some patients in advanced stages belonging to a poor risk group experience cancer-related death. Though conventional serum-based tumor markers, including α-fetoprotein (AFP), the ß-subunit of human chorionic gonadotropin (ß-hCG), and lactate dehydrogenase (LDH), are useful as prognostic and diagnostic biomarkers, unfortunately, these tumor markers only have a sensitivity of about 60%, and in pure seminoma even lower with about 20%. Therefore, the development of new tumor markers is an important and intensively ongoing issue. The analysis of epigenetic modification and non-coding RNA microRNAs (miRNAs) are carrying most promising potential as tumor markers in future. miRNAs are small RNAs secreted by testicular tumor cells and circulate and be measurable in body fluids. In recent years, miRNAs of the miR-371-373 cluster in particular have been identified as potentially superior tumor markers in testicular cancer patients. Studies showed that miR-371a-3p and miR-302/367 expression significantly differ between testicular tumors and healthy testicular tissue. Several studies including high prospective multi-center trials clearly demonstrated that these miRNAs significantly exceed the sensitivity and specificity of conventional tumor markers and may help to facilitate the diagnosis, follow-up, and early detection of recurrences in testicular cancer patients. In addition, other miRNAs such as miR-223-3p, miR-449, miR-383, miR-514a-3p, miR-199a-3p, and miR-214 will be discussed in this review. However, further studies are needed to identify the value of these novel markers in additional clinical scenarios, including the monitoring in active surveillance or after adjuvant chemotherapy, but also to show the limitations of these tumor markers. The aim of this review is to give an overview on the current knowledge regarding the relevance of non-coding miRNAs as biomarkers in testicular cancer.

Application of miRNAs in the diagnosis and monitoring of testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men and originate from the common precursor germ cell neoplasia in situ (GCNIS). For decades, clinical management of patients with TGCT has relied on classic serum tumour markers: α-fetoprotein, human chorionic gonadotropin subunit-ß and lactate dehydrogenase. In the past 10 years, microRNAs have been shown to outperform classic serum tumour markers in the diagnosis of primary tumours and in follow-up monitoring and prediction of relapse. miR-371a-3p is the most consistent marker and exhibits >90% diagnostic sensitivity and specificity in TGCT. However, miR-371a-3p cannot be used to diagnose GCNIS or mature teratoma. Future efforts must technically standardize the microRNA-based methods internationally and introduce miR-371a-3p as a molecular liquid biopsy-based marker for TGCTs in the clinic.

Lung Surfactant Accelerates Skin Wound Healing: A Translational Study with a Randomized Clinical Phase I Study.

Lung surfactants are used for reducing alveolar surface tension in preterm infants to ease breathing. Phospholipid films with surfactant proteins regulate the activity of alveolar macrophages and reduce inflammation. Aberrant skin wound healing is characterized by persistent inflammation. The aim of the study was to investigate if lung surfactant can promote wound healing. Preclinical wound models, e.g. cell scratch assays and full-thickness excisional wounds in mice, and a randomized, phase I clinical trial in healthy human volunteers using a suction blister model were used to study the effect of the commercially available bovine lung surfactant on skin wound repair. Lung surfactant increased migration of keratinocytes in a concentration-dependent manner with no effect on fibroblasts. Significantly reduced expression levels were found for pro-inflammatory and pro-fibrotic genes in murine wounds. Because of these beneficial effects in preclinical experiments, a clinical phase I study was initiated to monitor safety and tolerability of surfactant when applied topically onto human wounds and normal skin. No adverse effects were observed. Subepidermal wounds healed significantly faster with surfactant compared to control. Our study provides lung surfactant as a strong candidate for innovative treatment of chronic skin wounds and as additive for treatment of burn wounds to reduce inflammation and prevent excessive scarring.

Serum Level of microRNA-375-3p Is Not a Reliable Biomarker of Teratoma.

BACKGROUND/AIM: Clinical management of testicular germ cell tumours (GCT) is based upon the measurement of serum tumour markers. Recent studies have shown that the microRNA-371a-3p is a sensitive and specific serum biomarker for all subgroups of GCT, except teratoma. To close the diagnostic gap relating to teratoma, serum levels of microRNA-375-3p have recently been suggested to represent a specific serum marker of this histological subgroup. In the present study, we tested this hypothesis. MATERIALS AND METHODS: miRNA expression was analysed in serum of 21 GCT patients with teratoma, twelve patients with other GCT, and twelve male controls using the qPCR method. RESULTS: The serum miR-375-3p levels of teratoma patients were not different from other GCT patients or controls. The ROC analysis revealed an AUC of 0.524 for the discrimination between teratoma and other pathologies. CONCLUSION: The miR-375-3p does probably not qualify for a useful serum biomarker to distinguish teratoma from other GCTs and from controls.

Matrix Metalloproteinase-3 is Key Effector of TNF-α-Induced Collagen Degradation in Skin.

Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (p < 0.001) in KO skin explants compared to WT control skin but did not differ (p = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (p = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.

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.

Mechanical and migratory properties of normal, scar, and Dupuytren's fibroblasts.

Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor-ß1 (TGF-ß1) is known to induce expression of α-smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF-ß1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF-ß1 enhances the level of α-smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.

The Novel Biomarker of Germ Cell Tumours, Micro-RNA-371a-3p, Has a Very Rapid Decay in Patients with Clinical Stage 1.

BACKGROUND: Accumulating evidence suggests serum levels of microRNA (miR)-371a-3p to be a novel tumour marker of testicular germ cell tumours (GCTs). Presently, there is only limited information regarding the velocity of decline of serum levels in response to treatment. PATIENTS AND METHODS: Twenty-four patients with testicular GCT (20 seminoma, 4 nonseminoma, median age 40 years) with clinical stage 1 had measurements of serum levels of miR-371a-3p preoperatively and repeatedly on the following 3 days. Three had additional tests done within 24 h after surgery. Measurement results were analysed using descriptive statistical methods. RESULTS: Serum levels dropped to 2.62, 1.27, and 0.47% of the preoperative level within 1, 2, and 3 days, respectively. The computed half-life amounts to 3.7-7 h. The velocity of decay is significantly associated with tumour size. CONCLUSIONS: Serum-levels of miR-371a-3p have a short half-life of less than 12 h. The rapid decay after treatment represents a valuable feature confirming the usefulness of miR-371a-3p as a valuable serum biomarker of GCT.