Real-World Application of Pre-Orchiectomy miR-371a-3p Test in Testicular Germ Cell Tumor Management.

PURPOSE: Current serum tumor markers for testicular germ cell tumor are limited by low sensitivity. Growing evidence supports the use of circulating miR-371a-3p as a superior marker for malignant (viable) germ cell tumor management. We evaluated the real-world application of serum miR-371a-3p levels in detecting viable germ cell tumor among patients undergoing partial or radical orchiectomy. MATERIALS AND METHODS: Serum samples were collected from 69 consecutive patients before orchiectomy. Performance characteristics of serum miR-371a-3p were compared with conventional serum tumor markers (⍺-fetoprotein/ß-human chorionic gonadotropin/lactate dehydrogenase) between patients with viable germ cell tumor and those without viable germ cell tumor on orchiectomy pathology. Relative miR-371a-3p levels were correlated with clinical course. The Kruskal-Wallis test and linear and ordinal regression models were used for analysis. RESULTS: For detecting viable germ cell tumor, combined conventional serum tumor markers had a specificity of 100%, sensitivity of 58% and AUC of 0.79. The miR-371a-3p test showed a specificity of 100%, sensitivity of 93% and AUC of 0.978. Median relative expression of miR-371a-3p in viable germ cell tumor cases was more than 6,800-fold higher than in those lacking viable germ cell tumor. miR-371a-3p levels correlated with composite stage (p=0.006) and, among composite stage I cases, independently associated with embryonal carcinoma percentage (p=0.0012) and tumor diameter (p <0.0001). Six patients underwent orchiectomy after chemotherapy and were correctly predicted to have presence or absence of viable germ cell tumor by the miR-371a-3p test. CONCLUSIONS: If validated, the miR-371a-3p test can be used in conjunction with conventional serum tumor markers to aid clinical decision making. A positive miR-371a-3p test in patients after preoperative chemotherapy or with solitary testes could potentially guide subsequent orchiectomy or observation.

High-throughput oncogene mutation profiling in human cancer.

Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.

Sun exposure related methylation in malignant and non-malignant skin lesions.

We investigated the aberrant promoter methylation status of 12 genes in skin lesions, both malignant (basal cell carcinomas (BCCs), n=68 and squamous cell carcinomas (SCCs), n=35) and non-malignant (tags, n=58) skin lesions and compared the results of lesions from sun exposed (SE) and sun protected (SP) regions. Methylation was studied using a methylation specific PCR (MSP) and methylation of CDH1 was also measured using a semi-quantitative fluorescence based real-time MSP method. The methylation index (MI) was calculated as the methylated fraction of the genes examined. In this report, we found high frequencies of methylation of several known or suspected tumor suppressor genes in tags and skin cancers. Among the 12 genes, for the cadherin genes CDH1 and CDH3 and for two of the laminin 5 encoding genes LAMA3 and LAMC2 methylation frequencies greater than 30% were noted in one or more specimen types. We investigated whether methylation was tumor related. Surprisingly, the differences in the methylation profile of genes among the three specimen types were modest, and the MI, indicators of overall methylation frequencies, was nearly identical. However, significant differences were noted in the frequencies of methylation among the three specimen types for the genes RASSF1A (P=0.002), CDH1 (P=0.007) and one or more of three CAD genes (P=0.02). Methylation was highly significantly related to sun exposure, and sun protected specimens had little or no methylation. As methylation of CDH1 was completely SE specific we analyzed all the skin samples using a semi-quantitative real-time PCR assay for the CDH1 gene. The concordance between standard MSP and real-time MSP for all the samples (n=161) was 75% (P<0.0001). While weak signals were detected in the SP samples by real time PCR, the differences between SE and SP specimens were 148 fold for tags and 390 fold for BCCs. These differences were highly significant (P<0.0001). These findings suggest that methylation commences in UV exposed skin at a relatively early age and occurs in skin prior to the onset of recognizable preneoplastic changes.

An evaluation of cobalt chloride as an O2-sensitive chemoreceptor stimulant in channel catfish.

The effects of cobalt chloride on heart rate, blood pressure, ventilatory frequency and opercular pressure amplitude in channel catfish, Ictalurus punctatus were measured to evaluate the potential of cobalt as a histochemical probe to study mechanisms of oxygen chemoreception, as well as assess the general effects of cobalt on the cardioventilatory physiology of fishes. Cobalt, like cyanide, has been previously used to stimulate oxygen chemoreceptors and hypoxic reflexes in mammals but there is little information on the cardioventilatory effects of cobalt on fish. Catfish were exposed to increasing concentrations (1-20 mg/kg) of cobalt in the water (external) or injections into the dorsal aorta (internal) and the cardioventilatory effects recorded. Mean arterial pressure showed a significant, dose-dependent increase in response to cobalt injections. Heart rate increased slowly, but significantly after cobalt injections but the magnitude of change was not dose-dependent. There was a small increase in ventilatory rate but no effect on amplitude. External cobalt had similar effects but the responses were weaker. Although cobalt stimulated some cardioventilatory reflexes the pattern and magnitude of the responses were noticeably different from those of cyanide and hypoxia. The results suggest that the cardioventilatory reflexes stimulated by cobalt were not mediated by O(2)-sensitive chemoreceptors and that cobalt is not an effective O(2) receptor stimulant in fishes.

Somatic mutations of the HER2 kinase domain in lung adenocarcinomas.

Mutations in the epidermal growth factor receptor gene (EGFR) in lung cancers predict for sensitivity to EGFR kinase inhibitors. HER2 (also known as NEU, EGFR2, or ERBB2) is a member of the EGFR family of receptor tyrosine kinases and plays important roles in the pathogenesis of certain human cancers, and mutations have recently been reported in lung cancers. We sequenced the tyrosine kinase domain of HER2 in 671 primary non-small cell lung cancers (NSCLC), 80 NSCLC cell lines, and 55 SCLCs and other neuroendocrine lung tumors as well as 85 other epithelial cancers (breast, bladder, prostate, and colorectal cancers) and compared the mutational status with clinicopathologic features and the presence of EGFR or KRAS mutations. HER2 mutations were present in 1.6% (11 of 671) of NSCLC and were absent in other types of cancers. Only one adenocarcinoma cell line (NCI-H1781) had a mutation. All HER2 mutations were in-frame insertions in exon 20 and target the identical corresponding region as did EGFR insertions. HER2 mutations were significantly more frequent in never smokers (3.2%, 8 of 248; P=0.02) and adenocarcinoma histology (2.8%, 11 of 394; P=0.003). In 394 adenocarcinoma cases, HER2 mutations preferentially targeted Oriental ethnicity (3.9%) compared with other ethnicities (0.7%), female gender (3.6%) compared with male gender (1.9%) and never smokers (4.1%) compared with smokers (1.4%). Mutations in EGFR, HER2, and KRAS genes were never present together in individual tumors and cell lines. The remarkable similarities of mutations in EGFR and HER2 genes involving tumor type and subtype, mutation type, gene location, and specific patient subpopulations targeted are unprecedented and suggest similar etiologic factors. EGFR, HER2, and KRAS mutations are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers.