HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer.

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

Expression profiling on subclasses of primary parotid gland carcinomas.

INTRODUCTION: The underlying molecular mechanisms of parotid gland carcinomas (PGC) are still unknown. Knowledge about the tumor-driving signaling pathways is necessary either for diagnostics or developing new therapeutic options in this heterogeneous and rare entity. MATERIAL AND METHODS: 94 matching RNA formalin-fixed and paraffin-embedded tissue samples from PGC and the corresponding non-tumor area, RNA quality and quantity were sufficient for gene expression profiling of 770 genes using the NanoString's nCounter technology. Oncogenic and tumor suppressor genes were examined in the three common PGC tumor entities: adenoid cystic carcinoma (ACC), adenocarcinoma NOS (AC-NOS), and mucoepidermoid carcinoma (MEC). RESULTS: Expression profiling and subsequent hierarchical cluster analysis clearly differentiated between non-tumor gland tissue samples and PGC. In addition expression pattern of all three entities differed. The extensive pathway analysis proved a prominent dysregulation of the Wnt signaling pathway in the three PGC entities. Moreover, transcript upstream analysis demonstrated a pronounced activation of the PI3K pathway in ACC and MEC. DISCUSSION: Our findings revealed divergent molecular expression profiles in MEC, ACC and AC-NOS that are presently studied for their potential application in PGC diagnostics. Importantly, identification of Wnt and PI3K signaling in PGC revealed novel options of PGC therapy.

Evolution analysis of heterogeneous non-small cell lung carcinoma by ultra-deep sequencing of the mitochondrial genome.

Accurate assessment of tumour heterogeneity is an important issue that influences prognosis and therapeutic decision in molecular pathology. Due to the shortage of protective histones and a limited DNA repair capacity, the mitochondrial (mt)-genome undergoes high variability during tumour development. Therefore, screening of mt-genome represents a useful molecular tool for assessing precise cell lineages and tracking tumour history. Here, we describe a highly specific and robust multiplex PCR-based ultra-deep sequencing technology for analysis of the whole mt-genome (wmt-seq) on low quality-DNA from formalin-fixed paraffin-embedded tissues. As a proof of concept, we applied the wmt-seq technology to characterize the clonal relationship of non-small cell lung cancer (NSCLC) specimens with multiple lesions (N = 43) that show either different histological subtypes (group I) or pulmonary adenosquamous carcinoma as striking examples of a mixed-histology tumour (group II). The application of wmt-seq demonstrated that most samples bear common mt-mutations in each lesion of an individual patient, indicating a single cell progeny and clonal relationship. Hereby we show the monoclonal origin of histologically heterogeneous NSCLC and demonstrate the evolutionary relation of NSCLC cases carrying heteroplasmic mt-variants.

Expression of the apoptosis repressor with caspase recruitment domain (ARC) in liver metastasis of colorectal cancer and its correlation with DNA mismatch repair proteins and p53.

INTRODUCTION: Apoptotic signaling is one of the most important processes in the measurement of chemotherapeutic effectiveness. In apoptotic machinery, various pathways and proteins are involved (i.e., mismatch repair proteins, p53). One of the regulatory proteins is ARC, which can inhibit not only the extrinsic but also the intrinsic apoptotic signaling. MATERIALS AND METHODS: In this study, we investigated the expression levels of ARC in colorectal liver metastasis and compared them with the expression of mismatch repair proteins and p53. Furthermore, we investigated ARC expression level depending on sex, age, tumor grade, mucin production, tumor size and number of liver metastasis. RESULTS: ARC expression level in colorectal cancer liver metastasis was independent from clinical data (i.e., age, gender, tumor size, tumor number or mucin production) but strongly correlated with MSH2 and MSH6 expression, which further supported the evidence for the regulatory role of MSH2 and MSH6 in apoptosis; i.e., in case of sufficient MSH2 and MSH6 expression, significantly higher ARC level is required to suppress the apoptosis. A regulatory interaction between ARC and p53 has been described, but we found no correlation between p53 expression levels and ARC levels. CONCLUSION: Further studies are needed to define the exact role of ARC in apoptotic signaling and thus its role in chemoresistance and survival of tumor cells.

MDM2 gene amplification in esophageal carcinoma.

Esophageal cancer (EC) is one of the most common malignancies diagnosed in the Western world with an increasing incidence noted for esophageal adenocarcinoma (EAC). Despite improvements in staging, surgical procedures and postoperative treatments, the overall survival of patients with EC remains low. Murine double minute­2 (MDM2) acts as an oncogene by inducing the degradation of the tumor­suppressor protein TP53. In order to evaluate the MDM2 gene amplification status in EAC and squamous cell carcinoma (SCC), we established a quantitative PCR (qPCR) assay, screening a total of 127 esophageal carcinoma cases for MDM2 amplification. Esophageal carcinoma cases with enhanced MDM2 gene copy numbers were further analyzed by fluorescence in situ hybridisation (FISH) and MDM2 immunostaining. Among a total of 23 specimens (18%), identified by qPCR to possess elevated MDM2 gene copy numbers, one third (6.3%) showed a cluster­like fluorescence pattern by FISH analyses and marked MDM2 protein immunostaining. MDM2 gene amplifications did not correlate with the occurrence of TP53 mutations. Due to the high therapeutic relevance of MDM2 overexpression, but the high cost of FISH, we suggest a primary screening of MDM2 copy number variations by qPCR, followed by detailed FISH analysis of the identified ECs.

Targeted next generation sequencing of parotid gland cancer uncovers genetic heterogeneity.

Salivary gland cancer represents a heterogeneous group of malignant tumors. Due to their low incidence and the existence of multiple morphologically defined subtypes, these tumors are still poorly understood with regard to their molecular pathogenesis and therapeutically relevant genetic alterations.Performing a systematic and comprehensive study covering 13 subtypes of salivary gland cancer, next generation sequencing was done on 84 tissue samples of parotid gland cancer using multiplex PCR for enrichment of cancer related gene loci covering hotspots of 46 cancer genes.Mutations were identified in 22 different genes. The most frequent alterations affected TP53, followed by RAS genes, PIK3CA, SMAD4 and members of the ERB family. HRAS mutations accounted for more than 90% of RAS mutations, occurring especially in epithelial-myoepithelial carcinomas and salivary duct carcinomas. Additional mutations in PIK3CA also affected particularly epithelial-myoepithelial carcinomas and salivary duct carcinomas, occurring simultaneously with HRAS mutations in almost all cases, pointing to an unknown and therapeutically relevant molecular constellation. Interestingly, 14% of tumors revealed mutations in surface growth factor receptor genes including ALK, HER2, ERBB4, FGFR, cMET and RET, which might prove to be targetable by new therapeutic agents. 6% of tumors revealed mutations in SMAD4.In summary, our data provide novel insight into the fundamental molecular heterogeneity of salivary gland cancer, relevant in terms of tumor classification and the establishment of targeted therapeutic concepts.