A clinically applicable molecular classification of oncocytic cell thyroid nodules

Whole chromosome instability with near-whole genome haploidization (GH) and subsequent endoreduplication is considered a main genomic driver in the tumorigenesis of oncocytic cell thyroid neoplasms (OCN). These copy number alterations (CNA) occur less frequently in oncocytic thyroid adenoma (OA) than in oncocytic carcinoma (OCA), suggesting a continuous process. The current study described the CNA patterns in a cohort of 30 benign and malignant OCN, observed using a next-generation sequencing (NGS) panel that assesses genome-wide loss of heterozygosity (LOH) and chromosomal imbalances using 1500 single-nucleotide polymorphisms (SNPs) across all autosomes and the X chromosome in DNA derived from cytological and histological samples. Observed CNA patterns were verified using multiparameter DNA flow cytometry with or without whole-genome SNP array analysis and lesser-allele intensity-ratio (LAIR) analysis. On CNA–LOH analysis using the NGS panel, GH-type CNA were observed in 4 of 11 (36%) OA and in 14 of 16 OCA (88%). Endoreduplication was suspected in 8 of 16 (50%) OCA, all with more extensive GH-type CNA (P &lt; 0.001). Reciprocal chromosomal imbalance type CNA, characterized by (imbalanced) chromosomal copy number gains and associated with benign disease, were observed in 6 of 11 (55%) OA and one equivocal case of OCA. CNA patterns were different between the histopathological subgroups (P &lt; 0.001). By applying the structured interpretation and considerations provided by the current study, CNA–LOH analysis using an NGS panel that is feasible for daily practice may be of great added value to the widespread application of molecular diagnostics in the diagnosis and risk stratification of OCN.</p

High-resolution analysis of HLA class I alterations in colorectal cancer

BACKGROUND: Previous studies indicate that alterations in Human Leukocyte Antigen (HLA) class I expression are frequent in colorectal tumors. This would suggest serious limitations for immunotherapy-based strategies involving T-cell recognition. Distinct patterns of HLA surface expression might conceal different immune escape mechanisms employed by the tumors and are worth further study. METHOD: We applied four-color multiparameter flow cytometry (FCM), using a large panel of alloantigen-specific anti-HLA-A and -B monoclonal antibodies, to study membranous expression of individual HLA alleles in freshly isolated colorectal cancer cell suspensions from 21 patients. RESULTS: Alterations in HLA class I phenotype were observed in 8 (38%) of the 21 tumors and comprised loss of a single A or B alleles in 4 cases, and loss of all four A and B alleles in the other 4 cases. Seven of these 8 tumors were located on the right side of the colon, and those showing loss of both HLA-A and -B membranous expression were all of the MSI-H phenotype. CONCLUSION: FCM allows the discrimination of complex phenotypes related to the expression of HLA class I. The different patterns of HLA class I expression might underlie different tumor behavior and influence the success rate of immunotherapy

Genome Haploidisation with Chromosome 7 Retention in Oncocytic Follicular Thyroid Carcinoma

Contains fulltext : 108012.pdf (publisher's version ) (Open Access)BACKGROUND: Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). METHODS: Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. RESULTS: All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). CONCLUSIONS: We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. MICROARRAYS: Data are made available at GEO (GSE31828)

Lack of TNFα mRNA expression in cervical cancer is not associated with loss of heterozygosity at 6p21.3, inactivating mutations or promoter methylation

Infection with oncogenic human papillomavirus (HPV) is considered to be the major etiologic event for cervical cancer. Tumor necrosis factor α (TNFα), a proinflammatory cytokine, may be involved in orchestrating an antitumor immune response against human papillomavirus expressing cervical cancer cells. Hence, loss of TNFα could be advantageous for tumor cells to escape immune clearance. The aim of our study was to investigate TNFα gene expression and epigenetic characteristics associated with the loss of TNFα expression in cervical cancer. To this end, we examined TNFα expression, loss of heterozygosity (LOH) at 6p21.3, the locus of TNFα, mutational status of the TNFα locus, loss of the TNFα promoter variant 2 allele and CpG hypermethylation of the TNFα promoter. RNA in situ hybridization showed absence of TNFα expression in 45% of 63 tumors. LOH occurred in 57% of the tumors and was not concordant with absence of TNFα mRNA. No mutations in the TNFα gene were identified in 15 cases deficient in TNFα expression exhibiting LOH. Furthermore, lack of TNFα expression did not correlate with promoter methylation. In conclusion, TNFα mRNA expression is absent in nearly half of the cervical tumors analyzed. Neither promoter methylation nor genetic causes for lack of expression were evident

High human papillomavirus oncogene mRNA expression and not viral DNA load is associated with poor prognosis in cervical cancer patients

Purpose: Cervical cancer is now known to be caused by infection with an oncogenic type of the human papillomavirus (HPV). However, little is known about the continued role of HPV once cancer has been established. Here, we describe the quantitative relation between HPV DNA copy number and mRNA expression of the viral oncogenes (E6 and E7) and the prognostic value of both measures in cervical cancer patients. Experimental Design: We studied the number of viral DNA copies and the level of HPV E6/E7 mRNA expression in 75 HPV 16-positive or HPV 18-positive International Federation of Gynecology and Obstetrics stage Ib and IIa cervical cancer patients. Measurements were done with quantitative PCR. DNA copy number analysis was done on pure tumor cell samples enriched with flow sorting. mRNA expression data were compensated for the percentage of tumor cells included. Results: The number of viral DNA copies was not predictive of survival in cervical cancer patients. In contrast, high HPV E6/E7 mRNA expression was strongly related to an unfavorable prognosis (P = 0.006). In a multivariate Cox model for overall survival, including all known prognostic variables and stratified for HPV type, the level of E6/E7 mRNA expression was an independent prognostic indicator, second only to lymph node status. No correlation was observed between DNA copy number and the level of HPV E6/E7 mRNA expression, which reflects that not all DNA copies are equally transcriptionally active. Conclusions: Cervical cancer patients with high HPV E6/E7 oncogene mRNA expression have a worse survival independently from established prognostic factors

Differentiating Benign from Malignant Thyroid Tumors by Kinase Activity Profiling and Dabrafenib BRAF V600E Targeting

Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves’ disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers