Cross-hybridization between HPV genotypes in the Linear Array Genotyping Test confirmed by Next-Generation Sequencing

Background: Linear Array Genotyping Test (LA) is one of the gold standards used for Human Papillomavirus (HPV) genotyping, however, since its launching in 2006, new HPV genotypes are still being characterized with the use of high specificity techniques such as Next-Generation Sequencing (NGS). Derived from a previous study of the IMSS Research Network on HPV, which suggested that there might be cross-reaction of some HPV genotypes in the LA test, the aim of this study was to elucidate this point. Methods: Double stranded L1 fragments (gBlocks) from different HPVs were used to perform LA test, additionally, 14 HPV83+ and 26 HPV84+ cervical samples determined with LA, were individually genotyped by NGS. Results: From the LA HPV83+ samples, 64.3% were truly HPV83+, while 42.9% were found to be HPV102+. On the other hand, 69.2% of the LA HPV84+ samples were HPV84+, while 3.8, 11.5 and 30.8% of the samples were indeed HPV 86, 87 and 114 positive, respectively. Additionally, novel nucleotide changes in L1 gene from HPV genotypes 83, 84, 87, 102 and 114 were determined in Mexican cervical samples, some of them lead to changes in the protein sequence. Conclusions: We demonstrated that there is cross-hybridization between alpha3-HPV genotypes 86, 87 and 114 with HPV84 probe in LA strips and between HPV102 with HPV83 probe; this may be causing over or under estimation in the prevalence of these genotypes. In the upcoming years, a switch to more specific and sensitive genotyping methods that detect a broader spectrum of HPV genotypes needs to be implemented

Comparative Genomic Hybridization and Transcriptome Sequencing Reveal Genes with Gain in Acute Lymphoblastic Leukemia: <i>JUP</i> Expression Emerges as a Survival-Related Gene

Acute lymphoblastic leukemia (ALL) in children or adults is characterized by structural and numeric aberrations in chromosomes; these anomalies strongly correlate with prognosis and clinical outcome. Therefore, this work aimed to identify the genes present in chromosomal gain regions found more frequently in patients with acute lymphoblastic leukemia (ALL) and ALL-derived cell lines using comparative genomic hybridization (CGH). In addition, validation of the genes found in these regions was performed utilizing RNAseq from JURKAT, CEM, and SUP-B15 cell lines, as well as expression microarrays derived from a MILE study. Chromosomes with common gain zones that were maintained in six or more samples were 14, 17, and 22, in which a total of 22 genes were identified. From them, NT5C3B, CNP, ACLY, and GNB1L maintained overexpression at the mRNA level in the cell lines and in patients with ALL. It is noteworthy that SALL2 showed very high expression in T-ALL, while JUP was highly expressed in B-ALL lineages. Interestingly, the latter correlated with worse survival in patients. This provided evidence that the measurement of these genes has high potential for clinical utility; however, their expressions should first be evaluated with a sensitive test in a more significant number of patients

Glycogene expression profiles based on microarray data from cervical carcinoma HeLa cells with partially silenced E6 and E7 HPV oncogenes

Abstract Background Aberrant glycosylation is a characteristic of tumour cells. The expression of certain glycan structures has been associated with poor prognosis. In cervical carcinoma, changes in the expression levels of some glycogenes have been associated with lymph invasion. Human papillomavirus (HPV) infection is one of the most important factors underlying the development of cervical cancer. The HPV oncoproteins E6 and E7 have been implicated in cervical carcinogenesis and can modify the host gene expression profile. The roles of these oncoproteins in glycosylation changes have not been previously reported. Methods To determine the effect of the E6 and E7 oncoproteins on glycogene expression we partially silenced the E6 and E7 oncogenes in HeLa cells, we performed a microarray expression assay to identify altered glycogenes and quantified the mRNA levels of glycogenes by RT-qPCR. A protein-protein interaction network was constructed to identify potentially altered glycosylation pathways. Results The microarray analysis showed 9 glycogenes that were upregulated and 7 glycogenes that were downregulated in HeLa shE6/E7 cells. Some of these genes participate in glycosylation related to Notch proteins and O-glycans antigens. Conclusions Our results support that E6 and E7 oncoproteins could modify glycogene expression the products of which participate in the synthesis of structures implicated in proliferation, adhesion and apoptosis

Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages

CDH1 somatic alterations in Mexican patients with diffuse and mixed sporadic gastric cancer

Abstract Background Diffuse gastric cancer (DGC) is associated with the reduction or absence of the expression of the cell adhesion protein E-cadherin (encoded by the CDH1 gene). Molecular characteristics are less well described for mixed gastric cancer (MGC). The main somatic alterations that have been described in the CDH1 gene are mutations, loss of heterozygosity (LOH) and promoter methylation. The aim was to analyze CDH1 somatic alterations in Mexican patients with diffuse and mixed gastric cancer. Methods We searched for mutations in the CDH1 gene in tumor DNA from DGC (n = 13) and MGC (n = 7) patients by next generation sequencing (NGS). Validation of findings was performed using Sanger sequencing. LOH was analyzed using dinucleotide repeat markers surrounding the CDH1 gene, and methylation was investigated by DNA bisulfite conversion and sequencing. E-cadherin protein deficiency was analyzed by immunohistochemistry. Results Seventeen point variants were identified by NGS, 13 of them were validated by Sanger sequencing. Only 1/13 had not been previously reported (c.-137C > A), and 12/13 were already reported as polymorphisms. Two DGC cases presented LOH at the locus 16q22.1 (13.3%). CDH1 promoter methylation was positive in (7/11) 63.6% and (4/6) 66.6% of the cases with DGC and MGC, respectively. E-cadherin protein deficiency was observed in 58.3% of DGC cases while 100% in MGC cases. Conclusions While no pathogenic somatic mutations were found that could explain the diffuse histology of gastric cancer in DGC and MGC, methylation was the most common somatic inactivation event of the CDH1 gene, and LOH was rare. The previously unreported c.-137C > A variant modify the CDH1 gene expression since it alters the binding sites for transcription factors

Detection of Alpha, Beta, Gamma, and Unclassified Human Papillomaviruses in Cervical Cancer Samples From Mexican Women

International audienceBackground: Cervical cancer (CC) is associated to high-risk human papillomavirus (HPV) infections, for this reason it is crucial to have sensitive and accurate HPV diagnostic tests. To date, most research is focused on HPVs within the Alphapapillomavirus (α-PVs) genus and little attention has been paid to cervical infections with other HPV genotypes, like those of the Betapapillomavirus (β-PVs) and Gammapapillomavirus (γ-PVs) genera. The aim of this study was to determine the HPV genotypes from different genera in women with CC using Next-Generation Sequencing (NGS).Methods: The study comprised 48 HPV positive CC samples evaluated with the Linear Array HPV Genotyping test and individually sequenced by 454 NGS using PGMY09/11 and FAP primers. To determine the HPV genotypes present in each sample, the obtained sequences were compared with all HPV L1 gene reference sequences from the Papillomavirus Episteme database (PaVE). Moreover, 50 HPV positive low-grade cervical lesion samples individually genotyped with NGS were also included to determine the genotypes present preferentially in CC patients.Results: Among the 48 CC samples, 68.75% consisted of multiple HPV infections, 51 different genotypes were detected, of which 7 are still unclassified, 28 belong to α-PVs (6, 11, 16, 18, 26, 30, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 54, 59, 62, 66, 68, 69, 70, 71, 74, 81, 102, 114), 10 to β-PVs (5, 12, 21, 37, 38b, 47, 80, 107, 118, 122), and 6 to γ-PVs (101, 103, 123, 135, 147, 214). Among them, HPV16 was the most prevalent genotype (54.2%), followed by HPV18 (16.7%), HPV38b (14.6%), and HPVs 52/62/80 (8.3%). Some genotypes were exclusively found in CC when compared with Cervical Intraepithelial Neoplasia grade 1 (CIN1) samples, such as HPVs 5, 18, 38b, 107, 122, FA39, FA116, mSK_120, and mSK_136.Conclusions: This work demonstrates the great diversity of HPV genotypes detected by combining PGMY and FAP primers with NGS in cervical swabs. The relatively high attribution of β- and γ- PVs in CC samples suggest their possible role as carcinogenic cofactors, but deeper studies need to be performed to determine if they have transforming properties and the significance of HPV-coinfections

Supplementary Figure - Expression of NK Cell Surface Receptors in Breast Cancer Tissue as Predictors of Resistance to Antineoplastic Treatment

<p>Supplementary Figure for Expression of NK Cell Surface Receptors in Breast Cancer Tissue as Predictors of Resistance to Antineoplastic Treatment by Garcia-Chagollan Mariel, Carranza-Torres Irma Edith, Carranza-Rosales Pilar, Guzmán-Delgado Nancy Elena, Ramírez-Montoya Humberto, Martínez-Silva María Guadalupe, Mariscal-Ramirez Ignacio, Barrón-Gallardo Carlos Alfredo, Pereira-Suárez Ana Laura, Aguilar-Lemarroy Adriana, and Jave-Suárez Luis Felipe in Technology in Cancer Research & Treatment</p