Hierarchical evaluation of histology and p16-labeling can improve the risk assessment on cervical intraepithelial neoplasia progression.

OBJECTIVE: High-grade cervical lesions (HSIL) are associated with the presence of high-risk HPV types, tissue expression of p16, and increased chance of malignant progression, requiring surgical intervention. To improve risk evaluation, we assessed the discriminatory power of the histological findings associated with p16 immunohistochemistry (IHC) staining to classify the low-grade cervical lesion (LSIL) and HSIL. METHODS: We collected cervical biopsies from colposcopy-visible lesions and non-affected tissue (adjacent to the lesions) of 62 Brazilian women and labeled them with anti-p16 antibodies. In addition to the observational pattern and labeling to define the latent classes (affected vs. non-affected), a computational tool was used for semi-quantitative analysis of p16 expression. The intensity of staining of the nucleus or cytoplasm was captured using the Gimp 2.10 software. ROC curves were used to determine cutoff values for p16 expression in patients classified as LSIL and HSIL by latent class statistics for each labeling stratum. RESULTS: p16 nuclear labeling showed the best sensitivity and specificity to discriminate LSIL with low p16 expression (62%) and HSIL with high p16 expression (37%). Many patients whose lesions had intermediate levels of p16 nuclear staining were subsequently stratified according to the expression of p16 in the cytoplasm, indicating that five of 21 LSIL were at risk of progression, and 13 of 41 HSIL at risk of regression. CONCLUSIONS: We suggest a hierarchical analysis, with histology at the first level, followed by a labeling analysis in the nucleus and then in the cytoplasm to increase the accuracy of the HPV cervical lesion stratification.

Contribution of HLA-G and FOXP3 genes and proteins in the severity of cervical intraepithelial neoplasia during HPV infection.

Human Papillomavirus (HPV) persistence leads to the chronification of cervical inflammation, where HLA-G and Foxp3; immunomodulatory molecules, may contribute to the aggravation of the lesion and cancerization. Here, we evaluated the synergic effect of these two molecules in the worsening of the lesion in presence of HPV infection. Hundred and eighty (180) women cervical cells and biopsies were collected for (i) HLAG Sanger sequencing and gene expression, and (ii) HLA-G and Foxp3 molecule expressions by immunohistochemistry. 53 women were HPV+ against 127 women HPV-. HPV+ women were more at risk of having cytological changes (p ≤ 0.0123), histological changes (p < 0.0011), and cervical lesion (p = 0.0004). The HLA-G + 3142CC genotype predisposed women to infection (p = 0.0190), while HLA-G + 3142C and +3035 T alleles were associated with HLA-G5 transcript expression. Both sHLA-G (p = 0.030) and Foxp3 (p = 0.0002) proteins were higher in cervical lesion as well as in high-grade lesion. In addition, sHLA-G+ cells were positively correlated to Foxp3+ cells in presence of HPV infection and in cervical grade II/III injuries. In conclusion, HPV may use HLA-G and Foxp3 as a way of host immune escape contributing to the persistence of infection and inflammation, leading to the cervical lesion and the worsening of lesions.

The antigen processing-associated transporter gene polymorphism: Role on gene and protein expression in HPV-infected pre-cancerous cervical lesion.

Human papillomavirus (HPV) is the major pathogen for cervical lesions. The evasion mechanism of the immune response and persistence of HPV infection can be influenced by polymorphisms (SNPs) in genes associated with transporter associated with antigen processing (TAP), which may change the peptide binding affinity or the TAP expression impacting the efficiency of peptide transport in the secretory pathway, and the presentation of peptides to cytotoxic T lymphocytes. This study aimed to evaluate the role of the TAP1 and TAP2 polymorphisms, TAP1, and TAP2 genes expressions, and protein levels in cervical cells presenting different degrees of pre-cancerous lesions in 296 immunocompetent women infected or not by HPV. TAP SNPs were genotyped by Sanger sequencing, and gene expression by real-time PCR. Aneuploidy was determined by DNA index using flow cytometry. TAP-1 and TAP-2 tissue expressions were evaluated by immunohistochemistry. The Asp697Gly SNP of TAP1 presented a risk for cellular aneuploidy (P=0.0244). HPV+ women had higher TAP-2 mRNA (P=0.0212) and protein (P<0.0001) levels. The TAP2D and TAP2E haplotypes were associated with the risk for aneuploidy and pre-cancerous lesions. In conclusion, nucleotide variability at the peptide binding region of peptide transporter genes, particularly of the TAP2 gene, may influence the HPV-peptide transportation from the cytosol to the endoplasmic reticulum, increasing the susceptibility to the development of high-grade cervical lesions.

Increased PD-1 Level in Severe Cervical Injury Is Associated With the Rare Programmed Cell Death 1 (PDCD1) rs36084323 A Allele in a Dominant Model.

The high-risk oncogenic human papillomavirus (HPV) has developed mechanisms for evasion of the immune system, favoring the persistence of the infection. The chronic inflammation further contributes to the progression of tissue injury to cervical cancer. The programmed cell death protein (PD-1) after contacting with its ligands (PD-L1 and PD-L2) exerts an inhibitory effect on the cellular immune response, maintaining the balance between activation, tolerance, and immune cell-dependent lesion. We evaluated 295 patients exhibiting or not HPV infection, stratified according to the location (injured and adjacent non-injured areas) and severity of the lesion (benign, pre-malignant lesions). Additionally, we investigated the role of the promoter region PDCD1 -606G>A polymorphism (rs36084323) on the studied variables. PD-1 and PDCD1 expression were evaluated by immunohistochemistry and qPCR, respectively, and the PDCD1 polymorphism was evaluated by nucleotide sequencing. Irrespective of the severity of the lesion, PD-1 levels were increased compared to adjacent uninjured areas. Additionally, in cervical intraepithelial neoplasia (CIN) I, the presence of HPV was associated with increased (P = 0.0649), whereas in CIN III was associated with decreased (P = 0.0148) PD-1 levels, compared to the uninjured area in absence of HPV infection. The PDCD1 -606A allele was rare in our population (8.7%) and was not associated with the risk for development of HPV infection, cytological and histological features, and aneuploidy. In contrast, irrespective of the severity of the lesion, patients exhibiting the mutant PDCD1 -606A allele at single or double doses exhibited increased protein and gene expression when compared to the PDCD1 -606GG wild type genotype. Besides, the presence of HPV was associated with the decrease in PDCD1 expression and PD-1 levels in carriers of the -606 A allele presenting severe lesions, suggesting that other mediators induced during the HPV infection progression may play an additional role. This study showed that increased PD-1 levels are influenced by the -606G>A nucleotide variation, particularly in low-grade lesions, in which the A allele favors increased PDCD1 expression, contributing to HPV immune system evasion, and in the high-grade lesion, by decreasing tissue PD-1 levels.

Citologia de impressão conjuntival em crianças desnutridas graves hospitalizadas / Conjunctival impression cytology in children hospitalized with severe malnutrition

OBJETIVOS: avaliar os achados da citologia de impressão conjuntival (CIC) em crianças com desnutrição energético protéica (DEP) grave, hospitalizadas no Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). MÉTODOS: estudo descritivo, tipo série de casos, realizado no período de agosto de 2005 a maio de 2006, envolvendo 41 crianças de um a 60 meses, com desnutrição grave (peso/altura < -3DP e/ou edema - padrão NCHS). A técnica para a realização da CIC seguiu normas padronizadas segundo estudo de validação. A leitura das lâminas foi realizada por dois observadores independentes. RESULTADOS: a idade dos casos variou de um a 47 meses, com a mediana de nove meses. Observou-se padrão de anormalidade da CIC em 46,3 por cento das crianças. Entre os casos com CIC anormal (marginal (-) e deficiente), 40 por cento apresentaram edema. CONCLUSÕES: a prevalência elevada de resultados anormais de CIC, resumindo as classificações marginal (-) e padrão deficiente, justifica, na experiência aqui relatada, o cuidado normativo de se utilizar doses elevadas de vitamina A no tratamento hospitalar de desnutridos graves.

OBJECTIVES: to evaluate conjunctival impression cytology (CIC) findings in children hospitalized with severe energy protein malnutrition at the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). METHODS: a descriptive case series study was carried out between august 2005 and may 2006 with 41 children aged between 1 and 60 months with severe malnutrition (weight/height < -3DP and/or edema -NCHS standard). The CIC technique used followed the standard norms established by the validation study. The slides were examined by two independent observers. RESULTS: the age of cases varied from one to 47 months, with a median of nine months. An abnormal CIC pattern was observed in 46.3 percent of children. Forty percent of cases of abnormal CIC (marginal (-) and deficient), also presented edema. CONCLUSIONS: the high prevalence of abnormal CIC results found here justifies the standard treatment of high doses of vitamin A for cases of severely malnourished children admitted to hospital.