[The p16INK4A mRNA transcripts estimation in cervical smear with different degrees of cervical dysplasia]. / Stanovenie expresie p16INK4A mRNA transkriptu v steroch z krcka maternice s rôznymi stupnami cervikálnej dysplázie.

OBJECTIVE: To determine the presence of HPV infection and expression level of p16INK4A mRNA transcripts in cervical smears as adjunct biomarker in detection of cervical intraepithelial neoplasia or cancer. DESIGN: Prospective pilot clinical study assessing clinical utility and validity of ddCt method for qPCR mRNA expression of p16ink4a in comparison to immunohistochemistry. SETTING: Department of Molecular Biology, Department of Obstetrics and Gynecology, Jessenius Medical Faculty, Commenius University, Martin, Slovak Republic. METHODS: Cervical smears (OC) from patients with different cervical lesions (L-SIL, H-SIL, SCA; n=45) and from healthy controls (n=45) were tested for the presence of HPV infection and p16INK4A mRNA transcripts using relative quantification (RQ). Results were compared to H&E and IHC histological findings from biopsies (conization, hysterectomy). RESULTS: HPV 16 was the most frequent finding (53.3%) in the group of subjects with cervical dysplasia. The p16INK4A mRNA expression analysis revealed the slightly reduced expression in L-SIL group, 4-fold increased expression in H-SIL and 10-fold increase in women with SCA when compared to controls. The p16INK4A mRNA expression in OC was present in 30% of L-SIL, 75% of H-SIL and 85.7% of SCA samples, respectively. The test overall sensitivity was 81.48% (95% CI: 61.92-93.7) and specificity 60% (95% CI: 26.24- 87.84) with PPV of 84.62% and NPV of 54.55%. The likelihood ratio (LR) in case of test positivity was 2.04 and for negativity 0.31. The diagnostic accuracy of p16INK4A expression by RQ method in OC smears for prediction of p16 positivity in cervical dysplasia was 66.7% for the L-SIL lesions, 59.5% for H-SIL lesions, and 100% for SCA (r=0.9897, p<0.0913) when compared to IHC p16 positive findings in surgically treated samples. CONCLUSION: The relative quantification is able to determine the level of p16INK4A mRNA transcripts in cervical smear cells with active carcinogenesis nearly at the same level as IHC staining. The advance of biopsy sparing over IHC is qualifying this diagnostic approach for useful candidate in selective management of women with cervical dysplasia looking for cervix preservation or avoiding the unnecessary overtreatment.

One-year follow-up of kidney transplant recipients with increased expression of mRNA for granzyme B in urinary cells.

BACKGROUND: Measurement of mRNA encoding cytotoxic proteins in urinary cells is recognized as a potential noninvasive means to diagnose acute rejection in kidney allograft recipients. We sought to evaluate kidney graft function after 1 year follow-up without therapeutic intervention among patients with increased urinary expression of mRNA for granzyme B, albeit with stable graft function at the time of measurements. PATIENTS AND METHODS: The 29 randomly selected patients were at a median of 39 months (range, 10-156) after transplantation with stable graft function over the previous 3 months. The reference housekeeping gene GAPDH was used for expression measurement in a TaqMan Gene Expression Assay with the target granzyme B gene. Delta delta ct relative gene expression analysis compared results with reference samples from 10 healthy individuals. Kidney graft function was reassessed after 1 year follow-up; immunosuppression was not changed during this period. RESULTS: mRNA granzyme B expression was significantly higher among the group of randomly assessed out-clinic patients with stable graft function than among healthy volunteers (mean ± standard error of the mean, 6.18 ± 1.27; P < .01). Despite no therapeutic intervention, no significant changes were observed in delta glomerular filtration rate or quantitative proteinuria between groups with mRNA expression > 5× versus <2× higher than the healthy controls at 1 year of follow-up. CONCLUSION: Increased mRNA expression for granzyme B in urinary cells over the medium to long term among kidney transplant recipients did not predict changes in renal allograft function after 1 year follow-up.