Analysis of p53 gene and HPV infection in cervical and ovarian carcinomas

Uvod: Ginekološki maligniteti predstavljaju veoma raznoliku grupu kancera, među kojima su najučestaliji karcinomi grlića materice a najsmrtonosniji karcinomi jajnika. Podaci o povezanost p53 mutacija (TP53 po HUGO nomenklaturi), polimorfizma kodona 72 i infekcije humanim papiloma virusima (HPV) sa kliničko-histopatološkim karakteristikama, nastankom ovih maligniteta, kao i odgovorom na antikancersku terapiju su kontradiktorni. Malo se zna o međusobnoj povezanosti TP53 mutacija, polimorfizma kodona 72 i HPV infekcije. Zato postoji potreba za ispitivanjem pomenutih potencijalnih biomarkera ovih maligniteta. Cilj: Ispitivanje povezanosti TP53 mutacija, polimorfnih varijanti kodona 72 i HPV infekcije sa demografskim karakteristikama, kliničko-histopatološkim karakteristikama karcinoma grlića materice i karcinoma jajnika i karakteristikama bolesnica sa ovim karcinomima, kao i ispitivanje međusobne povezanosti pomenutih potencijalnih biomarkera i njihovog uticaja na antikancersku terapiju. Materijal i metode: U radu su analizirana 53 uzorka karcinoma grlića materice i 54 uzorka karcinoma jajnika. Kontrolnu grupu činilo je 95 uzoraka briseva grlića materice žena sa urednim ginekološkim i normalnim Papa nalazom, kao i odsustvom prethodne istorije postojanja prekancerskih i kancerskih lezija ginekološke regije. DNK je izolovana metodom isoljavanja. Egzoni 4-8 TP53 gena su amplifikovani lančanom reakcijom polimeraze (PCR). Preliminarni skrining mutacija vršen je metodom konformacionog polimorfizma jednolančane DNK (SSCP), a automatskim sekvenciranjem DNK je potvrđivano prisustvo i utvrđivan tip mutacija. Polimorfizam kodona 72 TP53 gena je ispitivan analizom polimorfizma dužine restrikcionih fragmenata (RFLP). Prisustvo HPV infekcije je detektovano putem amplifikacije dela L1 virusnog gena. HPV16 i HPV18 tipovi u karcinomima grlića materice su detektovani amplifikacijom dela E7, odnosno E1 virusnih gena, dok je genotipizacija HPV u karcinomima jajnika vršena sekvenciranjem DNK. Za statističku obradu podataka korišćeni su Fišerov egzaktni, χ2 , odds ratio i Log-Rank test...Introduction: Gynecological malignancies present a various group of cancers; among them the most frequent are carcinomas of cervix and the most aggressive are ovarian carcinomas. Conflicting data about correlation of TP53 mutations, codon 72 polymorphism and HPV infection with clinicopathological characteristics, the origin of these malignancies and with the response to anti-cancer therapy, as well as correlation between mentioned biomarkers, are pointing out the necessity of analysis of these biomarkers. Goal: Examination of correlation of TP53 mutations, codon 72 polymorphic variants and HPV infection with demographic features, clinicopathological characteristics of ovarian and cervical carcinomas, patient’s characteristics, as well as examination of interconnection of these biomarkers and their possible predictive values to anti-cancer therapy. Material and methods: 53 samples of cervical carcinomas and 54 samples of ovarian carcinomas were analyzed. Control group was consisted of 95 cervical smears of gynecological healthy women with normal Papa test results and without previous history of pre- and cancer lesion of gynecological region. DNA was extracted by salting-out procedure. Exons 4-8 of TP53 gene were amplified by polymerase chain reaction (PCR). Preliminary screening of mutations was done by single strand conformation polymorphism (SSCP) and automatic DNA sequencing was employed to confirm the presence and establish the type of mutation. Codon 72 polymorphism was assessed by restriction fragment-length polymorphism (RFLP). Presence of HPV infection was detected through amplification of one part of L1 viral gene. Types HPV16 and HPV18 in cervical carcinomas were detected by amplification of one part of E7 or E1 viral gene, while HPV genotyping in ovarian carcinomas were performed by DNA sequencing. Fisher exact, χ2, odds ratio and Log-Rank tests were employed for statistical analysis..

Impact of TGFB1 Leu10Pro polymorphism on acute radiotherapy-induced toxicity in prostate cancer patients

Background: Radiotherapy (RT)-induced acute toxicity associated with bladder and bowel injury has great impact the quality of life for prostate cancer (PCa) patients. TGFB1 is a key proinflammatory and profibrotic cytokine, but its role in acute toxicity is still unclear. TGFB1 T>C transition at codon 10 results in leucine to proline substitution and increased TGFB1 protein levels. The aim of this study was to examine impact of TGFB1 Leu10Pro (rs1800470) polymorphism on RT-induced acute toxicity in PCa patients. Patients and methods: Eighty two patients who had a localized or locally advanced PCa were treated with radical (72 Gy)(n=47) or postoperative/salvage (66 Gy)(n=35) RT without previous hormonal therapy. TGFB1 Leu10Pro was determined by PCR-RFLP analysis on DNA from PBMC. The differences in the distribution of genotypes for dominant, recessive, codominant and overdominant genetic model between patients with or without acute genitourinary (GU) or gastrointestinal (GI) toxicity as well as different grade of toxicity were tested by χ2 and Fisher’s exact test. P values ≤0.05 were considered statistically significant. Results: Heterozygote PCa patients had lower rate of acute GU and GI toxicity then homozygotes (LeuLeu, LeuPro, ProPro were: 100%, 90.7%, 100% for GU and 92.0%, 88.4%, 100%, respectively for GI). The frequency of toxicity grade ≥2 were higher in LeuPro then both homozygote carriers (41% vs. 28.2% for GU and 26.3% vs. 21.6% for GI acute toxicity). The differences were not statistically significant. Conclusion: The present study did not establish impact of TGFB1 Leu10Pro polymorphism on RT-induced acute toxicity in PCa patients.5th Congress of the Serbian Association for Cancer Research with International Participation SDIR-5, Virtual event, December 3, Belgrade, 2021

Association between TGFB1 C-509T polymorphism and acute toxicity after radiotherapy for prostate cancer

Background: Radiotherapy (RT) for prostate cancer (PCa) is associated with a spectrum of side effects (toxicity) in the surrounding normal tissues. TGFB1 is a key cytokine associated with inflammation and fibrosis, but its role in acute toxicity is unclear. The presence of T allele at −509 bp of the promoter region of TGFB1 gene is related to higher concentrations of TGFB1 than C allele. We aimed to investigate association between C-509T polymorphism (rs1800469) and RT-induced acute toxicity. Patients and methods: Eighty six patients who had a localized or locally advanced PCa were treated with radical (72 Gy)(n=49) or postoperative/salvage (66 Gy)(n=37) RT without previous hormonal therapy. TGFB1 C-509T was determined by PCR-RFLP analysis on DNA from PBMC. The differences in the distribution of genotypes between patients with or without acute genitourinary (GU) or gastrointestinal (GI) toxicity were tested by χ2 and Fisher’s exact test. P values ≤0.05 were considered statistically significant. The genotype-specific associations with toxicity were estimated as odds ratios (OR) with 95% confidence intervals (CIs) for dominant, recessive, codominant and overdominant genetic model. Results: Heterozygote carriers of TGFB1 C-509T had statistically significant lower rate of acute GU and GI toxicity then homozygotes (CC plus TT) (p=0.048; p=0.047). Additionally, the OR indicated lower risk for acute toxicity development in heterozygote than homozygote patients (OR (95%CI) were: 0.12 (0.01- 1.11) for GU and 0.19 (0.03- 1.02) for GI). Conclusion: The obtained data indicate that CT genotype of TGFB1 C-509T could be potential biomarkers of lower risk for acute RT-induced toxicity.5th Congress of the Serbian Association for Cancer Research with International Participation SDIR-5, Virtual event, December 3, Belgrade, 2021

Hypericum perforatum L. extracts exert cytotoxic effects and show different miRNA signatures in PC-3 and DU 145 prostate cancer cells

Phytochemicals and bioactive substances derived from a wide range of plant extracts have been reported to exert various anticancer effects. Prostate cancer is one of the leading causes of cancer-related deaths within the male population. Prostate cancer-specific miRNA signatures were associated with cancer formation and progression, with various subtypes, and response to therapy. MicroRNA levels of expression were shown to change after the treatment of various compounds and substances extracted from natural products. Natural herbal compounds were shown to induce variations in miRNA expression levels in cancer cells. The aims of this study were to investigate the cytotoxic effects of methanol, ethyl-acetate, and hexane extracts obtained from branch-body part and flowers of Hypericum perforatum L. against humane PC-3 and DU 145 and to test potential miRNA-128/133b/155/193a/206/21/335 signature changes and differences between the two prostate cancer cell lines. Cytotoxic activity of H. perforatum extracts, their effects on cell cycle distribution, and miRNA expression levels were examined in humane PC-3 and DU 145 prostate cancer cells by MTT cell survival assay, flow cytometry, and quantitative real-time PCR. Hexane extract of flowers showed the strongest intensity of cytotoxic activity against PC-3 and DU 145 cells. The highest increase in the percentage of PC-3 cells in the subG1 phase was observed in cell samples treated with hexane extract of flowers and branch-body part. Significant differences in miRNA-128/133b/155/193a/206/21/335 levels were observed between PC-3 and DU 145 cell lines, especially in samples treated with flower extracts compared with the branch-body part. Conclusions: Investigated extracts have significant anticancer potential not only from the aspects of cytotoxicity and cell cycle effects but also from the aspect of lowering oncogenic or increasing tumor-suppressive miRNAs. The best effect might be the increase of tumor-suppressive miR-128 (accompanied by miR-193a) induced by the hexane extract of the flowers, which also exerted the highest cytotoxic activity. Hexane extract of flowers may be the candidate for further investigation for improving the efficiency of standard therapies for PCa. A miRNA signature might be cell-type specific after the treatment with H. perforatum extracts

Association of polymorphisms in TGFB1, XRCC1, XRCC3 genes and CD8 T-lymphocyte apoptosis with adverse effect of radiotherapy for prostate cancer

The genetic background of each person might affect the severity of radiotherapy (RT)-induced normal tissue toxicity. The aim of study was to evaluate the influence of TGFB1 C-509T and Leu10Pro, XRCC1 Arg280His and XRCC3 Thr241Met polymorphisms as well as the level of radiation-induced CD8 T-lymphocyte apoptosis (RILA) on adverse effects of RT for prostate cancer (PCa). The study included 88 patients with localized or locally advanced PCa who were treated with RT. The polymorphisms were determined by PCR–RFLP analysis on DNA from peripheral blood mononuclear cells. RILA values were measured by flow cytometry. We found that CT genotype of TGFB1 C-509T could be protective biomarker for acute genitourinary (GU) and gastrointestinal (GI) radiotoxicity, while Thr variant of XRCC3 Thr241Met could predict the risk for acute GU radiotoxicity. Correlation between RILA values and toxicity was not detected. Univariate logistic regression analysis showed that Gleason score and risk group were risk factors for late GU, while for late GI radiotoxicity it was diabetes mellitus type 2. However, in multivariate model those were not proven to be significant and independent risk factors. Identification of assays combination predicting individual radiosensitivity is a crucial step towards personalized RT approach

Evaluation of cytokine expression and circulating immune cell subsets as potential parameters of acute radiation toxicity in prostate cancer patients

One of the challenges of radiation oncology in the era of personalized medicine is identification of biomarkers associated with individual radiosensitivity. The aim of research was to evaluate the possible clinical value of the associations between clinical, physical, and biological factors, and risk for development of acute radiotoxicity in patients with prostate cancer. The study involved forty four patients treated with three-dimensional conformal radiotherapy. The concentrations of IL-1β, IL-2, IL-6, IFN-γ and TGF-β1 were assessed before radiotherapy, after 5th, 15th and 25th radiotherapy fractions, at the end, and 1 month after the end of radiotherapy. Cytokine gene expression was determined in peripheral blood mononuclear cells. The univariate analysis of circulating cytokine levels during radiotherapy showed that increased serum concentrations of IL-6 were significantly associated with higher grade of acute genitourinary toxicity. The multivariate analysis demonstrated that increased level of IL-6 during the radiotherapy was significantly associated with higher grade of acute genitourinary toxicity across treatment. TGF-β expression levels significantly decreased during course of radiotherapy. Research indicates that changes in circulating cytokine levels might be important parameter of radiotoxicity in patients with prostate cancer. These findings suggest that future studies based on multi-parameter examination are necessary for prediction of individual radiosensitivity

XRCC3 Thr241Met gene polymorphism and acute radiotherapy induced toxicity for prostate cancer

Purpose or Objective About half of all prostate cancer (PCa) patients receive radiotherapy (RT) either as single curative treatment or as adjuvant/salvage treatment after radical prostatectomy. However, RT is associated with a spectrum of side effects (toxicity) in the surrounding normal tissues. Acute toxicity occurs within 90 days of treatment, is usually transient and affects skin and mucosa of bladder and intestine resulting in dermatitis, cystitis or diarrhea. XRCC3 gene encodes for protein that is involved in homologous recombination repair of double-strand breaks created by ionizing radiation. Disruption of these pathways has the potential to affect the normal tissue response to RT. The T variant of XRCC3 Thr241Met single nucleotide polymorphisms (SNP) in exon 7 (C>T, rs861539) was reported to be associated with elevated levels of DNA adducts, chromosomal deletions, sensitivity to ionizing radiation and cross-linking agents. The aim of this study was to examine association between this SNP and RT-induced normal tissue toxicity in PCa patients. Materials and Methods Eighty one patients who had a histologically confirmed localized or locally advanced PCa were included in the study. Patients were treated with 3DC RT (n=70) or ARC RT (n=11) with radical (72 Gy)(47 patients) or postoperative/salvage (66 Gy)(34 patients) RT without previous hormonal therapy. DNA from peripheral blood mononuclear cells was extracted by salting out method. XRCC3 Thr241Met SNP was determined by PCR-RFLP analysis. The restriction fragments were separated on 2100 Bioanalyzer using DNA 1000 kit. The differences in the distribution of genotypes of XRCC3 Thr241Met between patients with or without acute RT-induced genitourinary (GU) or gastrointestinal (GI) toxicity were tested by χ2 and Fisher’s exact test. P values ≤ 0.05 were considered statistically significant, while p values between 0.1 and 0.05 were pointed out as a statistical trend. Data were analyzed by SigmaStat 3.5. Results The acute GI toxicity appeared in 100%, 94.6% and 81.8% of ThrThr, ThrMet and MetMet PCa patients, raspectivelly. There was the statistical trend towards higher acute GU toxicity in carriers of Thr variant (ThrThr plus ThrMet) vs. MetMet (p=0.087) as well as ThrThr vs. MetMet (p=0.058). For acute GI toxicity, there was a similar distribution in genotypes: 90.9%, 91.9%, 90.9% for ThrThr, ThrMet, MetMet, respectively. PCa patients with ThrThr genotype had higher rate of acute GU toxicity grade ≥2 (45.5%) than ThrMet (28.6%) and MetMet (22.2%) while in GI toxicity MetMet had higher rate of grade ≥2 (40%) than ThrThr (23.3%) and ThrMet (23.5%) but without statistical significance. Conclusion The obtained data indicate that Thr variant of XRCC3 Thr241Met SNP is related to acute GU toxicity. Grade ≥2 acute GU toxicity could be associated with ThrThr while GI toxicity with MetMet genotype. Further study on larger group is necessary to confirm this date and to clarify mechanism underlying this observation.Learning from every patient; May 6-10, Onsite in Copenhagen & online, 2022

Association between miR-21/146a/155 level changes and acute genitourinary radiotoxicity in prostate cancer patients: A pilot study

Introduction: Nearly sixty percent of patients with prostate cancer (PCa) undergo radiation therapy (RT). During the course of treatment patients may experience normal tissue reactions. It is a well established fact that genetic and epigenetic mechanisms, such as microRNA (miRNA) level changes might be associated with radiotoxicity, as a response to irradiation. Materials and methods: This is the first study that has investigated levels of radiosensory miRNAs in association with acute genitourinary radiotoxicity extracted from peripheral blood mononuclear cells (PBCs), in three points; before RT (BRT), after RT (ART) and on the first control examination (FCONT). We measured levels of miR-21/146a/155 expression by quantitative real-time PCR (qRT-PCR), comparative ΔΔCt method, in fifteen patients with localized prostate cancer, treated with three-dimensional conformal radiotherapy (3DCRT). Nine subjects have experienced acute genitourinary (GU) radiotoxicity whereas six where without GU radiotoxicity. Results: Firstly, we detected the highest levels of miR-21 in ART group (p = 0.043) in the patients with acute GU radiotoxicity. Secondly, we found trend towards higher miR-21 levels and significantly higher levels of miR-146a/155 within the patients with acute GU toxicity than in patients without (p = 0.068, p = 0.016, and p = 0.010, respectively). Thirdly, we detected significant change in miR-146a/155 levels within the patients without acute GU radiotoxicity during RT p = 0.042, and p = 0.041, respectively). Conclusion: miR-21/146a/155 might be useful potential factors of radiosensitivity and acute genitourinary radiotoxicity in prostate cancer patients. miRNA might have great potential as predictors of various pathological conditions extracted from PBMCs. © 2018 Elsevier Gmb