Methyl CpG binding protein MBD2 has a regulatory role on the BRCA1 gene expression and its modulation by resveratrol in ER+, PR+ & triple-negative breast cancer cells.

BACKGROUND: Resveratrol has demonstrated its ability to regulate BRCA1 gene expression in breast cancer cells, and previous studies have established the binding of MBD proteins to BRCA1 gene promoter regions. However, the molecular mechanism underlying these interactions remains to be elucidated. The aimed to evaluate the impact of MBD proteins on the regulation of BRCA1, BRCA2, and p16 genes and their consequential effects on breast cancer cells. METHODS: Efficacy of resveratrol was assessed using the MTT assay. Binding interactions were investigated through EMSA, ChIP, & MeIP assay. Expression analyses of MBD genes and proteins were conducted using qRT-PCR and western blotting, respectively. Functional assays, including clonogenic, migratory, and sphere formation assays were used to assess cancer cells' colony-forming, metastatic, and tumor-forming abilities. The cytotoxicity of resveratrol on cancer cells was also tested using an apoptosis assay. RESULTS: The study determined an IC50 of 30µM for resveratrol. MBD proteins were found to bind to the BRCA1 gene promoter. Resveratrol exhibited regulatory effects on MBD gene expression, subsequently impacting BRCA1 gene expression and protein levels. Higher concentrations of resveratrol resulted in reduced colony and sphere formation, decreases migration of cancer cells, and an increases number of apoptotic cells in breast cancer cells. Impact Identification of MBD2-BRCA1 axis indicates their significant role in the induction of apoptosis and reduction of metastasis and proliferation in breast cancer cells. Further therapy can be designed to target these MBD proteins and resveratrol could be used along with other anticancer drugs to target breast cancer. CONCLUSIONS: In conclusion MBD2 protein interact to the BRCA1 gene promoter, and resveratrol modulates MBD2 gene expression, which in turn regulates BRCA1 gene expression, and inhibits cell proliferation, migration, and induces apoptosis in ER+, PR+ & Triple negative breast cancer cells.

Comparison of HPV 16/18 Genotyping and p16/Ki67 Dual Staining for Detection of High-Grade Cervical Lesion in Patients with Low-Grade Cervical Smears.

Objective: To triage low-grade cervical smears (ASCUS/LSIL) by HPV 16/18 genotyping and dual staining with p16/Ki67 and to compare the sensitivity and specificity of these two triage methods for detection of high-grade cervical intraepithelial neoplasia (HGCIN). Methods: In this prospective cross-sectional study, we evaluated a total of 89 women with low-grade smears (54 ASCUS, 35 LSIL) recruited from a tertiary care hospital. All patients underwent colposcopy guided cervical biopsy. Histopathology was used as gold standard. All samples were subjected to HPV 16/18 genotyping (excluding 9) using DNA PCR and p16/Ki67 dual staining (excluding 4) using Roche® kit. We then compared the two triage methods to detect high-grade cervical lesions. Results: Overall, in all low-grade smears sensitivity, specificity and accuracy of HPV 16/18 genotyping, was found to be 66.7%, 77.1% and 76.2% respectively (p = 0.03). In low-grade smears sensitivity, specificity and accuracy of dual staining, was found to be 66.7%, 84.8% and 83.5% respectively (p = 0.01). Conclusions: Overall, in all low-grade smears the sensitivity of the two tests was comparable. However, dual staining had a higher specificity and accuracy than HPV 16/18 genotyping. It was concluded that both are effective triage methods but dual staining had a better performance than HPV 16/18 genotyping.

Role of identified proteins in the proteome profiles of CDK4/6 inhibitor-resistant breast cancer cell lines.

Abemaciclib (Ab) and palbociclib (Pb) are CDK4/6 inhibitors used to cure advanced breast cancer (BC). However, acquired resistance is a major challenge. The molecular mechanisms and signature proteins of therapy resistance for Ab and Pb drugs need to be explored. Here we developed resistant cells for Ab and Pb drugs in MCF-7 cell lines and explored the mechanisms and signature proteins of therapy resistance in BC. Proteome profiling was performed using the label-free proteome-orbitrap-fusion-MS-MS technique. Gene ontology (GO)-terms, KEGG pathways and network analysis were performed for the proteome data. Drug-resistant cells showed increased drug tolerance, enhanced colony formation potential and an increased gap-healing tendency for the respective drug. Up-regulation of survival genes (BCL-2 and MCL-1) and down-regulation of apoptosis inducers were observed. Drug-resistance markers (MDR-1 and ABCG2 (BCRP)) along with ESR-1, CDK4, CDK6, and cyclin-D1 genes were up-regulated in resistant cells. A total of 237 and 239 proteins were found to be differentially expressed in the Ab and Pb-resistant cells, respectively. Down-regulated proteins induce apoptosis signalling and nucleotide metabolisms and restrict EGFR signalling; however, up-regulated proteins induce Erk, wnt-ß-catenin, VEGFR-PI3K-AKT, glucose transportation, and hypoxia signalling pathways and regulate hydrogen peroxide signalling pathways. The panel of identified proteins associated with these pathways might have characteristics of molecular signature and new drug targets for overcoming drug resistance in breast cancer.

In silico prediction and interaction of resveratrol on methyl-CpG binding proteins by molecular docking and MD simulations study.

Resveratrol enhances the BRCA1 gene expression and the MBD family of proteins bind to the promoter region of the BRCA1 gene. However, the molecular interaction is not yet reported. Here we have analyzed the binding affinity of resveratrol with MBD proteins. Our results suggest that resveratrol binds to the MBD proteins with higher binding affinity toward MeCP2 protein (ΔG = -6.5) by sharing four hydrogen bonds as predicted by molecular docking studies. Further, the molecular dynamics simulations outcomes showed that the backbones of all three protein-ligand complexes are stabilized after the period of 75 ns, constantly fluctuating around the deviations of 0.4 Å, 0.5 Å and 0.7 Å for MBD1, MBD2 and MeCP2, respectively. The inter-molecular hydrogen bonding trajectory analysis for protein-ligand complexes also support the strong binding between MeCP2-resveratrol complex. Further, binding free energy calculations showed binding energy of -94.764 kJ mol-1, -53.826 kJ mol-1 and -36.735 kJ mol-1 for MeCP2-resveratrol, MBD2-resveratrol and MBD1-resveratrol complexes, respectively, which also supported our docking results. Our study also highlighted that the MBD family of proteins forms a binding interaction with other signaling proteins that are involved in various cancer initiation pathways.

Schleichera oleosa Seed Extract Reduced the Proliferation of Breast Cancer by Regulating the BRCA1 and p16 Genes.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females worldwide. Schleichera oleosa (kusum tree) belongs to the Sapindaceae family commonly found in many states of India. This plant is traditionally being used in various pathological conditions. METHODS: In vitro studies were performed using seed extract of Schleichera oleosa. Different concentrations of seed extracts were treated on MCF-7 breast cancer cell line and its effect on migration and colony formation were observed. BRCA1 and p16 gene expression was analyzed by real-time PCR and Western blotting. RESULTS: We have analyzed anticancer and anti-metastatic effects of seed extract in breast cancer and IC50 was 140µg/ml concentration. Further, its inhibitory role in cell migration and colony formation was at 140µg/ml (P<0.0001) concentration and reduced significantly growth of sphere at 140 µg (P<0.0031) and 150µg (P<0.0010) concentration after 5 days of treatment. The apoptosis study was shown a significant increase at 140 µg (P<0.0001) in apoptotic cells. Expression of BRCA1 and p16 were found to be over-expressed as 1.4 and 1.7 fold, respectively, at 140µg/ml concentration after 24 h of treatment at the transcription level. BRCA1 protein was up-regulated but p16 expression down-regulated at 140 to 150µg/ml (One-Way ANOVA, P<0.0001) concentration. CONCLUSION: In this study, we found a significant role of S. Oleosa seed extract has an anti-cancer as well as anti-metastatic via up-regulation of BRCA1 and p16 genes in breast cancer cells.

Development of a pH-sensitive functionalized metal organic framework: in vitro study for simultaneous delivery of doxorubicin and cyclophosphamide in breast cancer.

Exploration of an efficient dual-drug based nanocarrier with high drug loading capacity, specific targeting properties, and long-term stability is highly desirable in cancer therapy. Metal-organic frameworks (MOFs) have proven to be a promising class of drug carriers due to their high porosity, crystalline properties with defined structure information, and their potential for further functionalization. To enhance the drug efficacy as well as to overcome the burst effect of drugs, here we synthesized a pH responsive folic acid (FA) and graphene oxide (GO) decorated zeolitical imidazolate frameworks-8 (GO-FA/ZIF-8), for targeted delivery of doxorubicin (DOX) and cyclophosphamide (CP), simultaneously. In this system, DOX molecules were encapsulated in the pores of ZIF-8 during in situ synthesis of ZIF-8 and CP molecules have been captured by the GO surface via hydrogen bonding and π-π interactions as well. Furthermore, the resulting pH-responsive nanocarrier (DOX@ZIF-8/GO-FA/CP) showed in vitro sustained release characteristics (76% of DOX and 80% of CP) by cleavage of chemical bonding and disruption of the MOFs structure under acidic condition (at pH 5.6). Moreover, DOX@ZIF-8/GO-FA/CP has synergistic cytotoxic effects as compared to the combination of both the drugs without ZIF-8/GO-FA when treating MCF-7 and MDA-MB-231 breast cancer cell lines (with a combination index of 0.29 and 0.75 for MCF-7 and MDA-MB-231 cell-lines, respectively). Hence this system can be applied as an effective platform for smart dual drug delivery in breast cancer treatment through its remarkable manageable multidrug release.

p16 promoter methylation, expression, and its association with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 subtype of breast carcinoma.

OBJECTIVES: The purpose of the study is to investigate p16 protein expression and promoter methylation of p16 gene and their association with molecular subtypes based on parameter such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). MATERIALS AND METHODS: A total of 114 breast cancer tissue biopsies were collected for methylation-specific polymerase chain reaction (MSP) and immunohistochemical (IHC) analysis. RESULTS: Seven tissue microarrays were constructed. p16 protein expression was studied in 114 cases, of which 35/114 (30.7%) cases showed strong expression and the majority of them had ER-positive tumor (57.6%), and it was statistically significant (P < 0.0074). Similarly, p16 expression was reduced in the majority of PR-negative tumors (83.9%) and the association was statistically significant (P = 0.0026). p16 methylation was studied in 114 cases and was positive in 71.0% cases. CONCLUSION: High p16 protein expression was associated with ER-positive, PR-negative, and HER2-negative tumors which is associated with poor prognosis. p16 protein expression may be used as a prognostic indicator to predict treatment response to hormonal therapy.

BRCA1 Promoter Methylation and Expression - Associations with ER+, PR+ and HER2+ Subtypes of Breast Carcinoma

Introduction: Considering the increasing trend in incidence rates, morbidity and mortality of breast cancer, there is an urgent need to identify and validate new biomarkers for early detection and better management. The purpose of the study was to investigate BRCA1 protein expression and promoter methylation of the BRCA1 gene and their association with molecular subtypes based on estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) positivity. Materials and Methods: A total of 114 breast cancer tissue biopsies were collected for methylation specific PCR (MSP) and immunohistochemical (IHC) analysis. Results: Seven tissue microarrays were constructed. BRCA1 protein expression was reduced in 55/114 (48.2%) and in the majority of ER-negative tumors (73.3%) (p<0.001). Similarly BRCA1 expression was reduced in the majority of PR-negative tumors (69.2%) but without statistical significance (p value=0.083). BRCA1 methylation was positive in 59.6% cases. A subset regarding ER+, PR+ and HER2+ was identified which consisted of 31.6% in which an inverse relationship between BRCA1 methylation and protein expression was noted. Conclusion: Reduced expression was associated with ER and PR negative status which is linked with a poor prognosis. BRCA1 protein expression might thus be used as a prognostic indicator to predict treatment response to hormone therapy.

Decreased expression of MGMT in correlation with aberrant DNA methylation in esophageal cancer patients from North India.

O-6-methylguanine-DNA methyltransferase, DNA repair gene, has been found to be involved with the pathogenesis of the esophageal cancer. DNA hypermethylation and other factors have been suggested to downregulate O-6-methylguanine-DNA methyltransferase. In this communication, the methylation status of O-6-methylguanine-DNA methyltransferase gene and the corresponding O-6-methylguanine-DNA methyltransferase protein expression in esophageal cancer from North India has been studied. In all, 80 samples of tumor tissue along with adjacent normal tissue as controls were analyzed for messenger RNA level of O-6-methylguanine-DNA methyltransferase gene, protein expression, and subcellular localization. The messenger RNA expression was studied using real-time quantitative polymerase chain reaction, protein expression, and its subcellular localization by Western blotting and immunohistochemistry. DNA methylation was assessed through methylation-specific polymerase chain reaction. Clinicopathological parameters were recorded and correlated with the O-6-methylguanine-DNA methyltransferase expression. O-6-methylguanine-DNA methyltransferase messenger RNA expression was found to be downregulated in 65% cases (52/80). The expression of O-6-methylguanine-DNA methyltransferase at the protein level was also found to be absent in 65% (52/80) cases. In all, 52 cases had low or no expression of the protein, whereas out of those 28 remaining cases, 11.25% (09/80) cases had high O-6-methylguanine-DNA methyltransferase protein expression. The absence of O-6-methylguanine-DNA methyltransferase protein coincided with the methylated cases in 84% (38/45), whereas in 07 cases, out of the 45 methylated, O-6-methylguanine-DNA methyltransferase protein was present. The aggressive esophageal cancer patients having methylated O-6-methylguanine-DNA methyltransferase had more than 50% cases with no/mild expression of the O-6-methylguanine-DNA methyltransferase protein ( p > 0.001). Loss of O-6-methylguanine-DNA methyltransferase protein was very frequent in the incidence of esophageal cancer from North Indian patients, and methylation of the promoter region of O-6-methylguanine-DNA methyltransferase was significantly associated in its downregulation.

Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer.

In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers.

Expression of BRCA1 and BRCA2 proteins and their correlation with clinical staging in breast cancer.

OBJECTIVES: The purpose of this study was to evaluate the level of expression of the BRCA1 and BRCA2 proteins in sporadic breast cancer cases to determine the functional role of these genes in breast carcinogenesis. MATERIALS AND METHODS: Paraffin embedded histologically proven invasive breast tissue sections that were obtained from 40 patients and the adjacent normal breast tissue sections used as controls to determine breast carcinoma specific changes in the expression of BRCA1 and BRCA2 by immunohistochemistry (IHC). RESULTS: Majority of the cases express either low or no detectable level of BRCA1 expression in tumor tissues in comparison with control; the decline in BRCA1 expression was found to be more prominent in advanced grade 3 disease. On the other hand, the expression of BRCA2 protein was moderate or low in breast cancer cases and its overall distribution did not show significant difference when compared with controls. Interestingly, those breast cancer cases, which were found to express low or no BRCA1 expression, demonstrated a higher protein level of BRCA2. The inverse correlation of BRCA1 and BRCA2 expression was more prominent in post-menopausal patients. CONCLUSIONS: Our results demonstrate in a subset of cases that decline in BRCA1 expression that may be associated with potentially compensatory increase in BRCA2 protein, which may depend on tumor grade as well as menopausal status.

Carcinogenic Helicobacter pylori in gastric pre-cancer and cancer lesions: association with tobacco-chewing.

AIM: To investigate the low gastric cancer incidence rate relative to the highly prevalent Helicobacter pylori (H. pylori) infection; data relevant to H. pylori infection during gastric carcinogenesis in Indian patients is currently lacking. METHODS: The present study examines the prevalence of H. pylori infection in DNA derived from 156 endoscopic gastric biopsies of different disease groups that represent gastric pre-cancer [intestinal metaplasia (n = 15), dysplasia (n = 15)], cancer [diffuse adenocarcinoma (n = 44), intestinal adenocarcinoma (n = 21)], and symptomatic but histopathologically-normal controls (n = 61). This was done by generic ureC polymerase chain reaction (PCR) and cagA-specific PCR that could specifically identify the carcinogenic H. pylori strain. RESULTS: Our analysis showed the presence of H. pylori infection in 61% of symptomatic histopathologically-normal individuals, however only 34% of control tissues were harboring the cagA(+) H. pylori strain. A similar proportion of H. pylori infection (52%) and cagA (26%) positivity was observed in the tumor tissue of the gastric cancer group. In comparison, H. pylori infection (90%) and cagA positivity (73%) were the highest in gastric pre-cancer lesions. In relation to tobacco and alcohol abuse, H. pylori infection showed an association with tobacco chewing, whereas we did not observe any association between tobacco smoking or alcohol abuse with prevalence of H. pylori infection in the tissue of any of the patient groups studied. CONCLUSION: High incidence of H. pylori infection and carcinogenic cagA positive strain in pre-cancer lesions during gastric carcinogenesis may be associated with the habit of chewing tobacco.

Physical state & copy number of high risk human papillomavirus type 16 DNA in progression of cervical cancer.

BACKGROUND & OBJECTIVES: High-risk human papilloma virus (HR-HPV) infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions. METHODS: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30) and high grade (HSIL, 30), and invasive carcinoma, (squamous cell carcinoma SCC, 70) cases. RESULTS: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60) and cancer cases (29/70), HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load. INTERPRETATION & CONCLUSIONS: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.

Value of high-risk human papillomavirus 16 deoxyribonucleic acid testing with cytological entities in peri and postmenopausal women.

BACKGROUND: Genital human papillomavirus (HPV) infection is a sexually transmitted disease that is caused by HPV. Some types of HPV, called high-risk (HR) types may cause cell changes that sometimes lead to cervical cancer. HPV screening has been proposed for symptomatic female population; however, Pap test is the main stay in low resource setting. AIM: To detect HR HPV 16 positivity in perimenopausal and postmenopausal women and its association with cytological entities diagnosed on Pap smear. MATERIALS AND METHODS: Pap smears and cervical scrapes were collected from 230 women consisting of 120 perimenopausal women approaching menopause and 110 postmenopausal women with a cervix after cessation of menstruation and processed as per routine procedure for detection of HR-HPV 16 deoxyribonucleic acid (DNA). Cytologically abnormal HPV 16 negative cases were also tested for other HR-HPV types. RESULTS: Among the perimenopausal women 12 (10%) cases were positive for HR-HPV 16 consisting of 6 (5%) abnormal cases and 108 (90%) were HPV 16 negative consisting of 5 (4.1%) abnormal cases. However, among 110 postmenopausal women 14 (12.7%) were positive for HPV 16 DNA consisting of 6 (5.4%) abnormal cases and 96 (87.2%) were HPV 16 negative consisting of 4 (3.6%) abnormal cases. HPV 16 negative abnormal cases (9) were positive for low risk-HPV 6/11 consisting of atypical squamous cells (3) and low-grade squamous intraepithelial lesions-HPV (6). CONCLUSIONS: There is not much variation in HPV 16 positive cases in peri and postmenopausal women. By combining HPV DNA testing with Pap smear more cases having potential for pre-cancer lesions may be detected; however, HPV test cannot replace the Pap smear in low resource setting.

GSTP1 methylation and polymorphism increase the risk of breast cancer and the effects of diet and lifestyle in breast cancer patients.

Glutathione S-transferases (GSTs) are an important group of isoenzymes that play an essential role in the detoxification of carcinogens. Polymorphism at exon 5 of the GST π family decreases the catalytic activity and affects the detoxification ability of the enzyme, GSTP1. GSTP1 promoter hypermethylation and loss of expression are frequently observed in various types of carcinoma. We hypothesized that somatic epigenetic modification in homozygous mutants increases the degree to which breast cancer risk is affected by lifestyle factors and dietary habits. The present study used tumor biopsies and blood samples from 215 breast cancer patients and 215 blood samples from healthy donors. GSTP1 polymorphism was studied using PCR-restriction fragment length polymorphism, methylation using methylation-specific PCR and loss of expression using immunohistochemistry and western blotting. No significant increase was observed in the breast cancer risk of individuals with the mutant (Val) allele [odds ratio (OR), 1.48; 95% confidence interval (CI), 0.97-2.26 for heterozygotes; OR, 1.42; 95% CI, 0.86-2.42 homozygous mutants]. GSTP1 promoter hypermethylation was detected in one-third of tumor biopsies (74/215) and was found to be associated with a loss of expression. Genotype and tumor methylation associations were not observed. Estrogen (ER) and progesterone (PR) receptor-positive tumors had a higher methylation frequency. GSTP1 polymorphism was not associated with increased promoter hypermethylation. The results suggest that GSTP1 methylation is a major event in breast carcinogenesis and may act as a tumor-specific biomarker.

Human papillomavirus infection among young adolescents in India: impact of vaccination.

High-risk human papillomaviruses (HR-HPVs) are the causative agents of cervical cancer and prophylactic HPV vaccination has been recommended for adolescents but no data are available on the prevalence of HPV infection among adolescents in India. Self-collected midstream urine samples from 940 healthy school children, aged 8-17 years, from 12 different schools in and around Noida and Delhi, India, were collected for HPV detection by PCR. Of 458 girls, 15 (3.2%) were positive for HPV and 10 (66.6%) were positive for high-risk human papillomavirus (HR-HPV) type16 and 2 (13.3%) for HPV 18. Of 342 boys, 7 (2.1%) were HPV positive, of which 5 (71.4%) had HPV type 6 but interestingly, none were positive for HR-HPV types 16 or 18. Among HPV positive girls, 13 (66.6%) were >13 years and the rest were <13 years (P = 0.004), while all seven HPV positive boys were >13 years (P = 0.007). The majority of HPV positive adolescents (80-86%) belonged to the Hindu and related communities, whereas only about 14-20% belonged to the Muslim community. A significant association (P < 0.001) was observed between the parent's education and the awareness of cervical cancer, which was significantly higher among adolescent girls from India, thereby exerting an immense psychosocial impact on vaccination programs. A lower prevalence of HR-HPV infection among adolescent girls will have significant positive effect on HPV vaccination and cancer control programs in India where education and awareness should go hand in hand.

Defining the validity of classical and non-classical cellular changes indicative of low-grade squamous intraepithelial lesion encompassing human papillomavirus infection in relation to human papillomavirus deoxyribonucleic acid testing.

BACKGROUND: Human papillomavirus (HPV) infection as of now has been beyond doubt to be the causative agent for cervical carcinoma. Its morphological identification in Pap smear is important. AIM: To define the validity of classical and non-classical cellular changes indicative of low-grade squamous intraepithelial lesion (SIL) encompassing HPV infection in relation to positivity for 'high risk' HPV16 as well as for 'low risk' HPV6/11. MATERIALS AND METHODS: A total of 3000 Papanicolaou smears were screened, of which 150 were reported as low grade-SIL encompassing HPV infection (LSIL-HPV). Subsequently cervical scrapes from these 150 subjects, along with equal number of normal women as controls, were collected and processed for HPV deoxy-ribonucleic acid testing by polymerase chain reaction (PCR). RESULTS: ON THE BASIS OF CYTOMORPHOLOGICAL CHARACTERISTICS IN PAP SMEARS, HPV INFECTION WERE CATEGORIZED INTO THE FOLLOWING TWO GROUPS: Classical (koilocytic) changes (CC) encountered in 30 women and non-classical changes (NCC) encountered in 120 women. It was observed that 21 (70%) CC and 46 (38.3%) NCC of HPV infection were positive for HR-HPV16; however only 12 cases (10%) of NCC and two cases (6.6%) of CC were positive for LR-HPV 6/11. Majority (41.7%) of HPV positive cases were reported in the age group of 25 to 30 years and HPV positivity decreased with the increasing age. CONCLUSION: Classical cellular changes are not the only diagnostic features for HPV infection in Pap smear, non-classical diagnostic features also support the diagnosis of HPV infection and may be positive for HR-HPV16.

Epidermal growth factor receptor expression in urinary bladder cancer.

OBJECTIVE: To evaluate the expression pattern of epidermal growth factor receptor (EGFR) in urinary bladder cancer and its association with human epidermal growth factor receptor 2 (HER2), epidermal growth factor (EGF), interleukin-6 (IL-6), and high risk human papilloma virus (HPV) types 16 and 18. MATERIALS AND METHODS: Thirty cases of urothelial carcinoma were analyzed. EGFR, HER2, EGF, and IL-6 expressions in the tissue were evaluated by immunohistochemical staining. For HPV, DNA from tissue samples was extracted and detection of HPV was done by PCR technique. Furthermore, evaluation of different intracellular molecules associated with EGFR signaling pathways was performed by the western blot method using lysates from various cells and tissues. RESULTS: In this study, the frequencies of immunopositivity for EGFR, HER2, EGF, and IL-6 were 23%, 60%, 47%, and 80%, respectively. No cases were positive for HPV-18, whereas HPV-16 was detected in 10% cases. Overall, expression of EGFR did not show any statistically significant association with the studied parameters. However, among male patients, a significant association was found only between EGFR and HER2. CONCLUSIONS: Overexpression of EGFR and/or HER2, two important members of the same family of growth factor receptors, was observed in a considerable proportion of cases. Precise knowledge in this subject would be helpful to formulate a rational treatment strategy in patients with urinary bladder cancer.

Breast cancer and human papillomavirus infection: no evidence of HPV etiology of breast cancer in Indian women.

BACKGROUND: Two clinically relevant high-risk HPV (HR-HPV) types 16 and 18 are etiologically associated with the development of cervical carcinoma and are also reported to be present in many other carcinomas in extra-genital organ sites. Presence of HPV has been reported in breast carcinoma which is the second most common cancer in India and is showing a fast rising trend in urban population. The two early genes E6 and E7 of HPV type 16 have been shown to immortalize breast epithelial cells in vitro, but the role of HPV infection in breast carcinogenesis is highly controversial. Present study has therefore been undertaken to analyze the prevalence of HPV infection in both breast cancer tissues and blood samples from a large number of Indian women with breast cancer from different geographic regions. METHODS: The presence of all mucosal HPVs and the most common high-risk HPV types 16 and 18 DNA was detected by two different PCR methods - (i) conventional PCR assays using consensus primers (MY09/11, or GP5+/GP6+) or HPV16 E6/E7 primers and (ii) highly sensitive Real-Time PCR. A total of 228 biopsies and corresponding 142 blood samples collected prospectively from 252 patients from four different regions of India with significant socio-cultural, ethnic and demographic variations were tested. RESULTS: All biopsies and blood samples of breast cancer patients tested by PCR methods did not show positivity for HPV DNA sequences in conventional PCRs either by MY09/11 or by GP5+/GP6+/HPV16 E6/E7 primers. Further testing of these samples by real time PCR also failed to detect HPV DNA sequences. CONCLUSIONS: Lack of detection of HPV DNA either in the tumor or in the blood DNA of breast cancer patients by both conventional and real time PCR does not support a role of genital HPV in the pathogenesis of breast cancer in Indian women.