Aberrant gene-specific DNA methylation signature analysis in cervical cancer.

Multicomponent molecular modifications such as DNA methylation may offer sensitive and specific cervical intraepithelial neoplasia and cervical cancer biomarkers. In this study, we tested cervical tissues at various stages of tumor progression for 5-methylcytosine and 5-hydroxymethylcytosine levels and also DNA promoter methylation profile of a panel of genes for its diagnostic potential. In total, 5-methylcytosine, 5-hydroxymethylcytosine, and promoter methylation of 33 genes were evaluated by reversed-phase high-performance liquid chromatography, enzyme-linked immunosorbent assay based technique, and bisulfate-based next generation sequencing. The 5-methylcytosine and 5-hydroxymethylcytosine contents were significantly reduced in squamous cell carcinoma and receiver operating characteristic curve analysis showed a significant difference in (1) 5-methylcytosine between normal and squamous cell carcinoma tissues (area under the curve = 0.946) and (2) 5-hydroxymethylcytosine levels among normal, squamous intraepithelial lesions and squamous cell carcinoma. Analyses of our next generation sequencing results and data from five independent published studies consisting of 191 normal, 10 low-grade squamous intraepithelial lesions, 21 high-grade squamous intraepithelial lesions, and 335 malignant tissues identified a panel of nine genes ( ARHGAP6, DAPK1, HAND2, NKX2-2, NNAT, PCDH10, PROX1, PITX2, and RAB6C) which could effectively discriminate among the various groups with sensitivity and specificity of 80%-100% (p < 0.05). Furthermore, 12 gene promoters (ARHGAP6, HAND2, LHX9, HEY2, NKX2-2, PCDH10, PITX2, PROX1, TBX3, IKBKG, RAB6C, and DAPK1) were also methylated in one or more of the cervical cancer cell lines tested. The global and gene-specific methylation of the panel of genes identified in our study may serve as useful biomarkers for the early detection and clinical management of cervical cancer.

Prevalence of human papillomavirus types and phylogenetic analysis of HPV-16 L1 variants from Southern India.

BACKGROUND: The human papillomavirus (HPV) and its variants show wide geographical distribution and have been reported to cause cervical lesions. With cervical neoplasia as the leading cancer in Indian women, the aim of the present study was to evaluate the multiple infection HPV type distribution and variant genotypes in cervical samples from the coastal Karnataka region, India. MATERIALS AND METHODS: A total of 212 samples were screened by nested polymerase chain reaction using PGMY9/11 and GP5+/6+ primers. HPV positive samples were sequenced to identify the types and a phylogenetic tree was constructed using the neighbor-joining method. RESULTS: Sequence analysis identified a total of 14 HPV types distributed in 20%, 73.3% and 82.5% of non-malignant, pre-malignant [low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL)] and cervical cancer samples. The distribution of high risk HPV in cancer samples was HPV 16, 76.4%, HPV18, 11.7%, HPV81, 2.9%, HPV31, 1.4%, HPV35, 1.4% and HPV 45, 1.4%. Multiple infections were observed in 11.8% of tumor samples with HPV 16 contributing to 62.5% of cases. In non-malignant samples, 20% of HPV positive samples were detected with HPV16, 82.3%, HPV33, 5.8% and HPV58, 5.8% and very low incidence of multiple infections. Comparative phylogenetic analysis of HPV variants identified 9 HPV sequences as new papillomavirus species, predominantly classified as European lineage type. CONCLUSIONS: The findings for HPV infections associated with progression of cervical cancer in coastal Karnataka region and HPV variant analysis provide baseline data for prevention and HPV vaccination programs.

Variables influencing the integrity of lower uterine segment in post-cesarean pregnancy.

BACKGROUND: There is significant increase in proportion of cases with previous cesarean delivery requiring obstetric care. The available literature fails to provide uniform opinion on each woman's characteristics to identify risk of uterine rupture while planning trial of labor after cesarean. OBJECTIVE: To study the association of abnormal lower uterine segment with some of the present and previous obstetric variables including patient characteristics and surgical techniques at previous cesarean operation. MATERIALS AND METHODS: Consenting consecutive 96 post-cesarean singleton pregnancies admitting after 36 weeks gestation at the same facility from July 2011 to December 2012 for repeat cesarean, were studied. Only the cases with cephalic presentation and vertex as presenting part, having no placenta previa, polyhydramnios, uterine anomaly or fibroid and those who had previous one lower segment cesarean were recruited. Based on the intra-operative finding the lower uterine segment (LUS) was categorized into those having a normal and abnormal (grades 2-4) LUS. Sonographic assessment of LUS thickness and any abnormalities if any were noted. The findings of abnormal LUS (direct observation at surgery and sonographic impression within a week before surgery) were looked for association with some of the present and previous obstetric variables including patient characteristics and surgical techniques at previous cesarean operation using Student t, Chi square or Fisher's exact test for analysis as appropriate. Receiver operating curve analysis was used to determine the optimal cut off value for prediction of LUS integrity by ultrasound. RESULTS: Of the women recruited for the study, 36 were admitted in early labor and ultrasound evaluation of LUS was performed in 48 of the remaining 60 women admitted antenatal for elective cesarean delivery. There were 38 abnormal LUS (39.6%) with 22 of them (57.9%) graded as 'thinned out LUS'. The incidence of scar dehiscence (grade 3, cases 5) was 5.2% of 96 cases and there were no cases of scar rupture. Proportion of cases with abnormal LUS was significantly high when primary cesarean was done in preterm (p = 0.02); it was a single layer uterine closure (p = 0.02), and inter-cesarean interval was 54 months (p = 0.01). Abnormal LUS was also seen to be associated with maternal age beyond 35 years (p = 0.2), when cesarean was performed in labor (p = 0.5), following 18 h of rupture of membranes (p = 0.75), for a baby weight more than 3 kg (p = 0.4), and different suture materials (polyglactin 910 and chromic catgut) were used to close uterus at primary cesarean delivery (p = 0.1), and also if they had post-partum fever (p = 0.3). Ultrasound measurement of LUS by abdominal scan correlated with the intra-operative LUS grading and a thickness of more than 3.2 mm within a week before delivery and was seen to be the safe cut off above which most of the women had a normal LUS (sensitivity 92.3%, specificity 81.1%). CONCLUSION: Factors at primary cesarean operation significantly influence the state of LUS at term in subsequent pregnancy .

DNA promoter methylation-dependent transcription of the double C2-like domain ß (DOC2B) gene regulates tumor growth in human cervical cancer.

Double C2-like domain ß (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

Mitochondrial DNA variation analysis in cervical cancer.

This study was undertaken to investigate the mitochondrial DNA (mtDNA) variation in non-malignant and malignant cervical tissue samples. We have identified 229 and 739 variations non-malignant and malignant tissues respectively distributed over 321 locations in the D-loop (50 in non-malignant and 166 in malignant; 216 variations), coding region (139 in non-malignant and 455 in malignant; 594 variations) tRNA and rRNA genes (39 in non-malignant and 119 in malignant; 158 variations). Besides, 77 novel and 34 various other disease associated variations were identified in non-malignant and malignant samples. A total of 236 tumor specific variations in 201 locations representing 30.1% in D-loop, 59.3% in coding regions and 10.6% in RNA genes were also identified. Our study shows that D loop (in 67 locations) is highly altered followed by ND5 (35 locations) region. Moreover, mtDNA alterations were significantly higher in malignant samples by two tailed Fisher's exact test (P≤0.05) with decreased mtDNA copy numbers. Bioinformatic analysis of 59 non-synonymous changes predicted several variations as damaging leading to decreased stability of the proteins. Taken together, mtDNA is highly altered in cervical cancer and functional studies are needed to be investigated to understand the consequence of these variations in cervical carcinogenesis and their potential application as biomarkers.

Plastic spatula with narrow long tip provides higher satisfactory smears for Pap test.

BACKGROUND: Ayre spatula for cervical smear collection is being used despite the suggestion that different modified spatulas provide more satisfactory sampling. AIMS: To see whether the cytological pickup improves with the use of long tipped spatula. SETTING AND DESIGN: Rurally based University Hospital; crossover study. MATERIALS AND METHODS: Pap smear using Ayre spatula in 500 and with plastic narrow long tip (Szalay) spatula in 500 clinic attending women was taken and analyzed. Crossover smears were taken with modified spatula in 163 and using Ayre spatula in 187 women after 2 weeks of initial smears. The same pathologist made cytological reporting for all smears and was unaware of the type of spatula used. RESULTS: Smears from Ayre spatula had significantly higher reports of inadequate smears (94 of 500 vs. 68 of 500 for Ayre and Szalay, respectively; P = 0.032) and it remained so even after crossover (94 of 187 vs. 70 of 163 for Ayre and Szalay, respectively; P = 0.2). Cellular quality appeared better with smears taken using Szalay spatula, but the overall abnormal smear detection rate remained similar with either collection tool (χ(2) = 1.5; P = 0.2). CONCLUSIONS: Proportion of satisfactory smears is higher when long tip plastic spatula is used for collection of sample.

Simultaneous detection of periodontal pathogens in subgingival plaque and placenta of women with hypertension in pregnancy.

BACKGROUND: There are many studies documenting increased prevalence of periodontal infection in women with preeclampsia. But, very few studies have attempted to establish causal relationship between the two. OBJECTIVE: To find out causal circumstantial evidence by isolating specific periodontal pathogens in oral and placental samples. MATERIALS AND METHODS: Antenatal periodontal screening and subgingival plaque collection was carried out in ten women with hypertension in pregnancy and ten normotensive controls on their hospital admission at term for cesarean delivery. Placental biopsy was obtained after aseptic placental collection at the time of elective cesarean delivery. Subgingival plaque and placental biopsy were studied for Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, Prevotella intermedia and Aggregatibacter actinomycetemcomitans using quantitative polymerase chain reaction technique. Periodontist and laboratory personnel were unaware of case or control status. Periodontal status was not informed to the obstetrician recruiting the cases and laboratory. Microbiology report was not revealed till end of the study. RESULTS: Periodontal pathogens were found to be high in the group with hypertension than the controls. P gingivalis was found in all the samples from subgingival plaque and placenta, irrespective of the periodontal disease status. CONCLUSION: In cases with hypertension, periodontal pathogens are present in higher proportion in subgingival plaque and placenta.

Pregnancy associated breast cancer--the obstetrician's role.

Breast cancer is the most common cancer in many geographic areas, the most frequent cause of cancer deaths in women, and is also the cancer most likely to be seen during pregnancy and lactation. Delay in diagnosis appears to be the primary reason for the generally worse prognosis overall for all patients with breast cancer diagnosed during pregnancy and lactation. In this context, the patient's family physician or obstetrician who performs the routine antenatal examinations can play an important role by performing a vital breast examination which might bring to light and prompt timely investigation of otherwise asymptomatic breast masses.

Diagnosis of ovarian cancer by Raman spectroscopy: a pilot study.

OBJECTIVE: To assess the efficacy of conventional Raman spectroscopy in combination with discriminating parameters, Mahalanobis distance, spectral residuals, and "limit test" methodology in differentiation of normal and malignant ovarian tissues. BACKGROUND DATA: Ovarian cancer is the second most common cancer among women and the leading cause of death among gynecologic malignancies. Initial laparotomy and subsequent frozen section analysis can influence the surgical management of ovarian cancers. Although frozen section pathology is sensitive and specific enough, interpretation is often subjective, time consuming, and requires highly skilled personnel. Raman spectroscopy is sensitive to biochemical variations in the samples, rapid, more objective, and amenable to multivariate statistical tools. It can therefore be an ideal tool for discrimination between normal and malignant ovarian tissues. METHODS: 72 Spectra from eight normal and seven malignant ovarian tissues were recorded by conventional near-infrared (NIR) Raman spectroscopy (excitation wavelength of 785 nm). Spectral data were analyzed by principal components analysis (PCA) and other discriminating parameters such as Mahalanobis distance, spectral residuals, and a multiparametric limit test approach. RESULTS: A mean malignant spectrum exhibits a broader amide I band, a stronger amide III band, a minor blue shift in the delta CH2 band, and a hump around 1480 cm(-1) compared to a normal spectrum. The normal spectra show relatively stronger peaks around the 855 and 940 cm(-1) region. Scores of factor 1 as well as Mahalanobis distance and spectral residuals gave good classification among the tissue types. The limit test approach provided unambiguous and objective discrimination. CONCLUSION: The findings of this study demonstrate the efficacy of conventional Raman spectroscopy and our statistical methodologies in discrimination of normal from malignant ovarian tissues. Prospectively, by evaluating the models and developing suitable fiberoptic probes, this technique could be useful in diagnosis during initial laparotomy.

Ovarian crescent sign and sonomorphological indices in preoperative determination of malignancy in adnexal masses.

OBJECTIVE: To compare the value of ovarian crescent sign (OCS) and various sonomorphologic scoring systems in consolidating pre-operative suspicion of ovarian malignancy in adnexal masses. MATERIALS AND METHODS: A prospective study was carried out in 60 consenting women with an undiagnosed adnexal mass requiring operative intervention. OCS was considered to be present if normal ovarian tissue was seen adjacent to the tumor area on ultrasound. Various other sonological parameters were noted to calculate five morphological scoring systems. Doppler velocimetry study values were available in 27 cases. The results were compared for correctness of suspicion with histopathologic examination report of the tumor obtained at surgery. RESULTS: Eleven of 60 specimens showed histopathologic diagnosis of malignancy. OCS was identified in 97% of the benign masses. The sign was not seen in 10 of the 11 cases with malignancy. Sensitivity and negative predictive value of crescent sign was better than values obtained for the compared sonomorphological indices and Doppler velocimetry studies. CONCLUSION: Ovarian crescent sign is a reliable and simple sonographic indicator comparable to sonomorphological indices and Doppler flow velocimetric studies for the preoperative detection of malignancy in adnexal masses.

FTIR and Raman microspectroscopy of normal, benign, and malignant formalin-fixed ovarian tissues.

Ovarian cancer is the sixth most common cancer among women worldwide, and mortality rates from this cancer are higher than for other gynecological cancers. This is attributed to a lack of reliable screening methods and the inadequacy of treatment modalities for the advanced stages of the disease. FTIR and Raman spectroscopic studies of formalin-fixed normal, benign, and malignant ovarian tissues have been undertaken in order to investigate and attempt to understand the underlying biochemical changes associated with the disease, and to explore the feasibility of discriminating between these different tissue types. Raman spectra of normal tissues indicate the dominance of proteins and lower contents of DNA and lipids compared to malignant tissues. Among the pathological tissues studied, spectra from benign tissues seem to contain more proteins and less DNA and lipids compared to malignant tissue spectra. FTIR studies corroborate these findings. FTIR and Raman spectra of both normal and benign tissues showed more similarities than those of malignant tissues. Cluster analysis of first-derivative Raman spectra in the 700-1700 cm(-1) range gave two clear groups, one corresponding to malignant and the other to normal+benign tissues. At a lower heterogeneity level, the normal+benign cluster gave three nonoverlapping subclusters, one corresponding to normal and two for benign tissues. Cluster analysis of second-derivative FTIR spectra in the combined spectral regions of 1540-1680 and 1720-1780 cm(-1) resulted into two clear clusters corresponding to malignant and normal+benign tissues. The cluster corresponding to normal+benign tissues produced nonoverlapping subclusters for normal and benign tissues at a lower heterogeneity level. The findings of this study demonstrate the feasibility of Raman and FTIR microspectroscopic discrimination of formalin-fixed normal, benign, and malignant ovarian tissues.

Evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by Raman microspectroscopy.

A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging.