Rashmi Seth

Organization of Human Papillomavirus Productive Cycle during Neoplastic Progression Provides a Basis for Selection of Diagnostic Markers

ABSTRACT The productive cycle of human papillomaviruses (HPVs) can be divided into discrete phases. Cell proliferation and episomal maintenance in the lower epithelial layers are followed by genome amplification and the expression of capsid proteins. These events, which occur in all productive infections, can be distinguished by using antibodies to viral gene products or to surrogate markers of their expression. Here we have compared precancerous lesions caused by HPV type 16 (HPV16) with lesions caused by HPV types that are not generally associated with human cancer. These include HPV2 and HPV11, which are related to HPV16 (supergroup A), as well as HPV1 and HPV65, which are evolutionarily divergent (supergroups E and B). HPV16-induced low-grade squamous intraepithelial lesions (CIN1) are productive infections which resemble those caused by other HPV types. During progression to cancer, however, the activation of late events is delayed, and the thickness of the proliferative compartment is progressively increased. In many HPV16-induced high-grade squamous intraepithelial lesions (CIN3), late events are restricted to small areas close to the epithelial surface. Such heterogeneity in the organization of the productive cycle was seen only in lesions caused by HPV16 and was not apparent when lesions caused by other HPV types were compared. By contrast, the order in which events in the productive cycle were initiated was invariant and did not depend on the infecting HPV type or the severity of disease. The distribution of viral gene products in the infected cervix depends on the extent to which the virus can complete its productive cycle, which in turn reflects the severity of cervical neoplasia. It appears from our work that the presence of such proteins in cells at the epithelial surface allows the severity of the underlying disease to be predicted and that markers of viral gene expression may improve cervical screening.

The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133−expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause “off-target” effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133− populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133− populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133− population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133− population of SW480. Prolonged culture of a pure CD133− population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features.

Quantitative analysis of IL‐10 and IFN‐γ mRNA levels in normal cervix and human papillomavirus type 16 associated cervical precancer

Human papillomavirus type 16 is a major factor in cervical carcinogenesis. Inappropriate cytokine synthesis may direct the local immune response away from a type‐1 (cellular) pattern and may subsequently contribute to the development and progression of precancer. Quantitative reverse transcription–polymerase chain reaction (RT‐PCR) using a competitive mimic was carried out to determine type‐1 (interferon gamma (IFN‐γ)) and type‐2 (interleukin‐10 (IL‐10)) cytokine mRNA levels in whole cervical specimens (without microdissection) from seven normal and nine HPV‐16 positive CIN formalin‐fixed paraffin‐embedded tissues. Microdissection was used to measure separately the epithelial and sub‐epithelial levels of IFN‐γ and IL‐10 mRNAs in 11 specimens of normal cervix and 25 HPV‐16 positive CIN (nine CIN 1, seven CIN 2 and nine CIN 3). IFN‐γ mRNA was lower in CIN than normal (p=0.04). IL‐10 mRNA level in CIN was significantly higher (p=0.005) than in normal cervix (before microdissection). Epithelial IFN‐γ mRNA showed a significant decrease in all grades of CIN (median=3.58) compared with normal (7.74) (p<0.05), but there was no significant difference between the grades. A significant decrease in sub‐epithelial IFN‐γ mRNA was found in CIN 1(9.81), CIN 2 (3.82) and CIN 3 (4.62) compared with normal cervix (27.35) (p<0.05). Also, sub‐epithelial IFN‐γ mRNA was significantly lower in CIN 2 and CIN 3 than in CIN 1 (p=0.005 and 0.0005, respectively). IL‐10 was detected in the epithelium of only one of 11 normal and one of 25 CIN, but sub‐epithelial IL‐10 was significantly higher in CIN 2 (0.08) and CIN 3 (0.26) than in normal (0.00) (p=0.036 and 0.0032, respectively). There was no significant difference in the sub‐epithelial level of IL‐10 between normal and CIN 1 (0.00) (p=0.96). Our results suggest that reduced epithelial and sub‐epithelial IFN‐γ, as well as increased sub‐epithelial IL‐10 synthesis may play a role in the development and progression of HPV‐16 associated cervical precancer. Copyright

INTERLEUKIN‐8 AND INDUCIBLE NITRIC OXIDE SYNTHASE mRNA LEVELS IN INFLAMMATORY BOWEL DISEASE AT FIRST PRESENTATION

Interleukin‐8 (IL‐8) and nitric oxide (NO) may be important mediators in the pathogenesis of chronic idiopathic inflammatory bowel disease (CIIBD), but their roles in disease activity in ulcerative colitis (UC) and Crohns disease (CD) are uncertain. The aim of this study was to measure mRNA for IL‐8 and inducible NO synthase (iNOS) in small mucosal biopsies from untreated patients at first presentation and to relate these measurements to the histological levels of polymorph infiltration graded on a ten‐point scale. For this purpose, a sensitive enzyme‐linked oligonucleotide chemiluminescent assay (ELOCA) was developed to quantitate reverse transcription‐polymerase chain reaction (RT‐PCR) products amplified from RNA from paired biopsy samples. The levels of IL‐8 and iNOS mRNAs were calculated as ratios of the RT‐PCR products to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) RT‐PCR product. In UC patients, median values of IL‐8/GAPDH and iNOS/GAPDH were significantly elevated compared with controls and CD. However, in both UC and CD, the IL‐8/GAPDH and iNOS/GAPDH ratios correlated significantly with polymorph infiltration. ELOCA enabled quantitation of multiple mRNAs in small mucosal biopsies from untreated patients with CIIBD and supported a role for IL‐8 and iNOS in acute inflammation in both UC and CD.

After-effects reported by women following colposcopy, cervical biopsies and LLETZ: results from the TOMBOLA trial.

OBJECTIVE Few studies have investigated physical after-effects of colposcopy. We compared post-colposcopy self-reported pain, bleeding, discharge and menstrual changes in women who underwent: colposcopic examination only; cervical punch biopsies; and large loop excision of the transformation zone (LLETZ). DESIGN Observational study nested within a randomised controlled trial. SETTING Grampian, Tayside and Nottingham. POPULATION Nine hundred-and-twenty-nine women, aged 20-59, with low-grade cytology, who had completed their initial colposcopic management. METHODS Women completed questionnaires on after-effects at approximately 6-weeks, and on menstruation at 4-months, post-colposcopy. MAIN OUTCOME MEASURES Frequency of pain, bleeding, discharge; changes to first menstrual period post-colposcopy. RESULTS Seven hundred-and-fifty-one women (80%) completed the 6-week questionnaire. Of women who had only a colposcopic examination, 14-18% reported pain, bleeding or discharge. Around half of women who had biopsies only and two-thirds treated by LLETZ reported pain or discharge (biopsies: 53% pain, 46% discharge; LLETZ: 67% pain, 63% discharge). The frequency of bleeding was similar in the biopsy (79%) and LLETZ groups (87%). Women treated by LLETZ reported bleeding and discharge of significantly longer duration than other women. The duration of pain was similar across management groups. Forty-three percent of women managed by biopsies and 71% managed by LLETZ reported some change to their first period post-colposcopy, as did 29% who only had a colposcopic examination. CONCLUSIONS Cervical punch biopsies and, especially, LLETZ carry a substantial risk of after-effects. After-effects are also reported by women managed solely by colposcopic examination. Ensuring that women are fully informed about after-effects may help to alleviate anxiety and provide reassurance, thereby minimising the harms of screening.

Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer

Background and aims: KRAS and BRAF mutations occur in colorectal cancers (CRCs) and are considered mutually exclusive methods of activating the RAS/RAF/MEK/ERK pathway. This pathway is a therapeutic target and KRAS mutation may predict tumour responsiveness. The purpose of this study was to investigate the relationship between KRAS and BRAF mutations in 24 CRC cell lines and 29 advanced CRCs. Methods: KRAS and BRAF mutations were detected using high resolution melting and sequencing. Expression of mutations was confirmed by reverse transcription- PCR (RT-PCR) and sequencing. CpG island methylator phenotype (CIMP) was tested by methylation-specific PCR. Results: KRAS or BRAF mutation occurred in 79% of cell lines and 59% of CRCs. In the cell lines, KRAS mutations occurred in 54% of cases (with 62% in codons 12/13 and 38% in other codons). Four cell lines had a homozygous mutation. Only heterozygous BRAF mutations were detected in 29% cell lines. The V600E mutation occurred most commonly and was associated with CIMP+ status (p = 0.005). Mutations at codons 529 and 581 were also found and, in one case, BRAF and KRAS mutation co-occurred. Unexpectedly, BRAF splice variants (with a predicted kinase-dead protein) were found in 5/24 (21%) cell lines. In advanced CRCs, KRAS mutations occurred in 48% of cases (64% codons 12/13, 36% other codons) and BRAF mutations in 10% (66% V600E, 33% exon 11). A compound KRAS/BRAF mutation was not seen. Conclusions: Disrupted Ras/Raf signalling is common in CRC. Homozygous KRAS mutations and concomitant KRAS/BRAF mutations may be indicative of a gene dosage effect. The significance of BRAF splice variants is uncertain but may represent another layer of complexity. Finally, if KRAS mutation is to be used for predictive testing, then the whole gene may need to be screened as mutations occur outside codons 12/13.

Cervical Human Papillomavirus Screening among Older Women

HPV-negative women >50 years of age still require cervical screening

Lifestyle and socio-demographic factors associated with high-risk HPV infection in UK women

The world age-standardised prevalence of high-risk HPV (hrHPV) infection among 5038 UK women aged 20–59 years, with a low-grade smear during 1999–2002, assessed for eligibility for TOMBOLA (Trial Of Management of Borderline and Other Low-grade Abnormal smears) was 34.2%. High-risk HPV prevalence decreased with increasing age, from 61% at ages 20–24 years to 14–15% in those over 50 years. The age-standardised prevalence was 15.1, 30.7 and 52.7%, respectively, in women with a current normal, borderline nuclear abnormalities (BNA) and mild smear. In overall multivariate analyses, tertiary education, previous pregnancy and childbirth were associated with reduced hrHPV infection risk. Risk of infection was increased in non-white women, women not married/cohabiting, hormonal contraceptives users and current smokers. In stratified analyses, current smear status and age remained associated with hrHPV infection. Data of this type are relevant to the debate on human papillomavirus (HPV) testing in screening and development of HPV vaccination programmes.

C‐terminal Tensin‐like (CTEN) is an oncogene which alters cell motility possibly through repression of E‐cadherin in colorectal cancer

The Tensin gene family encodes proteins thought to modulate integrin function. C‐terminal Tensin‐like (CTEN) is a member of the Tensin gene family which lacks the N‐terminus actin‐binding domain. Cten is reported to have both oncogenic and tumour‐suppressor functions. We investigated the role that Cten may play in colorectal cancer (CRC). By quantitative RT–PCR CTEN is up‐regulated (i.e. > two‐fold increase) in 62% of cell lines and 69% of tumours compared with normal mucosa, consistent with CTEN being a possible oncogene. Stable transfection of HCT116 and SW480 (CRC cell lines with low endogenous Cten expression) with a Cten expression vector gave identical results in both cell lines. Forced Cten expression did not cause change in cell numbers, although it did confer resistance to staurosporine‐induced apoptosis (p < 0.005). Cten also induced epithelial–mesenchymal transition (EMT) in tumour cells accompanied by a significant increase in both cell migration (transwell migration and cell wounding assays, p < 0.001 and p < 0.05, respectively) and cell invasion (invasion through Matrigel, p < 0.001). Given the observed EMT, we investigated the levels of E‐cadherin. Cten induction was associated with a reduction in E‐cadherin protein expression but not levels of E‐cadherin mRNA. These data suggest that CTEN is an oncogene in CRC which stimulates EMT, cell migration and invasion and may therefore have a role in tumour invasion/spread. Furthermore, Cten induction is associated with post‐transcriptional repression of E‐cadherin. Copyright

Decreased synthesis and expression of TGF-β1, β2 and β3 in epithelium of HPV 16-positive cervical precancer: a study by microdissection, quantitative RT-PCR, and immunocytochemistry

Cervical carcinogenesis is a multistep process initiated by ‘high‐risk’ human papillomaviruses (HR‐HPVs), most commonly HPV 16. Transforming growth factor‐beta (TGF‐β) inhibits epithelial proliferation and down‐regulates transcription of E6/E7 genes of HPV. Altered TGF‐β expression may be important in carcinogenesis. Quantitative RT‐PCR was used to investigate TGF‐β1, β2, and β3 mRNA levels in nine specimens of normal cervix and 15 cervical precancers (eight HPV‐positive, including five HPV 16‐positive). Immunocytochemical expression of TGF‐β1, β2, and β3 was examined in cervical intraepithelial neoplasia (CIN) positive for HPV 16 (26), and in HPV‐negative, normal ectocervical epithelium (9); reserve cell hyperplasia (12); and immature (7) and mature (15) squamous metaplasia. The intensity of staining for TGF‐β1 was measured using grey‐scale image analysis. Microdissection was used to investigate epithelial and stromal (excluding crypts) levels of TGF‐β1 mRNA in HPV 16‐positive cervical precancer. Normal cervix, including reserve cells and immature and mature metaplasia, showed strong immunocytochemical expression of all TGF‐β isoforms. Expression was decreased in the basal third of the epithelium in CIN 1, in the basal and middle thirds in CIN 2, and in all layers in CIN 3. Quantitative analysis of TGF‐β1 expression showed that the changes in CIN compared with normal ectocervix and mature metaplasia were statistically highly significant (p<0.001, ANOVA). TGF‐β1, β2, and β3 mRNA levels showed a significant decrease only in the five HPV 16‐positive CIN samples when compared with normal (p=0.0034, 0.0033, and 0.029, respectively). TGF‐β mRNA levels in HPV 16‐positive epithelium also decreased from normal through low‐grade to high‐grade precancer. Stromal TGF‐β1 was absent or very low compared with epithelial production and was not altered in HPV 16 precancer. Progressive loss of epithelial TGF‐β expression and synthesis may be important in HPV 16‐associated human cervical carcinogenesis. Copyright