Intussusception After Roux-en-Y Gastric Bypass: Correlation Between Radiological and Operative Findings.

INTRODUCTION: Intussusceptions diagnosed on computed tomography (CT) scans in Roux-en-Y gastric bypass (RYGB) patients could cause serious small bowel obstruction (SBO) or be an incidental finding. The objective of this study was to correlate radiological findings with clinical outcomes to differentiate intussusceptions requiring emergent surgery for SBO. METHODS: A search for acute abdominal CT scans reporting intussusceptions in RYGB patients between 2012 and 2019 at Skåne University Hospital, Malmö, Sweden, retrieved 35 scans. These were independently reevaluated by two radiologists for the length and location of the intussusception, whether oral contrast passed through, proximal bowel dilatation, and signs of internal herniation. Clinical outcome in terms of emergency surgery and the diagnosis was determined through chart review. RESULTS: Out of 35 acute patients, 9 patients required emergency surgery within 24 h. Intussusception caused SBO in five patients, and one patient had an internal herniation, while three patients had unremarkable findings. Eight patients were evaluated for intermittent pain with five unremarkable laparoscopies, while 18 patients had intussusceptions as incidental findings. Intussusception length on CT as measured by radiologists O.E. and D.L. predicted acute bowel obstruction (p = .014 and p < .001). A 100 mm threshold predicted bowel obstruction with a sensitivity of 80% and 100% and a specificity of 93% and 86% by radiologists O.E. and D.L., respectively. Proximal bowel dilatation predicted SBOs of any cause as well as SBO caused by an intussusception (all p < .05). CONCLUSION: Intussusception length > 100 mm on CT in RYGB patients is an easy and valuable sign indicating SBO that may require emergent surgery.

Age-group differences in human papillomavirus types and cofactors for cervical intraepithelial neoplasia 3 among women referred to colposcopy.

BACKGROUND: Recommendations for high-risk human papillomavirus (HR-HPV) testing as an adjunct to cytology for cervical cancer screening differ by age group, because HR-HPV tests lack adequate specificity in women aged <30. Here, we assess age-group differences in HPV types and other risk factors for cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) versus CIN0-2 in women from four colposcopy clinics. METHODS: Women ages 18 to 69 (n = 1,658) were enrolled and completed structured interviews to elicit data on behavioral risk factors prior to their examinations. HPV genotyping was done on exfoliated cervical cell samples. We estimated relative risks (RR) for HPV types and cofactors for CIN3+, overall and stratified by age group. RESULTS: After 2 years of follow-up, we identified 178 CIN3+, 1,305 CIN0-2, and 175 indeterminate outcomes. Nonvaccine HR-HPV types were only associated with CIN3+ among women ≥ 30 (RR = 2.3, 95% CI: 1.5-3.4; <30: RR = 0.9). Among all HR-HPV-positive women, adjusting for age, significant cofactors for CIN3+ included current smoking (RR = 1.5), former smoking (RR = 1.8), regular Pap screening (RR = 0.7), current regular condom use (RR = 0.5), and parity ≥ 5 (RR = 1.6, P(trend) for increasing parity = 0.07). However, the parity association differed by age group (≥ 30: RR = 1.8, P(trend) = 0.008; <30: RR = 0.9; P(trend) =.55). CONCLUSION: Subgroup variation by age in the risk of CIN3+ points to the importance of the timing of exposures in relation to CIN3+ detection. IMPACT: Future screening strategies need to consider natural history and secular trends in cofactor prevalence in the pursuit of appropriately sensitive and specific screening tools applied to appropriate age groups.

Lack of HPV 16 and 18 detection in serum of colposcopy clinic patients.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) types is necessary for the development of high-grade cervical dysplasia and cervical carcinoma. The presence of HPV DNA in the blood of cervical cancer patients has been reported; however, whether HPV DNA is detectable in the blood of patients with pre-invasive cervical disease is unclear. OBJECTIVES: The objectives of this study were to determine if HPV 16 and HPV 18 DNA could be detected in the serum of colposcopy clinic patients, and if serum HPV detection was associated with grade of cervical disease and HPV cofactors. STUDY DESIGN: Samples were selected from a biorepository collected from non-pregnant, HIV-negative women ages 18-69 attending colposcopy clinics at two urban public hospitals. Cervical disease status was based on review of colposcopy, biopsy and cytology findings. Serum HPV DNA detection was conducted using a novel PCR and mass spectroscopy-based assay. RESULTS: Of the 116 adequate serum samples, all (100%) were negative for HPV 16 and HPV 18. Over half (51.7%) of participants had cervical HPV 16 and/or HPV 18 infection. Nearly one-third (31.1%) had high grade, 10.3% had low grade, and 50.9% had no cervical disease. Nearly one-third (28.5%) had ever regularly smoked cigarettes, 70.7% had early onset of sexual intercourse, and 75% had ever used oral contraceptives. CONCLUSIONS: In this colposcopy clinic population with a range of clinical characteristics and established HPV cofactors, HPV DNA was undetectable in their serum. Our findings suggest that serum HPV DNA detection is not a cervical cancer screening tool.

Optimization of SELDI-TOF protein profiling for analysis of cervical mucous.

Cervical mucous, produced in the region where cervical neoplasia occurs, is thought to be a good choice for discovery of biomarkers to improve cervical cancer screening. In this study, SELDI-TOF MS analysis was used to evaluate parameters for protein profiling of mucous. Proteins were extracted from mucous collected with Weck-Cel sponges. Several parameters like extraction reagent, loading protein concentration, matrix type, bind/wash conditions and sample fractionation, on different protein chip surfaces were evaluated. SELDI peak number and consistency in the resulting spectra were used to evaluate each condition. Analysis of spectra generated by different protein chips revealed an average of 30 peaks in the 2.5-30 kDa mass range using sinnapinic acid in the unfractionated sample. Sample concentration and buffer conditions evaluated did not lead to large alterations in the profiles. Quality control spectra were reproducible with intra- and inter-assay intensity CV for CM10, H50 and Q10 arrays being less than 20% and 30% respectively. IMAC30-Cu chips had higher intra- and inter-assay CV's at 25% and 35%. Current data showed that optimizing pre-analytical parameters can help in standardization and reproducibility of protein profiles produced by cervical mucous, and thus can be used for protein biomarker discovery with the SELDI platform.

Evaluation of RNA markers for early detection of cervical neoplasia in exfoliated cervical cells.

Numerous molecular biomarkers have been suggested for early detection of cervical cancer, but their usefulness in routinely collected exfoliated cells remains uncertain. We used quantitative reverse transcription-PCR to evaluate expression of 40 candidate genes as markers for high-grade cervical intraepithelial neoplasia (CIN) in exfoliated cervical cells collected at the time of colposcopy. Samples from the 93 women with CIN3 or cancer were compared with those from 186 women without disease matched (1:2) for age, race, and high-risk human papillomavirus status. Normalized threshold cycles (C(t)) for each gene were analyzed by receiver operating characteristics to determine their diagnostic performance in a split sample validation approach. Six markers were confirmed by an area under the curve >0.6 in both sample sets: claudin 1 (0.75), minichromosome maintenance deficient 5 (0.71) and 7 (0.64), cell division cycle 6 homologue (0.71), antigen identified by monoclonal antibody Ki-67 (0.66), and SHC SH2-domain binding protein 1 (0.61). The sensitivity for individual markers was relatively low and a combination of five genes to a panel resulted in 60% sensitivity with 76% specificity, not positively increasing this performance. Although the results did not indicate superiority of RNA markers for cervical cancer screening, their performance in detecting disease in women referred for colposcopy suggests that the genes and pathways they highlight could be useful in alternative detection formats or in combination with other screening indicators.

Quantitation of site-specific HPV 16 DNA methylation by pyrosequencing.

Human papillomavirus (HPV) is a necessary but insufficient cause of cervical cancer. Factors influencing transcription, such as epigenetic silencing through viral DNA methylation, may impact neoplastic progression. Pyrosequencing technology was applied to quantify methylation at 19 cytosine guanine dinucleotide (CpG) sites in the L1 3' and long control region (LCR) of HPV 16 DNA using cell lines, CaSki ( approximately 400 integrated copies of HPV 16) and SiHa (1-2 integrated copies of HPV 16) that differ in their transcriptional activity. Methylation levels ranged from 20 to 100% in CaSki and from 0 to 85% in SiHa over the entire 19 CpG sites, with a >40-fold difference in the methylation levels of their promoter and enhancer regions (SiHa<2% and CaSki 79%). The method was successful at a limiting dilution of 1-4 HPV 16 DNA copies/3000 cells, a level compatible with most clinical samples. The results were not affected by fixation in methanol-based liquid cytology collection fluid or method of extraction. Conditions optimized with cell lines were applicable to fixed exfoliated cervical cells. Pyrosequencing provides a quantitative site-specific assessment of methylation at multiple CpG sites without cloning, and is thus suited to large-scale molecular epidemiologic studies.

Human papillomavirus in amniotic fluid.

BACKGROUND: There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. METHODS: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. RESULTS: There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23-55 years. All samples were beta-globin positive. HPV was not detected in any of the paired samples. CONCLUSION: Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.

Gene expression profiling of dysplastic differentiation in cervical epithelial cells harboring human papillomavirus 16.

Molecular events occurring with high-risk human papillomavirus (HPV)-associated dysplastic differentiation of cervical epithelial cells are largely unknown. This study used differential display PCR to identify expression changes between nondifferentiating monolayer and differentiated organotypic (raft) cultures of W12 keratinocytes. These cells were originally derived from a clinical biopsy of HPV 16-positive dysplastic cervical epithelium and retain high-risk HPV 16 and the ability to differentiate, albeit with dysplastic morphology. Using this model system we identified 84 genes with changed expression during dysplastic differentiation. Most (70/84, approximately 80%) were down-regulated with differentiation, consistent with a restriction of expression during terminal differentiation. Twenty-two genes had no known function and 6 novel expressed sequence tags were identified among this group. Of the 62 genes with known functions, 25 belonged to transcription-, translation-, and posttranslation-related categories and 30 had functions associated with neoplastic initiation/progression, calcium signaling, epithelial differentiation, and structure remodeling. Some of the genes with altered expression identified in this model of dysplastic differentiation may be useful biomarkers for early detection of cervical neoplasia and other HPV-associated oropharyngeal and anogenital cancers.

Gene expression profile of cervical tissue compared to exfoliated cells: impact on biomarker discovery.

BACKGROUND: Exfoliated cervical cells are used in cytology-based cancer screening and may also be a source for molecular biomarkers indicative of neoplastic changes in the underlying tissue. However, because of keratinization and terminal differentiation it is not clear that these cells have an mRNA profile representative of cervical tissue, and that the profile can distinguish the lesions targeted for early detection. RESULTS: We used whole genome microarrays (25,353 unique genes) to compare the transcription profiles from seven samples of normal exfoliated cells and one cervical tissue. We detected 10,158 genes in exfoliated cells, 14,544 in the tissue and 7320 genes in both samples. For both sample types the genes grouped into the same major gene ontology (GO) categories in the same order, with exfoliated cells, having on average 20% fewer genes in each category. We also compared microarray results of samples from women with cervical intraepithelial neoplasia grade 3 (CIN3, n = 15) to those from age and race matched women without significant abnormalities (CIN1, CIN0; n = 15). We used three microarray-adapted statistical packages to identify differential gene expression. The six genes identified in common were two to four fold upregulated in CIN3 samples. One of these genes, the ubiquitin-conjugating enzyme E2 variant 1, participates in the degradation of p53 through interaction with the oncogenic HPV E6 protein. CONCLUSION: The findings encourage further exploration of gene expression using exfoliated cells to identify and validate applicable biomarkers. We conclude that the gene expression profile of exfoliated cervical cells partially represents that of tissue and is complex enough to provide potential differentiation between disease and non-disease.

Epidemiologic and viral factors associated with cervical neoplasia in HPV-16-positive women.

While infection with high-risk HPV is the most important risk factor for cervical cancer, HPV alone is insufficient. Our purpose was to identify viral and epidemiologic factors associated with cervical disease in HPV-16 DNA-positive women referred to colposcopy. We used a standardized interview to collect epidemiologic data from consenting women. Total nucleic acids from exfoliated cervical cells were used for all viral assays (HPV detection and typing using L1 consensus PCR with line probe hybridization, variant classification by sequencing, viral load and transcript copy determination by quantitative PCR and transcript pattern by nested RT-PCR). Cervical disease was based on colposcopic biopsy. Logistic regression was used to calculate ORs with 95% CIs. There were 115 HPV-16 positive women among 839 enrollees. By univariate analyses, age >25 years (OR = 3.05, 95% CI 1.20-7.76), smoking (OR = 3.0, 95% CI 1.19-7.56), high viral load (OR = 5.27, 95% CI 2.05-13.60), detection of both E6 and E6*I transcripts (OR = 10.0, 95% CI 2.1-47.58) and high transcript copies (OR = 5.56, 95% CI 2.05-13.60) were significant risk factors for CIN III with reference to No CIN/CIN I. Less than a third of the women (31.5%) had prototype HPV-16 detected, and variants showed no association with disease, viral load or transcription. Viral DNA and transcript copies were highly correlated, and the ratio of transcript copies to DNA copies was not changed with disease status. While viral load, transcript copies and transcript pattern were statistically associated with CIN III, none of these measures effectively discriminated between HPV-16 women with disease requiring treatment and those who could be followed. Cellular proliferation and differentiation pathways affected by HPV should be investigated as biomarkers for cervical cancer screening.

Molecular quality of exfoliated cervical cells: implications for molecular epidemiology and biomarker discovery.

The biologic sample collected in molecular epidemiology studies must accurately reflect the disease being studied and have sufficient molecular quality for the intended assays. Noninvasive sampling methods, such as scrapes or brushes, are increasingly used. In this study, we evaluate the impact of sample collection media and extraction methods on the quality and yield of RNA from routine exfoliated cervical cytology. Excess cellular material remaining on the cytologic collection device after preparation of the routine screening Papanicolaou smear was placed in a variety of collection media and extracted using two commercial kits. The collection media had the largest impact on the yield and quality of RNA as evaluated by denaturing agarose gel electrophoresis and image analysis. Two collection media, PAXgene and PreservCyt, yielded RNA from most samples. The RNA showed some degree of degradation as evidenced by the reduced size of the higher molecular weight ribosomal band. However, with a sensitive gold particle-based detection method, reproducible microarray results were obtained using this RNA.