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Nursing education is transforming from traditional systems paradigms to concept-based curricula. We explored how this change impacts faculty using focus groups with questions adapted from Bridges' transitions model. Results revealed that many faculty did not feel there was a clear vision for the transition and felt inadequately prepared for the change. Uncertainty about their new role led some to feel displaced. To ease future transitions, we suggest that leadership clearly communicate the rationale for the change, ensure that there is a shared vision, provide an appropriate timeline for the transition, and support faculty as their roles are redefined.

Objective: Early-onset colorectal cancer (CRC) is increasing in the USA despite rapid declines in older ages. Similar patterns are reported in Australia and Canada, but a comprehensive global analysis of contemporary data is lacking., Design: We extracted long-term data from Cancer Incidence in Five Continents and supplemental sources to report on worldwide CRC incidence rates and trends by age (20-49 years and ≥50 years) through diagnosis year 2012 or beyond (Australia, Finland, New Zealand, Norway, Sweden, USA)., Results: During 2008-2012, age-standardised CRC incidence rates in adults <50 ranged from 3.5 per 100 000 (95% CI 3.2 to 3.9) in India (Chennai) to 12.9 (95% CI 12.6 to 13.3) in Korea. During the most recent decade of available data, incidence in adults <50 was stable in 14 of 36 countries; declined in Austria, Italy and Lithuania; and increased in 19 countries, nine of which had stable or declining trends in older adults (Australia, Canada, Denmark, Germany, New Zealand, Slovenia, Sweden, UK and USA). In Cyprus, Netherlands and Norway, inclines in incidence in young adults were twice as rapid as those in older adults (eg, Norway average annual per cent change (AAPC), 1.9 (95% CI 1.4 to 2.5) vs 0.5 (95% CI 0.3 to 0.7)). Among most high-income countries with long-term data, the uptick in early-onset disease began in the mid-1990s. The steepest increases in young adults were in Korea (AAPC, 4.2 (95% CI 3.4 to 5.0)) and New Zealand (AAPC, 4.0 (95% CI 2.1 to 6.0))., Conclusion: CRC incidence increased exclusively in young adults in nine high-income countries spanning three continents, potentially signalling changes in early-life exposures that influence large bowel carcinogenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Constipation often begins in the first year of life. The aim of this study was to assess the effect of fructooligosaccharides (FOS) in the treatment of infants with constipation. This randomized, double-blind, placebo-controlled clinical trial included infants with constipation who were randomly assigned to one of two parallel groups: FOS or placebo. Either the FOS supplement or the placebo was added to the infant formula. Thirty-six infants completed the 4-week intervention. Therapeutic success occurred in 83.3% of the FOS group infants and in 55.6% of the control group infants ( p = 0.073; one-tailed test). Compared with the control group, the FOS group exhibited a higher frequency of softer stools ( p = 0.035) and fewer episodes of straining and/or difficulty passing stools ( p = 0.041). At the end of the intervention, the mouth-to-anus transit time was shorter (22.4 and 24.5 h, p = 0.035), and the Bifidobacterium sp. count was higher ( p = 0.006) in the FOS group. In conclusion, the use of FOS in infants with constipation was associated with significant improvement in symptoms, but the results showed no statistical significance regarding the success of the therapy compared with the control group. FOS was associated with reduced bowel transit time and higher counts of the genus Bifidobacterium in the stool.

Objective: Although widely recommended, Lynch syndrome (LS) testing with tumor microsatellite instability (MSI) and/or immunohistochemistry (IHC) is infrequently performed in early-onset colorectal cancer (CRC), and CRC generally. Reasons are poorly understood. Hence, we conducted a national survey focusing on gastroenterologists, as they are frequently first to diagnose CRC, assessing testing barriers and which specialist is felt responsible for ordering MSI/IHC. Additionally, we assessed factors influencing timing of MSI/IHC ordering; testing on colonoscopy biopsy, opposed to post-operative surgical specimens, assists decisions on preoperative germline genetic testing and extent of colonic resection (ECR)., Methods: A 21-question web-based survey was distributed through an American College of Gastroenterology email listing., Results: In total 509 completed the survey. 442 confirmed gastroenterologists were analyzed. Only 33.4% felt gastroenterologists were responsible for MSI/IHC ordering; pathologists were believed most responsible (38.6%). Cost, unfamiliarity interpreting results and unavailable genetic counseling most commonly prevented routine ordering (33.3%, 29.2%, 24.9%, respectively). In multivariable analysis, non-academic and rural settings were associated with cost and genetic counseling barriers. Only 46.1% felt MSI/IHC should always be performed on colonoscopy biopsy. Guideline familiarity predicted whether respondents felt surgical resection should be delayed until results returned given potential effect on ECR decisions., Conclusion: Inconsistencies in who is felt should order MSI/IHC may lead to diffusion of responsibility, preventing consistent testing, including preoperatively. Assuring institutional universal testing protocols are in place, with focus on timing of testing, can optimize care. Strategies addressing cost barriers and genomic service availability in rural and non-academic settings can enhance testing. Greater emphasis on guideline familiarity is required.

This study aimed to investigate the association between twelve gene polymorphisms and body weight loss, 12 months after Roux-en-Y gastric bypass (RYGB) surgery. Three hundred and fifty-one obese women participated in this study. The statistical software WEKA was used to identify which gene polymorphisms were potential predictors of postoperative percentage of excess weight loss (%EWL). Our results indicate that the only gene polymorphism that predicted %EWL was rs3813929, which is related to the serotonin receptor gene (5-HT2C). Therefore, the 5-HT2C gene polymorphism rs3813929 (more specifically, the TT genotype) predicted greater %EWL 12 months after RYGB surgery among female patients., (Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.)

Persons with a family history (FH) of colorectal cancer (CRC) or adenomas that are not due to known hereditary syndromes have an increased risk for CRC. An understanding of these risks, screening recommendations, and screening behaviors can inform strategies for reducing the CRC burden in these families. A comprehensive review of the literature published within the past 10 years has been performed to assess what is known about cancer risk, screening guidelines, adherence and barriers to screening, and effective interventions in persons with an FH of CRC and to identify FH tools used to identify these individuals and inform care. Existing data show that having 1 affected first-degree relative (FDR) increases the CRC risk 2-fold, and the risk increases with multiple affected FDRs and a younger age at diagnosis. There is variability in screening recommendations across consensus guidelines. Screening adherence is <50% and is lower in persons under the age of 50 years. A provider's recommendation, multiple affected relatives, and family encouragement facilitate screening; insufficient collection of FH, low knowledge of guidelines, and poor family communication are important barriers. Effective interventions incorporate strategies for overcoming barriers, but these have not been broadly tested in clinical settings. Four strategies for reducing CRC in persons with familial risk are suggested: 1) improving the collection and utilization of the FH of cancer, 2) establishing a consensus for screening guidelines by FH, 3) enhancing provider-patient knowledge of guidelines and communication about CRC risk, and 4) encouraging survivors to promote screening within their families and partnering with existing screening programs to expand their reach to high-risk groups. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2633-2645. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Background: Although screening for colorectal cancer (CRC) is a widely accepted concept nationally and screening rates are increasing, there are differences in screening rates between states and within states., Methods: In an effort to increase screening rates and ensure equal access with respect to race/ethnicity, the New York City Department of Health and Mental Hygiene formed a coalition of stakeholders in 2003, with its primary focus on colonoscopy, to develop and implement strategies across the city to achieve this goal., Results: From a screening colonoscopy rate of only 42% in 2003, these concerted efforts contributed to achieving a screening rate of 62% by 2007 and a screening rate of almost 70% in 2014 with the elimination of racial and ethnic disparities., Conclusions: This article provides details of how this program was successfully conceived, implemented, and sustained in the large urban population of New York City. The authors hope that by sharing the many elements involved and the lessons learned, they may help other communities to adapt these experiences to their own environments so that CRC screening rates can be maximized. Cancer 2016;122:269-277. © 2015 American Cancer Society., (© 2015 American Cancer Society.)

Background & Aim: n-3 fatty acid intake has been associated with inflammatory benefits in cardiovascular disease (CVD). Functionalising meat may be of great interest. The aim of the present study was to assess the effect of functional meat containing n-3 and rosemary extract on inflammatory and oxidative status markers in subjects with risk for CVD., Methods and Results: A randomised, double-blind, cross-over study was undertaken to compare the effects on the above markers of consuming functional or control meat products. 43 volunteers with at least two lipid profile variables showing risk for CVD were randomly assigned to receive functional meat (FM) or control meat (CM) over 12-weeks with a 4-week wash-out interval before crossover. Functional effects were assessed by examining lipid profile, CRP, PAI-1, TNF-alpha, IL-6, fibrinogen (inflammatory markers), and TBARS, FRAP and 8-iso-PGF2 (oxidative status markers). 33 subjects (24 women) aged 50.7±8.8 years completed the study. In FM treatment, PAI-1, fibrinogen and 8-iso-PGF2 decreased significantly after 12 weeks, while FRAP significantly increased. In contrast, in CM treatment, a significant increase was seen in PAI-1, while FRAP significantly declined. Significant differences were also seen between the FM and CM treatments after 12 weeks in terms of the change observed in PAI-1, FRAP and 8-iso-PGF2 values. No significant differences were seen in anthropometric variables nor were adverse effects reported., Conclusion: The consumption of FM containing n-3 and rosemary extract improved oxidative and inflammatory status of people with at least two lipid profile variables showing risk for CVD. The inclusion of such functional meat in a balanced diet might be a healthy lifestyle option., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: The aim of the study was to test the clinical efficacy and effect on colonic transit time (CTT) of a dietary fiber mixture given to children with controlled chronic constipation (CC) after the withdrawal of stool softeners and enemas., Methods: This randomized, placebo-controlled, double-blind clinical trial involved 54 patients aged 4 to 12 years and had CC that was controlled by the use of low-dose stool softeners. The use of these softeners was discontinued when the patients were admitted to the clinical trial. The patients were randomized into 2 groups for the 4-week study period. One group received a dietary fiber mixture and the other group received a placebo (maltodextrin). The primary outcome was therapeutic failure (oral stool softeners or enemas was required to prescribe during the trial). Secondary outcomes included defecation frequency, stool consistency (measured using the Bristol Stool Form Scale), and CTT., Results: Therapeutic failure was observed in 34.6% (9/26) of the patients in the dietary fiber mixture group and in 35.7% (10/28) in the control group (P = 0.933). The mean increase in daily bowel movements was 0.53 in the dietary fiber mixture group and 0.23 in the control group (P = 0.014). The patients in the dietary fiber mixture group (60.0%) passed nonhardened stools more frequently than did those in the control group (16.7%, P = 0.003). The CTT was similar for both groups., Conclusions: The fiber mixture did not prevent the suspension of stool softeners or lead to reduced CTT; however, the mixture promoted an increased frequency of defecation and an improvement in the stool consistency.

The bacterial colonization is defined immediately after birth, through direct contact with maternal microbiota and may be influenced during lactation. There is emerging evidence indicating that quantitative and qualitative changes on gut microbiota contribute to alterations in the mucosal activation of immune system leading to intra- or extra-intestinal diseases. A balance between pathogenic and beneficial microbiota throughout childhood and adolescence is important to gastrointestinal health, including protection against pathogens, inhibition of pathogens, nutrient processing (synthesis of vitamin K), stimulation of angiogenesis, and regulation of host fat storage. Probiotics can promote an intentional modulation of intestinal microbiota favoring the health of the host. This paper is a review about modulation of intestinal microbiota on prevention and adjuvant treatment of pediatric gastrointestinal diseases.

Objectives: In 2003, in response to low colonoscopy screening rates and significant sociodemographic disparities in colonoscopy screening in New York City (NYC), the NYC Department of Health and Mental Hygiene, together with the Citywide Colon Cancer Control Coalition, launched a multifaceted campaign to increase screening. We evaluated colonoscopy trends among adult New Yorkers aged 50 years and older between 2003 and 2007, the first five years of this campaign., Methods: Data were analyzed from the NYC Community Health Survey, an annual, population-based surveillance of New Yorkers. Annual prevalence estimates of adults who reported a timely colonoscopy, one within the past 10 years, were calculated. Multivariate models were used to analyze changes over time in associations between colonoscopy screening and sociodemographic characteristics., Results: Overall, from 2003 to 2007 the proportion of New Yorkers aged 50 years and older who reported timely colonoscopy screening increased from 41.7% to 61.7%. Racial/ethnic and sex disparities observed in 2003 were eliminated by 2007: prevalence of timely colonoscopy was similar among non-Hispanic whites, non-Hispanic blacks, Hispanics, men, and women. However, Asians, the uninsured, and those with lower education and income continued to lag in receipt of timely colonoscopies., Conclusions: The increased screening colonoscopy rate and reduction of racial/ethnic disparities observed in NYC suggest that multifaceted, coordinated urban campaigns can improve low utilization of clinical preventive health services and reduce public-health disparities.

There is concern regarding the possible negative effects of ingestion of dietary fibre on growth and intestinal Fe absorption in infants. The aim of the present study was to compare the effect of a fibre mixture on the growth and the intestinal absorption of Fe in rats with Fe-deficiency anaemia with that of a diet without fibres. Faecal weight and caecal pH were also evaluated. According to the Hb depletion-repletion model, twenty-two male weaned Wistar rats were fed the AIN93-G diet without Fe until Fe-deficiency anaemia was induced with Hb < 70 g/l. The anaemic rats were divided into two groups: (1) fibre mixture group--fed 100 g of fibre mixture/kg of diet (soya polysaccharide, inulin, resistant starch, Arabic gum, fructo-oligossaccharide and cellulose) (n 11); (2) control group--fed without fibres (n 11). All diets had 157 mg of ferric citrate (30 mg of elemental Fe) added to lead to recovery from anaemia. Fe intestinal absorption was measured by Hb repletion efficiency (HRE) and apparent Fe intestinal absorption. The HRE was 44.8 (SD 9.5) % in the fibre mixture group and 43.0 (SD 9.5) % in the control group (P = 0.664). The apparent Fe absorption was 46.2 (SD 16.5) and 47.2 (SD 10.2) % (P = 0.861) in the fibre mixture and control groups, respectively. The faecal weight median was 6.17 g in the fibre mixture group and 2.11 g in the control group (P < 0.001). The caecal pH was in the same order: 6.11 (SD 0.59) and 7.07 (SD 0.34) (P < 0.001). Both the groups consumed similar quantities of diet, and growth was similar in both the groups. The fibre mixture had no influence either on growth or on Fe intestinal absorption in rats recovering from anaemia. This mixture favoured an increase in faecal weight and a decrease in caecal pH.

This secondary analysis was conducted to determine the relationship between test weights and days of breastfeeding. Test weights were performed with a scale (accurate to 2 grams) before and after feeding. Days of breastfeeding was measured by self-reporting. All test weights were interrelated and were associated with higher days of breastfeeding. Test weights at day 2 to 4 and at 2 weeks were not correlated significantly with either intention or self-efficacy at any time. However, 6-week test weights were correlated with intention at all 3 times, and self-efficacy at 6 weeks. Women with higher breastfeeding self-efficacy scores tended to report higher intention to breastfeed for 6 months.

The use of colonoscopy in colorectal cancer (CRC) screening has increased substantially in recent years. Media messages and changes in insurance reimbursement, as well as new screening guidelines from the American Cancer Society and the US Preventive Services Task Force, have contributed to this increase. Primary care providers (PCPs) are frequently responsible for making the recommendation and referral for screening. The process of successfully referring a patient for screening colonoscopy can be cumbersome and requires a coordinated effort between the PCP and the endoscopist. In recognition of the potential complexity of this process, the National Colorectal Cancer Roundtable has issued a report to describe the components of a quality screening colonoscopy referral system in primary care practice. The elements of a quality program include an optimal scheduling and referral system, the appropriate patient preparation information, consistent reporting and follow-up systems, and a detailed approach to dealing with special situations.

Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3'-untranslated region (3'-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3'-UTR mutant MSI cell lines. Cell lines with an EGFR 3'-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3'-UTR mutations were more sensitive to EGFR inhibition than EGFR 3'-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors.

Objective: To investigate how well the parents of children on cow's milk free diets perform at recognizing whether or not expressions describe and foods contain cow's milk proteins., Methods: Interviews were conducted with 24 parents of children on cow's milk and by-products exclusion diets and 23 parents of children with no need for any type of exclusion diet. They were asked if they recognized 12 expressions relating to cow's milk. They were then asked to classify 10 commercial food products in terms of whether or not they contained cow's milk proteins., Results: Terms that included the word milk were more often recognized by both groups of parents. The parents of children on exclusion diets recognized the terms cow's milk protein, traces of milk and milk formulation or preparation most frequently (p < 0.05). Less than 25.0% of those interviewed recognized casein, caseinate, lactalbumin and lactoglobulin. Both groups correctly identified more of the commercial products containing cow's milk than those free from milk. The median number of products containing cow's milk (total = 5) correctly identified by the parents of children on exclusion diets (4.0) was greater than for the control group (3.0; p = 0.005). Reading at least one label was associated with a greater chance of correctly identifying more than five of the 10 products (odds ratio = 8.0)., Conclusions: Despite having received guidance, the parents of children on exclusion diets were not fully prepared to manage these diets, indicating a need for improvements to the instruction provided when indicating exclusion diets.

Background: We have previously published results indicating that decreased expression of CDK2-AP1 in MSI human colorectal cancer is associated with deletion mutations in the poly (T) 8 repeat sequence within the 3'-UTR of the CDK2-AP1 gene. In this study, we test the hypothesis that the del T mutation results in decreased CDK2-AP1 expression by causing reduced mRNA stability., Methods: We introduced wild-type and mutant 3'-UTR sequences fused to a green fluorescent protein (GFP) gene separately into human CRC cell lines and quantified the expression of the GFP gene. Native CDK2-AP1 mRNA stability was measured in human CRC cell lines, using an actinomycin D assay and the mRNA structure folding software mfold 3.2., Results: Mutant GFP-3'-UTR samples demonstrated significantly reduced GFP expression compared with wild-type GFP-3'-UTR as measured by both FACS and real-time PCR. Both the actinomycin D assay and mfold software demonstrated significantly reduced mRNA stability for the del T poly (T) 8 transcript compared with the wild type., Conclusions: In summary, these novel results support our hypotheses that the del T poly (T) 8 observed in the 3'-UTR of the CDK2-AP1 gene in human MSI CRC is functionally significant and results in decreased CDK2-AP1 expression. The results also indicate the mechanism of this decreased expression is caused at least in part by decreased mRNA stability.

Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been shown to have potent antitumor effects against human non-small-cell lung cancer (NSCLC) cell growth; however, the mechanism of such an effect is not elucidated. Here, we demonstrate that erlotinib-induced cell growth inhibition in EGFR high-expressing human H322 NSCLC cells was accompanied by G1/S phase arrest, which was largely caused by a decrease in expression of G1/S-related cyclins, suppression of activities of cyclin-dependent kinase (CDK) 2 and CDK4, induction of CDK inhibitor p27(KIP1), and retinoblastoma hypophosphorylation. To further understand the role of p27(KIP1) in G1/S arrest and cell growth inhibition by erlotinib, we determined its effect on the expression of p27(KIP1) at transcriptional and posttranscriptional levels. Studies using real-time reverse transcription-polymerase chain reaction analysis and p27 promoter-driven luciferase reporter showed that erlotinib treatment resulted in the promotion of p27 gene transcription. In addition, erlotinib treatment led to an increase in p27(KIP1) half-life by inhibiting p27(KIP1) phosphorylation at Thr187 and by down-regulating Skp2 expression. Furthermore, immunofluorescence staining and cell fractionation showed that erlotinib treatment led to p27(KIP1) translocation to the nucleus. Knockdown of p27(KIP1) expression with p27(KIP1) small interfering RNA significantly abrogated erlotinib-induced G1 phase arrest and cell growth inhibition, suggesting that induction of p27(KIP1) is required for G1 arrest and cell growth inhibition by erlotinib. It is noteworthy that we found that G1 arrest and p27(KIP1) up-regulation by erlotinib occurred in the tested sensitive cell lines but to a lesser extent in the resistant cell lines. Taken together, these results suggest that erlotinib inhibits human NSCLC cell growth predominantly by inducing p27(KIP1) expression and by suppressing cell-cycle events involved in the G1/S transition.

Background: Recently, Denaturing High-Performance Liquid Chromatography (DHPLC) has been widely used for mutation detection hMLH1 and hMSH2 genes due to reduced cost of analysis, accuracy, and high sample throughput. Unfortunately, one major drawback in screening the hMLH1 and hMSH2 genes with any analysis technique involves sample preparation. Additionally, there are limitations to this technique which include: (1) amplicons for hMLH1 and hMSH2 exons cannot be generated under the same PCR thermal cycler condition due to differences in the annealing temperatures of the traditional primer sets which drastically increases sample preparation time; (2) due to minimal changes in the DHPLC chromatogram when compared to the corresponding wild-type amplicon, there is a possibility to not detect a homozygous mutation; and (3) lack of specialized mutation analysis software for automated screening of the hMLH1 and hMSH2 genes with the Transgenomic Wave system., Methods: To overcome these limitations, the hMLH1 and hMSH2 condition-oriented-PCR primer-embedded-reactor (COPPER) plate was developed to reduce the sample preparation time and technological skill required for analysis, as well as standardize a mutation screening technique for hMLH1 and hMSH2 analysis using the Transgenomic Wave system for research and clinical genetics investigations. In this study, we validated the COPPER plate for simultaneous amplification of the exons of the hMLH1 and hMSH2 genes coupled to DHPLC detection for colorectal cancer (CRC) patients., Results and Conclusions: Our results suggest that the COPPER plate DHPLC approach is a simple, cost effective, accurate, universal, and reproducible technology for screening hMLH1 and hMSH2 genes which are associated with human CRC. We also believe that the COPPER plate DHPLC approach is amenable for characterization of other germline alterations in clinical genetics and pharmacogenetics.

We have previously demonstrated an association between microsatellite instability and decreased CDK2-AP1 (p12(DOC-1)) expression in human colorectal cancer (CRC) cell lines. In those same studies, induction of CDK2-AP1 expression promoted both cell cycle arrest and apoptosis. The goals of our present study were to better understand the mechanisms leading to reduced CDK2-AP1 expression in microsatellite unstable (MSI) CRC and to study further the effect of CDK2-AP1 modulation on cell proliferation and apoptosis utilizing RNA interference (RNAi) techniques. We used direct sequencing to screen for mutations of the poly (T)8 microsatellite-like region in the 3' end of the CDK2-AP1 gene in 24 CRC cell lines. We then utilized an in vitro human mismatch repair (MMR) recombinant system to assess for correction of the mutation and changes in CDK2-AP1 expression secondary to hMLH1 transfection. We also investigated the effect of CDK2-AP1 modulation in four settings: (1) native CDK2-AP1 absence, (2) endogenous CDK2-AP1 expression, (3) RNAi-induced CDK2-AP1 inhibition and (4) induced CDK2-AP1 over expression. The mutation - del T poly (T)8 - at the 3' end of the CDK2-AP1 gene was found in 3/12 (25%) of MSI CRC cell lines, but in none of the microsatellite stable samples (0/12). Interestingly, when wild-type MMR protein - MLH1 - was induced in an in vitro human recombinant system, the del T poly (T)8 mutation was reversed and CDK2-AP1 expression increased. RNAi-mediated CDK2-AP1 inhibition was associated with decreased apoptosis and increased cell proliferation in CDK2-AP1-non deficient CRC cell lines. We conclude that mutations in the microsatellite-like sequence of the CDK2-AP1 gene in MSI CRC are associated with decreased CDK2-AP1 expression. In addition, modulation of CDK2-AP1 expression in human CRC alters cell proliferation and apoptosis.

Background: We previously reported differential expression of the growth suppressor, deleted in oral cancer-1 (DOC-1), in microsatellite-unstable (MSI+) versus microsatellite-stable colorectal cancer (CRC) cell lines. MSI+ CRC cell lines demonstrated decreased DOC-1 expression and decreased apoptosis. Transfection of wild-type DOC-1 into an MSI+ cell line (SW48) resulted in increased apoptosis. We undertook our current experiment to identify specific elements modulated by DOC-1 expression that result in increased apoptosis., Methods: SW48 is an MSI+ CRC cell line that does not constitutively express DOC-1. SW48 was suspended in culture medium and incubated to 60% confluence. Half the plates were transfected with cytomegalovirus (CMV)-DOC-1. At 30 hours, RNA and protein were isolated with Trizol. Complementary DNA microarray was performed to compare SW48(CMV-DOC-1) with SW48, which lacks DOC-1. Signal intensity was analyzed by GenePix Pro 3.0 software. Expression ratios / 1.5 were considered significant. Poor-quality spots were flagged and excluded from analysis. Real-time polymerase chain reaction was performed to determine DOC-1 levels in both cell lines., Results: Successful transfection of DOC-1 was confirmed by real-time polymerase chain reaction and by Western blot. Microarray revealed significant differential expression of DOC-1, as expected. Increased DOC-1 expression in SW48(CMV-DOC-1) was associated with significantly increased expression of proapoptosis components of the caspase cascade (CASP7, CASP9) and bcl2/bax pathway (BNIP3, BNIP3L, BID)., Conclusions: DOC-1 expression promotes apoptosis by upregulation of specific elements of the caspase cascade and bcl2/bax pathways. DOC-1 therefore deserves further study as a candidate for the therapeutic modulation of apoptosis in MSI+ CRC.

The precise genetic mechanism of malignant transformation in DNA mismatch repair deficient, microsatellite-unstable colorectal cancer (CRC) has yet to be elucidated. We employed cDNA microarray to identify patterns of gene expression among CRC cell lines and to compare directly lines with and without microsatellite instability. This study was undertaken to test the hypothesis that microsatellite-unstable CRC cell lines demonstrate specific patterns of gene expression that differ significantly from those observed among microsatellite-stable CRC. Multiple differential expression patterns were identified. Genes demonstrating differential expression included deleted-in-oral-cancer-1 (DOC-1), a highly conserved growth suppressor. DOC-1 expression correlated with microsatellite status, with significantly decreased expression in microsatellite-unstable cell lines and constitutive expression in microsatellite-stable cell lines. We also observed alterations in the biologic behavior of p12(DOC-1)-deficient cell lines, with increased S phase and decreased apoptosis compared to microsatellite-stable (DOC-1+) cell lines. Transfection of p12(DOC-1) into SW48, which lacks p12(DOC-1) expression, resulted in cell cycle and apoptosis profiles similar to other p12(DOC-1)+ cell lines. These results support the hypothesis that microsatellite-unstable CRC is characterized by novel patterns of gene expression different from those associated with microsatellite-stable CRC, and demonstrate that p12(DOC-1) has tumor suppressor potential in colon epithelial cells.

Telomerase activity is present in most malignant tumors and provides a mechanism for unlimited replication of neoplastic cells. Expression of the gene encoding human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene, is associated with telomerase activity, and it is overexpressed in most colorectal carcinomas. In the present study we assayed telomerase activity by the telomeric repeat amplification protocol (TRAP) and used in situ hybridization (ISH) and the reverse transcription polymerase chain reaction (RT-PCR) to study hTERT expression in colorectal carcinomas and adjacent normal tissues. Telomerase activity was found in 30/35 (85.7%) of normal mucosae and 35/35 (100%) of adenocarcinomas, and RT-PCR detected hTERT in 33/35 (94.3%) of the carcinomas. ISH, on the other hand, detected weak but significant expression of hTERT in a significant percentage of lymphocytes infiltrating normal colorectal mucosa. hTERT gene expression was detected in the nuclei of adenocarcinoma cells in 27/35 (77.1%) of the lesions. The results of our comparison of telomerase activity and hTERT gene expression by RT-PCR-based ISH appeared contradictory, but a careful review suggested that the discrepancy was attributable to contamination by infiltrating lymphocytes. Our findings suggest that ISH-based analysis of hTERT gene expression is superior to both TRAP telomerase activity and hTERT mRNA RT-PCR analysis as a means of determining telomerase status during carcinogenesis.

The status of the porta hepatis lymph nodes in patients with hepatic metastases from colorectal cancer affects their prognosis and management. Lymphatic mapping with isosulfan blue dye is well established in breast cancer and melanoma. An animal model consisting of three dogs receiving general anesthesia was utilized. Each dog underwent a laparotomy and increasing doses of isosulfan blue dye were injected into the right medial segment of the liver. Intraoperatively, the presence of blue dye in the porta hepatis region was determined and the lymph node identified. Continuous physiological monitoring was performed. Serum determination of liver function tests, amylase levels, and white blood cell count were performed preoperatively and on postoperative days 1, 2, 4, and 7. The animals were sacrificed on day 7. A portal lymph node was identified in each case and there was no perioperative morbidity or mortality. There were no significant alterations in blood pressure or heart rate in the animals. There was a dose-responsive decrease in the O2 saturation as measured by transcutaneous monitoring, but arterial blood gas analysis showed that pO2 levels remained stable. There were no significant changes in the liver function tests, amylase levels, or white blood cell counts. There was a small increase in alkaline phosphatase, which normalized by postoperative day 7. Hepatic injection of isosulfan blue dye appears to be safe and effective in identifying porta hepatis lymph nodes in the animal model and sets the basis for further study in human subjects.

Hypothesis: Absorbable mesh slings can prevent radiation-induced bowel injury when adjuvant pelvic radiotherapy is given in the early postoperative period. We hypothesized that the mesh sling technique is similarly effective during "sandwich" sequence adjuvant chemoradiation., Design: Retrospective review., Setting: Tertiary care comprehensive cancer center., Patients: Nonrandomized series of 19 consecutive patients who underwent abdominoperineal resection and received postoperative sandwich sequence chemoradiation at Roswell Park Cancer Institute, Buffalo, NY, between January 1994 and September 1999., Interventions: Twelve patients had an absorbable mesh sling placed at the completion of abdominoperineal resection. Seven patients did not have an absorbable mesh sling placed., Main Outcome Measures: Radiotherapy dose and gastrointestinal toxic effects., Results: All 12 patients in the "mesh" group were able to receive full-dose radiotherapy with tumor bed boost (total dose, 54 Gy, 11 patients; 59.4 Gy, 1 patient). Only 3 of 7 patients in the "no mesh" group were able to receive a tumor bed boost (total dose, 46.8 Gy, 1 patient; 50.4 Gy, 3 patients; 54 Gy, 3 patients). Acute gastrointestinal toxic effects were minimal in the mesh group (grade 1, 10 patients; grade 2, 2 patients) compared with the no mesh group (grade 2, 6 patients; grade 3, 1 patients). None of the patients in the mesh group have shown evidence of late gastrointestinal toxic effects. One patient in the no mesh group required surgery for complications of chronic radiation enteritis., Conclusions: The protective effects of an absorbable mesh sling extend beyond the life expectancy of the mesh itself. Sandwich sequence chemoradiation should not preclude the use of the mesh sling technique.

Background: Mutations of the p53 tumor suppressor gene play an integral role in sporadic colorectal carcinogenesis but prior studies have failed to show their prognostic significance consistently., Methods: Fifty-six consecutive sporadic colorectal tumors were analyzed for their p53 status. Polymerase chain reaction amplification with primers for exons 5-9 was conducted and these products were subjected to single strand conformation polymorphism analysis. Suspected mutations were confirmed with DNA sequencing. p53 status was entered into a colorectal clinical database and these patients then were followed prospectively. Patient status with regard to disease recurrence and survival was updated every 6 months. Survival and disease free survival were calculated according to the method of Kaplan and Meier. The association between p53 status and clinical and pathologic factors with survival and recurrence was statistically determined using univariate analysis and the Cox proportional hazards model for multivariate analysis., Results: p53 mutations were detected in 28 of 56 patients (50%). The median follow-up time was 45 months (range, 3-72 months). There were 33 patients (59%) who were alive at last follow-up. Fifteen of the 23 patients who died (65%) had p53 mutations and 8 (35%) had wild-type p53. Thirteen patients developed a disease recurrence, 9 of whom (69%) had tumors with p53 mutations. Overall 4-year survival rates for patients with wild-type p53 and mutant p53 were 71% and 54%, respectively (P = 0.05). The 4-year disease free survival rates for patients with wild-type p53 and mutant p53 were 83% and 62%, respectively (P = 0.09). p53 status and stage were found to be independent significant predictors for survival (p53 negative: P = 0. 02; stage: P = 0.0002.) Stage was found to be the sole significant predictor for disease free survival (P = 0.006)., Conclusions: In this group of colorectal carcinoma patients, p53 mutations were a significant negative prognostic indicator for overall survival. This finding holds prognostic and therapeutic implications for the management of colorectal carcinoma patients., (Copyright 2000 American Cancer Society.)

Purpose: The aim of this study was to evaluate the efficacy of photodynamic therapy in the management of residual neoplasms of the perianal skin., Methods: This is a retrospective review. Five patients with pathologic confirmation of residual perianal neoplasms were treated with photodynamic therapy. There were three females. The mean age was 52 (range, 33-79) years. Pathology consisted of Bowen's disease in two patients, squamous-cell carcinoma in two patients, and extramammary Paget's disease in one patient. Four patients received one photodynamic therapy treatment and one patient received two treatments three months apart., Results: Treatment was followed by immediate perianal erythema, subsequent blister formation in 36 to 48 hours, and sloughing of the treated area in 72 hours. With a mean follow-up of 5.2 (range, 1-8) years, there were two recurrences. One recurrence was in a patient four years after treatment for Paget's disease, and the other was in a patient nine months after treatment for Bowen's disease. The latter was managed successfully with wide local excision. Treatment-related toxicities included significant perianal pain in four patients, controlled with analgesia management., Conclusions: Photodynamic therapy can successfully be used after wide local excision for residual neoplasms of the perianal skin. Treatment can be rendered with acceptable morbidity.

Background: This study assessed the presurgical and preradiation discussion of the risk of posttherapy sexual dysfunction among patients who underwent potentially curative therapy for rectal cancer. The incidence of sexual dysfunction after treatment for rectal cancer was then determined., Methods: A retrospective review of the medical records of 52 consecutive patients who underwent potentially curative procedures for rectal cancer within 15 cm from the anal verge was performed., Results: Presurgical discussion of the risk of sexual dysfunction was not documented in the consent in 37 of 52 patients (71%). Among the 5 males who underwent local excision, none reported posttherapy sexual dysfunction. Of the 6 males who were treated by low anterior resection, only 1 had a postoperative complaint of sexual dysfunction. Five of 15 males (33%) treated with abdominoperineal resection (APR) alone reported postprocedure sexual dysfunction, whereas 6 of 8 males (75%) treated with APR and radiation reported dysfunction. Of the entire female cohort, only 1 of the 16 reported sexual dysfunction posttherapy., Conclusion: A discussion of the risks of posttherapy sexual dysfunction was documented for fewer than one third of the patients. Among males after APR, the use of postoperative radiation showed a trend toward an increase in sexual dysfunction. Surgery and/or radiation therapy did not impact on sexual dysfunction in females.

Context: Germline mutations of the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 have been shown to cosegregate with the colorectal cancer phenotype in multiple hereditary nonpolyposis colorectal cancer (HNPCC) pedigrees. However, the frequency of these mutations among African American patients with colorectal cancer is unknown., Objective: To investigate the frequency of germline alterations of the DNA MMR genes hMLH1 and hMSH2 among African Americans affected by HNPCC and early-age onset colorectal cancer., Design, Setting, and Patients: Forty unrelated African American HNPCC and early-age onset colorectal cancer patients (8 women, 3 men) were identified from the cancer registry at a National Cancer Institute-designated referral center, 11 of whom were available for and agreed to study participation from January 1997 to February 1998. The mean age of the subjects was 44 years. An additional 50 age- and sex-matched African Americans without personal or family history of colorectal, endometrial, ovarian, urinary tract, or upper gastrointestinal tract malignancy were also studied as a polymorphism control population. In all subjects, genomic DNA was amplified by polymerase chain reaction for all hMLH1 and hMSH2 exons and screened using single-strand conformation polymorphism (SSCP) analysis. Samples demonstrating significant SSCP shifts underwent automated nucleotide sequencing analysis., Main Outcome Measure: Frequency of hMLH1 and hMSH2 germline alterations in the affected and control subjects., Results: Germline hMLH1 and hMSH2 mutations were detected in 3 (27%) of the African American colorectal cancer probands studied. Each mutation was novel. Two hMLH1 (an A-->T transversion at codon 26 and a GG-->AT substitution across codons 177 and 178) mutations and 1 hMSH2 mutation (a C-->T transition at codon 389) were identified in 3 female study subjects. Six other hMLH1 and hMSH2 alterations were detected but were presumed to be polymorphisms. Neither missense mutation (at codons 26 and 389) was detected in the control population., Conclusions: The results of our analysis support an association between the 3 mutations reported and predisposition to colorectal cancer. Further studies are needed to define DNA MMR gene-associated colorectal cancer in African Americans, an understudied population at increased risk of fatal colorectal cancer.

Purpose: An increased incidence of multiple (synchronous and metachronous) colorectal carcinomas has been reported in hereditary nonpolyposis colorectal cancer. This review was undertaken to determine the clinical implications of multiple colorectal carcinomas in hereditary nonpolyposis colorectal cancer., Methods: A retrospective review of the records of patients in the hereditary nonpolyposis colorectal cancer registry at Roswell Park Cancer Institute who had either synchronous or metachronous colorectal carcinomas was conducted., Results: Twenty-five of 93 patients with documented pathology were found to have multiple colorectal carcinomas. The mean age at diagnosis of the index colorectal carcinoma was 46.7 (range, 28-65) years. There were 7 (7.5 percent) patients with synchronous colorectal carcinomas and 20 (21.5 percent) patients with metachronous colorectal carcinomas. Two of the seven (28.6 percent) patients with synchronous colorectal carcinomas developed a metachronous colorectal carcinoma. In the patients with metachronous colorectal carcinomas, 29 metachronous events were noted: colon (23) and rectum (6). The mean and median time interval for metachronous colorectal carcinomas were 10.9 and 11.8 (range, 1.5-43.8) years, respectively. The mean times to first, second, and third events were 11.7 (range, 1.5-43.5), 7.9 (range, 2.7-18.7), and 12.3 (range, 11.8-12.7) years, respectively. The majority of patients with metachronous colorectal carcinomas did not have stage progression at the diagnosis of the metachronous colorectal carcinomas: 13 patients had lower or same stage at first event, 4 had lower or same stage at second event, and 2 patients had lower stage at third event. Three of 20 patients with metachronous colorectal carcinomas died of their disease., Conclusion: Multiple colorectal cancers are common in hereditary nonpolyposis colorectal cancer. Even though stage progression may not be evident at diagnosis of metachronous colorectal cancer, some of these patients will nevertheless die of their disease.

This study evaluates prognostic factors that may influence survival in patients who present with carcinomatosis from colorectal cancer. Patients may present with carcinomatosis as the pattern of metastases at the initial diagnosis of colorectal cancer. Little is known about the natural history of carcinomatosis and the prognostic factors affecting outcome. All patients treated at Roswell Park Cancer Institute from 1988 to 1994 who presented with carcinomatosis at the initial diagnosis of colorectal cancer were identified. A retrospective review of the medical records for patient and tumor demographics was performed. Estimated survival distributions were calculated by the method of Kaplan and Meier. Tests of significance with respect to survival distribution were based on the log-rank test. Cox proportional hazards model was used for the multivariate analysis. There were 31 males and 38 females. The median age was 61 years (range, 26-80). The primary cancers were in the sigmoid with 24 patients (35%), cecum with 14 patients (20%), and transverse colon with 11 patients (16%). The remainder were distributed throughout the colon and rectum. The most common presentation was large bowel obstruction in 29 patients (42%). T3 and T4 cancers were present in 39 (57%) and 13 patients (19%), respectively. Lymph nodes were positive in 39 patients (57%), and mucin-producing tumors were also present in 39 patients (57%). Twelve patients (17%) had one site of disease, 17 patients (25%) had two sites of disease, and 37 (54%) patients had three or more sites of disease. Ascites was present in 29 patients (42%). Residual disease was present at the completion of surgery in 45 patients, absent in 13 patients, and status unknown in 11. The presence of residual disease (p = 0.0001), presence of ascites (p = 0.02), stage greater than T3 (p = 0.02), and increasing number of carcinomatosis sites (p = 0.006) were found to have a negative impact on survival on univariate analysis. On multivariate analysis, only the presence of residual disease at the completion of surgery was found to be an independent predictor of survival (p = 0.04). Overall median survival was 14 months with a 26% estimated 2-year survival. The presence of gross residual disease at the completion of surgery was shown to be the only independent factor negatively affecting survival. This has potential implications for the operative management of patients presenting with colorectal carcinomatosis.

Colorectal cancer remains a significant public health challenge, despite our increased understanding of the genetic mechanisms involved in the initiation and progression of this disorder. It has become clear that multiple mechanisms lead to the tumorigenic phenotype, with familial predisposition syndromes accounting for less than 15% of all colorectal cancers. A genome-wide scan for loss of heterozygosity (LOH) was carried out with 150 highly polymorphic markers in an effort to identify additional loci involved in colorectal tumorigenesis in DNA samples from 42 colorectal cancer patients. The results confirm earlier observations that tumor DNAs from patients with hereditary nonpolyposis colon cancer (HNPCC) either maintain heterozygosity or exhibit altered or additional alleles. DNAs from patients with early onset colorectal carcinomas (diagnosed prior to age 50) revealed a higher overall degree of LOH than DNAs from patients with sporadic colorectal cancers diagnosed later in life (after age 50). While regions on 1p, 10q and 14q are suggestive, statistical analysis of LOH at these regions failed to reach significance. However, LOH at 9p did reveal a statistically significant increase in the early onset patient group, compared to the greater than age 50 group. LOH on 9p may involve inactivation of p16/CDKN2 through aberrant DNA methylation on the remaining chromosome, resulting in a situation analogous to a homozygous deletion of p16 and providing a selective growth advantage to these cells. This marker may prove to be a useful prognostic indicator for patient stratification in the design of therapy for early onset colorectal cancer patients.