Genome-wide association studies and Mendelian randomization analyses provide insights into the causes of early-onset colorectal cancer.

BACKGROUND: The incidence of early-onset colorectal cancer (EOCRC; diagnosed <50 years of age) is rising globally; however, the causes underlying this trend are largely unknown. CRC has strong genetic and environmental determinants, yet common genetic variants and causal modifiable risk factors underlying EOCRC are unknown. We conducted the first EOCRC-specific genome-wide association study (GWAS) and Mendelian randomization (MR) analyses to explore germline genetic and causal modifiable risk factors associated with EOCRC. PATIENTS AND METHODS: We conducted a GWAS meta-analysis of 6176 EOCRC cases and 65 829 controls from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), the Colorectal Transdisciplinary Study (CORECT), the Colon Cancer Family Registry (CCFR), and the UK Biobank. We then used the EOCRC GWAS to investigate 28 modifiable risk factors using two-sample MR. RESULTS: We found two novel risk loci for EOCRC at 1p34.1 and 4p15.33, which were not previously associated with CRC risk. We identified a deleterious coding variant (rs36053993, G396D) at polyposis-associated DNA repair gene MUTYH (odds ratio 1.80, 95% confidence interval 1.47-2.22) but show that most of the common genetic susceptibility was from noncoding signals enriched in epigenetic markers present in gastrointestinal tract cells. We identified new EOCRC-susceptibility genes, and in addition to pathways such as transforming growth factor (TGF) ß, suppressor of Mothers Against Decapentaplegic (SMAD), bone morphogenetic protein (BMP) and phosphatidylinositol kinase (PI3K) signaling, our study highlights a role for insulin signaling and immune/infection-related pathways in EOCRC. In our MR analyses, we found novel evidence of probable causal associations for higher levels of body size and metabolic factors-such as body fat percentage, waist circumference, waist-to-hip ratio, basal metabolic rate, and fasting insulin-higher alcohol drinking, and lower education attainment with increased EOCRC risk. CONCLUSIONS: Our novel findings indicate inherited susceptibility to EOCRC and suggest modifiable lifestyle and metabolic targets that could also be used to risk-stratify individuals for personalized screening strategies or other interventions.

Markers of tyrosine kinase activity in eosinophilic esophagitis: a pilot study of the FIP1L1-PDGFRα fusion gene, pERK 1/2, and pSTAT5.

The pathogenesis of eosinophilic esophagitis (EoE) is incompletely understood. In certain eosinophilic diseases, activation of tyrosine kinase after fusion of the Fip1-like-1 and platelet-derived growth factor receptor-α genes (F-P fusion gene) mediates eosinophilia via downstream effectors such as extracellular-regulated kinase (ERK1/2) and signal transducers and activators of transcription (STAT5). This mechanism has not been examined in EoE. Our aim was to detect the F-P fusion gene, pERK1/2, and pSTAT5 in esophageal tissue from patients with EoE, gastroesophageal reflux disease (GERD), and normal controls. We performed a cross-sectional pilot study comparing patients with steroid-responsive and steroid-refractory EoE, to GERD patients and normal controls. EoE cases were defined by consensus guidelines. Fluorescence in situ hybridization (FISH) was performed to detect the F-P fusion gene and immunohistochemistry (IHC) was performed to detect pERK1/2 and pSTAT5 in esophageal biopsies. Twenty-nine subjects (median age 30 years [range 1-59]; 16 males; 24 Caucasians) were included: eight normal, six GERD, and 15 EoE (five steroid-refractory). On FISH, 98%, 99%, and 99% of the nuclei in the normal, GERD, and EoE groups, respectively, were normal (P= 0.42). On IHC, a median of 250, 277, and 479 nuclei/mm(2) stained for pERK 1/2 in the normal, GERD, and EoE groups, respectively (P= 0.07); the refractory EoE patients had the highest degree pERK 1/2 staining (846 nuclei/mm(2); P= 0.07). No trend was seen for pSTAT5. In conclusion, the F-P fusion gene was not detected with increased frequency in EoE. Patients with EoE had a trend toward higher levels of pERK 1/2, but not STAT5, in the esophageal epithelium, with highest levels in steroid-refractory EoE patients.

Statistical strategies to improve the efficiency of molecular studies of colorectal cancer prognosis.

The evaluation of tumour molecular markers may be beneficial in prognosis and predictive in therapy. We develop a stopping rule approach to assist in the efficient utilisation of resources and samples involved in such evaluations. This approach has application in determining whether a specific molecular marker has sufficient variability to yield meaningful results after the evaluation of molecular markers in the first n patients in a study of sample size N (n

Disposable versus reusable biopsy forceps for colorectal epithelial cell proliferation in humans.

The performance of various measures of rectal mucosal proliferation has been evaluated in the literature, but the performance of the forceps used to obtain the tissue has received little attention. We used data from two large studies of proliferation at a single institution to compare reusable and disposable endoscopic forceps. Endoscopic pinch biopsies were taken 10 cm from the anal verge using either reusable or disposable, oval-cupped, sheathed forceps. The specimens were fixed, embedded, and sectioned, taking care to orient the specimens longitudinally. Five sections were placed on each slide. We determined how many slides did not contain eight scorable crypts (inadequate) and how many sections were necessary to identify eight complete crypts. There were 395 subjects who had biopsies taken with reusable forceps and 185 subjects who had biopsies taken with disposable forceps. The specimens were inadequate in 27.6% of the reusable forceps specimens versus 2.7% of the disposable forceps (P < 0.0001). The mean number of tissue sections necessary to identify eight scorable crypts for the reusable forceps was 3.82 (SD, 0.87) compared with 3.17 (SD, 0.83) for disposable forceps (P = 0.0001). The specimens taken with the disposable forceps were better, probably because the forceps were sharper. We believe that the better quality of the specimens and the sterility justify the higher cost of disposable forceps. We would urge investigators in proliferation studies to evaluate the biopsy equipment as carefully as they evaluate other aspects of their methods.

Rectal mucosal proliferation, dietary factors, and the risk of colorectal adenomas.

Colorectal cancer arises from a series of precursor stages, the so called adenoma-carcinoma sequence. Increased rectal mucosal proliferation may be an early step in this sequence. Because dietary factors are implicated in the etiology of colorectal cancer, one might predict that diet would also be associated with proliferation. We conducted this study to examine the association of diet with rectal mucosal proliferation. Rectal mucosal proliferation was measured in endoscopic biopsy specimens by proliferating cell nuclear antigen (PCNA) immunohistochemistry and whole crypt mitotic counts (WCMCs). Diet was evaluated using a validated quantitative food frequency questionnaire. The correlation between PCNA labeling index (LI) and WCMCs was determined using Kendall's tau, a nonparametric measure of correlation. Logistic regression was used to examine the effect of proliferation on adenoma status, controlling for confounders. The relationship between proliferation and dietary and demographic factors was examined using linear regression. There were 308 patients who had one or both measures of proliferation. There was no significant correlation between PCNA LI and WCMCs (Kendall's tau = 0.04; P = 0.35). Neither measure of proliferation was predictive of adenoma status, even after adjusting for potential confounders. Body mass index and calories per day were significant predictors of WCMC (P = 0.01 and P = 0.03, respectively). PCNA labeling index was not associated with any dietary variables, although its association with dietary fat nearly reached statistical significance (P = 0.09). The association between proliferation and diet were generally inconsistent. There appears to be no simple relationship between colorectal cancer risk factors, colorectal adenomas, and these two measures of rectal mucosal proliferation. We need simpler, more reliable intermediate markers for use in etiological and intervention studies.

In vivo activation of mitogen-activated protein kinases in rat intestinal neoplasia.

BACKGROUND & AIMS: To investigate whether mitogen-activated protein kinase (MAPK) cascades might play a role in the progression of colon cancer, c-Jun N-terminal kinase (JNK) and extracellular signal regulating kinase (ERK) activity during colonic tumorigenesis were examined. METHODS: The 1,2-dimethylhydrazine (DMH)-induced colon carcinoma model was used to study the activation of these kinases during intestinal carcinogenesis. Male Sprague-Dawley rats were injected with DMH for 24 weeks. Normal-appearing intestinal mucosa from control and treated animals and DMH-induced intestinal tumors were assayed for JNK and ERK activity using solid phase in vitro kinase assays. Tumors were typed for mutations in the K-ras gene. RESULTS: There was little or no difference in JNK and ERK activity in hyperproliferative mucosa from DMH-treated animals compared with normal mucosa from control animals. However, in 16 colonic neoplasms, an average of 23-fold and 29-fold increases in JNK and ERK activities were observed, respectively, over control levels. In addition, activating protein-1 binding was strongly induced in the colonic tumors. Activation did not correlate with the presence of mutations in K-ras. CONCLUSIONS: Both the JNK and ERK MAPKs are highly activated during late progression of colorectal carcinoma. This change is dependent on the tumorigenic state rather than changes in proliferation.

The in vitro HL-60 cell--Trypanosoma brucei rhodesiense culture system: a rapid in vitro drug screen.

The in vitro culture system is described in which Trypanosoma brucei rhodesiense (LOUTat.1) was grown with the human feed layer cell HL-60. The use of this system in determining the 50% growth Inhibitory Concentration (IC50) of unknown compounds for both the trypanosomes and the host cell was demonstrated. The data shows that several analogues of pentamidine have significantly reduced host cell toxicity but maintain or have increased typanocidal activity. The value of the trypanosome/HL-60 in vitro culture system as a rapid primary in vitro drug screen is discussed. Based upon the ability of this primary screen to predict potential drug efficacy, several analogues screened in vitro were then tested in vivo. The results of the in vivo tests confirmed the ability of the in vitro screen to predict drug efficacy, and also suggests that better analogues of pentamidine (less host toxicity and greater trypanocidal activity) can be obtained to treat human trypanosomiasis.

Comparison of rectal mucosal proliferation measured by proliferating cell nuclear antigen (PCNA) immunohistochemistry and whole crypt dissection.

Rectal mucosal proliferation has been promoted as an intermediate marker for risk of colorectal neoplasia. Proliferating cell nuclear antigen (PCNA) immunohistochemistry has become a standard method to measure cell proliferation. Whole-crypt dissection may provide a technically simpler method for determining proliferation within an entire crypt. We conducted a study to assess the reliability (reproducibility) of whole-crypt dissection in 10 subjects. Reliability of whole-crypt dissection with the subject as the unit of observation was excellent. The intraclass correlation coefficient for subjects was 0.93. Biopsy-to-biopsy reliability was lower (r=0.86) and crypt-to-crypt reliability lower still (r = 0.35). There was poor correlation between measures of proliferation index using the two techniques (Kendall's tau = 0.13; P = 0.08). Compartment analysis based on the percentage of the total number of labeled cells appearing in each crypt quartile also did not demonstrate a significant correlation between the two measures. We conclude that PCNA labeling index and whole-crypt mitotic count are not comparable measures of rectal mucosal proliferation.

Reproducibility and variability of the rectal mucosal proliferation index using proliferating cell nuclear antigen immunohistochemistry.

Rectal mucosal proliferation has been shown to be increased in patients with neoplastic lesions of the large bowel and may serve as a marker of risk for colorectal malignancy. We conducted analyses to determine reliability and components of variability that might suggest optimal analysis strategies for studies of proliferation. Endoscopic pinch biopsies were obtained from 17 adult patients, labeled using proliferating cell nuclear antigen, scored using strict rules, and then rescored. Labeling index, defined as the proportion of labeled cells in a crypt, was calculated for each crypt, biopsy, subject, and group. There was excellent reproducibility. The technician was able to select previously scored crypts 95% of the time. The overall labeling index was identical on repeat. There was considerable variability in labeling index among crypts from a single biopsy and between biopsies of a single subject. Variance component estimates suggested that 20% of the variability of labeling index was due to subject, 30% due to the biopsy within a subject, and 50% due to crypts within a biopsy. There were substantial gains in statistical power by scoring two biopsies rather than one. There was less gain from further increases in biopsy number. There was little statistical advantage for counting more than 8 crypts/biopsy. Demonstrating a decrease of 25% in the mean labeling index with 90% power could require more than 100 subjects/group. We conclude that proliferating cell nuclear antigen is an extremely reproducible method to determine proliferation index. There is considerable variability among subjects, biopsies, and crypts.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosoma brucei rhodesiense: the inhibition of HL-60 cell growth by the African trypanosomes in vitro.

Trypanosomiasis is of major public health importance in Africa where the disease affects man and livestock. In order to explore the underlying mechanisms of pathogenesis in African trypanosomiasis, we studied the inhibition of host cell (human promyelocytic HL-60 cells) growth by Trypanosoma brucei rhodesiense using an in vitro system. This inhibition was not due to changes in pH or nutritional depletion of the culture medium by the trypanosomes as inhibitory activity was still observed in cultures that had been supplemented with glucose or fresh culture medium. Our study suggests that the African trypanosomes produce a soluble factor which inhibits the growth of HL-60 cells. This growth inhibitor does not appear to kill the HL-60 cells as determined by the trypan blue dye exclusion test. The production of this factor does not require host cell contact nor does it require a host cell cofactor. The trypanosome growth inhibitor is strictly a trypanosome product. Estimation of the molecular weight of the trypanosome growth inhibitor with Amicon filters revealed that the factor is greater than 30,000 Da in size. Protease and heat treatment of the factor resulted in the depletion of inhibitory activity. These results indicate that the African trypanosomes produce a large-molecular-weight protein growth inhibitory factor which could play a role in the pathogenesis of the disease.

Mechanisms of HCl-induced lowering of intracellular pH in rabbit esophageal epithelial cells.

BACKGROUND: For acid injury to occur in esophageal epithelium, extracellular HCl must lower intracellular pH. Therefore, we sought to define the mechanisms for translation of low extracellular pH (pHo) into low intracellular pH (pHi). METHODS: To define the mechanisms, primary cultures of rabbit esophageal epithelial cells were loaded with the pH-sensitive fluorescent dye, 2'7''-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which enabled pHi to be recorded by microfluorimetry. RESULTS: Lowering pHo to 6.0 with HCl caused pHi to decline at 0.07-0.08 units/min and produced an average cellular H+ load of 3-6 mmol/L at 1 minute and of 11 mmol/L at 5 minutes. This degree of acidification was primarily attributable to increased H+ entry via a 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive Clo-dependent mechanism because DIDS or exposure to Clo-free solution blocked cell acidification. Furthermore a contribution to low pHi at low pHo of abolition of acid extrusion via Na+/H+ and Na(+)-dependent Cl-/HCO3- exchangers was sought but not established because exposure at low pHo to either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or Na(+)-free solution + EIPA increased the degree of acid loading over untreated cells. CONCLUSIONS: Low pHo results in low pHi in esophageal cells primarily because of increased H+ entry via a DIDS-sensitive, Clo-dependent mechanism consistent with the known acid-loading Na(+)-independent Cl-/HCO3- exchanger.

Studies of pHi in rabbit esophageal basal and squamous epithelial cells in culture.

Rabbit esophageal epithelium grown in primary culture enabled the study of intracellular pH (pHi) regulation at two distinct stages in the life cycle of the epithelial cell: basal and mature squamous. pHi was measured in single basal and mature squamous cells after loading with the fluorescent probe 2',7'-bis(carboxyethyl)-5(and -6)carboxyfluorescein at 25 degrees C in a nominally bicarbonate-free HEPES buffer. The results revealed that the resting pHi was higher and the intrinsic buffer capacity lower (at pH values less than or equal to 7.6) in basal compared with mature squamous cells. In addition, both types recovered from an acid load (NH4Cl prepulse) by an Na(+)-dependent, amiloride-inhibitable process consistent with an Na+/H+ antiporter. However, hydrogen ion extrusion rates by the Na+/H+ antiporter, even after taking into account buffer capacity and acid loading, were two to four times as fast for basal as for mature squamous cells. Further, mature squamous cell but not basal cell recovery from an acid load deteriorated with time (3 weeks) in culture. These results establish the use of primary cultures for studying pHi regulation at different stages in the epithelial cell life cycle and document that basal and mature squamous cells show Na+/H+ antiport activity for extrusion of an acid load but that this activity diminishes in effectiveness as the cell matures.