[Clinicopathological and molecular genetic features of Crohn's disease].

Objective: To investigate the clinicopathological and molecular genetic characteristics of Crohn's disease (CD). Methods: A retrospective analysis was conducted on 52 CD patients who underwent surgical resection at the First Affiliated Hospital of Nanjing Medical University between January 2014 and June 2023. Clinical presentations and histopathological features were assessed. Whole-genome sequencing was performed on 17 of the samples, followed by sequencing and pathway enrichment analyses. Immunohistochemistry was used to assess the expression of frequently mutated genes. Results: Among the 52 patients, 34 were males and 18 were females, male-to-female ratio was 1.9∶1.0, with a median age of 45 years at surgery and 35 years at diagnosis. According to the Montreal classification, A3 (51.9%,27/52), B2 (61.5%, 32/52), and L3 (50.0%,26/52) subtypes were the most predominant. Abdominal pain and diarrhea were the common symptoms. Histopathological features seen in all 52 patients included transmural inflammation, disruption of cryptal architecture, lymphoplasmacytic infiltration, varying degrees of submucosal fibrosis and thickening, increased enteric nerve fibers and neuronal proliferation. Mucosal defects, fissure ulcers, abscesses, pseudopolyps, and adenomatous proliferation were also observed in 51 (98.1%), 38 (73.1%), 28 (53.8%), 45 (86.5%), and 28 (53.8%) cases, respectively. Thirty-one (59.6%) cases had non-caseating granulomas, and 3 (5.8%) cases had intestinal mucosal glandular epithelial dysplasia. Molecular analysis showed that 12/17 CD patients exhibited mutations in at least one mucin family gene (MUC2, MUC3A, MUC4, MUC6, MUC12, MUC17), and MUC4 was the most frequently mutated in 7/17 of cases. Immunohistochemical stains showed reduced MUC4 expression in epithelial cells, with increased MUC4 expression in the epithelial surface, particularly around areas of inflammatory cell aggregation; and minimal expression in the lower half of the epithelium. Conclusions: CD exhibits diverse clinical and pathological features, necessitating a comprehensive multidimensional analysis for diagnosis. Mutations and expression alterations in mucin family genes, particularly MUC4, may play crucial roles in the pathogenesis of CD.

[The relationships between functional gastrointestinal diseases and psychological factors, diet and lifestyles: a network analysis].

Objective: To investigate the relationship between common functional gastrointestinal diseases symptoms with psychological factors, diet and lifestyles by using the network analysis method which has achieved great success in the field of psychology in recent years. Method: A questionnaire survey was conducted in two military units using the cluster sampling method during July 2020, and a total of 1 805 subjects were included. Functional gastrointestinal disease symptoms were evaluated with the Gastrointestinal Symptom Rating Scale (GSRS). The state, trait anxiety scale and stress response scale were used to evaluate the mental and psychological state by self-evaluation. R was used to build the network and calculate statistical parameters. Results: 1 486 of the 1 805 subjects (82.3%) had experienced functional gastrointestinal diseases symptoms within 2 weeks, but most of them were mild. Network analysis shows that there was a strong interaction between digestive system symptoms with different clinical manifestations (Spearman coefficient ranges 0.31-0.56). There was a clear relationship between functional gastrointestinal symptoms and mental and psychological factors (Spearman coefficient ranges 0.16-0.27), but there was no clear interaction with diet, age, education level, body mass index, etc. Functional gastrointestinal diseases symptoms were connected with mental and psychological factors through two nodes: stress and indigestion. The stability coefficient of node strength correlation was 0.75, indicating that the network was stable. Conclusions: The current study revealed the network structure and features of functional gastrointestinal diseases symptoms with mental and psychological factors. The key linking nodes provided potential interfering target for controlling functional gastrointestinal symptoms related to mental and psychological factors.

Insurance status and risk of suicide mortality among patients with cancer: a retrospective study based on the SEER database.

OBJECTIVE: Given that the presence of insurance may affect the risk of suicide mortality in cancer patients, we aimed to examine the association in a population-based study using the Surveillance, Epidemiologic, and End Results (SEER) database. STUDY DESIGN: A retrospective analysis of data from the SEER database. METHODS: We conducted a retrospective study using the SEER database. Hazard ratios (HRs), adjusted HRs (aHRs), and 95% confidence intervals (95% CIs) of suicide death were calculated using Cox proportional hazard models to evaluate the risk of suicide mortality among the cohorts. RESULTS: Multivariable analysis revealed that cancer patients without insurance had an increased risk of suicide death compared with patients with private insurance (aHR, 1.37; 95% CI, 1.01-1.72), whereas no significant result was observed in patients with any Medicaid (aHR, 1.10; 95% CI, 0.93-1.30; P = 0.27). In addition, the stratified analysis indicated that the risk of suicide death in patients in the uninsured and Medicaid groups presented with localized stage of disease (aHR, 1.32; 95% CI, 1.02, 1.69), White (aHR, 1.34; 95% CI, 1.05, 1.71), and American Indian/Alaska Native and Asian/Pacific Islander (aHR, 1.89; 95% CI, 1.08, 3.30) were greater than insured patients. CONCLUSION: Overall, our results indicated that insurance status was a statistically significant predictor of suicide death in patients with cancer. Healthcare providers should identify those patients at high risk of suicide and provide appropriate mental health and psychosocial oncology services in time.

[Efficacy and safety of Changsulin® compared with Lantus® in type 2 diabetes: a phase â ¢ multicenter, randomized, open-label, parallel, controlled clinical trial].

Objective: To compare the efficacy and safety of Changsulin® with Lantus® in treating patients with type 2 diabetes mellitus (T2DM). Methods: This was a phase Ⅲ, multicenter, randomized, open-label, parallel-group, active-controlled clinical trial. A total of 578 participants with T2DM inadequately controlled on oral hypoglycemic agents were randomized 3∶1 to Changsulin® or Lantus® treatment for 24 weeks. The efficacy measures included changes in glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG), 2h postprandial plasma glucose (2hPG), 8-point self-monitoring of blood glucose (SMBG) profiles from baseline, and proportions of subjects achieving targets of HbA1c and FPG. The safety outcomes included rates of hypoglycemia, adverse events (AEs) and anti-insulin glargine antibody. Results: After 24 weeks of treatment, mean HbAlc decreased 1.16% and 1.25%, FPG decreased 3.05 mmol/L and 2.90 mmol/L, 2hPG decreased 2.49 mmol/L and 2.38 mmol/L in Changsulin® and in Lantus®, respectively. No significant differences could be viewed in above parameters between the two groups (all P>0.05). There were also no significant differences between Changsulin® and Lantus® in 8-point SMBG profiles from baseline and proportions of subjects achieving the targets of HbA1c and FPG (all P>0.05). The rates of total hypoglycemia (38.00% and 39.01% for Changsulin® and Lantus®, respectively) and nocturnal hypoglycemia (17.25% and 16.31% for Changsulin® and Lantus®, respectively) were similar between the two groups (all P>0.05). Most of the hypoglycemia events were asymptomatic, and no severe hypoglycemia were found in both groups. No differences were observed in rates of AEs (61.77% vs.52.48%) and anti-insulin glargine antibody (after 24 weeks of treatment, 6.91% vs.3.65%) between the two groups (all P>0.05). Conclusions: Changsulin® shows similar efficacy and safety profiles compared with Lantus® and Changsulin® treatment was well tolerated in patients with T2DM.

[A preliminary study on resting-state functional magnetic resonance imaging of brain after anterior cruciate ligament preservation reconstruction with autologous tendon].

Objective: To preliminarily study on the possible mechanism of cerebral cortical dysfunction pattern after anterior cruciate ligament (ACL) preservation reconstruction with autologous tendon through resting-state functional magnetic resonance imaging (fMRI). Methods: From June 2015 to February 2019, 18 patients (10 males and 8 females with an average age of (36±10) years) with left anterior cruciate ligament rupture and treated with arthroscopic preservation reconstruction with autologous tendon were enrolled in this study, and 17 comparable healthy controls were included in Tongji Hospital of Tongji University. fMRI was performed after the postoperative period (2 to 12 weeks). The fMRI data were preprocessed by SPM8 software package and RESTplus software. The amplitude of low-frequency fluctuation (ALFF) and the fractional amplitude of low-frequency fluctuation (fALFF) in those two groups were calculated. Two-sample t-test was performed on ALFF and fALFF of the two groups, and multiple test corrections were performed by using AlphaSim. These methods were used for contrast studies on the characteristic activities of the brain dysfunction. Results: Compared with those in the control, ALFF in the central cingulate gyrus (cingulum_mid_bilateral), involving the auxiliary movement zone (supp_motor_ area) were significantly higher in the patients (P<0.01 before correction, P<0.05 after AlphaSim correction). The fALFF in activation cluster 1 was significantly higher in the right central gyrus (postcentral_R), the right lower lobule (parietal_inf_R), and the right upper margin (supramarginal_R) in the patients than that in the normal control group, respectively (P<0.01 before correction, P<0.05 after AlphaSim correction); the fALFF in activation cluster 2 in the right central cingulate gyrus (cingulum_mid_R), involving the right auxiliary movement zone (supp_motor_area_R) was significantly higher in the patients than that in the normal control group, respectively (P<0.01 before correction, P<0.05 after AlphaSim correction). Conclusion: The patients' cerebrum cortical function associated with the kinesthesis and their regulations are abnormally changed after anterior cruciate ligament preservation reconstruction with autologous tendon.

[Normal values for solid state high resolution anorectal manometry in healthy adult volunteers].

Objective: To explore the normal values for two-dimension solid state high resolution anorectal manometry (HRAM) in healthy adult volunteers. Methods: The healthy adult volunteers were recruited by advertisement and underwent solid state HRAM in the left lateral position. Anorectal pressures and rectal sensation were recorded and analyzed. Results: (1) A total of 126 Chinese healthy adult volunteers (male: 50 cases (39.7%); age: (37.5±14.2) years old ) were recruited in this study. (2) Mean anal resting pressure (MERP) was (71.8±17.3) mmHg (1 mmHg=0.133 kPa). Maximum anal resting pressure (MARP) was (79.3±17.8) mmHg, Maximum anal squeeze pressure (MSP) was (178.7±52.8) mmHg. Anal high pressure zone (HPZ) length was (3.4±0.6) cm. During simulated evacuation, residual anal pressure (RAP) was (63.8±20.5) mmHg, and anal relaxation rate (ARR) was (37.0±11.5)%. Rectal threshold volume for first sensation (FST), desire to defecate (DDT), urgency to defecate (UDT) and maximum discomfort (MDT) was (47.4±10.0) ml, (84.5±18.2) ml, (125.8±28.5) ml, and (175.5±36.1) ml, respectively. (3) Compared with female subjects, male subjects had higher MSP[(211.0±50.7) mmHg vs (157.5±42.5) mmHg], RAP[(71.6±18.1) mmHg vs (58.8±20.5) mmHg]and rectal MDT[(187.0±36.4) mmHg vs (168.0±34.1)mmHg], but lower ARR[(32.1±8.0)% vs (40.2±12.3)%], all P<0.01. (4) MERP, MARP, MSP and rectal MDT were higher in young group (≤40 years old), all P<0.05. Conclusions: These observations provide normal values for two-dimension solid state HRAM, which have significant difference between genders and different age groups.

[Research advances on venous thromboembolism in burn patients].

Venous thromboembolism (VTE) has become a serious medical problem faced by medical personnel all over the world, due to its high incidence, high fatality, and easily missed and misdiagnosed. Patients with severe burns are at high risk for VTE due to the presence of blood hypercoagulability, central venous catheterization, repeatedly received surgical procedures, and prolonged bed rest. Identifying the risk factors of VTE in burn patients and taking targeted preventive measures are the key to reduce the incidence of VTE. However, there are no risk assessment tools or prevention guidelines for VTE in burn patients at home and abroad, and scholars from various countries are actively exploring the occurrence, influencing factors, and prevention of VTE in burn patients. This paper reviews the research progress of the occurrence situation, related risk factors, risk assessment, and prevention of VTE in burn patients in recent years, and discusses the existing problems and future research directions in this field.

[Analysis of etiology and complications in children with stage 5 chronic kidney disease].

Objective: To investigate the etiology, complications, and prognostic factors of stage 5 chronic kidney disease (CKD5) in children. Methods: A case series study was conducted to retrospectively analyze the general situation, clinical manifestations, laboratory tests, genetic testing, and follow-up data (until October 2022) of 174 children with CKD5 who were diagnosed and hospitalized at the Children's Hospital of Chongqing Medical University from April 2012 to April 2021. The characteristics of complications in the children were compared based on age, gender, and etiology. Based on the presence or absence of left ventricular hypertrophy (LVH), patients were divided into LVH group and non LVH group for analyzing the influencing factors of cardiovascular disease. Patients were also divided into death group and survival group, peritoneal dialysis group and hemodialysis group based on the follow-up data for analyzing the prognostic factors. The chi-square test, independent sample t-test, Fisher exact probability test, Mann-Whitney U test and Kruskal Wallis test were used to analyze data among different groups. Multivariate Logistic regression analysis was used to identify the prognostic factors. Results: A total of 174 children with CKD5 were enrolled in the study (96 boys and 78 girls), aged 11.2 (8.2, 13.0) years. Congenital kidney and urinary tract malformations (CAKUT) were the most common causes of the CKD5 (84 cases, 48.3%), followed by glomerular diseases (83 cases, 47.7%), and among which 28 cases (16.1%) were hereditary glomerular diseases. The common complications of CKD5 included anemia (98.2%, 165/168), mineral and bone disorder in chronic kidney disease (CKD-MBD) (97.7%, 170/174), lipid metabolism disorders (87.5%, 63/72), hypertension (81.4%, 127/156) and LVH (57.6%,57/99). The incidences of hypertension in primary glomerular disease were higher than that in CAKUT(93.8%(30/32) vs.73.7%(56/76),χ2=5.59,P<0.05). The incidences of hypertension in secondary glomerular disease were higher than that in CAKUT and that in hereditary kidney disease (100.0%(20/20) vs. 73.7%(56/76), 68.2%(15/22), both P<0.05). The incidence of hypocalcemia in CAKUT, primary glomerular disease, and hereditary kidney disease was higher than that in secondary glomerular disease (82.1%(69/84), 88.2%(30/34), 89.3%(25/28) vs. 47.6%(10/21), χ2=10.21, 10.75, 10.80, all P=0.001); the incidence of secondary hyperparathyroidism in women was higher than that in men (80.0%(64/80) vs. 95.0%(57/60), χ2=6.58, P=0.010). The incidence of LVH in children aged 6-<12 was higher than that in children aged 12-18 (73.5%(25/34) vs. 43.1%(22/51), χ2=7.62, P=0.006). Among 113 follow-up children, the mortality rate was 39.8% (45/113). Compared to the survival group, the children in the death group had lower hemoglobin, higher blood pressure, lower albumin, lower alkaline phosphatase and higher left ventricular mass index ((67±19) vs. (75±20) g/L, 142 (126, 154) vs. 128(113, 145) mmHg(1 mmHg=0.133 kPa), (91±21) vs. (82±22) mmHg, 32 (26, 41) vs. 40 (31, 43) g/L, 151 (82, 214) vs. 215 (129, 37) U/L, 48 (38, 66) vs. 38(32, 50) g/m2.7,t=2.03, Z=2.89, t=2.70, Z=2.49, 2.79, 2.29,all P<0.05), but no independent risk factors were identified (all P>0.05). The peritoneal dialysis group had better alleviation for anemia, low calcium, and high phosphorus than the hemodialysis group ((87±22) vs. (72±16) g/L, (1.9±0.5) vs. (1.7±0.4) mmol/L, (2.2±0.7) vs. (2.8±0.9) mmol/L, t=2.92, 2.29, 2.82, all P<0.05), and the survival rate of the peritoneal dialysis group was significantly higher than that of the hemodialysis group (77.8% (28/36) vs. 48.4% (30/62), χ2=8.14, P=0.004). Conclusions: CAKUT is the most common etiology in children with CKD 5, and anemia is the most common complication. The incidence of complications in children with CKD 5 varies with age, gender and etiology. Anemia, hypertension, hypoalbuminemia, reduced alkaline phosphatase and elevated LVMI may be the prognostic factors in children with CKD5. Peritoneal dialysis may be more beneficial for improving the long-term survival rate.

[Clinical characteristics and survival analysis of primary intestinal extranodal NK/T-cell lymphoma, nasal type].

Objective: To retrospectively analyze the clinical features and survival analysis of primary intestinal extranodal NK/T-cell lymphoma nasal type(ENKTL). Methods: Patients with intestinal lymphoma at the First Affiliated Hospital of Air Force Military Medical University were collected from January 2009 to December 2019, and those with primary intestinal ENKTL screened. The general situation, main symptoms, ECOG scale, lactic dehydrogenase(LDH), and ß(2)-microglobulin(ß(2)-MG)in the serum, lesion site and form, numbers of extranodal invasion, Lugano stage, pathological features, and lifetime and survival outcomes were evaluated. Results: In total, 34 patients with confirmed diagnosis of primary intestinal ENKTL were identified. The incidence rate of primary intestinal ENKTL is 16.2% (34/210). A total of 26 patients were men and 8 were women with the median age at diagnosis of 45 (range, 20-69)years and patients younger than 60 years account for 85.3%. It included abdominal pain (76.5%), diarrhea(20.6%), hematochezia(29.4%), and abdominal mass(2.9%), 85.3% with B-symptoms, and abnormally elevated LDH in the serum(73.5%). The lesion sites included large intestine(52.9%), small intestine(29.4%), and both of them(17.7%). The lesion forms were featured by mass(8.8%), ulcer(32.4%), diffuse infiltration(2.9%), and hybrid(55.9%). Lugano stage included stage Ⅰ/Ⅱ (70.6%)24 patients and stage Ⅳ(29.4%)10 patients. About 94.1% patients were EBER positive. The median survival time was 60 days. The overall survival rate had significant differences on ECOG scale, LDH in the serum, IPI score and complications(P=0.037, 0.009, 0.002, and 0.000, respectively). Conclusion: Primary intestinal ENKTL was commonly observed in men at young or middle age, and the most common symptom was abdominal pain, often with B-symptoms, abnormally elevated LDH in the serum. The most common site was the large intestine. The lesion forms were mainly featured by the hybrid and ulcer. It seemed that patients with IPI low-risk group and without any complication would have longer survival time.

[Clinical and genetic characteristics of 62 children with mitochondrial epilepsy].

Objective: To summarize the clinical and genetic characteristics of children with mitochondrial epilepsy. Methods: Clinical data of 62 children who were clinically and genetically diagnosed with mitochondrial epilepsy by the Department of Neurology, Beijing Children's Hospital from October 2011 to December 2018 were analyzed retrospectively, and the control of epilepsy was followed up. T test or χ(2) test were used to analyze the related factors affecting the prognosis of epilepsy between the effective group and the ineffective group. Results: Of the 62 patients, 33 were male and 29 were female. The age of onset was 3.38 (0-12.00) years; for the type of seizures, 68% (42/62) of the patients had focal seizures, generalized or secondary generalized tonic-clonic seizures were seen in 32% (20/62), myoclonic seizures in 23% (14/62), spastic seizures in 7 cases, tonic seizures in 4 cases, absence seizure, atonic seizure and clonic seizure in 1 case each; 16 cases (26%) had status epilepticus, of whom 6 cases had epilepsia partialis continua; 52% (32/62) had 2 or more types of seizures. The clinical phenotypes were mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) in 29 cases, Leigh syndrome (LS) in 11 cases, combined oxidative phosphorylation deficiency in 6 cases, myoclonus epilepsy with ragged-red fibers in 5 cases, Alpers syndrome in 4 cases, pontocerebellar hypoplasia type 6 and mitochondrial DNA depletion syndrome 9 in 2 cases each, mitochondrial complex Ⅰ deficiency nuclear type 20, progressive cavitating leukoencephalopathy, and biotinidase deficiency in 1 case each. Of the 62 cases, 40 cases (65%) had mitochondrial DNA (mtDNA) variations, of which 26 cases had m.3243A>G variants, 6 cases had m.8344A>G variants, and 3 cases had m.8993T>G/C variants, m.3271T>C, m.3481G>A, m.3946G>A, m.13094T>C, m.14487T>C variant was in 1 case each; nuclear DNA (nDNA) variations were identified in 22 cases (35%), of which 7 cases carrying variations in mitochondrial ammonia acyl tRNA synthetase coding gene, mutations in POLG and the gene encoding complex Ⅰ were in 4 cases each, variations in SUCLG1 and SDHA genes were in 2 cases each, and variations in PDHA1, BTD and TRIT1 genes were in 1 case each. Forty-three patients were followed up, and the follow-up time was 20 (3-84) months. According to the follow-up results, the anti-epilepsy treatment was effective in 19 cases (44%) and ineffective in other 24 cases (56%). The onset age of the effective group was 3.42 (0-11.50) years and that of the ineffective group was 0.92 (0-9.50) years. The onset duration of the effective group was 0 (0-7.00) years and that of the ineffective group was 0 (0-4.83) years. There was no significant difference between the effective group and the ineffective group (t=1.662, 0.860; P=0.104, 0.395). In the effective group and the ineffective group, 12 cases and 9 cases used less than 2 kinds of antiepileptic drugs, 7 cases and 15 cases used more than or equal to 2 kinds of antiepileptic drugs, 13 and 15 cases had first epilepsy, 6 and 9 cases had non-first epilepsy, 14 and 11 cases had mtDNA variation, 5 and 13 cases had nDNA variation, respectively. There was no significant difference between the two groups (χ(2)=2.794, 0.164, 3.380; P=0.095, 0.686, 0.066). Conclusions: The types of seizures with mitochondrial epilepsy in children varied, with focal motor seizures being the most common, followed by generalized or secondary generalized tonic-clonic seizures. Most children have more than two types of seizures. MELAS is the most common clinical phenotype, followed by LS; mtDNA variation is the dominant gene variation, of which m.3243A>G variation is the most common hotspot variation, followed by gene variation encoding mitochondrial aminoacyl tRNA synthase.

An audit of outcomes in colorectal cancer in China.

AIM: This study was designed to analyses the correlation between the extent of lymph node excision and the prognosis of colorectal cancer and to discuss the clinical significance of excision of lymph nodes adjacent to mesenteric artery pedicle. METHODS: A total of 1409 patients with colorectal cancer who had the primary radical operation in our hospital during 1985 to 2000 was analyzed in this study. They were divided into two groups according to whether undergone an excision of lymph nodes adjacent to mesenteric artery pedicle. There were 857 patients in the excision group and 552 patients in the non-excision group. Comparison of prognoses was based on the follow-up results. RESULTS: The 1-year, 3-year and 5-year overall survival rates of the excision group were 90%, 81% and 77%, while those of the non-excision group were 91%, 84% and 79%, respectively. The 1-year, 3-year and 5-year tumor-free survival rates of excision group were 90%, 79% and 75%, while those of the non-excision group were 90%, 83% and 76%. Kaplan-Meier analysis did not show any difference in overall survival rate or tumor-free survival rate between these two groups (P>0.05). After follow-up of 12 to 289 months there were 42 cases of local recurrence and 79 cases of metastases in the excision group. In the non-excision group local recurrence occurred in 30 cases and 60 patients had metastases. There was no difference in the rates of local recurrence and metastasis between these two groups. Multivariate correlation analyses showed that the excision of lymph nodes adjacent to mesenteric artery pedicle was not statistically correlated to recurrence, metastasis and survival time after radical operation of colorectal cancer. CONCLUSION: In radical operation of colorectal cancer it is not necessary to perform excision of lymph nodes adjacent to mesenteric artery pedicle which is not correlated to prognosis.

Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.

Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.

Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors.

The E2F group of transcription factors transactivates genes that promote progression through the G1-S transition of the cell cycle. Members of the retinoblastoma (Rb) family of proteins bind to E2Fs and inhibit this function. E2F-4, one example of the E2F group, functions as an oncogene when transfected into nontransformed cells in vitro. On the other hand, mice that are homozygously lacking a normal E2F-1 gene develop cancers, consistent with a tumor-suppressive role for this gene. The exact function of E2Fs has thus been unclear; moreover, direct involvement of this gene in primary human tumorigenesis has not been shown. We, therefore, investigated mutation within the E2F-4 coding region in 16 primary gastric adenocarcinomas, 12 ulcerative colitis-associated neoplasms, 46 sporadic colorectal carcinomas, 9 endometrial cancers, and 3 prostatic carcinomas. We limited our investigation to the serine repeat within E2F-4, reasoning that this tract might be altered in genetically unstable tumors (replication error-positive, or RER+). All tumors were RER+, with the exception of a control group of 15 RER- sporadic colorectal carcinomas. PCR with incorporation of [32P]dCTP was performed using primers flanking the serine trinucleotide (AGC) repeat. Twenty-two of 59 gastrointestinal tumors (37%) contained E2F-4 mutations; these comprised 5 of 16 gastric tumors (31%), 4 of 12 ulcerative colitis-associated neoplasms (33%, including 1 dysplastic lesion), and 13 of 31 sporadic colorectal cancers (42%). No mutation was present in any of the endometrial, prostate, or RER- colorectal tumors. Of note, homozygous mutations occurred in three cases, and two of seven informative patients showed loss of one E2F-4 allele in their tumors. Furthermore, the RER+ sporadic colorectal tumors were evaluated at trinucleotide repeats within the genes for N-cadherin and B-catenin; no tumors demonstrated mutation of these genes. These data suggest that E2F-4 is a target of defective DNA repair in these tumors.

FHIT gene alterations in esophageal cancer and ulcerative colitis (UC).

FHIT (fragile histidine triad gene), a candidate tumor suppressor gene, was recently identified and cloned at chromosome 3p14.2. Alterations of this gene have been reported in a number of primary human tumors, including colorectal, esophageal, gastric and lung carcinomas. However, some reports have found no abnormalities in this gene. We investigated a total of 63 primary esophageal tumors, nine esophageal cancer cell lines and 17 ulcerative colitis-associated neoplasms (UCANs) for alterations of FHIT. In 13 esophageal tumors, we employed overlapping reverse transcriptase-PCRs (RT-PCRs) to amplify and sequence the complete open reading frame of FHIT. One of 13 primary esophageal tumors analysed by RT-PCR expressed no detectable FHIT transcript; the remaining 12 expressed normal-sized transcripts with wild-type open reading frame sequences. In an additional 50 esophageal tumors, the polymorphic microsatellite loci D3S1300 and D3S1313 were used to evaluate loss of heterozygosity (LOH) at 3p14.2. Eleven of these 50 tumors showed LOH at one or both loci. In all these 11 tumors, genomic PCR and direct sequencing of FHIT exons 5-9 was performed. This analysis revealed that none of these 11 primary esophageal tumors contained any alterations in the FHIT open reading frame or adjacent intron sequences. Finally, among 17 UCANs, the in vitro synthesized protein (IVSP) assay detected no truncated protein products, nor were there any abnormalities in size or DNA sequence of FHIT RT-PCR products. However, in six of nine esophageal carcinoma cell lines, no FHIT RT-PCR product was detectable using either of the overlapping primer sets. Genomic PCR and direct sequencing of exons 5-9, also performed in these nine cell lines, revealed wild-type sequence in eight cell lines; however, one cell line contained no exon 5 PCR product. This cell line also lacked detectable FHIT transcript. These data suggest that the open reading frame of FHIT is not important in the development or progression of most primary esophageal carcinomas or UCANs, although lack of expression of the FHIT transcript may be common in esophageal cancer-derived cell lines. The possibility of an additional tumor suppressor gene at chromosome 3p14.2 remains to be evaluated.

Apparent protection from instability of repeat sequences in cancer-related genes in replication error positive gastrointestinal cancers.

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.