The value of right ventricular ultrasound assessment and cardiac biomarkers in the prognosis of sepsis.

OBJECTIVE: The aim of this study was to explore the risk factors affecting the prognosis of patients with sepsis using a prospective design. PATIENTS AND METHODS: From January 2022 to March 2023, a prospective study was conducted in the Intensive Care Unit (ICU) of Cangzhou Central Hospital, including 58 patients who met the diagnostic criteria for sepsis. Patients were divided into a survival group (39 cases) and a death group (19 cases) based on outcome. Within 24 hours, the following indicators were collected: gender, age, underlying diseases, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score, cardiac troponin I (cTnI), B-type natriuretic peptide (BNP), lactate, procalcitonin, ejection fraction (EF), tricuspid annular plane systolic excursion (TAPSE), systolic velocity (S'), and global longitudinal strain/strain rate (GLS/GLSr) and global circumferential strain/strain rate (GCS/GCSr) by speckle tracking. Logistic regression analysis was used to evaluate the risk factors for mortality in septic patients, and receiver operating characteristic (ROC) curves were used to evaluate the predictive value of various risk factors for sepsis-related death. RESULTS: There was no significant difference in gender, age, underlying diseases, BNP, procalcitonin, EF, TAPSE, S', GLSr, GCS, or GCSr between the two groups (p>0.05). There were statistically significant differences in APACHE II score, SOFA score, cTnI, lactate, and GLS between the two groups (p<0.05). Logistic regression analysis showed that SOFA score (OR=2.32, 95% CI: 1.067-5.289, p<0.05), cTnI (OR=1.19, 95% CI: 1.001-1.312, p<0.05), and GLS (OR=1.58, 95% CI: 1.012-2.721, p<0.05) were risk factors for sepsis-related death (p<0.05). The areas under the ROC curves for SOFA score, cTnI, and GLS were 0.769, 0.757, and 0.846, respectively. CONCLUSIONS: SOFA score, cTnI, and GLS are independent risk factors for mortality in patients with sepsis. Among these factors, GLS has the highest predictive value for patient prognosis. Therefore, when predicting the prognosis of patients with sepsis, the assessment of right ventricular ultrasound can be used in clinical practice.

[A clinical study of targeted immunotherapy combined with hepatic arterial chemoembolization in the treatment of liver injury associated with primary liver cancer].

Objective: To investigate the conditions of occurrence and factors influencing liver injury caused by molecular targeted drugs and immune checkpoint inhibitors combined with hepatic arterial chemoembolization (TACE) in the treatment of primary liver cancer. Methods: 105 cases of primary liver cancer admitted to the Third Hospital of Hebei Medical University from January 2020 to June 2023 were selected. Patients liver biochemical indicators conditional changes before and after treatment with targeted drugs+TACE and targeted drugs+immune checkpoint inhibitors (ICIs)+TACE were analyzed. Liver injuries above grade 2 and its independent risk factors to predict and evaluate model accuracy were established. Independent samples t-test, analysis of variance, and rank sum test were used for comparison of measurement data between groups. Count data were compared with a χ(2) test between groups. Results: A total of 50 (47.62%) of the 105 cases developed liver injury during the treatment course, with 26 (52%) cases of first-grade liver injury, 16 (32%) cases of second-grade liver injury, 8 (16%) cases of third-grade liver injury, and none of fourth-grade liver injury. There was no statistically significant difference in the incidence of liver injury between the two groups of patients (χ(2)=1.299, P = 0.637). Multivariate logistic regression analysis showed that total bilirubin, prealbumin, and prothrombin activity were independent risk factors for the occurrence of liver injury. The total bilirubin-prealbumin-prothrombin activity (TAP) model was established. TAP diagnosis of grade 2 or higher liver injury had an area under the receiver characteristic curve of 0.935, sensitivity of 84.35%, and specificity of 92.31% at a cut-off value of 1.24, and significantly better diagnostic performance than albumin-bilirubin (ALBI) grade. Conclusion: The occurrence of severe liver injury is minimal and well tolerated in the targeted drug + TACE treatment group and targeted drug + ICIs + TACE treatment group. The TAP model can be used as a new method to assess the risk of liver injury above grade 2 in patients treated with targeted immunotherapy combined with TACE.

[Clinical cohort study of total hysterectomy via transvaginal natural orifice transluminal endoscopic surgery and transumbilical laparoendoscopic single site surgery].

Objective: To study the safety and feasibility of total hysterectomy by transvaginal natural orifice transluminal endoscopic surgery (vNOTES) and transumbilical laparoendoscopic single site surgery (TU-LESS). Methods: The study was a retrospective cohort analysis. In West China Second University Hospital, from October 1, 2018 to June 30, 2019, the patients with an indication for hysterectomy due to benign uterine diseases were enrolled, and were grouped by the surgical procedure. Various post-operative surgical outcomes, including the success rate of operation, uterine volume, operative time, blood loss, intraoperative complications, postoperative complications, the first time of flatus, the preserved time of catheter after operation, and the length of hospitalization were measured. The data were assessed by the software of SPSS. Results: Totally 30 patients were included in the vNOTES group, and 77 patients were included in the TU-LESS group, all of the procedures were completed successfully without conversion to traditional multiport laparoscopy or laparotomy. Non differences were observed between two groups, including uterine volume [(165±36) vs (235±38) cm3, P=0.243], operation time [(150±41) vs (169±48) minutes, P=0.063], blood loss [(54±15) vs (54±14) ml, P=0.985], the rate of intraoperative complications [3% (1/30) vs 4% (2/77), P=1.000], the rate of postoperative complications [13% (4/30) vs 4% (3/77), P=0.095] and the rate of bilateral salpingo-oophorectomy [50% (15/30) vs 31% (24/77), P=0.069]. Patients in the vNOTES group had significantly less recovery time of intestine function [(1.7±0.5) vs (2.3±0.6) days, P=0.001], shorter time of indwelling catheter [(1.9±0.4) vs (2.3±0.6) days, P=0.004], and duration of hospitalization [(3.2±0.8) vs (3.6±0.9) days, P=0.045]. Conclusions: vNOTES and TU-LESS are both safe and feasible for total hysterectomy. Compared with TU-LESS, vNOTES may be a promising approach with earlier recovery, less injury and better cosmetic.

[Risk factor analysis on anastomotic leakage after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy and establishment of a nomogram prediction model].

Objective: To investigate the risk factors of anastomotic leakage (AL) after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy and construct a nomogram prediction model. Methods: This study was a retrospective case-control study that collected and reviewed the clinicopathological data of 359 patients who underwent laparoscopic surgery from January 2012 to January 2018, including 202 patients from the Department of General Surgery, Nanfang Hospital of Southern Medical University and 157 patients from the Department of Gastrointestinal Surgery of Fujian Provincial Cancer Hospital. Inclusion criteria: (1) age ≥ 18 years old; (2) diagnosis as rectal cancer by biopsy before treatment; (3) distance from tumor to anus within 12 cm; (4) locally advanced stage (T3-T4 or N+) diagnosed by imaging (CT, MRI, PET or ultrasound); (5) standardized neoadjuvant therapy followed by laparoscopic radical operation. Exclusion criteria: (1) previous history of colorectal cancer surgery; (2) short-term or incomplete standardized neoadjuvant therapy; (3) Miles, Hartmann, emergency surgery, palliative resection; (4) conversion to open surgery. Clinicopathological data, including age, gender, body mass index (BMI), preoperative albumin, distance from tumor to anus, operation hospital, American Society of Anesthesiologists score (ASA score), operation time, T stage, N stage, M stage, TNM stage, pathological complete response (pCR) were analyzed with univariate analysis to identify predictors for AL after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy. Then, incorporated predictors of AL, which were screened by multivariate logistic regression, were plotted by the "rms" package in R software to establish a nomogram model. According to the scale of the nomogram of each risk factor, the total score could be obtained by adding each single score, then the corresponding probability of postoperative AL could be acquired. The area under ROC curve (AUC) was used to evaluate the predictive ability of each risk factor and nomogram on model. AUC > 0.75 indicated that the model had good predictive ability. The Bootstrap method (1000 bootstrapping resamples) was applied as internal verification to show the robustness of the model. The discrimination of the nomogram was determined by calculating the average consistency index (C-index) whose rage was 0.5 to 1.0. Higher C-index indicated better consistency with actual risk. The calibration curve was used to assess the calibration of prediction model. The Hosmer-Lemeshow test yielding a non-significant statistic (P>0.05) suggested no departure from the perfect fit. Results: Of 359 cases, 224 were male, 135 were female, 189 were ≥ 55 years old, 98 had a BMI > 24 kg/m(2), 176 had preoperative albumin ≤ 40 g/L, 128 had distance from tumor to anus ≤ 5 cm, 257 were TNM 0-II stage, 102 were TNM III-IV stage, and 84 achieved pCR after neoadjuvant therapy. The incidence of postoperative AL was 9.5% (34/359). Univariate analysis showed that gender, preoperative albumin and distance from tumor to the anus were associated with postoperative AL (All P<0.05). Multivariate logistic regression analysis revealed that male (OR=2.480, 95% CI: 1.012-6.077, P=0.047), preoperative albumin ≤40 g/L (OR=5.319, 95% CI: 2.106-13.433, P<0.001) and distance from tumor to anus ≤ 5 cm (OR=4.339, 95% CI: 1.990-9.458, P<0.001) were significant independent risk factors for postoperative AL. According to these results, a nomogram prediction model was constructed. The male was for 55 points, the preoperative albumin ≤ 40 g/L was for 100 points, and the distance from tumor to the anus ≤ 5 cm was for 88 points. Adding all the points of each risk factor, the corresponding probability of total score would indicated the morbidity of postoperative AL predicted by this nomogram modal. The AUC of the nomogram was 0.792 (95% CI: 0.729-0.856), and the C-index was 0.792 after internal verification. The calibration curve showed that the predictive results were well correlated with the actual results (P=0.562). Conclusions: Male, preoperative albumin ≤ 40 g/L and distance from tumor to the anus ≤ 5 cm are independent risk factors for AL after laparoscopic surgery in rectal cancer patient with neoadjuvant therapy. The nomogram prediction model is helpful to predict the probability of AL after surgery.

[Investigation of dose-dependent association between bedtime routines and sleep outcomes in infants and toddlers].

Objective: To investigate the current bedtime routine among Chinese children less than 3 years of age and explore its dose-dependent association with sleep duration and sleep quality. Method: Healthy full-term born children aged 0-35 months were selected by stratified cluster random sampling method from 8 provinces in China following the "Hospital of Province-City-County" sampling technical route during 2012-2013.Brief Infant Sleep Questionnaire(BISQ) was used to assess sleep conditions of these children.Children's personal and family information was obtained by Shanghai Children's Medical Center Socio-demographic Questionnaire.Both of these questionnaires were filled in by parents. The effects of bedtime routine on children's sleep duration and quality were analyzed by multivariate analysis of variance. Result: The children's average age was(12±10) months(n=1 304), of whom 689 were males (52.8%, 689/1 304). There were 48.5%(632/1 304)of the parents reported that their children had not established regular sleep routines. There was a consistent dose-dependent association between bedtime routine and sleep duration, as well as other indicators for sleep quality (all P<0.05). The more regular the sleep routines, the longer the sleep duration, the earlier the children went to sleep, the shorter the sleep onset latency, the fewer the nighttime wakeup and the shorter the nighttime waking.The nighttime sleep duration was significantly longer for those with a bedtime routine 'every night' than those who 'never' had a bedtime routine (9.5(95%CI: 9.4-9.6)vs. 8.9(95%CI: 8.6-9.3)h, t=3.345, P=0.001). Compared with children who never had bedtime routines, children with regular bedtime routines had fewer night wakeup (1.3(95%CI: 1.2-1.4) vs. 2.4( 95%CI: 2.0-2.9), t=3.182, P=0.001) and shorter night waking duration(16.6(95%CI: 14.6-18.8) vs. 59.2 (95%CI: 47.0-72.7)min, t=6.383, P<0.01). Conclusion: The percentage of children who have established regular bedtime routine is low in China. There is significant dose-dependent association between regular bedtime routine and sleep outcomes, especially sleep quality. The more regular the sleep routines, the better the sleep quality.

Sex-specific, rapid neuroestrogen fluctuations and neurophysiological actions in the songbird auditory forebrain.

Recent evidence shows that brain-derived steroids such as estrogens ("neuroestrogens") are controlled in a manner very similar to traditional neurotransmitters. The advent of in vivo microdialysis for steroids in songbirds has provided new information about the spatial and temporal dynamics of neuroestrogen changes in a region of the auditory cortex, the caudomedial nidopallium (NCM). Here, experiments using in vivo microdialysis demonstrate that neuroestradiol (E(2)) fluctuations occur within the auditory NCM during presentation of naturalistic auditory and visual stimuli in males but only to the presentation of auditory stimuli in females. These changes are acute (within 30 min) and appear to be specific to the NCM, because similar treatments elicit no changes in E(2) in a nearby mesopallial region or in circulating plasma. Further experiments coupling in vivo steroid microdialysis with extracellular recordings in NCM show that neuroestrogens rapidly boost auditory responses to song stimuli in females, similar to recent observations in males. We also find that the rapid actions of estradiol on auditory responses are fully mimicked by the cell membrane-impermeable estrogen biotinylestradiol, consistent with acute estrogen actions at the neuronal membrane. Thus we conclude that local and acute E(2) flux is regulated by convergent multimodal sensory input, and that this regulation appears to be sex-specific. Second, rapid changes in local E(2) levels in NCM have consequences for the modulation of auditory processing in females and males. Finally, the rapid actions of neuroestrogens on NCM auditory processing appear to be mediated by a nonclassical, membrane-bound estrogen receptor.

Global analysis of CpG methylation reveals epigenetic control of the radiosensitivity in lung cancer cell lines.

Epigenetic regulation by CpG methylation has an important role in tumorigenesis as well as in the response to cancer therapy. To analyze the mechanism of epigenetic control of radiosensitivity, the CpG methylation profiles of radiosensitive H460 and radioresistant H1299 human non-small cell lung cancer (NSCLC) cell lines were analyzed using microarray profiling. These analyses revealed 1091 differentially methylated genes (DMG) (absolute difference of mean beta-values, |Deltabeta |>0.5), including genes involved in cell adhesion, cell communication, signal transduction and transcriptional regulation. Among the 747 genes hypermethylated in radioresistant H1299 cells, CpG methylation of SERPINB5 and S100A6 in radioresistant H1299 cells was confirmed by methylation-specific PCR. Reverse transcriptase-PCR showed higher expression of these two genes in radiosensitive H460 cells compared with radioresistant H1299 cells. Downregulation of SERPINB5 or S100A6 by small interfering RNA in H460 cells increased the resistance of these cells to ionizing radiation. In contrast, promoter CpG sites of 344 genes, including CAT and BNC1, were hypomethylated in radioresistant H1299 cells. Suppression of CAT or BNC1 mRNA expression in H1299 cells also reduced the resistance of these cells to ionizing radiation. Thus, we identified DMGs by genome-wide CpG methylation profiling in two NSCLC cell lines with different responses to ionizing radiation, and our data indicated that these differences may be critical for epigenetic regulation of radiosensitivity in lung cancer cells.

Mutual exclusiveness between PIK3CA and KRAS mutations in endometrial carcinoma.

In endometrial carcinomas (ECs), previous report suggested that PIK3CA mutations do not coexist with KRAS mutations, but the significant mutual exclusiveness has not been demonstrated. In this study, we examined the mutation frequency of PIK3CA in EC and its mutual exclusiveness with KRAS mutation. We performed mutational analysis of PIK3CA through a polymerase chain reaction single-strand conformation polymorphism assay in 44 cases of endometrial cancer and analyzed the correlation with loss of PTEN, KRAS mutation, and RASSF1A hypermethylation. Somatic mutations of PIK3CA were detected in 14 of 44 (31.8%) of endometrial cancers. In exon 9, seven PIK3CA mutations were located, while seven mutations were located in exon 20. The most common mutation was E545A (35.7%), followed by H1047R (28.6%). Concomitant loss of PTEN expression and PIK3CA mutation was found in four cases of endometrial cancer. KRAS mutations were mutually exclusive with PIK3CA mutations, and those mutations were inversely correlated with statistical significance (P = 0.039). Also, we found that mutations in ERBB2 were mutually exclusive with PIK3CA mutations. RASSF1A and hMLH1 methylation were not correlated with the presence of PIK3CA mutations. PIK3CA was frequently mutated in endometrial cancers. KRAS and PIK3CA mutations are inversely correlated, suggesting that genetic alterations of KRAS and PIK3CA may play equivalent roles in endometrial carcinogenesis.

Concurrent hypermethylation of multiple genes is associated with grade of oligodendroglial tumors.

Current evidence suggests that epigenetic changes play an important role in the evolution of human cancers. In this study, we evaluated whether hypermethylation of CpG islands at the gene promotor regions of several tumor-related genes is involved in the carcinogenesis of oligodendroglial tumors. We examined the methylation status of 11 genes in a series of 43 oligodendroglial tumors (19 oligodendrogliomas, 13 anaplastic oligodendrogliomas, 9 oligoastrocytomas, and 2 anaplastic oligoastrocytomas) by methylation-specific polymerase chain reaction. Our results showed that hypermethylation of CpG islands was detectable in 8 of 11 genes studied and 74% of tumors were hypermethylated in at least 1 gene. Promotor hypermethylations were detected in O6-methylguanine-DNA methyltransferase (MGMT), RB1, estrogen receptor, p73, p16INK4a, death-associated protein kinase, p15INK4b, and p14ARF at 60%, 34%, 30%, 16%, 12%, 10%, 7%, and 2%, respectively. No hypermethylation was detected in the promotors of glutathione-S-transferase P1, von Hippel-Lindau or the DNA mismatch repair (hMLH1) genes. Statistical analysis revealed that concordant hypermethylation of at least 2 genes, p16INK4a and p15INK4b were significantly associated with anaplastic oligodendroglial tumors, and hypermethylation of MGMT was significantly associated with loss of chromosome 19q and with combined loss of chromosomes 1p and 19q. More importantly, several candidate tumor suppressor genes such as p16INK4a, p15INK4b, and p73 that were previously reported as unmutated in oligodendroglial tumors were found to be hypermethylated in their CpG islands. Taken together, we conclude that hypermethylation of CpG islands is a common epigenetic event that is associated with the development of oligodendroglial tumors.

Molecular detection of cervical intraepithelial neoplasia and cervical carcinoma by microsatellite analysis of Papanicolaou smears.

Carcinoma of the uterine cervix is one of the most common malignancies worldwide, yet it is clearly preventable by population screening. The Papanicolaou (Pap) smear has proved to be the most successful test for the detection of precancerous lesions and is largely responsible for the reduction of cervical cancer mortality and morbidity rates. However, the Pap smear is not perfect; false-negative results of various rates are reported. To improve the diagnostic efficacy of cervical cytology, we performed microsatellite analysis on paired Pap smear samples from cervical lesions. Nine microsatellite markers were chosen from chromosomal regions commonly displaying loss of heterozygostity (LOH) in cervical cancer and those displaying microsatellite instability (MI) in other squamous cell cancer. Microsatellite alterations were detected in 16/21 (76%) Pap smear DNA samples including 11 of 13 (85%) smears from invasive squamous cell carcinomas (SCCs) and 5 of 8 (63%) from squamous intraepithelial lesions (SILs). Microsatellite alterations detected in the Pap smear DNA were identical to those identified in seven paired primary tumors available for analysis. Moreover, this molecular approach detected genetic alterations in two cases apparently negative by cytologic examination. None (0/25) of the control patients displayed microsatellite alterations in paired Pap smears. Microsatellite analysis of cervical cytologic samples may provide a complementary method to analyze suspicious but not diagnostic cytologic samples further.

Methylation status in the promoter region of the human PGP9.5 gene in cancer and normal tissues.

PGP 9.5 is a neurospecific peptide that functions to remove ubiquitin from ubiquitinated cellular proteins, thereby preventing them from targeted degradation by the proteasome-dependent pathway or regulating their localization, activity or structure. Using the serial analysis of gene expression method (SAGE), we initially found that the PGP9.5 transcript and protein was highly expressed in more than 50% of primary lung cancers and nearly all lung cancer cell lines but was not detectable in the normal lung. This increased expression could be the result of transcriptional regulation accompanied by methylation changes at the CpG island of the promoter region. We studied the methylation status of the cytosines at the promoter region of human PGP9.5 using sodium bisulfite genomic sequencing in normal and neoplastic cells. Although no methylation of PGP9.5 promoter was observed in the normal lung, normal cervical tissue, and lung cancer cell lines, this region was densely methylated in the HeLa cell line. Exposure to HeLa cells to the demethylating agent, 5-aza-2'-deoxycytidine, led to re-expression of PGP9.5. This data suggested that while other mechanisms may be involved in the frequent overexpression of PGP9.5 gene in lung tumors and lung cancer cell lines, promoter methylation may play a role in the transcriptional suppression of PGP9.5 gene expression in the cervical tissue-derived HeLa cell line.

Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix.

Promoter hypermethylation is an important pathway for the repression of gene transcription in cancer. We investigated promoter hypermethylation of six genes, p16, APC, HIC-1, death-associated protein kinase (DAPK), O(6)-methylguanine-DNA-methyltransferase (MGMT), and E-cadherin, in uterine cervical carcinoma from 53 patients including 31 cases of squamous cell carcinoma (SCC) and 22 cases of adenocarcinoma (AC). Aberrant methylation of at least one of these genes was detected in 79% (42 of 53) of cases including 71% (22 of 31) of SCC and 91% (20 of 22) of AC cases. No aberrant methylation was detected in normal cervical tissue from 24 control hysterectomy specimens. There was no correlation between promoter hypermethylation at any gene and the presence of human papillomavirus-16 or -18 E7 DNA. In AC cases, promoter hypermethylation of the APC and HIC-1 genes was detected at a statistically significant higher frequency than in the SCC cases (APC, 60% versus 13%, P < 0.001; HIC-1, 63% versus 32%, P < 0.03). Conversely, promoter hypermethylation of p16 and DAPK was more common in SCC cases than in AC cases. Our results suggest that promoter hypermethylation is a frequent epigenetic event in cervical carcinoma. The pattern of gene promoter hypermethylation is distinctly different between AC and SCC. The absence of these epigenetic alterations in normal cervical tissue suggests that they may also be valuable as cancer markers.

Detecting colorectal cancer in stool with the use of multiple genetic targets.

BACKGROUND: Colorectal cancer cells are shed into the stool, providing a potential means for the early detection of the disease using noninvasive approaches. Our goal was to develop reliable, specific molecular genetic tests for the detection of colorectal cancer in stool samples. METHODS: Stool DNA was isolated from paired stools and primary tumor samples from 51 colorectal cancer patients. Three genetic targets-TP53, BAT26, and K-RAS-were used to detect tumor-associated mutations in the stool prior to or without regard to the molecular analyses of the paired tumors. TP53 gene mutations were detected with a mismatch-ligation assay that detects nine common p53 gene mutations. Deletions within the BAT26 locus were detected by a modified solid-phase minisequencing method. Mutations in codons 12 and 13 of K-RAS were detected with a digital polymerase chain reaction-based method. RESULTS: TP53 gene mutations were detected in the tumor DNA of 30 patients, all of whom had the identical TP53 mutation in their stools. Tumors from three patients contained a noninherited deletion at the BAT26 locus, and the same alterations were identified in these patients' stool specimens. Nineteen of 50 tumors tested had a K-RAS mutation; identical mutations were detected in the paired stool DNA samples from eight patients. In no case was a mutation found in stool that was not also present in the primary tumor. Thus, the three genetic markers together detected 36 (71%) of 51 patients (95% confidence interval [CI] = 56% to 83%) with colorectal cancer and 36 (92%) of 39 patients (95% CI = 79% to 98%) whose tumors had an alteration. CONCLUSION: We were able to detect the majority of colorectal cancers by analyzing stool DNA for just three genetic markers. Additional work is needed to determine the specificity of these genetic tests for detecting colorectal neoplasia in asymptomatic patients and to more precisely estimate the prevalence of the mutations and sensitivity of the assay.

Immunohistochemical analysis of Fas ligand expression in normal human tissues.

Cross-linking of Fas and Fas ligand (FasL) induces apoptosis in Fas-bearing cells and regulates apoptosis. Fas is widely expressed in normal human tissues, but FasL expression has been considered to be restricted to lymphoid tissues. Recent studies have demonstrated that FasL is also expressed in some nonlymphoid tissues. To screen the in situ expression of FasL in normal human tissues, immunohistochemistry was performed using paraffin-embedded human tissues. FasL immunostaining was easily detected in testis, neurons, trophoblasts, tonsil, lymph node, Paneth cells, hepatocytes, renal tubular epithelium and bronchial epithelium, consistent with previous reports. Surprisingly, FasL was also expressed in many other cell types, including thymic medulla, skeletal muscle, cardiac muscle, pituitary gland, parathyroid gland, prostate glands, oocytes, epithelium of fallopian tube, endometrial glands, and gastric parietal cells. These findings demonstrate that FasL is widely expressed in human tissues and suggest that wide but cell-type specific expression of FasL may not only be implicated in the regulation of immune homeostasis but also in the regulation of cell death and life in many cell types in vivo.

Alterations of Fas (APO-1/CD95) gene in transitional cell carcinomas of urinary bladder.

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas in cancer patients have been described solely in lymphoid-lineage malignancies. We analyzed somatic mutations and loss of heterozygosity of Fas gene in 43 transitional cell carcinomas of urinary bladder. Overall, 12 tumors (28%) were found to have Fas mutations, including 11 missense mutations and 1 frameshift mutation. Ten of the 12 mutations were located in the death domain known to be involved in the transduction of an apoptotic signal, and 8 of these 10 mutations showed an identical G to A transition at bp 993, indicating a potential hotspot in bladder cancers. Three of eight (38%) informative tumors carrying Fas mutations showed LOH at polymorphic sites in the promoter region. This is the first report on the Fas gene mutations in nonlymphoid malignancies, and our data suggest that alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some bladder cancers.

Alterations of Fas (Apo-1/CD95) gene in non-small cell lung cancer.

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas gene in cancer patients have been described solely in lymphoid-lineage malignancies. However, many non-lymphoid tumor cells have been found to be resistant to Fas-mediated apoptosis, which suggests that Fas mutations, one of the possible mechanisms for Fas-resistance, may be involved in the pathogenesis of non-lymphoid malignancies as well. In this study, we have analysed the entire coding region and all splice sites of the Fas gene for the detection of the gene mutations in 65 human non-small cell lung cancers by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing. Overall, five tumors (7.7%) were found to have the Fas mutations, which were all missense mutations. Four of the five mutations identified were located in the cytoplasmic region (death domain) known to be involved in the transduction of an apoptotic signal and one mutation was located in the transmembrane domain. This is the first report on the Fas gene mutations in non-lymphoid malignancies, and the data presented here suggests that alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human lung cancers.

In vivo expression of soluble Fas and FAP-1: possible mechanisms of Fas resistance in human hepatoblastomas.

Many tumour cells express both Fas and its ligand (FasL) on their surface and it has remained a mystery why such cells do not simply kill themselves. It remains to be determined whether Fas and FasL are expressed in human hepatoblastomas and if so, what is responsible for the possible Fas resistance of these tumours. In this study, the expression of Fas and FasL was examined in 23 cases of human hepatoblastoma by immunohistochemical staining. To elucidate possible Fas resistance in hepatoblastomas, Fas-resistance pathways including the expression of bcl-2 and Fas-associated phosphatase-1 (FAP-1), and the expression of soluble Fas (sFas) mRNA, were analysed by immunohistochemistry and in situ reverse transcription-polymerase chain reaction (in situ RT-PCR). Fas gene mutation in the death domain was also examined. Fas and FasL were expressed in all hepatoblastomas analysed. Twenty (87 per cent) and 18 (78 per cent) cases of hepatoblastoma were positive for sFas mRNA and FAP-1, respectively, but none of the hepatoblastomas expressed bcl-2. Mutation in the death domain of the Fas gene was not found in hepatoblastomas. Taken together, these findings demonstrated that Fas, a death receptor, and its ligand are co-expressed in hepatoblastomas in vivo, but some inhibitors of Fas-mediated apoptosis are also expressed in these tumours. These results suggest that it is probably due to the action of inhibitory molecules of the Fas pathway that the tumour cells of hepatoblastomas do not kill themselves in an autocrine-driven cycle and that in this manner hepatoblastomas avoid apoptosis.

Alterations of Fas (Apo-1/CD95) gene in cutaneous malignant melanoma.

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas gene in cancer patients have been described solely in lymphoid-lineage malignancies. However, many nonlymphoid tumor cells have been found to be resistant to Fas-mediated apoptosis, which suggests that Fas mutations, one of the possible mechanisms for Fas resistance, may be involved in the pathogenesis of nonlymphoid malignancies as well. In this study, we have analyzed the entire coding region and all splice sites of the Fas gene for the detection of the gene mutations in 44 human malignant melanomas in skin by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Overall, 3 tumors (6.8%) were found to have the Fas mutations, which were all missense variants and identified in the cytoplasmic region (death domain) known to be involved in the transduction of an apoptotic signal. The data presented here suggest that somatic alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human malignant melanomas.

Detection of p16 gene alteration in cervical cancer using tissue microdissection and LOH study.

The p16 gene was identified as cyclin-dependent kinase inhibitor (CDKI) and this may negatively regulate the cell cycle by acting as a tumor suppressor. Using tissue microdissection, the molecular changes at p16 and Rb genes were analysed in the spectrum of disease from dysplasia to invasive cancer of the uterine cervix. Six of 27 (22%) cases informative for D9S171 and IFNA of 9p21-22 marker (p16INK4a) showed loss of one or both alleles in at least one of these loci. LOH of pRb was detected in 29% (5/17). Gene alterations at p16 and pRb loci were only detectable in some cases of HPV-16/18 DNA positive cervical cancer. Three cases demonstrated mutational changes of p16INK4a, and the alterations were determined to be G to T shift, suggesting transitional missense mutation. In summary, the inactivation of the p16/cdk-cyclin/Rb cascade may play an additional role during the malignant progression in HPV-16/18 positive cervical cancers.