Right gastroepiploic artery length determined anastomotic leakage after minimally invasive esophagectomy for esophageal cancer: a prospective cohort study.

BACKGROUND: This prospective cohort study, conducted at a high-volume esophageal cancer center from July 2019 to July 2022, aimed to investigate the link between the right gastroepiploic artery (RGEA) length and anastomotic leakage (AL) rates following minimally invasive esophagectomy (MIE). Real-world data on stomach blood supply in the Chinese population were examined. MATERIALS AND METHODS: A total of 516 cases were enrolled, categorized into two groups based on the Youden index-determined optimal cut-off value for the relative length of RGEA (length of RGEA/length of gastric conduit, 64.69%) through ROC analysis: Group SR (short RGEA) and Group LR (long RGEA). The primary observation parameter was the relationship between AL incidence and the ratio of direct blood supply from RGEA. Secondary parameters included the mean length of the right gastroepiploic artery, greater curvature, and the connection type between right and left gastroepiploic vessels. Patient data were prospectively recorded in electronic case report forms. RESULTS: The study revealed median lengths of 43.60 cm for greater curvature, 43.16 cm for the gastric conduit, and 26.75 cm for RGEA. AL, the most common postoperative complication, showed a significant difference between groups (16.88 vs. 8.84%, P =0.01). Multivariable binary logistic regression identified Group SR and LR (odds ratio: 2.651, 95% CI: 1.124-6.250, P =0.03) and Neoadjuvant therapy (odds ratio: 2.479, 95% CI: 1.374-4.473, P =0.00) as independent predictors of AL. CONCLUSIONS: The study emphasizes the crucial role of RGEA length in determining AL incidence in MIE for esophageal cancer. Preserving RGEA and fostering capillary arches between RGEA and LGEA are recommended strategies to mitigate AL risk.

[Assessing heart function of patients with single left ventricular valvalar insufficiency leision using 64-slice multi-detector row CT angiography].

OBJECTIVE: To evaluate the accuracy of results of heart functions determined by 64-slice multidetector row computed tomography (64-MDCT) in patients with single valvular insufficiency leision in left ventricle. METHODS: 58 patients with single valvular insufficiency leision in left ventricle were enrolled in this study. Their heart functions were assessed by magnetic resonance imaging (MRI), 64-MDCT and echocardiography (Echo) respectively. The assessed parameters included left ventricular end-diastolic and end-systolic volume (LVEDV, LVESV), stroke volume (LVSV), ejection fraction (LVEF), and effective ejection fraction (eLVEF). The correlations between eLVEF and some clinical indicators, such as cardiopulmonary bypass time (CPBT), ventilation time (VT), vasoactive drug used time (VDUT), and length of ICU stay (ICUST) were analyzed. RESULTS: No significant differences were found in the parameters measured by 64-MDCT, Echo and MRI. A strong correlation between 64-MDCT and MRI (r: 0.79-0. 92) was found with all of the parameters. The eLVEF measured by 64-MDCT and MRI correlated with CPBT, VT, VDUT and ICUST well (r: 0.56-0. 84). CONCLUSION: 64-MDCT is a rapid, accurate and cheap choice for assessing heart functions of patients with single valvular insufficiency leision in left ventricle. eLVEF is a good predictor for the outcomes of operations.

[Establishment and characterization of a nude mice model of human diffuse large B-cell lymphoma].

OBJECTIVE: To establish a diffuse large B-cell lymphoma (DLBCL)-mice model using human DLBCL cell line LY8, to investigate its characteristics of growth and to provide a model for in vivo study of DLBCL pathogenesis and treatment. METHODS: LY8 cells were injected subcutaneously into the right flank of nude mice. Harvested tumor tissues were cut into small pieces of 1.5 mm × 1.5 mm × 1.5 mm and implanted subcutaneously into nude mice. Tumor growth was visualized and the histologic characteristics were documented. Expression of LCA, CD20, CD79α, Ki-67, CD3, CD45RO, bcl-6, MUM-1, CD10 and bcl-2 were examined by using immunohistochemistry. IgH clonal rearrangement and status of three microsatellite loci (D14S68, D18S69, D20S199) in the xenografted tumor samples and the parental cell line LY8 were detected using PCR amplification followed by PAGE. RESULTS: The subcutaneous xenograft DLBCL model was successfully established by using cell line LY8, and a stable growth was achieved up to the 9th generation. The tumor in each generation showed similar growth characteristics and the rate of subcutaneous tumor formation was 91.9% (114/124). The tumor growth was observed from the 2nd week after implantation, reaching 1.3 cm in major diameter at the 3rd week and 2.0 cm at the 4th week. The tumor had identical morphological characteristics with those of human DLBCL, and expressed LCA, CD20, CD79α, bcl-6, MUM-1, CD10 and bcl-2. The tumor of xenograft mice and cell line LY8 showed identical IgH rearrangement and microsatellite length. CONCLUSIONS: A human DLBCL bearing mouse model was successfully established. The mice model is similar to human counterpart with high stability and repeatability. Therefore, it provides an ideal animal model for in vivo studies of the biological characteristics and treatment of DLBCL.

Germline mutation analysis of hPMS2 gene in Chinese families with hereditary nonpolyposis colorectal cancer.

AIM: To study the germline mutation of hPMS2 gene in 26 unrelated Chinese hereditary nonpolyposis colorectal cancer (HNPCC) probands and to fulfill the screening strategy for HNPCC in Chinese. METHODS: Genomic DNA was extracted from the peripheral blood. To avoid the interference of pseudogene in detection of the remaining 11 exons (exon 1-5, 9, 11-15), long-range polymerase chain reaction (PCR) was conducted to amplify the complete coding region of hPMS2 gene firstly. Then 1/8 of the PCR products were used as template to amplify the individual exon respectively and DNA sequencing was done. Direct DNA sequencing of the conventional PCR products of exon 6, 7, 8 and 10 of hPMS2 gene was performed. The same analysis was made in 130 healthy persons without family histories of HNPCC to further investigate the pathological effects of the detected missense mutation. RESULTS: One HNPCC proband fulfilled Bethesda guidelines and was found to carry the germline mutation of hPMS2 gene, which has not been reported in Chinese HNPCC families. It was a missense mutation at c.1532C>T of exon 11. It was detected in three controls as well with an occurrence rate of 2.3% (3/130). Since it could not be found in the PMS2-single nucleotide polymorphism (SNP) database, this missense mutation is a new SNP unreported up to date. Meanwhile, 260 reported SNPs of hPMS2 gene were detected in the 26 HNPCC probands. The 2nd and 5th exons were probably the hot SNP regions of hPMS2 gene in Chinese HNPCC families involving 53.1% of all reported SNP. CONCLUSION: The germline mutation of hPMS2 gene may be rare in Chinese HNPCC families. The 2nd and 5th exons are hot SNP regions of hPMS2 gene.

[Immunoglobulin heavy chain gene rearrangement study in difficult cases of B-cell lymphoproliferative disorder].

OBJECTIVE: To evaluate the ancillary diagnostic value of IgH gene rearrangements in those B-cell lymphoproliferative disorder cases whom are difficult in making a final diagnosis. METHODS: IgH gene clonal rearrangements were retrospectively analyzed in a total of 77 diagnostically difficult B-cell lympho-proliferative patients. Standardized BIOMED-2 system IgH gene clonality assay kit targeting FR1, FR2, FR3 was used, followed by heteroduplex-polyacrylamide gel electrophoresis (PAGE) and silver nitrate staining. RESULTS: The final diagnoses of the 77 cases were: 12 cases of reactive lymphoid hyperplasia, 20 cases of atypical lymphoid hyperplasia or suspicious lymphoma, and 45 cases of B-cell lymphoma. Detection rates of at least one positive reaction were 2/12, 11/20 (55%), 36/45 (80%) in the three groups, respectively. In B-cell lymphomas, the clonality detection rate of FR1, FR2 and FR3 was 60% (27/45), 60% (27/45) and 56% (25/45), respectively. The type distribution were: 20 marginal zone lymphomas, including 18 extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, 7 diffuse large B-cell lymphomas, 7 follicular lymphomas, 1 mantle-cell lymphoma, 1 Burkitt's lymphoma, 4 plasma cell neoplasms and 5 unclassified B-cell lymphomas. Rearrangements of FR1, FR2 or FR3 were not detected in 9 (20%) of the B cell lymphoma cases, nevertheless, one of them had developed liver lesion later, and was confirmed finally to be B cell lymphoma. Fourteen patients of reactive lymphoid hyperplasia with positive IgH gene clonal rearrangements, and atypical lymphoid hyperplasia had follow-up history available. Four of them were diagnosed as lymphoid malignancies upon further biopsy, and in three of them, clonal IgH gene rearrangements were detected. CONCLUSIONS: B-cell lymphoproliferative disorder requiring a detection of clonal IgH gene rearrangement for making a final diagnosis. Combined detections of three IgH FR1, FR2 and FR3 rearrangements provide important ancillary diagnostic value in confirming suspected B-cell lympho-proliferative disorders. It is important to take an additional biopsy or to follow-up those patients who that have a detectable IgH gene clonal rearrangement but without apparent morphological evidence of lymphoma. For cases with a negative IgH gene rearrangements, it might be necessary to perform clonality analysis for other forms of gene rearrangements including IgH or IgK and IgL in order to further improve the detection sensitivity.

[Clinical significance in detection of immunoglobulin heavy chain clonal rearrangement in bone marrow of patients with B cell lymphoma].

OBJECTIVE: To explore the feasibility of semi-nested PCR technique for detection of immunoglobulin heavy chain (IgH) clonal rearrangement in bone marrow of B-cell lymphoma patient and to further evaluate its clinicopathological value. METHODS: Gene clonal rearrangement of IgH was detected by semi-nested PCR using primers of FR2 & FR3A in 105 bone marrow samples of patients with B-cell lymphoma. The PCR detection results were compared with the cytomorphology of bone marrow aspiration biopsy. The correlation between PCR detection results and clinicopathological factors were evaluated. RESULTS: Among 105 cases of B-cell lymphoma, bone marrow involvement was detected by PCR technique in 48 cases (45.7%), while only 22 cases (21.0%) were detected by bone marrow cytological analysis. There was a significant difference between two methods (P < 0.05), and the concordance rate was 71.4%. The incidence of bone marrow involvement at the time of initial diagnosis detected by PCR technique was 30.8% for diffuse large B cell lymphoma (DLBCL), 25.0% for follicular lymphoma (FL), and 100.0% for small lymphocytic lymphoma (SLL), respectively. Bone marrow involvement detected by PCR detection correlated with Ann Arbor stage. Rate of clonal IgH gene rearrangement by PCR in early B-cell lymphoma was lower than that in advanced stage B-cell lymphoma patients (P = 0.02). There was no statistically significant difference in efficacy between patients with positive and negative results detected by PCR (P > 0.05). But difference in complete response (CR) rate (23.3% and 46.3%) had significant difference (P = 0.019). CONCLUSION: Semi-nested PCR analysis may be an effective method for detection of abnormalities in bone marrow in patients with B-cell lymphoma and is superior to cytomorphology. The positive rate in patients with advanced Ann Arbor stage is higher than that in patients with early Ann Arbor stage, and patients with PCR negative result have more chances to achieved CR after treatment.

Isolated mitral regurgitation: quantitative assessment with 64-section multidetector CT--comparison with MR imaging and echocardiography.

PURPOSE: To evaluate the accuracy of 64-section multidetector computed tomography (CT) for the assessment of the severity of isolated mitral regurgitation by measuring ventricular volumetrics compared with those at magnetic resonance (MR) imaging and echocardiography. MATERIALS AND METHODS: This study was approved by an institutional review board; patient informed consent was obtained. Forty-nine patients (22 men, 27 women; mean age, 39 years +/- 11 [standard deviation]) with isolated mitral regurgitation underwent retrospective electrocardiographically (ECG) gated 64-section CT, echocardiography, and MR imaging for the assessment of the severity of mitral regurgitation. Stroke volumes of the left and right ventricles were measured at 64-section CT and MR imaging. With these measurements, regurgitant volumes (RVs) and regurgitant fractions (RFs) were calculated and compared. The agreement between CT and MR imaging was tested by using linear regression and Bland-Altman analyses. RFs were compared with corresponding echocardiographic grades (grades I-IV) based on the absolute area of the regurgitation jet and in relation to the size of the left atrium by using Spearman rank order correlation and a weighted kappa test. RESULTS: No significant differences were found in calculated RV and RF between 64-section CT and MR imaging (P = .56 and .87, respectively; paired t test). Regression analysis showed that 64-section CT correlated well with MR imaging for the measurement of RV (r = 0.89; 95% confidence interval [CI]: 0.81, 0.94) and for calculating the RF (r = 0.91; 95% CI: 0.84, 0.95). Bland-Altman analysis showed no significant differences in RV (bias, -1.0 mL) and RF (bias, 0.2%) between 64-section CT and MR imaging. The severity of regurgitation estimated by using echocardiography correlated well with that estimated by using 64-section CT (r = 0.95; 95% CI: 0.92, 0.97) and MR imaging (r = 0.94; 95% CI: 0.91, 0.96). CONCLUSION: ECG-gated 64-section CT helps provide quantitative information with high accuracy for determining the severity of isolated mitral regurgitation.

Sixty-four-slice multidetector computed tomography for preoperative evaluation of left ventricular function and mass in patients with mitral regurgitation: comparison with magnetic resonance imaging and echocardiography.

Quantitative values of left ventricular (LV) function and muscle mass in patients with mitral regurgitation are independent predictors of cardiac morbidity and mortality. The aim of this study was to prospectively evaluate whether 64-MDCT can assess the LV function in patients with mitral regurgitation with high accuracy when compared with the MRI and echocardiography results. Fifty-one patients with mitral regurgitation underwent retrospectively ECG-gated 64-MDCT, echocardiography, and MRI for assessing the global ventricular function. End-diastolic and end-systolic volume, stroke volume, ejection fraction, and mass were measured on 64-MDCT and echocardiography, and compared with the results measured on MRI which served as the reference standard. Intertechnique agreement was tested by using Pearson's correlation and Bland-Altman analyses. No significant differences were revealed in calculated LV function and mass between the 64-MDCT and MRI (paired t test, p = 0.07-0.53). Pearson's correlation analysis showed the functional parameters and mass correlated closely between the 64-MDCT and MRI (r = 0.89-0.96, p < 0.001). When compared with MRI, echocardiography underestimated the volumetric parameters of LV (paired t test, p = 0.0003-0.004), but significantly overestimated the EF values (p = 0.003), and moderate correlations of functional parameters were obtained (r = 0.78, 0.60, 0.81, and 0.62, respectively). ECG-gated 64-MDCT allows for accurate and reliable assessment of LV function in patients with mitral regurgitation, whereas LV volumes measured by two-dimensional echocardiography were underestimated and the ejection fraction was overestimated when compared with those achieved by using MRI.

MLH1 promoter germline-methylation in selected probands of Chinese hereditary non-polyposis colorectal cancer families.

AIM: To detect the MLH1 gene promoter germline-methylation in probands of Chinese hereditary nonpolyposis colorectal cancer (HNPCC), and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC. METHODS: The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR (MSP) in 18 probands from unrelated HNPCC families with high microsatellite-instability (MSI-H) phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were collected with microsatellite-stability (MSS) phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C > A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MLH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein. RESULTS: Five probands with MLH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria (JC) and the Amsterdam criteria (AC), respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines (BG), 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group (22.2%, 4/18) was higher than that in the JC/BG groups (5.6%, 1/18) in all HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype (excluding family H42 without tumor tissue), indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein. CONCLUSION: Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.

Two novel germline mutations of MLH1 and investigation of their pathobiology in hereditary non-polyposis colorectal cancer families in China.

AIM: To detect germline mutations of MLH1, and investigate microsatellite instability and expression of MLH1 in tumor tissues of hereditary non-polyposis colorectal cancer (HNPCC) with two novel germline mutations, and further investigate the pathobiology of the two novel mutations of MLH1. METHODS: RNA was extracted from the peripheral blood of 12 patients from 12 different families that fulfilled the Amsterdam II Criteria for HNPCC. Germline mutations of MLH1 were determined by RT-PCR, followed by cDNA sequencing analysis. PCR-GeneScan analysis was used to investigate microsatellite instability with a panel of five microsatellite markers (BAT26, BAT25, D5S346, D2S123 and mfd15), along with immunohistochemical staining to detect the expression of MLH1 protein in two patients' tumor tissues with novel mutations. RESULTS: Three germline mutations were found in four patients, one of the mutations has previously been reported, but the other two, CGC right arrow TGC at codon 217 of exon 8 and CCG right arrow CTG at codon 581 of exon 16, have not been reported. The two patients' tumor tissues with novel mutations had high-frequency microsatellite instability that showed more than two unstable loci, and both tumors lost their MLH1 protein expression. CONCLUSION: The two novel germline mutations of MLH1 in HNPCC families i.e. CGC right arrow TGC at codon 217 of exon 8 and CCG right arrow CTG at codon 581 of exon 16, are very likely to have pathological significance.

[Detection of t (14; 18) chromosomal translocation in paraffin-embedded tissues of follicular lymphoma and its clinical significance].

OBJECTIVE: To study the genetic aberrations and their pathologic significance in follicular lymphoma (FL). METHODS: Paraffin-embedded tissue samples of 55 cases of FL, 28 cases of other small B-cell lymphomas and 10 cases of reactive follicular hyperplasia were retrieved. Nested polymerase chain reaction (PCR) was used to detect clonal rearrangement of immunoglobulin heavy chain gene (IgH) in FL and other small B-cell lymphomas. The translocation t (14; 18) was studied by PCR and dual-color fluorescence in-situ hybridization (FISH) in FL. Cases of reactive follicular hyperplasia were used as controls. RESULTS: Amongst the 55 cases studied, 49 cases were nodal and 6 cases were extranodal. There were 33 males and 22 females. The male-to-female ratio was 1.5:1. The median age of the patients was 57 years. Twenty-five cases belonged to histologic grade 1, while 19 cases were grade 2 and 11 cases were grade 3. Beta-actin DNA was detected in 50 cases of FL. Amongst those 50 cases, clonal IgH rearrangement was present in 34 (68%). Twenty-four cases (48%) and 25 cases (50%) were positive for FR3A and FR2 respectively. Fifteen cases (30%) showed dual positivity for both FR3A and FR2. Thirty-four cases (68%) demonstrated clonal IgH rearrangement. As for other small B-cell lymphomas, 25 cases were positive for beta-actin. FR3A and FR2 were detected in 18 and 17 cases respectively. Clonal IgH rearrangement was demonstrated in 24 cases. In contrast, none of the 4 cases of reactive follicular hyperplasia showed the clonal rearrangement pattern. Amongst the 44 cases of nodal FL analyzed, t (14; 18) was detected in 15 cases (with 14 cases in MBR and 1 case in mcr). In general, FISH was superior to PCR in detecting t (14; 18) using paraffin-embedded tissue samples. CONCLUSIONS: The detection rate of clonal IgH rearrangement in FL is lower than that in other small B-cell lymphomas. Demonstration of t (14; 18) in paraffin-embedded tissue samples by FISH helps in diagnosis of FL. FISH is superior to PCR, as the technique is more sensitive and less labor intensive.

[Study on the germline mutation of MSH6 gene in Chinese hereditary nonpolyposis colorectal cancer pedigrees using PCR based sequencing].

OBJECTIVE: To detect the germline mutation of mismatch repair gene (MSH6) in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds fulfilling different clinical criteria. METHODS: The germline mutations of MSH6 gene were detected by PCR based DNA sequencing in 39 unrelated HNPCC probands fulfilling different clinical criteria in which MSH2 and MLH1 mutations were excluded. The exons with missense mutations were analyzed using PCR sequencing in the germline genomic DNA of 137 healthy persons. The expression of MSH6 protein was detected by Envision immunohistochemistry staining in the tumor tissues of the mutational probands. RESULTS: Six germline mutations of MSH6 gene were detected in 39 probands of Chinese HNPCC kindreds, and the mutations distributed in the exon 4, 6, 9 and 10. Four out of six mutations were missense mutation, one was nonsense mutation and the remaining one was insertion mutation in splice site. The results of sequecing for the exons with above four missense mutations in 137 healthy persons' genomic DNA showed that 5 of 137 persons had the missense mutation of c.3488 A to T at codon 1163 of the 6th exon. The mutational rate was approximately 3.65% (5/137), so the mutation could be a single nucleotide polymorphism (SNP). The remaining missense mutations were not found in any germline genomic DNA of 137 healthy persons. Positive expression of MSH6 protein had been identified in the tumor of the SNP proband while the tumors had negative MSH6 protein expression in the rest probands of germline mutation MSH6 gene. The types of mutations and their potential significance were determined by comparing the following databases: http://www.ncbi.nlm.nih.gov/, http://www.ensembl.org/homo-sapies, and http://www.insight-group.org. Five out of the six mutations had not been reported previously and they were new pathological mutations, the rest one was a new SNP. CONCLUSION: Germline mutations of MSH6 gene may play an important role in Chinese HNPCC kindreds fulfilling different clinical criteria. It is necessary to analyze the germline mutations of MSH6 gene using sequencing to identify HNPCC families in the probands in which MSH2 and MLH1 mutation were excluded.

Three novel missense germline mutations in different exons of MSH6 gene in Chinese hereditary non-polyposis colorectal cancer families.

AIM: To investigate the germline mutations of MSH6 gene in probands of Chinese hereditary non-polyposis colorectal cancer (HNPCC) families fulfilling different clinical criteria. METHODS: Germline mutations of MSH6 gene were detected by PCR-based DNA sequencing in 39 unrelated HNPCC probands fulfilling different clinical criteria in which MSH2 and MLH1 mutations were excluded. To further investigate the pathological effects of detected missense mutations, we analyzed the above related MSH6 exons using PCR-based sequencing in 137 healthy persons with no family history. The clinicopathological features were collected from the Archive Library of Cancer Hospital, Fudan University and analyzed. RESULTS: Four germline missense mutations distributed in the 4(th), 6(th) and 9(th) exons were observed. Of them, three were not found in international HNPCC databases and did not occur in 137 healthy controls, indicating that they were novel missense mutations. The remaining mutation which is consistent with the case H14 at c.3488A>T of exon 6 of MSH6 gene was also found in the controls, the rate was approximately 3.65% (5/137) and the type of mutation was not found in the international HNPCC mutational and SNP databases, suggesting that this missense mutation was a new SNP unreported up to date. CONCLUSION: Three novel missense mutations and a new SNP observed in the probands of Chinese HNPCC families, may play an important role in the development of HNPCC.

[Nodal versus extranodal diffuse large B-cell lymphoma: comparison of clinicopathologic features, immunophenotype and prognosis].

OBJECTIVE: To study the clinicopathologic features and outcome of patients with diffuse large B-cell lymphoma (DLBCL), and to compare the differences between DLBCL of nodal and extranodal origins. METHODS: One hundred and forty-two cases of de novo DLBCL collected during a 10-year period were reviewed. The clinicopathologic features and follow-up (2 - 108 months) data were analyzed. Tissue microarray blocks were performed and immunohistochemical studies using antibodies against CD10, bcl-6 and MUM1 were carried out. The cases were then further categorized into germinal center B cell-like (GCB) and non-GCB subtypes. RESULTS: Primary gastrointestinal DLBCL often presented as early-stage disease (stage I or II) and was associated with low international prognostic index. They showed better prognosis than DLBCL of nodal and other extranodal origins. The positivity rates of CD10, bcl-6 and MUM1 were 19%, 51% and 58%, respectively. 36% of the cases belonged to GCB, while the remaining 64% were non-GCB. In general, DLBCL of extranodal origin showed more frequent bcl-6 expression than nodal DLBCL. As for extranodal DLBCL, GCB immunophenotype was often seen in thyroid and breast tumors, while testicular DLBCL usually carried a non-GCB immunophenotype. CONCLUSIONS: DLBCL of various origins show a diversified GCB and non-GCB differentiation. Nodal and extranodal DLBCL, as well as extranodal DLBCL from different primary sites, carry different biologic characteristics and prognostic implications.

[Effects of AKT protein kinase activation on biologic behavior of diffuse large B cell lymphoma cells].

OBJECTIVE: To observe the status of AKT and phospho-AKT (pAKT) in three diffuse large B cell lymphoma (DLBCL) cell lines, and to investigate the effects of AKT activation on biologic behavior of DLBCL cells. METHODS: Three DLBCL cell lines, ly1, ly8 and ly10 were maintained in 10% FBS or serum free culture medium. The expression of AKT and status of pAKT were detected by Western blotting. LY294002, an inhibitor of PI3K, was used to suppress the level of pAKT. Flow cytometry combined with PI staining, AnnexinV-FITC assay and Brdu incorporation assay were used to analyze the parameters of the cell cycle, apoptosis and proliferation respectively. RESULTS: There was constitutive activation of AKT in three DLBCL cell lines and the levels of pAKT were altered in the different environments. In 10% FBS culture medium, pAKT was higher than that in serum free culture medium in ly8 and ly10, however, pAKT in ly1 maintained in serum free culture medium was mildly higher than that in 10% FBS culture medium. When the cell lines ly1, ly8, ly10 were maintained in 10% FBS culture medium, the inhibitor LY294002 suppressed the level of pAKT efficiently in three DLBCL cell lines. The percentage of cells at S phase and the proliferation index were significantly decreased (P < 0.05) without an increase of apoptosis (P > 0.05). CONCLUSIONS: Activation of AKT may play an important role in the development of DLBCL. It is closely related to the control of cell cycle and proliferation, but is not associated with apoptosis. LY294002 can inhibit cell growth by decreasing the levels of pAKT in DLBCL cell lines.

Elimination of matrix effect and simultaneous determination of multi-components in complex systems by matrix coefficient non-linearity multivariate calibration based on single point response signals.

In order to deal with the matrix effect in the simultaneous determination of multi-components in a complex system, we have developed a novel method named matrix coefficient multivariate calibration method (MCMCM) for simultaneously determining n analytes in complex systems. The calibration models of n analytes, which are based on the experimental data of known samples, are first transformed into n linear equations, and then the equations are solved to obtain matrix calibration coefficients of the analytes in congeneric samples. In this way, the concentrations of n analytes in the unknown sample could be obtained easily and simultaneously by solving another n-variate linear equations with the help of the matrix calibration coefficients obtained-above. The method proposed in this work has been tested by voltammetry and atomic absorption spectrometry (AAS) with satisfactory results. On determining the elements such as Cu, Pb, Cd, Ni, Zn, Fe, Mn, Co, Ca, Mg, etc. in synthetic samples, the relative standard deviations (RSDs) of the results were 0.91 - 4.5%, and the recoveries were 95.8 - 105%. For actual samples, the RSDs and the recoveries were 1.5 - 6.9 and 92.0 - 110%, respectively.

[Real-time PCR analysis of beta-catenin mRNA in sporadic colorectal cancers].

OBJECTIVE: To detect beta-catenin mRNA levels in sporadic colorectal cancers (SCRC) and adjacent normal colorectal mucosa, and to investigate the association between the beta-catenin mRNA level and its aberrant expression and clinicopathological parameters. METHODS: The concentration of beta-catenin mRNA in 81 SCRCs and 28 adjacent normal colorectal mucosa specimens was determined by TaqMan real-time quantitative RT-PCR. The ratio of beta-catenin cDNA copies/GAPDH cDNA copies was used to represent the mRNA expression level in different tissues. The beta-catenin protein expression was determined by the EnVision two-step immunohistochemical method. RESULTS: beta-catenin mRNA levels in SCRCs (2.527 +/- 2.284) were lower than those in the adjacent normal colorectal mucosa (5.003 +/- 3.326), P < 0.05. In addition, beta-catenin mRNA levels in lymph node-positive cases and tumors with ulcerative and infiltrating growth types were significantly lower (1.827 +/- 1.288, 2.202 +/- 2.035) than those in lymph node-negative cases and polypoid growth type tumors (3.359 +/- 2.881, 3.108 +/- 2.610), P < 0.05. No significant difference of beta-catenin mRNA level was found between cases with aberrant beta-catenin cytoplasm or nuclear expression and those without. CONCLUSIONS: SCRCs express lower levels of beta-catenin mRNA than normal colorectal mucosa. Such lower level expression is associated with lymph node metastasis and tumors with ulcerative and infiltrative growth pattern. Aberrant cytoplasmic and nuclear expression of beta-catenin appears unrelated to the lower mRNA levels. Quantitative detection of beta-catenin mRNA may be a useful approach to monitor the biological behavior of SCRCs.

[Two novel germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer family].

OBJECTIVE: To explore germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer (HNPCC), and to investigate the pathobiology of novel detectable mutations of MLH1. METHOD: RNA was extracted from the peripheral blood of 12 patients from 12 different families fulfilling the Amsterdam II Criteria of HNPCC. Germline mutations of MLH1 were determined by RT-PCR with gene specific primers, heat-resistance reverse transcriptase and long-template PCR polymerase, followed by cDNA sequencing analysis. PCR-Genescan analysis was used to further investigate microsatellite instability with a panel of 5 microsatellite markers (BAT26, BAT25, D5S346, D2S123 and Mfd15), along with immunohistochemistry staining to detect the expression of MLH1 protein in the tumor tissues. RESULTS: Four germline mutations were found in 4 patients, 2 of which were previously reported GTT-->GAT mutation at codon 384 of exon 12, and the other two were novel mutations: CGC-->TGC at codon 217 of exon 8 and CCG-->CTG at codon 581 of exon 16. Two tumors with the novel mutations had high frequency microsatellite instability showing more than 2 instable loci (RER + phenotype), and both tumors lost their MLH1 protein expression. CONCLUSION: The two novel germline mutations of MLH1 identified in this study, i.e. CGC-->TGC at codon 217 of exon 8 and CCG-->CTG at codon 581 of exon 16, are very likely to have pathological significance.

[Expressions of wildtype-RET and RET/PTC rearrangements in sporadic adult papillary thyroid carcinoma and their clinicopathologic correlation].

OBJECTIVE: To evaluate the expressions of wildtype-RET (WT-RET) and RET/PTC in sporadic adult papillary thyroid carcinoma and to investigate their clinicopathologic correlation. METHODS: Sixty-six papillary thyroid carcinomas (PTC) and thirty-six control cases with frozen and paraffin-embedded tissues were analyzed for the expressions of WT-RET and oncogene RET/PTC1 or RET/PTC3 by nested RT-PCR. RESULTS: (1) 62 percent (41/66) of PTC patients were above 40 years of age. Thirty-eight percent (25/66) of the tumors showed lymphocytic thyroiditis. Lymph node and distant metastasis were seen in 59% (39/66) and 7.6% (5/66) respectively. (2) Forty-five cases (68.1%) of PTCs expressed RET tyrosine kinase domain (RET-TK). Simultaneous expressions of RET-BP and TK were seen in nineteen PTCs (28.8 %). One of eight adenomas (12.5 %) expressed wild-type RET (WT-RET). (3) Fourteen PTCs (21.2%) expressed RET/PTC, including five cases expressing RET/PTC1 and nine cases expressing RET/PTC3. Six cases (9%) expressed both RET/PTC and WT-RET. (4) Statistic analysis did not show any correlation between the expression of WT-RET or RET/PTC and clinicopathologic parameters. CONCLUSIONS: The expression of RET/PTC was specific to PTC. However, its prevalence was low and, therefore, of limited diagnostic utility. The expression patterns of WT-RET in PTC and adenoma suggest that there are different molecular mechanisms in activating RET proto-oncogene in thyroid tumors.