Expression of Six1 and Six4 in esophageal squamous cell carcinoma and their correlation with clinical prognosis / 中华病理学杂志

<p><b>OBJECTIVE</b>Six1 and Six4 are expressed in several tumors, and associated with tumor progress and poor prognosis. The aim of this study was to investigate the expression of Six1 and Six4 in esophageal squamous cell carcinoma (ESCC), and to evaluate their correlation with the clinicopathological factors and prognosis.</p><p><b>METHODS</b>Tissue microarray technology and immunohistochemical method (EnVision) were used to detect the expression of Six1 and Six4 in the tumor tissues and corresponding adjacent normal epithelium of esophagus from 292 ESCC patients.</p><p><b>RESULTS</b>Among the 292 ESCC patients, the positive rates of Six1 and Six4 protein expression in tumor tissues were 72.9% (213/292) and 56.2% (164/292), respectively, significantly higher than the expression rate of 33.2% (97/292) and 32.5% (95/292) in adjacent normal epithelium of esophagus (P < 0.05). Chi square test showed that the expression of Six1 protein was related to tumor size, depth of tumor invasion and patient survival status; higher Six4 protein expression level was related to poor differentiation and increased depth of invasion. Single factor Log-rank analysis revealed that gender, TNM stage, Six1 protein expression level were related to the overall survival of ESCC patients (P < 0.05), while the five-year survival rate was significantly higher in the Six1-negative group than the Six1-positive group [51.9% (41/79) vs. 43.7% (93/213)]. Multi-factor Cox proportional risk model analysis showed that TNM stage and positive expression of Six1 were independent prognostic factors for ESCC patients (P < 0.05).</p><p><b>CONCLUSIONS</b>Six1 and Six4 are highly expressed in ESCC. Their expression levels are closely related to the progress and prognosis of ESCC. Over-expression of Six1 is related to poor prognosis in ESCC patients. Thus, Six1 could be used as an important prognostic indicator for ESCC patients.</p>

Detection of epidermal growth factor receptor mutations in non-small-cell lung carcinoma by direct sequencing and correlations with clinicopathological characteristics and sample types / 中华病理学杂志

<p><b>OBJECTIVE</b>To retrospectively analyze epidermal growth factor receptor (EGFR) gene mutation frequencies and distribution characteristics in Chinese patients with non-small-cell lung carcinoma (NSCLC) by direct gene sequencing.</p><p><b>METHODS</b>Clinical samples from 443 NSCLC patients were obtained for EGFR gene mutation analysis, including 299 surgical specimens, 59 core biopsies and 85 fine needle aspiration and pleural effusion cytology specimens. All samples were processed from paraffin embedded blocks and microdissection was performed to enrich tumor cells. PCR based direct gene sequencing was used to investigate tyrosine kinase domain coding region involving exon 18 through 21.</p><p><b>RESULTS</b>(1) Among 443 samples, 193 mutations were detected in 189 patients (42.7%) and 4 patients possessed two mutations involving two different exons in their tumor samples. The percentage of mutations involving exon 18 to 21 were 2.0% (4/193), 48.7% (94/193), 6.7% (13/193) and 42.5% (82/193) respectively. (2) There was no significant correlation of EGFR mutation with age, however, mutation rate (50.9%, 54/106) of exon 21 in patients over median age 57 was higher than that of the younger patients (32.2%, 28/87; P<0.01). (3) EGFR mutation rate was remarkably higher in female patients (53.5%, 107/200) than in male patients (33.7%, 82/243; P<0.01). (4) Mutation rate in adenocarcinomas (46.5%, 161/346) was much higher than in squamous cell carcinomas (13.3%, 4/30) and poorly differentiated carcinomas (24.1%, 7/29; P<0.01, P<0.05), while the adenosquamous carcinomas shared a mutation rate similar to that of adenocarcinoma (7/13, P>0.05). (5) In surgical samples, core biopsies and cytological samples, the EGFR mutation detection rates were 49.5% (148/299), 35.6% (21/59) and 23.5% (20/85) respectively. The fine needle aspiration and cytological samples showed much lower EGFR mutation detection rates (23.5%, 20/85) than that of surgical samples (49.5%, 148/299; P<0.01).</p><p><b>CONCLUSIONS</b>(1) Direct gene sequencing is a reliable and effective method for the detection of EGFR mutations in NSCLC, particularly for unknown EGFR mutations. (2) EGFR mutations are more frequent in female patients and patients with adenocarcinoma NSCLC, involving mainly exon 19 and 21. (3) The mutation distribution in exons of EGFR gene appears age-related. (4) Detection rate of EGFR mutation varies in different sample types. Small biopsy and fine needle aspiration specimens are valuable materials for analyzing EGFR mutation in NSCLC, although rare false negativity may occur using such samples.</p>

The analysis for mRNA mutation of MLH1, MSH2 genes and the gene diagnosis for hereditary nonpolyposis colorectal cancer / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify hereditary nonpolyposis colorectal cancer (HNPCC) families based on the germline mutations of MLH1 and MSH2 mRNA.</p><p><b>METHODS</b>RNA was extracted from the peripheral blood of the 14 members from 12 different families fulfilling Amsterdam Criteria II. The germline mutations of MLH1 and MSH2 mRNA were detected by cDNA sequencing analysis following reverse transcription-PCR(RT-PCR) with special primers, heat-resistance reverse transcriptase, and expand long template PCR. DNA was extracted from the peripheral blood of the 14 members, the corresponding exons, in which mutations were found using the above method, were amplified with Taq enzyme, sequencing analysis was followed.</p><p><b>RESULTS</b>Six germline mutations were detected and identified from the 6 different families based on mRNA, 4 of them to be in MLH1, the other 2 in MSH2. The MLH1 mutations distribute in the exon 8, 12, 16, and 19. The MSH2 mutations distribute in exons 1 and 2. The 6 mutations were identified from the corresponding exons respectively in genomic DNA sequencing analysis. The mutation types involve in 4 missense, 1 silent, and 1 non-coding area mutations. Five out of the 6 mutations have not been reported previously. Five out of the 6 mutations were pathological, involving in 5 different families. The five families were identified to HNPCC families.</p><p><b>CONCLUSION</b>HNPCC family can be identified with RNA-based sequencing of MLH1 and MSH2 from peripheral blood, which has the advantages of both cost, time saving and high sensitivity.</p>

Two novel germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer family / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer (HNPCC), and to investigate the pathobiology of novel detectable mutations of MLH1.</p><p><b>METHOD</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Microsatellite instability of sporadic colorectal carcinomas and its clinicopathological significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the incidence of microsatellite instability (MSI) in sporadic colorectal carcinoma (CRC) using BAT-25 and BAT-26 loci, and its association with clinicopathological features.</p><p><b>METHODS</b>Microsatellite analysis was performed on tissue samples from 73 primary and 53 metastatic tumors collected at the Department of Pathology, Fudan University Cancer Hospital in 2002. Genomic DNA was extracted from paraffin-embedded tissues. Microsatellite alterations of BAT-25 and BAT-26 were detected using fluorescent PCR followed by fragment analysis on automatic DNA sequencer with GeneScan 3.1 software. A case of hereditary nonpolyposis colorectal cancer syndrome (HNPCC) with known high-frequency MSI (MSI-H) was included as positive control.</p><p><b>RESULTS</b>Eleven out of 73 samples from primary tumors (15.1%) were MSI-positive and significantly associated with patient age, tumor site, differentiation and prognosis (P < 0.05). There was no significant difference between the positive rate of MSI in tissue samples from primary and metastatic sites among the 53 metastatic tumors, being 17.0% and 13.2%, respectively, P > 0.05. Two cases with negative MSI at the primary site but positive at the metastatic sites were observed.</p><p><b>CONCLUSION</b>MSI is a frequent molecular event that may serve as a useful parameter for studying tumor biological behavior. MSI plays a partial role in the metastasis of sporadic CRC, but the role of mismatch repair genes and its exact mechanism remains to be determined. The classification of sporadic CRC according to MSI may be of importance both theoretically and practically.</p>

Detection of cyclin D1 mRNA by reverse transcription-polymerase chain reaction in paraffin-embedded tissues and its diagnostic significance for mantle cell lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of detecting cyclin D1 mRNA in paraffin-embedded tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and competitive RT-PCR and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL).</p><p><b>METHODS</b>Paraffin-embedded samples of 36 cases of MCL, 71 cases of other small B-cell lymphomas and 20 cases of lymphoid reactive hyperplasia as control group were retrieved from archival materials. Cyclin D1 protein and its mRNA was detected by EnVision and RT-PCR and competitive RT-PCR in all samples. House-keeping gene PGK was choosen as internal control.</p><p><b>RESULTS</b>(1) Cyclin D1 protein was expressed in 27 of the 38 MCL (71.1%). No cyclin D1 expression was found in the control group. (2) PGK was detected in 103 of the 116 cases (88.8%) and also detected in 34 of 36 MCL cases (94.7%). (3) cyclin D1 mRNA was detected in 34 nodal mantle cell lymphoma cases by RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA was 94.4% in mantle cell lymphomas after exclusion of the 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia, except 1 case of B-SLL. Sequencing analysis showed that sequences were identical to cyclin D1. (4) Cyclin D1 mRNA overexpression was detected in 27 cases of nodal mantle cell lymphoma by competitive RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA overexpression was 75.0% in mantle cell lymphomas after exclusion of 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA overexpression was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia.</p><p><b>CONCLUSION</b>RT-PCR and competitive RT-PCR detection of cyclin D1 mRNA overexpression could be used for the diagnosis and differential diagnosis of mantle cell lymphoma in paraffin-embedded blocks.</p>

Detection of FUS-CHOP fusion gene in paraffin-embedded tissues and its clinicopathologic significance for myxoid/round cell liposarcomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of detecting FUS-CHOP fusion gene in formalin-fixed, paraffin-embedded tissue and its application in the diagnosis and differential diagnosis of myxoid/round cell liposarcomas (MRCLs).</p><p><b>METHODS</b>Forty-four formalin-fixed, paraffin-embedded MRCL samples and 60 control cases (atypical/well-differentiated liposarcoma, pleomorphic liposarcoma, low-grade myofibrosarcoma, etc.) retrieved from the archival files were studied. Nested reverse transcription-polymerase chain reaction (RT-PCR) technique was employed to detect the FUS-CHOP mRNA expression, followed by DNA sequencing confirmation of the PCR product. Housekeeping gene PGK was used to assess the quality of the mRNA templates.</p><p><b>RESULTS</b>PGK mRNA was detected in 93 of 104 tumor cases (89.4%), including 39 MRCLs cases (39/44, 88.6%) and 90% of the negative control cases. Type II FUS-CHOP fusion transcript was successfully detected in 20 out of 39 (51.3%) MRCL cases. Type I FUS-CHOP fusion transcript was not detected in any MRCLs in this study. All 60 negative control cases were negative for the FUS-CHOP fusion gene transcripts.</p><p><b>CONCLUSIONS</b>(1) Nested RT-PCR can be used to detect FUS-CHOP mRNA in formalin-fixed, paraffin-embedded tissues. (2) FUS-CHOP is considered a specific molecular and genetic hallmark for MRCLs. Nested RT-PCR is a sensitive and specific technique in detecting FUS-CHOP gene, and can be used in the diagnosis and differential diagnosis of MRCLs.</p>

Analysis of bcl-6 protein expression and gene rearrangement in diffuse large B-cell lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate bcl-6 protein expression and gene rearrangement patterns in diffuse large B-cell lymphoma (DLBCL) and their clinicopathologic significance.</p><p><b>METHODS</b>Immunohistochemical studies for bcl-6 and CD10 proteins were performed on 51 cases of DLBCL paraffin-embedded tissues (including 22 nodal samples and 29 extranodal samples) and 10 cases of reactive lymphoid hyperplasia (RLH) paraffin-embedded tissues. Interphase fluorescence in-situ hybridization (FISH) with dual color breakapart probe was also used to identify rearrangement of bcl-6 gene in 32 cases of nodal DLBCL tissues (including 22 paraffin-embedded samples and 10 fresh samples) and 5 cases of RLH paraffin-embedded tissues.</p><p><b>RESULTS</b>(1) The rates of bcl-6 protein expression in nodal DLBCL, extranodal DLBCL and RLH were 72.7% (16/22), 75.9% (22/29) and 100.0% (10/10) respectively. The rates of CD10 expression were 40.9% (9/22), 41.4% (12/29) and 100.0% (10/10) respectively. All lymphoma samples which expressed CD10 also showed co-expression of bcl-6 protein. (2) The co-expression of bcl-6 and CD10 was observed in 40.9% (9/22) nodal DLBCL and 41.4% (12/29) extranodal DLBCL. Low clinical stage (stage I and II) was more frequently observed in cases with co-expression of bcl-6 and CD10 (P < 0.05). (3) The rates of bcl-6 gene rearrangement in nodal DLBCL was 28.1% (9/32), with 27.3% (6/22) in paraffin-embedded tissues and 30.0% (3/10) in fresh tissues. There was no statistically significant difference found between the two groups (P > 0.05). Bcl-6 gene rearrangement was not found in all the 5 cases of RLH, and there was a significant difference between RLH and DLBCL (P < 0.05).</p><p><b>CONCLUSIONS</b>The rate of bcl-6 protein expression is high in DLBCL cases, and the detection of bcl-6 and CD10 protein co-expression may help in the diagnosis and differential diagnosis of DLBCL. Those DLBCL cases with co-expression of bcl-6 and CD10 may also have a better prognostic implication. On the other hand, bcl-6 gene rearrangement can be identified by interphase FISH with dual color breakapart probe in both paraffin-embedded and fresh lymphoma tissues.</p>

Analysis of c-kit gene mutations in gastrointestinal stromal tumors / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To define the frequency and spectrum of c-kit gene mutations in gastrointestinal stromal tumors (GIST).</p><p><b>METHODS</b>Fifty two cases of GIST and 28 cases of other tumors were examined for mutations in exon 11, 9 and 13 of c-kit gene using PCR amplification and DNA sequencing.</p><p><b>RESULTS</b>Fourteen out of 25 malignant GIST (56%), while 2 of 27 benign and borderline GIST (7.4%) revealed mutations in exon 11 of c-kit gene (P < 0.01). Most of the mutations consisted of in-frame deletion or replication from 3 to 48 bp in heterozygous and homozygous fashions, but none of the mutations disrupted the downstream reading frame of the gene. Point mutation and deletion concentrated at 550 - 570 codons but replication clustered within 570 - 585 codons. The mutation pattern in recurrence tissues was the same as the primary ones. Normal tissues adjacent to GIST with or without c-kit gene mutations showed wild type c-kit gene sequence. No mutation was found in exon 9 and 13. Neither c-kit gene expression nor gene mutations was found in 3 leiomyomas, 8 leiomyosarcomas, 2 schwannomas, 2 intra-abdomenal fibromitoses and 8 adenocarcinomas.</p><p><b>CONCLUSION</b>The mutations in exon 11 of c-kit gene might partially represent one of the molecular mechanisms of GIST. It can be used as a marker for distinguishing benignancy and malignancy of GIST. The mutations did not involve the reading frame. Except for long frame deletion, most mutations also did not affect protein expression. Mutation of c-kit gene in GIST provides a new genotypic marker to distinguish GIST from authentic leiomyomas, leiomyosarcomas, schwannomas and etc.</p>

Study of mutations in 5'-noncoding region of BCL-6 gene in B-cell non-Hodgkin's lymphomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate BCL-6 gene mutations in B-cell non-Hodgkin lymphomas (B-NHL) and their implications in lymphoma pathogenesis.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) and direct DNA sequencing methods were used to identify mutations in the 5'-noncoding region of BCL-6 gene in 135 cases of B-NHL, 5 cases of T-NHL, 5 cases of nodular lymphocyte predominance Hodgkin's lymphoma (NLPHL) and 10 cases of reactive hyperplasia of lymph node.</p><p><b>RESULTS</b>Mutations were identified in 6 cases of nodal DLBCL (27.3%), 4 cases of FL (22.2%), 4 cases of MALT lymphoma (22.2%), 4 cases of extranodal DLBCL (20.7%) and 2 cases of LRH (20%). No mutations were detected in T-NHL and NLPHL (P < 0.05). There were no significant differences in incidences of BCL-6 gene mutations between nodal and extranodal DLBCL (P > 0.05). All mutations were base substitutions and the frequency of single-base change was 0.14 x 10(-2)/bp approximately 0.68 x 10(-2)/bp.</p><p><b>CONCLUSIONS</b>Mutations of the 5'non-coding region of BCL-6 gene may be involved in the pathogenesis and progression of B-NHL. Molecular demonstration of such mutations may provide a marker of lymphomas derived from the germinal center-related B cells.</p>

Detection of EWS-FLI1 fusion transcript in Ewing's sarcoma/peripheral primitive neuroectodermal tumors by one-step RT-PCR using paraffin-embedded tissues / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of EWS-FLI1/ERG fusion transcript resulting from t(11;12)(q24;12) in paraffin-embedded tissues and its diagnostic implication for Ewing's sarcoma/peripheral primitive neuroectodermal tumors (ES/pPNET).</p><p><b>METHODS</b>One-step reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect a characteristic EWS-FLI1/ERG fusion transcript in 25 cases of ES/pPNET and 15 cases of other small round cell tumors (including 8 cases of rhabdomyosarcoma, 4 cases of synovial sarcoma, 2 cases of neuroblastoma and 1 case of lymphoma) using formalin-fixed and paraffin-embedded tissues.</p><p><b>RESULTS</b>EWS-FLI1/ERG fusion transcript was detected in 20 of the 25 ES/pPNET cases (80%). The 15 non-ES/pPNET control cases were negative for EWS-FLI1/ERG fusion transcript.</p><p><b>CONCLUSIONS</b>Detection of EWS-FLI1/ERG fusion transcript is a reliable index for molecular diagnosis of ES/pPNET. One-step RT-PCR is a practical method for such analysis in routine paraffin-embedded tumor tissues.</p>

Clinical analysis and molecular genetic study of hereditary nonpolyposis colorectal cancer kindreds / 中华外科杂志

<p><b>OBJECTIVE</b>To study the clinicopathological and molecular genetic characteristics of hereditary nonpolyposis colorectal cancer (HNPCC), to enable the early diagnosis and to evaluate the treatment.</p><p><b>METHODS</b>We analyzed 12 families of HNPCC from Wenzhou, Zhejiang province, China. Mismatch repair genes hMSH2 and hMLH1 expression and microsatellite instability of tumor tissue were studied using microdissection, microsatellite analysis, immunohistochemical staining and Gene Scan analysis. Direct DNA sequencing of hMSH2 and hMLH1 were performed subsequently.</p><p><b>RESULTS</b>Altogether 32 patients with colorectal cancer were recognized in 12 HNPCC families, with the median age of 45.2 years (75.0% before the age of 50 years). The proximal tumors accounted for 51.1%, while multiple colorectal cancers accounted for 34.4%. Poor differentiation cancers occupied half of the patients (53.1%). And 68.8% of the patients had the tumor of Dukes A and B. Among 12 HNPCC families, 7 cases in 6 HNPCC families developed extracolonic cancer. 13 cases died during follow up of 1 - 23 years. The median survival time was 6.4 years. 19 alive cases followed up from 1 to 28 years. All tumors (9/9) displayed microsatellite instability, with the half losing hMSH2 or hMLH1 expression. In the 5 genetic analyzed kindreds 3 possessed germline mutation. Two of three mutations have not been reported in the worldwide database previously.</p><p><b>CONCLUSION</b>HNPCC showed distinct clinicopathological characteristics. Microsatellite instability analysis and immunohistochemical staining might be the effective screening methods before direct DNA sequencing for the detection of mutation in mismatch repair genes. It is important to analyze the members of affected families.</p>

Mismatch pair defective phenotype in hereditary nonpolyposis colorectal cancer in the Chinese / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the protein expression pattern of DNA mismatch repair genes hMSH(2), hMLH(1) and the microsatellite instability (MSI) status in the tumor tissue from hereditary nonpolyposis colorectal cancer in the Chinese.</p><p><b>METHODS</b>Fifty-eight families fulfilling different clinical criteria including Amsterdam Criteria (AC) (22/24 families, 38 tumors), Japanese Criteria (JC) (12/15 families, 16 tumors) and Bethesda Guidelines (BG) (12/19 patients, 13 tumors) were studied. Monoclonal antibodies against hMSH(2), hMLH(1) proteins and a panel of microsatellite markers (5 loci) including BAT26, BAT25, D2S123, D5S346 and D17S250 were used for study.</p><p><b>RESULTS</b>MSI-H was identified in all 22 (100%) AC tumors, with 81.8% (18/22) showing altered hMSH(2) or hMLH(1) expression; in 14/15 (93.8%) JC cancer, 1/1 (100%) JC adenoma, with 45.5% (5/11) showing altered hMSH(2) or hMLH(1) expression; and in 7/13 (53.8%) BG tumors, with 4/7 showing loss of hMSH(2) or hMLH(1) gene expression.</p><p><b>CONCLUSION</b>The frequency of MSI-H and loss of mismatch repair protein are different in the families fulfilling different clinical criteria. Amsterdam Criteria and Japanese Criteria are the two most useful criterion systems for identifying mismatched repair defective tumors. However, Bethesda Guidelines should also be used for detecting more such tumors. The combination of immunohistochemical methods and microsatellite instability analysis is an effective strategy to detect the mismatch repair defective tumors. A close correlation does exist between hMSH(2), hMLH(1) protein expression pattern and MSI status.</p>

Large deletion in mismatch repair genes uncovered by quantitative multiplex PCR-high performance liquid chromatography system / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Establishing a new method on the basis of multiplex PCR-high performance liquid chromatography (HPLC) for screening a large deletion in mismatch repair genes.</p><p><b>METHODS</b>Thirty-five pairs of primers were used to amplify all 16 exons of MSH2 and all 19 exons of MLH1 gene in 8 multiplex PCR. The products of multiplex PCR were analysed for the large deletion with Double Strand DNA Analysis System of HPLC. Firstly, validation of the method was tested on positive and negative controls in blind analysis. Secondly, 14 blood cell DNA samples from hereditary nonpolyposis colorectal cancer (HNPCC) patients and 13 colorectal cancer (CRC) tissues DNA samples from sporadic CRC patients were checked with the new developed method.</p><p><b>RESULTS</b>(1) the genomic deletions in all 4 of positive controls were identically uncovered with the new method; (2) a novel germline and a novel somatic large deletions were unveiled in 1/14 HNPCC patients and in 1/13 CRC tissues.</p><p><b>CONCLUSION</b>The method developed on multiplex PCR-HPLC is reliable for uncovering large genomic deletion in mismatch repair genes, and can be taken as a valuable addition to mutation screening system.</p>

Mutation of 5'noncoding region of the bcl-6 gene in diffuse large B cell lymphomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To observe the mutation of 5'noncoding region of bcl-6 gene in diffuse large B cell lymphoma (DLBCL) and its effect on lymphoma pathogenesis.</p><p><b>METHODS</b>38 DLBCL, 2 reactive hyperplasias, 5 follicular lymphomas and 5 T cell lymphomas were chosen for PCR direct sequence analysis using two sets of primers in 5'noncoding region of the bcl-6 gene.</p><p><b>RESULTS</b>No mutation was found in the marginal region of reactive hyperplasias, T cell lymphomas, and follicular lymphomas but detected in 1/2 of the follicular center cells, and 7/38 cases of DLBCL. The incidence is less than that seen in other reports. Basepairs substitution and point insertion were the main mutation types.</p><p><b>CONCLUSIONS</b>The positive rate of mutation of 5'noncoding region of bcl-6 gene in DLBCL is 18.7%, less frequent than the published data of DLBCL reported in other countries. It may, in some extent, participate in the pathogenesis and progression of DLBCL.</p>

Study on the origin and differentiation of gastrointestinal stromal tumors / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the origin and differentiation of gastrointestinal stromal tumors (GISTs).</p><p><b>METHODS</b>Immunohistochemistry staining and electron microscopy were adopted.</p><p><b>RESULTS</b>In 212 cases of primary GISTs, the positive rates of CD117, CD34, alpha-SMA, MSA, desmin, S-100, PGP9.5 were 96.7%, 77.3%, 19.3%, 15.6%, 1.9%, 16.3%, and 12.3% respectively. Among them, GISTs showed a diffuse and strong positivity for CD117. Electron microscopy of tumor cells demonstrated numerous mitochondria, prominent perinuclear Golgi complex, smooth and rough endoplasmical reticulum and intermediate filaments. Irregular caveolae, dense plaque, incontinuous basal lamina were observed occasionally. Cytoplasmic processes were often observed accompanying with local adhesion present between the processes or between the processes and the cell membrane.</p><p><b>CONCLUSIONS</b>Data from both immunophenotype and electron microscopy suggest that GIST might originate from the mesenchymal cells, differentiating to be ICC afterwards, and possessing myoid characteristics in various extent.</p>

Mutation analysis of hMSH2 and hMLH1 genes in Chinese hereditary nonpolyposis colorectal cancer families / 中华病理学杂志

<p><b>OBJECTIVES</b>To determine the germ-line mutations of hMSH2 and hMLH1 genes in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) families' probands or in patients fulfilling different clinical criteria or guidelines; to clarify the nature and distribution of the mutations; to evaluate the sensitivity of different clinical criteria in mutation prediction.</p><p><b>METHODS</b>The entire coding regions (35 exons including exon-intron boundaries) of hMSH2 and hMLH1 genes were directly sequenced in 24 Amsterdam criteria (AC) probands, 15 Japanese criteria (JC) probands (except AC kindreds) and 19 Bethesda guidelines (BG) patients (except two former groups). All available affected and unaffected members from families of those with mutations were screened for mutation.</p><p><b>RESULTS</b>In 16 unrelated families selected by the different clinical criteria, 17 germ-line mutations were found with 11 (64.7%) of hMLH1 and 6 (35.3%) of hMSH2. Two mutations were identified in one of the families. Among the 17 germ-line mutations, 12 had not been reported previously. A diversified mutation spectrum was found, but 6 hMLH1 mutations were found to be concentrated in the region encompassing exon 14, 15 and 16. There was a wide spectrum of mutation type including frame shift, nonsense, splice site mutation, in frame insertion or deletion and missense mutations. The mutation detection rate of hMSH2 and hMLH1 in the AC group was significantly higher than that in the JC group (12/24 vs. 3/15). On the other hand, a low mutation rate (1/19) was detected in 19 BG patients. The mutation cosegregated with disease. Besides, three different genotypes in tumors from probands of mutation-positive families were found.</p><p><b>CONCLUSIONS</b>hMSH2 and hMLH1 mutations in Chinese HNPCC families show a wide spectrum. It seems that hMLH1 gene is involved more frequently than hMSH2 gene in Chinese HNPCC families. Different clinical criteria predict mutations with different sensitivities. The Amsterdam Criteria are most sensitive, while Japanese Criteria are highly practical and the Bethesda Guidelines are also practical to some extent. Gene mutations cosegregate with the disease phenotype. Carriers with no symptom in HNPCC families are most vulnerable groups, follow-ups are required for this group to get early diagnosis and to prevent the development of CRCs.</p>

Detection of cyclin D1 protein expression and t(11;14) chromosomal translocation in paraffin-embedded tissues and its clinicopathologic significance for mantle cell lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of detecting cyclin D1 protein expression and t(11;14) chromosomal translocation in paraffin-embedded tissues and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL).</p><p><b>METHODS</b>Paraffin-embedded samples of 36 cases of MCL and a control group of 71 cases of small B-cell lymphomas were retrieved from archive materials. Immunohistochemical staining for cyclin D1 and semi-nested PCR for t(11;14) were detected in all samples. House-keeping gene beta-actin was used to detect the quality of DNA.</p><p><b>RESULTS</b>(1) Cyclin D1 was expressed in 26 of the 36 MCL (72.2%). There was no cyclin D1 expression in the control group. (2) beta-actin DNA was detected in 101 of the 107 tumor cases (94.4%). t(11;14) was detected in 22 of the 36 MCL. Translocation was not found in control group. The positive rate for t(11;14) was 64.7% in MCL after exclusion of 2 cases which were negative for both t(11;14) and beta-actin. (3) 29 cases were positive for cyclin D1 and/or t(11;14), the positive rate reached 80.5%.</p><p><b>CONCLUSION</b>The combined detection of cyclin D1 and t(11;14) in paraffin-embedded tissues is found to be a specific and feasible method for diagnosis and differential diagnosis of mantle cell lymphoma.</p>

Clinicopathological, immunohistochemical and molecular genetic study of intra-abdomen extra-gastrointestinal stromal tumors / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the clinicopathological, immunohistochemical and molecular genetic features of intra-abdomen extra-gastrointestinal stromal tumors (EGISTs) and their differential diagnosis.</p><p><b>METHODS</b>Nine cases of EGISTs from the abdominal cavity or retroperitoneum which were previously diagnosed as leiomyoma, leiomyoblastoma, or leiomyosarcoma etc. by a panel of antibodies such as CD117, CD34, alpha-SMA, MSA, desmin, S-100, and PGP9.5 from which five cases were detected for c-kit gene mutation.</p><p><b>RESULTS</b>The tumors occurred in 5 men and 4 women, the age ranged from 38 to 72 years (mean 61.7 years). Four cases arose from the mesentery, two from omentum, two from retroperitoneum and one located at the hilus of the spleen. The size of tumors ranged from 5 cm to 23 cm (mean 12.9 cm) in diameter and the tumor cell components varied: mainly spindle cells (seven cases), epithelioid cells (one case), mixed cells (one case). Tumors expressed CD117 (8/9), CD34 (5/9), alpha-SMA (3/9), MSA (4/9), desmin (0), S-100 protein (1/9) and PGP9.5 (1/9). Of the five cases examined for heterozygous deletion mutation of 11 exon of the c-kit gene two were found positive. Two borderline cases showed long-term survival of 8 years and 11 years, respectively. In seven malignant cases, two showed adverse outcome, one survived 4 years without recurrence, two were lost in follow up and two new cases were still being in followed.</p><p><b>CONCLUSIONS</b>GIST-type stromal tumors can also occur in the abdomen, most cases were borderline or malignant, tumor coagulative necrosis, mitoses >or= 5 per 50 high-power fields and obvious nuclear atypia indicating malignancy. Differential diagnosis of EGIST including benign or malignant smooth muscle tumors, benign or malignant nerve sheath tumors etc.</p>