Clinical application of NanoString fluorescent barcode technology in the molecular subtyping of patients with diffuse large B-cell lymphoma / 白血病·淋巴瘤

Objective:To explore the clinical application of NanoString fluorescent barcode technology in the molecular subtyping of diffuse large B-cell lymphoma (DLBCL), and to analyze the correlation between the cell-of-origin subtype and prognosis of patients.Methods:The tumor tissue samples of 12 patients with DLBCL at the Third People's Hospital of Datong of Shanxi Province and 8 patients with DLBCL at Peking University, Health Science Center between January 2014 and December 2019 were collected. According to Hans algorithm, all patients were divided into 1 case of germinal center-derived B-cell (GCB) type and 19 cases of non-GCB type. NanoString platform was used to analyze the expression level differences of 15 genes-related to Lymph2Cx molecular subtyping of all samples at mRNA level. Hierarchical clustering was used to subgroup 20 DLBCL cases and to contrast the prognosis in different subgroups according to the subtyping.Results:NanoString fluorescent barcode technology was used to detect samples of 20 DLBCL cases and hierarchical clustering analysis was performed, and then subtyping results showed that 11 cases were GCB-like type and 9 cases were activated B cell (ABC)-like type. Based on Hans algorithm, 10 GCB-like cases were non-GCB type. According to the survival analysis, GCB-like group had a better overall survival compared with that in ABC-like group ( P=0.019). Conclusion:NanoString fluorescent barcode technology can be successfully applied to the cell-of-origin subtyping of DLBCL, and the molecular subtyping strategy can effectively predict the prognosis of patients.

Implication of BRAF V600E and CTNNB1 gene mutations in the pathological classification of craniopharyngioma / 中华病理学杂志

Objective@#To investigate the clinicopathological significance of BRAF V600E and CTNNB1 gene mutations in adamantinomatous craniopharyngiomas (ACP) and papillary craniopharyngiomas (PCP).@*Methods@#The retrospective study included a total of 67 craniopharyngiomas diagnosed from October 2009 to August 2018 at Xuanwu Hospital, Capital Medical University. The immunohistochemical staining for β-catenin and BRAF V600E expression, Sanger sequencing of exon 3 of CTNNB1, BRAF mutation analysis by scorpions amplification refractory mutation system (ARMS) fluorescence quantitative PCR were performed. Univariate survival analysis was used to correlate with tumor recurrence.@*Results@#Of the 67 patients, 53 were ACPs and 14 were PCPs. Four patients underwent multiple operations and one of them presented with malignant transformation into squamous cell carcinoma. Histologically, ACPs were characterized by whorl-like cell clusters, peripheral palisaded layer, stellate reticulum, finger-shaped protrusions, ghost cells and wet keratinous substances. While PCPs usually consisted of mature squamous epithelium associated with fibrovascular stroma resulting in papillary appearance. The nuclear immunopositivity for β-catenin was observed in 73.6% (39/53) of ACPs, and it was absent in PCPs (0/14). The nuclear translocation of β-catenin usually presented at whorl-like structures or around ghost cells. Of all the cases, mutations analysis in exon 3 of β-catenin gene CTNNB1 were successful in 46 cases and 42.1% (16/38) of ACP showed CTNNB1 gene mutation, while none of the PCPs harbored CTNNB1 gene mutation (0/8). The cytoplasmic immunopositivity for BRAF V600E mutant protein was found in all PCPs (14/14) and negative in all ACPs (0/53). ARMS-PCR results showed that BRAF V600E mutations were observed in 13/14 of PCPs but not seen in ACPs (0/53). Follow-up data were available in 35 patients with duration of 2 to 120 months. Ten patients experienced recurrences after the first surgery. Upon univariate survival analysis, only subtotal excision was found to be associated with increased recurrence (P=0.032), while pathological type, postoperative radiotherapy and CTNNB1 gene mutation were not (P>0.05).@*Conclusions@#There is significant difference in the expression of BRAF V600E and CTNNB1 genes between ACP and PCP, and their immunohistochemical and molecular detection therefore can be used in the diagnosis and differential diagnoses of craniopharyngiomas.

BRAF V600E mutation and clinicopathologic characteristics in 250 cases of brain tumors associated with epilepsy / 中华病理学杂志

Objective@#To investigate the clinicopathologic characteristics and BRAF V600E mutation of brain tumors associated with epilepsy.@*Methods@#Totally 250 patients with brain tumors associated with epilepsy were included from March 2008 to August 2017 retrospectively at Sanbo Brain Hospital, Capital Medical University.The clinical manifestations, histological features and BRAF V600E mutation results were collected and analyzed.@*Results@#There were 132 males and 118 females, and the male to female ratio was 1.1∶1.0. The age of patients ranged from 2 to 67 years(mean 22 years). The tumors had obvious local space occupying effect on MRI. The temporal lobe was the most common site (44.4%, 111/250). There were 58.4% (146/250) of ganglioglioma (GG), 24.0% (60/250) of dysembryoplastic neuroepithelial tumor (DNT), 12.8% (32/250) of pleomorphic xanthoastrocytoma(PXA), 4.0% (10/250) of angiocentric glioma (AG) and 0.8% (2/250) of papillary glioneuronal tumor (PGNT). Mixed GG, PXA and DNT morphological structures were found in 9 of patients. Among 250 cases, 35 cases were accompanied by focal cortical dysplasia(FCD). BRAF V600E was seen in 43 of 74 (58.1%) GG and 13 of 28 (46.4%) PXA. The most common pathologic grade of GG, DNT, AG and PGNT was WHO I. Some of the tumor cells from GG (34 cases) showed higher proliferative activity (WHO Ⅱ/Ⅲ). Most cases of PXA were WHOⅡand high proliferative activity was seen in nine cases.@*Conclusions@#The association of low-grade glioneuronal tumors with intractable epilepsy was well-recognized. The most common low-grade glioneuronal tumors were GG.GG may occur in any part of the central nervous system, with a predilection for temporal lobe. Each type of low-grade glioneuronal tumors has its own unique histological morphology, but some may show complex features with 2 or 3 mixed components. The occurrence of BRAF V600E mutations in GG is common, and their detection may be valuable for the diagnosis and treatment in GG.

Association between radiographic and clinicopathological features and prognosis in patients with extranodal NK/T cell lymphoma / 肿瘤研究与临床

Objective To explore the CT and MRI imaging and clinicopathological features of extranodal NK/T cell lymphoma (NK/TCL). Methods Sixty-six patients with NK/TCL diagnosed from 2002 June to 2016 April in Beijing Tongren Hospital with intact CT and/or MRI imaging results were enrolled in this study. All the patients had tailed clinical information and follow-up. The imaging and clinicopathological features were analyzed retrospectively and their prognostic value on overall survival was analyzed. Results There were 49 males and 17 females with median age of 42 years. The median follow-up time was 18 months. The cases showed surrounding invasions including 10 cases (15.2 %) in soft palate, 5 cases (7.6 %) in hard palate, 2 cases(3.0 %) in tonsil, 8 cases(12.1 %) in upper lip, 13 cases(19.7 %) in maxillofacial soft tissue, 9 cases (13.6 %) in eyelid, 10 cases (15.2 %) in orbital, 3 cases (4.5 %) in maxilla, 6 cases (9.1 %) in pterygopalatine fossa,6 cases(9.1 %)in infratemporal fossa,3 cases(4.5 %)in skull base, 3 cases(4.5 %) in eyeball and 2 cases (3.0 %) in brain tissue. Kaplan-Meier survival analysis found that the 2-year overall survival rates of the patients with the involvement of hard palate, upper lip, maxillofacial soft tissue, eyelid, orbital, maxillary, eyeball and brain organizer were lower than those of the patients without the involvement of these sites(χ2values were 4.470,4.041,4.456,13.933,8.986,4.000,44.121,6.527,16.822,respectively, all P< 0.05). Further multivariate Cox regression analysis showed that maxilla and brain involvement were independent adverse factors (RR=34.717, 95 % CI 3.404-354.035, P=0.003; RR=37.545, 95 % CI 3.188-442.187, P= 0.004). Conclusions MRI and CT examinations are of great value in diagnosis and prognostic assessment of NK/TCL. Clinicians can make correct and timely diagnosis by comprehensive clinical, radiological and pathological features and can make a detailed clinical assessment to give patients appropriate treatment,thus improving the outcome of the NK/TCL patients.

Effect of Yiqi Huoxue Tablets on Blood Coagulation and Fibrinolysis System of Cerebral Apoplexy Rats Induced by Artificial Cold Wave / 广州中医药大学学报

Objective To study the effect of Yiqi Huoxue Tablets on blood coagulation and fibrinolysis system of rats with cerebral apoplexy induced by artificial cold wave. Methods SD rats were randomized into 4 groups. The apoplexy-prone renovascular hypertensive rat model was established by the bilateral renal arteries clamping method. From the 14th day after the modeling operation, the treatment group was given Yiqi Huoxue Tablets (1.89 g·kg-1·d-1), the positive control group was given aspirin (80 mg·kg-1·d-1), and the model group and the blank group were given the same volume of distilled water, once a day. After 112 days, the rats were put into the artificial cold wave box staying for 3 days to inducing apoplexy, and then thrombin-activatable fibrinolysis inhibitor (TAFI) and protein C ( PC) , thrombin-antithrombin complexes ( TAT) and plasmin-antiplasmin ( PAP) levels were measured. Results The plasma levels of TAFI, PC, TAT, and PAP in Yiqi Huoxue Tablets group were lower than the model group, the difference being statistically significant ( P<0.05) . Conclusion The preventive and therapeutic mechanism of Yiqi Huoxue Tablets for cerebral apoplexy is probably related with the regulation of the balance between blood coagulation and fibrinolytic system of apoplexy-prone renovascular hypertensive rats.

Abnormality of anaplastic lymphoma kinase gene and its expression in pediatric neuroblastoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To correlate the abnormal expression of anapastic lymphoma kinase (ALK) protein with the genetic and epigenetic changes of ALK, and to analyze its clinical application in pediatric neuroblastoma.</p><p><b>METHODS</b>Three neuroblastoma (NB) cell lines (two ALK positive: SH-SY5Y and SK-N-SH, one ALK negative: SK-N-AS) and 43 paraffin-embedded NB tissues were included in the study. In both cell lines and clinical cases, immunohistochemistry was used to detect ALK protein expression; PCR and Sanger sequencing were used to detect ALK point mutation; fluorescence in situ hybridization (FISH) was used to detect ALK abnormality and bisulfite sequencing PCR (BSP) was used to detect methylation of CpG island in the promoter area of ALK.</p><p><b>RESULTS</b>The cell lines SH-SY5Y and SK-N-SH were positive for ALK expression (cytoplasm), while the SK-N-AS was negative; among the 43 cases of NB, 26 (60.5%, 26/43) were positive for ALK protein (membrane and cytoplasm), and the rest were negative. Survival analysis showed ALK protein expression was related to survival time, with ALK positive cases having shorter survival time than ALK negative cases (P = 0.020). But ALK protein expression had no association with tumor differentiation (P = 0.503), tumor sites (P = 1.000) and age of patients (P = 0.063). FISH showed ALK amplification in two cases (4.6%, 2/43), ALK gain was found in 30 cases (69.7%, 30/43), and the remaining cases had normal ALK copy (25.6%, 11/43). The presence of extra copies (amplification and gain) of ALK was associated with ALK positive protein expression (P = 0.020), but there was no association with tumor differentiation (P = 1.000), tumor sites (P = 0.775) and age of patients (P = 0.328). No point mutation was found in all three cell lines. Of the 43 NB cases, only one case (2.3%, 1/43) showed point mutation in exon 23, and was a synonymous mutation [A1200A (G4552C)]. The case was ALK negative, but the patient died two months after diagnosis. BSP analysis showed that CpG island in ALK promoter region were all unmethylated in three cell lines and 6 NB cases (including 3 ALK positive, 3 ALK negative).</p><p><b>CONCLUSIONS</b>ALK protein is expressed in most NB, and the expression indicates poor outcome. ALK expression is associated with extra copies of ALK, but there is no association with the methylation status of CpG island of ALK; the presence of extra copies of ALK is the most common genetic aberration in NB. Point mutation of ALK is rare, and may predict poor prognosis in pediatric NB.</p>

Genetic abnormalities and their relationship with the clinical features in pediatric Burkitt lymphoma / 白血病·淋巴瘤

Objective To analyze the molecular cytogenetic abnormalities and pathogenesis of pediatric Burkitt lymphoma (BL) by array comparative genomic hybridization (aCGH).Methods First,immunophenotype,molecular genetics and EB virus (EBV) infection status were detected using immunohistochemistry and fluorescence in situ hybridization in 21 pediatric BL patients.Second,in addition to detecting genome-wide genetic gain/deletion status,aCGH results with EBV infection status were also correlated.Results aCGH results showed genetic alterations in 19 cases (90.5 ％).Generally,frequency of chromosomal gain was higher than chromosomal deletion.The regions of frequently-occurring small DNA genomic fragment gains (≥40 ％ cases) were 3q21.1,5p13.2,19q13.32,12q23.1,14q32.33,6q27,20p13 and 20p11.21.Large DNA fragment gains and deletions could be detected in 42.9 ％ (9/21) cases in the 14q24.2 and 14q32.33 regions.There was no significant difference in genetic alterations between EBV (+) and EBV (-) BL cases (P≥0.05).Conclusion aCGH results show that BL cases have complex genetic alterations,which have no significant difference between EBV(+) and EBV(-) cases.Most BL cases show large DNA segment deletion or acquisition of 14q,indicating that 14q gene alteration plays an important role in the pathogenesis of BL.

A20 and TNF genes aberration in ocular adnexal MALT lymphomas / 白血病·淋巴瘤

Objective To investigate the copy number changes of A20 and TNF genes,and determine the contribution of the two genes in the development of ocular adnexal MALT lymphoma.Methods Forty-one cases of archive paraffin-embedded ocular adnexal MALT lymphoma tissues were detected by interphase fluorescence in situ hybridization (FISH) using the commercial chromosome 6 centromere probe (CEP6),and house-made site-specific probe of A20 and TNF. Results Of the 41 ocular adnexal MALT lymphoma cases,loss of heterozygosity (LOH)in A20 locus was detected in 2 cases (4.88 ％).TNF extra copies were found in 5 cases (12.20 ％),of which three cases simultaneously had extra CEP6 signals.No A20 deletion were found coexistence with TNF extra copies in any case.Conclusion A20 gene deletion is present in the small part of ocular adnexal MALT lymphoma, and might contribute to the development of Chinese ocular adnexal MALT lymphoma.A20 deletion is not associated with extra copies of TNF locus.

Clinicopathologic and molecular genetics features of mature aggressive B-cell lymphomas in pediatrics / 白血病·淋巴瘤

Objective To investigate the immunophenotype and molecular genetics of mature aggressive B-cell lymphomas in Chinese pediatric patients and provide the criteris for the diagnosis of them.Methods We collected 97 paraffin-embeded tissue samples of pediatric cases of mature aggressive B-cell lymphomas including 81 Burkitt lymphoma (BL) cases, 8 diffuse large B cell lymphoma (DLBCL) cases and 8unclassifiable B cell lymphoma with featares intermediate between BL and DLBCL (BL/DLBCL) cases. The immunophenotype and genetic features of them were detected by immunohistochemistry and interphase FISH.Results The expression of bcl-2 [3 %(2/66) in BL, 50 % (4/8) in DLBCL, 50 % (4/8) in BL/DLBCL], MUM1 [17 % (12/71) in BL, 63 % (5/8) in DLBCL, 63 % (5/8) in BL/DLBCL] and mean Ki-67 proliferation index [(93±4.4)% in BL, (83±14.3)% in DLBCL, (80±11.5)% in BL/DLBCL] were significantly different between BL and DLBCL and between BL and BL/DLBCL. The frequency of c-myc rearrangement [98 % (79/81) in BL,38 % (3/8) in DLBCL, 50 % (4/8) in BL/DLBCL] and an extra copy of bcl-6 [0 % in BL, 38 % (3/8) in DLBCL, 25 % (2/8) in BL/DLBCL] were also significantly different between BL and DLBCL and between BL and BL/DLBCL. Conclusion Diagnosis of the mature aggressive B cell lymphomas in pediatrics should be based on the comprehensive review and integration of morphologic, immunohistochemical and molecular genetic features. BL/DLBCL is more likely a subgroup of the DLBCL in pediatric population. The expression of CD10 and bcl-6 but not bcl-2, a high Ki-67 PI (＞90 %) and a c-myc rearrangement but not bcl-2 or bcl-6rearrangement are the features of BL. Regardless of the expression of CD10 and bcl-6, positive staining for bcl2, Ki-67 PI below 90 % and an extra copy of the bcl-6 favor a diagnosis of DLBCL or BL/DLBCL.

ALK and c-myc gene of anaplastic large cell lymphoma / 白血病·淋巴瘤

Objective To investigate the molecular genetic changes of anaplastic lymphoma kinase (ALK) gene and c-myc gene in anaplastic large cell lymphoma (ALCL). Methods The structural aberrations and changes of copy numbers in ALK and c-myc genes in 72 paraffin-embedded ALCL specimens were detected by interphase fluorescence in situ hybridization (FISH). Results Among 72 ALCL specimens, ALK protein was expressed in 42, ALK gene translocation was detected in 40 specimens in which extra copies of ALK gene were detected in 17. ALK gene translocation was not found in all 30 ALK negative specimens, but extra copies of ALK gene were detected in 14 cases. The difference of incidence rates of extra copies in ALK gene between ALK positive and ALK negative specimens was not significant (P＞0.05). c-myc gene translocation was not found in any of 72 ALCL specimens, but extra copies were detected in 24 cases.Conclusion Most (75.0%) ALCL have ALK gene aberration, in which ALK gene translocations are most common (55.6%), and the extra copies of ALK gene are relatively common genetic changes (43.1%). The ALK gene aberration is only detected in ALK positive ALCL and the gene translocations are in either ALK positive and negative ALCL. There is no or rare c-myc gene translocation in ALCL, but extra copies of c-myc gene are relatively common (33.3%).

Clinical and pathological features of the sporadic Burkitt's lymphoma / 白血病·淋巴瘤

Objective To investigate the clinical and pathological features of the sporadic Burkitt's lymphoma(BL),as well as its immunohistochemical and molecular characteristics.Methods 20 cases of sporadic BL were retrospectively studied by analyzing their light microscopy features,immunohistochemical expression,EBV infection detected by in stiu hybridization,chromosomal breakage of c-myc and/or lgH genes by interphase fluorescence in stiu hybridization (FISH),and their clinical manifestation.Results All the 20 cases of sporadic BL occurred in children(3-14 y)including 16 males and 4 females.Microscopically,the medium-sized tumor cells were monomorphic and proliferated in a diffuse pattern showingstarry-skywith numerous karyorrhectic debris.Mitotic figures were frequently seen.Immunohistochemically,the tumor cells were positive for CD20 and CD10,over 95%positive for Ki-67 and negative for TdT,CD99,MPO. No EBV infection wag found by in situ hybridization in 18 cases.Interphase FISH analysis detected c-myc gene breakage or amplification and/or IgH/c-myc gene breakage in all detected 15 cases.Conclusion Sporadic BL is a high-grade malignant B cell lymphoma.The tumor cell proliferation index is very high.Molecular and immunohistochemical analysis could reduce the incidence of misdiagnosis and thus phys a vital role on its correct diagnosis and appropriate therapy.

A clinical study of chromosome translocations in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue in Chinese patients / 中华内科杂志

Objective To investigate the genetic aberrations in extranodal marginal zone lymphoma of mueosa-associated lymphoid tissue (MALT) lymphomas from different sites of the body in Chinese patients. Methods Two hundred and seventeen paraffin-embedded MALT lymphoma specimens from 11 major sites were studied with interphase fluorescence in situ hybridization (FISH) to detect t(11; 18) (q21;q21)/API2-MALT1, t(1; 14) (p22; q32)/IGH-BCL10, (14; 18) (q32; q21)/IGH-MALT1 and BCL6 gene involved chromosome translocations. Results These translocations were mutually exclusive and detected in 21% (46/217) of the cases, including t(11;18) (q21;q21) API2-MALT1 13% (29/217), t (1;14)(p22 ;q32) IGH-BCLIO in 1% (3/217), t(14;18) (q32;q21) IGH-MALT1 1% (2/217), BCL6 involved translocation in 2% (4/217) and IGH-unknown translocation partner in 4% (8/217). t(11; 18) (q21;q21)API2-MALT1 was found with the highest frequency in MALT lymphoma from lungs (47% , 8/17) and small intestine (29%, 4/14), followed by salivary gland (17%, 1/6), stomach (14%, 12/84) and ocular adnexae (6% , 4/68). t(1 ;14) (p22;q32) was only detected in lungs (12%, 2/17) and stomach (1%, 1/84). t(14;18) (q32;q21) was mainly detected in lungs (6%, 1/17) and ocular adnexae (2%, 1/68). BCL6 gene involved translocation was detected in salivary gland (17% , 1/6) and stomach (4%, 3/84). Conclusions It is demonstrated that the four translocatidns occur with markedly variable frequencies in MALT lymphoma of different sites in Chinese patients. The distributions of these chromosome translocations in Chinese patients are slightly different from those reported in western patients.

Development of Chromatographic Fingerprint of Rhizoma Alismatis Orientalitis by HPLC-ELSD / 中药新药与临床药理

Objective A fingerprint of Rhizoma Alismatis Orientalitis' decoction pieces was established by HPLC-ELSD. Methods The gradient elution mode was applied in chromatographic separation by mobile phase of methanol and water at flow rate of 0.5 mL? min-1 in a Diamonsil TM C18 chromatographic column (250 mm ? 4.6 mm, 5 ? m), and recording time was 70 minute. The parameters of evaporative light scattering detector were set as follows: the temperature of the drift tube was 82.5 ℃ and gas flow rate was 1.9 L ? min-1.The data was analyzed with cluster analysis and similarity calculation software. Results The fingerprint showing 15 common peaks was obtained. The results of cluster analysis and similarity calculation showed there existed differences in fingerprints of 16 batchers of medicinal slices of Rhizoma Alismatis Orientalitis. Conclusion The method is effective and reproducible for the quality control of medicinal slices of Rhizoma Alismatis Orientalitis.

Studies on Quality control for Rhizoma Alismatis / 中药新药与临床药理

Objective To establish a method for the quality control of Rhizoma Alismatis. Methods The samples of Rhizoma Alismatis were identified with TLC and the content of alisol B-23-acetate in Rhizoma Alismatis was determined by HPLC-ELSD. Results The spots of TLC chromatogram of Rhizoma Alismatis was clear. A good linearity was in the range of 4.4～110.0 ?g/mL of alisol B-23-acetate, r=0.999 2(n=5), and the average recovery rate was 98.98 %(n=9), RSD=1.3 %. Conclusion This method was simple, accurate and with a good reproducibility and can be used for the quality control of Rhizoma Alismatis.