Frequent CDKN2B/P15 and DAPK1 methylation in duodenal follicular lymphoma is related to duodenal reactive lymphoid hyperplasia.

Duodenal type follicular lymphoma (DFL), a rare entity of follicular lymphoma (FL), is clinically indolent and is characterized by a low histological grade compared with nodal follicular lymphoma (NFL). Our previous reports revealed that DFL shares characteristics of both NFL and mucosa-associated lymphoid tissue (MALT) lymphoma in terms of clinical and biological aspects, suggesting its pathogenesis may involve antigenic stimulation. In contrast to NFL, the genomic methylation status of DFL is still challenging. Here, we determined the methylation profiles of DNAs from patients with DFL (n = 12), NFL (n = 10), duodenal reactive lymphoid hyperplasia (D-RLH) (n = 7), nodal reactive lymphoid hyperplasia (N-RLH) (n = 5), and duodenal samples from normal subjects (NDU) (n = 5) using methylation specific PCR of targets previously identified in MALT lymphoma (CDKN2B/P15, CDKN2A/P16, CDKN2C/P18, MGMT, hMLH-1, TP73, DAPK, HCAD). DAPK1 was frequently methylated in DFL (9/12; 75%), NFL (9/10; 90%), and D-RLH (5/7; 71%). CDKN2B/P15 sequences were methylated in six DFL samples and in only one NFL sample. Immunohistochemical analysis showed that p15 expression inversely correlated with methylation status. Genes encoding other cyclin-dependent kinase inhibitors (CDKN2A/P16, CDKN2C/P18) were not methylated in DFL samples. Methylation of the genes of interest was not detected in DNAs from D-RLH, except for DAPK1, and the difference in the extent of methylation between NDU and D-RLH was statistically significant (P = 0.013). Our results suggest that D-RLH serves as a reservoir for the development of DFL and that methylation of CDKN2B/P15 plays an important role in this process.

Diagnosis of Lyme Borreliosis With a Novel, Seminested Real-Time Polymerase Chain Reaction Targeting the 5S-23S Intergenic Spacer Region: Clinical Features, Histopathology, and Immunophenotype in 44 Patients.

ABSTRACT: Lyme borreliosis (LB) is the most common tick-borne infection in Europe and North America. Polymerase chain reaction (PCR) is an important tool to confirm the diagnosis, but not always successful, especially when organisms are sparse. We developed a novel, seminested real-time PCR assay [target: 5S-23S intergenic spacer region (IGS)] and compared it with 3 well-established conventional PCR assays (IGS/OspA/real-time IGS) on 596 formalin-fixed, paraffin-embedded routine skin biopsies. The seminested real-time assay identified 46 cases of borreliosis while 25, 27, and 38 were identified by the 3 other assays, respectively (P 0.01, P 0.02, and P 0.42; significance P < 0.05). Clinicopathologic and immunophenotypic analysis of PCR-positive cases revealed 38 erythema migrans (EM), 6 Borrelia lymphocytomas, and 2 acrodermatitis chronica atrophicans (ACA). In the 44 PCR-confirmed cases, plasma cells were present in only a third of EM cases. By contrast, CD123-positive plasmacytoid dendritic cells were common (74%) and therefore are unlikely to be helpful in the differential diagnosis between EM and tumid lupus erythematosus. A loss of CD34 in a third of all LB specimens limits its diagnostic value in the differential diagnosis with morphea. Interstitial macrophages were common in cutaneous LB (42/43) forming interstitial granulomas in a third of all cases, and 3/38 EM, 3/6 Borrelia lymphocytomas, and 1/2 ACA were only identified by the new seminested real-time assay, suggesting that it is especially helpful in confirming the diagnosis of Borrelia lymphocytoma.

Analysis of clonality in cutaneous B-cell lymphoma and B-cell pseudolymphoma using skin flow cytometry: Comparison of immunophenotyping and gene rearrangement studies.

To identify clonal neoplastic cells in skin affected by B-cell lymphoma using skin flow cytometry (FCM) techniques, we investigated light-chain restriction using skin FCM with clonality assessed by polymerase chain reaction and light-chain restriction by in situ hybridization (ISH). We retrospectively analyzed 16 cases of B-cell lymphoma with cutaneous involvement: primary cutaneous diffuse large B-cell lymphoma, leg type (pcDLBCL-LT) (n = 7), DLBCL-not otherwise specified (DLBCL-NOS) (n = 6), primary cutaneous follicle center lymphoma (pcFCL) (n = 1), and follicular lymphoma (n = 2), as well as cutaneous B-cell pseudolymphoma (n = 9). Results of skin FCM light-chain restriction analyses were compared with immunoglobulin H (IgH) gene rearrangement and κ/λ ISH findings. Skin FCM detected light-chain restriction in 11 of 14 B-cell lymphoma patients but none of the B-cell pseudolymphoma patients. The sensitivity of skin FCM for distinguishing B-cell lymphoma and B-cell pseudolymphoma was 79%, and the specificity was 100%. Eleven of 13 B-cell lymphoma patients exhibited gene rearrangement (sensitivity 85%), whereas six of seven pseudolymphoma patients were negative (specificity 86%). ISH was positive in three of 16 B-cell lymphoma cases (sensitivity 19%) but none of the B-cell pseudolymphoma cases (specificity 100%). ISH sensitivity was 29% for pcDLBCL-LT, 17% for DLBCL-NOS, and 0% for pcFCL and follicular lymphoma. Skin FCM therefore appears to be more sensitive than ISH in detecting light-chain restriction in DLBCL and follicular lymphoma, and as sensitive as IgH gene rearrangement analysis in detecting clonality. Skin FCM is thus a promising diagnostic tool for identifying monoclonal neoplastic B-cell populations.

Transcription factor 3 (TCF3) combined with histone deacetylase 3 (HDAC3) down-regulates microRNA-101 to promote Burkitt lymphoma cell proliferation and inhibit apoptosis.

To explore the function of transcription factor 3 (TCF3) on the proliferation and apoptosis of Burkitt lymphoma cells and its mechanism. qRT-PCR was performed to determine the expression of TCF3, histone deacetylase 3 (HDAC3), and microRNA-101 (miR-101) in the Burkitt lymphoma (BL) tumor tissues and lymph node tissues with reactive lymph node hyperplasia (RLNH). We found that the expression of TCF3 and HDAC3 was up-regulated in BL tumor tissues and lymphoma cells, and the miR-101 expression was down-regulated. And TCF3 and HDAC3 were negatively correlated with the expression of miR-101, respectively. In addition, knockdown of TCF3 can inhibit BL cell proliferation, reduce cell viability and promote cell apoptosis, retain the cell cycle in the G0/G1 phase, and inhibit the expression of Akt/mTOR pathway-related proteins (p-Akt and p-mTOR). When miR-101 was overexpressed, the results were the same as when TCF3 was knocked down. Moreover, we used Co-immunoprecipitation (Co-IP) to detect the interaction between TCF3 and HDAC3, and performed the Chromatin immunoprecipitation (ChIP) experiment to detect the enrichment of TCF3 and HDAC3 in the promoter region of miR-101. We found that TCF3 can interact with HDAC3 and is enriched in the miR-101 promoter region. In conclusion, TCF3 combined with HDAC3 down-regulates the expression of miR-101, thereby promoting the proliferation of BL cells and inhibiting their apoptosis.

Pseudolymphomatous Atypical Fibroxanthoma.

Atypical fibroxanthoma is a rare mesenchymal skin tumor of intermediate malignancy that typically occurs on sun-damaged skin of elderly patients. Histologically, it is composed of pleomorphic cells with hyperchromatic nuclei and abundant cytoplasm, commonly arranged in a spindle cell pattern. Different histologic variants have been described during the past years. We present a case of atypical fibroxanthoma containing a dense inflammatory infiltrate, which in conjunction with the existence of immunoblast-like and Reed-Sternberg-like neoplastic cells could be misinterpreted as a lymphoid neoplasm. Immunohistochemical studies revealed strong positivity of tumor cells for CD10 and negativity for cytokeratins, p63, p40, S100, SOX10, ERG, actin, desmin, B and T-cell markers, BCL6, CD15, and CD30. The inflammatory infiltrate contained a mixed reactive T- and B-cell population with negative T-cell receptor and immunoglobulin heavy rearrangements. We discuss the differential diagnosis of this entity in which clinical, immunohistochemical, and molecular features are essential to avoid the diagnosis of a lymphoproliferative disease.

Expression and clinical significance of ubiquitin­specific­processing protease 34 in diffuse large B­cell lymphoma.

Ubiquitin­specific­processing protease 34 (USP34) is a deubiquitinase that is involved in the pathogenesis of various cancers. Its roles in diffuse large B­cell lymphoma (DLBCL) are unknown. The present study aimed to determine the level of USP34 expression and to explore its association with clinicopathological features and prognosis in patients with DLBCL; a total of 30 cases of reactive lymphoid hyperplasia and 131 cases of DLBCL were included in this study. The level of USP34 expression was examined by immunohistochemistry (IHC), and correlations between USP34 expression and clinicopathological features and prognosis were analyzed. In addition, mutations, expression and clinical significance of USP34 in DLBCL were evaluated using data from The Cancer Genome Atlas (TCGA). USP34 expression was significantly higher in DLBCL compared with expression in reactive lymphoid hyperplasia. In DLBCL, overexpression of USP34 was associated with older age, germinal center B cell­like (GCB) subtype, multiple extranodal involvements and higher International Prognostic Index (IPI) scores. No significant association was identified between USP34 protein level and patient survival. In the TCGA dataset, low USP34 mRNA expression was demonstrated to be associated with a poor disease­free survival (DFS), but not with overall survival (OS) in patients with DLBCL. In conclusion, high expression of USP34 protein in DLBCL was associated with older age, GCB subtype, multiple extranodal involvement and high IPI scores of DLBCL. USP34 may be a valuable marker for the assessment of patients with DLBCL, and further studies are needed to clarify USP34 expression on DLBCLs.

[Clinicopathologic features of primary hepatic marginal zone lymphoma of mucosa-associated lymphoid tissue and hepatic pseudolymphoma].

Objective: To study the clinicopathological features of primary hepatic extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT lymphoma) and hepatic pseudolymphoma, and to discuss their differential diagnosis, treatment and prognosis. Methods: Three primary hepatic MALT lymphomas and two hepatic pseudolymphomas collected from January 2012 to March 2017 in the First Affiliated Hospital of Nanjing Medical University were evaluated by HE and immunohistochemistry(IHC), in-situ hybridization and immunoglobulin (Ig) gene rearrangement detection, and the relevant literature reviewed. Results: In the three MALT lymphomas, tumor cells infiltrated the portal areas with nodular pattern, and invaded the surrounding normal liver with serpiginous configuration and formation of confluent sheets. A number of bile ducts were entrapped within the lesions, and showed lymphoepithelial lesion. Reactive lymphoid follicles were present and surrounded by tumor cells, consisting of predominantly centrocyte-like cells and monocytoid B cells. There were clusters of epithelioid histiocytes in one case. The tumor cells were positive for CD20, PAX5 and negative for CD5, CD23, CD10, bcl-6, and cyclin D1. In the two hepatic pseudolymphomas, the lesions presented as solitary nodules well-demarcated from the surrounding liver tissue; one case was partially encapsulated with fibrous tissue. Entrapped bile ducts were only found at the edge of the lesions without lymphoepithelial lesion. The lesions comprised of massive lymphoid proliferation consisting predominantly of reactive lymphoid follicles, but not monocytoid B-cells or atypical cells. By IHC, a mixture of B- and T-cell population was identified. A monoclonal rearrangement of the Ig gene was detected in all three MALT lymphomas but not in two pseudolymphomas. Interphase fluorescence in situ hybridiazation test for MALT1 break-apart gene was positive in two cases of MALT lymphomas and EBER was negative in all studied cases. Conclusions: Primary heptic MALT lymphoma and pseudolymphoma are both rare lymphoid proliferative lesions of liver. These two lesions have overlapping histological and IHC features and are top differential diagnosis to each other. A combination analysis of morphology, immunophenotype and Ig gene rearrangement is helpful to distinguish between them.

Long non-coding RNA H19 promotes proliferation of Hodgkin's lymphoma via AKT pathway.

PURPOSE: To explore whether lncRNA (Long non-coding RNA) H19 could promote the development of Hodgkin's lymphoma (HL) by regulating cell proliferation via AKT pathway. METHODS: H19 expressions in 60 HL tissues, 40 RH (reactive hyperplasia of lymph nodes) tissues, L428, A20 and Ly1 cell lines were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). H19 siRNA and pcDNA-H19 were constructed. Cell viability after altering H19 expression was detected by EdU and cell counting kit-8 (CCK-8) assay. The mRNA level of AKT in HL tissues and RH tissues was detected by qRT-PCR. The relationship between AKT and H19 was further detected by Western blot. RESULTS: H19 was overexpressed in HL tissues and cell lines compared with those of controls. HL patients with huge lump and in Ann Arbor stage III-IV presented higher expression of H19. Besides, H19 expression was negatively correlated to overall survival (OS) of HL patients. In vitro experiments suggested that H19 downregulation decreased proliferation and viability of HL cells. AKT expression was upregulated in HL tissues compared with RH tissues, and was positively regulated by H19. Western blot results also indicated that H19 overexpression upregulated protein expression of AKT in HL cells. CONCLUSIONS: Overexpressed lncRNA H19 promotes HL development by stimulating proliferation of HL cells via AKT pathway.

Clinical significance of PCDH10 promoter methylation in diffuse large B-cell lymphoma.

BACKGROUND: PCDH10, one of the non-clustered protocadherins, is identified as a tumor suppressor gene in many tumors. Recently, promoter methylation of PCDH10 was found in diffuse large B-cell lymphoma (DLBCL) but not in normal lymph nodes, suggesting that its epigenetic aberrance is essential to the lymphomagenesis. However, there are few studies on the clinicopathological relevance and prognostic significance of PCDH10 methylation status in DLBCL. METHODS: One hundred-seven cases of DLBCL between Jan 2009 and Jul 2010 were selected to extract genomic DNA and perform bisulfite modification. Their methylation status of PCDH10 promoter were accessed by methylation-specific PCR (MSP) with methylated and unmethylated primers. Analysis of overall survival and clinicopathological correlation were conducted. RESULTS: PCDH10 hypermethylation were found in 54.2% (58/107) of DLBCL cases, but only 12.5% (1/8) in reactive lymph node/follicular hyperplasia. In RCHOP-treated cohort, promoter methylation of PCDH10 is an independent prognostic indicator of worse overall survival (p = 0.017; HR 4.045; 95%CI 1.287-12.711) and worse progress-free survival (p = 0.014; HR 2.977; 95%CI 1.245-7.119). Whereas, PCDH10 hypermethylation wasn't correlated with MYC translocation and cell of origin classification using Hans model. CONCLUSIONS: PCDH10 methylation status could serve as a valuable biomarker for risk classification, and a potential therapeutic target for demethylating drugs in DLBCL in the future.

Flow cytometric sorting coupled with exon capture sequencing identifies somatic mutations in archival lymphoma tissues.

The enormous number of archived formalin-fixed paraffin-embedded (FFPE) tissues available are a valuable resource of material for research. However, the use of such tissues poses many challenges, among which is the difficulty of isolating different cell populations within the tissue. In this study, we used tissue from two types of non-Hodgkin lymphoma as a model to demonstrate a method we have established and optimized to separate FFPE samples into distinct tumor and nonmalignant populations. Using FFPE reactive tonsil sections, various approaches for antigen retrieval and labeling, and the effectiveness of flow cytometric sorting were tested. We found that, among the 11 cell surface or intracellular antigen markers investigated, CD3ɛ, CD79A, LAT, PD-1, and PAX5 could be successfully labeled after antigen retrieval in Tris-EDTA buffer (pH 8.0) at 65 °C for 60 min, and 1.8-2.7 µg DNA per million cells could be extracted after sorting with DNA quality similar to that of tissue without staining or sorting. To test whether we could perform next-generation sequencing using a custom capture platform on sorted cells, we used three lymphoma cases with FFPE tissues which had been stored for 1 to 4 years. We demonstrated that the DNA from sorted cells was adequate for exon capture sequencing. By comparing the sequencing results between neoplastic and normal populations, somatic mutations could be clearly identified in the tumor population with variant frequencies as low as 11.7%.The corresponding normal fraction clearly helps in the analysis of somatic mutations and the exclusion of artifacts. This study provides an approach using flow cytometric sorting to separate different cellular populations in paraffin-embedded tissues and to unambiguously distinguish somatic mutations from germline variants or artifacts. This approach is also useful in enriching the tumor component in samples with heterogeneous components and low tumor content.

Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma.

BACKGROUND: Klotho, is a transmembrane protein, performs as a circulating hormone and upstream modulator of the insulin-like growth factor-1 receptor (IGF-1R), fibroblast growth factor (FGF), and Wnt signaling pathways. These pathways are involved in the development and progression of B cell lymphoma. We aimed to explore the expression pattern and functional mechanism of Klotho in diffuse large B cell lymphoma (DLBCL). METHODS: Immunohistochemistry (IHC) and western blotting were performed to detect the expression level of Klotho in DLBCL patients and cell lines. Tumor suppressive effect of Klotho was determined by both in vitro and in vivo studies. Signaling pathway activity was assessed by western blotting. RESULTS: Remarkable lower expression levels of Klotho were observed in DLBCL patients and cell lines. Enforced expression of Klotho could significantly induce cell apoptosis and inhibit tumor growth in DLBCL. Upregulation of Klotho resulted in declined activation of IGF-1R signaling, accompanied with decreased phosphorylation of its downstream targets, including AKT and ERK1/2. Moreover, xenograft model treated with either Klotho overexpression vector or recombinant human Klotho administration presented restrained tumor growth and lower Ki67 staining. CONCLUSIONS: Our findings establish that Klotho performs as a tumor suppressor and modulator of IGF-1R signaling in DLBCL. Targeting Klotho may provide novel strategies for future therapeutic intervention.

MicroRNA profiling in peripheral T-cell lymphoma, not otherwise specified.

BACKGROUND: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is an obviously heterogeneous and highly invasive malignancy without definite phenotype. MicroRNAs (miRNAs) are powerful gene regulators and have been reported as biomarkers in many malignancies. OBJECTIVE: To discover potential signaling miRNAs in PTCL-NOS distinguished from reactive lymphoid hyperplasia (RLH) and to explore the molecular characteristics based on the discovery. METHODS: We measured the expression profile of miRNAs in PTCL-NOS and RLH from our patients using a panel PCR array. We used GO-Analysis to evaluate the function of the targets regulated by the detected miRNAs. Then using pathway enrichment analysis, we defined potential pathways connected with the selected miRNAs. RESULTS: We found 20 miRNAs which were remarkably up/down-regulated in PTCL-NOS. GO-Analysis and pathway analysis indicated 61 GOs and 34 signaling pathways that were significantly increased or decreased regarding tumorigenesis, tumor progression, invasion, metastasis, and drug resistance. CONCLUSIONS: Briefly, our results suggest that 20 miRNAs have the potential to be used as biomarker for identification of patients with PTCL-NOS.

Molecular clonality assessment shows high performance to predict malignant B-cell non-Hodgkin's lymphoma using cytological smears.

AIMS: Molecular PCR-based clonality analysis is helpful for identification of monoclonal B-cell or T-cell populations and to distinguish malignant lymphoma from reactive lymphoid hyperplasia. Typically, clonality assessment on fine-needle aspiration cytology (FNAC) requires freshly obtained aspirates, but the collection and processing of these samples are often challenging in daily practice. In this study, we assessed the routine diagnostic value of the EuroClonality/BIOMED-2 assay for B-cell clonality on air-dried archived Giemsa-stained smears. METHODS: This study comprised a retrospective analysis of a consecutive diagnostic cohort of 192 FNAC samples from 184 patients with at least 2-year follow-up. The results from the clonality assay were integrated with cytomorphological assessment and evaluated for their accuracy in detecting malignant disease. EuroClonality expert re-evaluation was performed for all cases with ambiguous results and for cases in which the diagnosis did not match the follow-up data. RESULTS: The clonality assay showed a high accuracy of 93% for detection of malignancy, with a sensitivity of 93% and a specificity of 92%. All 64 cases with monoclonal Ig heavy chain (IGH)/Ig kappa chain (IGK) rearrangements were confirmed malignant by histology or clinical follow-up. Expert re-evaluation changed the definite diagnosis for five cases (3%), mainly because of low signals or no proper duplicate results. We discuss and elucidate all cases for which the clonality results did not match the disease follow-up. CONCLUSIONS: This study showed that EuroClonality/BIOMED-2 assay can successfully be performed on cytological Giemsa-stained smears and inclusion in daily practice can assist in better identification of malignant lymphoma.

Reactive lymphoid hyperplasia of the liver mimicking hepatocellular carcinoma: incidental finding of two cases.

Reactive lymphoid hyperplasia is a rare disease that forms a mass-like lesion and is characterized by the proliferation of non-neoplastic, polyclonal lymphocytes forming follicles. We recently encountered 2 cases of reactive lymphoid hyperplasia of liver, both of which were asymptomatic and mimicked hepatocellular carcinoma by various imaging modalities. Based on the clinical impression of hepatocellular carcinoma, surgical resections were performed. Microscopic findings revealed that both lesions consisted of an aggregation of lymphocytes consisting of predominantly B-cells, with multiple lymphoid follicles positive for CD10 and negative for bcl-2, consistent with the diagnosis of reactive lymphoid hyperplasia. Polyclonality of both lesions was further confirmed by B cell receptor gene rearrangement study. The incidence of reactive lymphoid hyperplasia in the liver is exceedingly rare, and it is difficult to differentiate such lesions from hepatic malignancies based upon clinical grounds. The clinicopathological findings and literature review of this report may be helpful to improve the clinical decision-making.

Genome-Wide Analysis of Ocular Adnexal Lymphoproliferative Disorders Using High-Resolution Single Nucleotide Polymorphism Array.

PURPOSE: We identified the genomic signature of ocular adnexal lymphoproliferative disorders (LPDs), especially ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma, IgG4-related ophthalmic disease (IgG4-ROD), reactive lymphoid hyperplasia (RLH), and diffuse large B-cell lymphoma (DLBCL). METHODS: We included 52 subjects with ocular adnexal LPDs (13 orbital MALT lymphomas, 16 conjunctival MALT lymphomas, 13 IgG4-RODs, 4 RLHs, and 6 DLBCLs) who had been treated at the Tokyo Medical University Hospital from 2008 to 2012. Genomic DNA was extracted from the tumor tissues and subjected to high-resolution single nucleotide polymorphism array (SNP-A) karyotyping using GeneChip Human Mapping 250K SNP arrays. The array data were investigated using Copy Number Analysis for GeneChips (CNAG) software. RESULTS: In ocular adnexal MALT lymphomas, the most frequent copy number (CN) gain region was trisomy 3 detected in 31% (9/29), followed by trisomy 18 in 17% (5/29), and 6p and 21q in 14% (4/29). The most frequent CN loss regions were 6q and 9p, detected in 7% (2/29). Uniparental disomy was detected on 6q in 14% (4/29), followed by 3q in 10% (3/29). Copy number variations (CNVs) were not detected in IgG4-RODs and RLHs. Conversely, CNVs were more frequent in DLBCLs than in ocular adnexal MALT lymphomas. Copy number variations were detected in 77% (10/13) of orbital MALT lymphomas and in 67% (11/16) of conjunctival MALT lymphomas. CONCLUSIONS: High-resolution single nucleotide polymorphism array is a useful method for discriminating ocular adnexal lymphomas from benign LPDs. The differences in the chromosomal abnormality patterns may reflect the activity of ocular adnexal LPDs.

[Detection and significance of BRAF gene in mature T/NK cell lymphoma].

OBJECTIVE: we aimed to investigate the mutation and expression of BRAF gene in mature T/NK cell lymphoma tissues and cell lines, explore the correlation between gene alterations and clinicopathological features and clinical outcomes of mature T/NK cell lymphoma. METHODS: Firstly, we detected common mutant sites of BRAF (locus 1 799 mutation in exon 15 and loci 463, 465 and 468 mutation in exon 11) in lymphoma Jurkat, Hut-78 and YTS cell lines, normal peripheral blood lymphocytes, different types of mature T/NK cell lymphoma and reactive hyperplasia lymph nodes by direct sequencing. Then we measured the expression of BRAF in Jurkat, Hut-78, YTS cells and normal peripheral blood lymphocytes by real time-PCR and Western-blot detection. We also used immunohistochemistry (IHC) to detect the expression of BRAF in mature T/NK cell lymphoma tissues and reactive hyperplasia lymph nodes, and to analyze the correlation between the expression of BRAF and clinocopathological features and clinical outcomes. RESULTS: We did not find common BRAF mutation in mature T/NK cell lymphoma tissues and cell lines, and the relatively expression of BRAF gene mRNA in normal peripheral blood lymphocytes, YTS, Hut-78 and Jurkat cells were 1.000, 5.207±0.013, 8.412±0.615 and 36.720±1.797, respectively, and protein expressions were 0.051±0.003, 0.102±0.013, 0.113±0.017 and 0.304±0.010, respectively, and the expression of BRAF in peripheral T cell lymphoma Jurkat cells was significantly higher than that of Hut-78, YTS cells and normal lymphocytes (P<0.05). Only 6 of 58 peripheral T cell lymphomas (10.3%) had positive BRAF expression, and were the subgroups of peripheral T cell lymphoma-unspecified type. The statistical data did not show any correlation between positive expression of BRAF and gender, age, clinical stage, location, lactate dehydrogenase in the 21 cases of peripheral T cell lymphoma-unspecified type (P<0.05), but the positive rate of BRAF in the effective treatment group (8.3%) was significantly lower than that of the invalid group (55.6%, P<0.05). CONCLUSION: The expression of BRAF gene may become a marker of malignant biological characteristics and clinical therapeutic target of peripheral T cell lymphoma.

Aberrant phenotypes in Kikuchi's disease.

Initial reports emphasized the immunophenotypic similarities between benign and malignant T cell populations, while some previous studies indicating that aberrant T-cell antigen loss is a good marker for detecting malignant T-cell proliferation. Recently, we found a very interesting and thought-provoking phenomenon: In benign disease-28 of 38 (73.7%) cases of Kikuchi's disease also showed aberrant phenotypes with loss of pan-T cell antigens, which makes the differential diagnosis between Kikuchi's disease and T cell lymphoma more challenging. In our study, 38 cases of Kikuchi's disease and 30 cases of reactive lymphoid hyperplasia (RLH) were studied by EliVision immunohistochemical staining. As well as TCR gene rearrangement using PCR was negative in 10 tested cases of the Kikuchi's disease. Among these cases, the most common antigen deficiency was CD5 (22 cases), then CD7 (11 cases), CD2 (8 cases) and CD3 (2 cases). Compared with proliferative and xanthomatous types of Kikuchi's disease, antigens tended to be lost in necrotizing type. Based on follow-up data, a correlation was not found between the occurrence of aberrant phenotypes and prognosis. In RLH, obvious pan-T cell antigen loss was also not found. In conclusion, this is the first study to demonstrate distinct patterns of antigen loss in Kikuchi's disease, suggesting that T cell antigen loss is not reliable as an auxiliary diagnostic standard for T cell lymphoma.

Reactive lymphoid hyperplasia of the terminal ileum: a benign (lymphoma-like) condition that may harbor aberrant immunohistochemical patterns or clonal immunoglobulin heavy chain gene rearrangements.

Small endoscopic biopsies of the terminal ileum may be difficult to assess for early involvement by lymphoma. Immunophenotypic and genotypic analyses are often utilized, but the performance of these studies in this setting is not well defined. Terminal ileal biopsies from 66 patients with prominent lymphoid hyperplasia and abnormal "lymphoma-like" morphology were evaluated by immunohistochemistry (IHC) for CD3, CD5, CD43, CD20, CD21, and CD10 expression and for IGH@ gene rearrangement by polymerase chain reaction using BIOMED-2 primers. Patients ranged in age from 3 to 80 years. Indications for endoscopy included inflammatory bowel disease (29), diarrhea and/or abdominal pain (28), history of lymphoma (13), and others (4). Four biopsies with abnormal morphology had abnormal IHC and a clonal IGH@ peak; all were obtained from patients with a history of lymphoma and determined to be recurrent lymphoma. Three biopsies with abnormal morphology and abnormal IHC but no clonal IGH@ peak were obtained from patients with a history of lymphoma (2) and chronic diarrhea (1); all showed symptom resolution or remission of disease (mean follow-up, 37 mo). Eight biopsies with abnormal morphology but no abnormal IHC expression also had abnormal IGH@ results (4 clonal and 4 borderline). IGH@ evaluation of follow-up biopsies for these cases were nonclonal (7) or clonal, but with a different clone from the prior biopsy (1); follow-up of the 8 patients showed no evidence of lymphoma (mean, 37.8 mo). Abnormal IHC expression pattern or clonal IGH@ rearrangement in endoscopic biopsies of the lymphoid-rich terminal ileum do not necessarily warrant a diagnosis of lymphoma. To prevent misdiagnosis, B-cell clonality studies should only be performed when there is strong clinical suspicion for lymphoma and compelling IHC data; the absence of a reproducible clone in repeat biopsy specimens may be useful in patients that do not have other clinical evidence of lymphoma.

PTEN in smooth muscle cells is essential for colonic immune homeostasis.

Colonic immune homeostasis is essential for normal gastrointestinal tract functioning. In this study, we report that specific gene targeting of phosphatase and tensin homolog (PTEN) in smooth muscle cells caused age-related colonic lymphoid hyperplasia followed by global immune activation in mice. Beginning at 5 weeks of age, these mutant mice displayed massive neutrophil infiltration in the colonic lamina propria. The gene expression levels of pro-inflammatory cytokines and chemokines, including those code for monocyte chemotactic protein-1 (Mcp-1), stromal cell-derived factor 1α (Sdf-1α), and chemokine (C-C motif) ligand 28 (Ccl-28), were upregulated in the colon. Accordingly, permeability and proliferation of the colonic epithelium was compromised. These abnormalities were alleviated to a great extent when the mutants were crossed with Akt1-null mice, indicating that the pathogenesis was mediated by Akt1 signaling. Our results suggest that in smooth muscle cells, PTEN is crucial for maintaining colonic immune homeostasis.