Regulation of Jak2 Kinase Gene by the Mir-135 Family in Classical Hodgkin Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 518-518
Author(s):  
Alfons Navarro ◽  
Tania Diaz ◽  
Antonio Martinez ◽  
Anna Gaya ◽  
Aina Pons ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Cell Line ◽  
Hodgkin Lymphoma ◽  
Disease Free Survival ◽  
Mirna Precursor ◽  
Negative Control ◽  
Classical Hodgkin Lymphoma ◽  
Protein Levels ◽  
Post Transfection ◽  
Specific Subset

Abstract Alterations of JAK/STAT signaling molecules have been reported in several lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis, with Jak kinase overexpression promoting in vitro cell transformation. JAk2 is a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. Activating mutation has been found in myeloid neoplasms but the expression of JAK2 in primary mediastinal large B-cell lymphomas and classical Hodgkin lymphomas is not due to mutations. On the other hand, miRNAs play an important role in cHL, where they regulate the expression of genes that are crucial for the transformation process and to rescue Hodgkin/Reed Sternberg cells from apoptosis. We have previously shown that miR-135a, a putative miRNA involved in JAK2 expression, is present in the miRNA signature of cHL. The aims of the study were to analyze the role of miR-135a in Hodgkin cells and to determine if Jak2 is a target gene of this miRNA. Moreover, we examined whether the expression of miR-135a in diagnostic samples predicted clinical outcome in cHL. To that aim, 50nM and 100nM of pre-miR-135 and pre-miRNA precursor Negative Control (Ambion) were transfected in the L-428 cHL cell line using nucleofection (AMAXA, Solution L and program X-001). MiR-135a levels were analyzed by stem-loop-RT-PCR and Real-time (TaqMan MicroRNA Assays, Applied Biosystems) in a 7500 Sequence Detection system. Apoptosis levels were assayed by Caspase-Glo 3/7 luminescent assay that measures caspase-3 and -7 activities. Jak2 protein levels were analyzed by Western Blot (Abcam moAb). Eighty-nine adult patients (median age, 29 yrs [range, 13–89]; males 47%) diagnosed with cHL at a single institution between September 1995 and June 2005 were studied. Seven patients (7.8%) were HIV+. Histological subtypes: nodular sclerosis (79%) and mixed cellularity (21%). Epstein-Barr Virus (EBV) was present in 28% of the samples. First-line treatment consisted of ABVD type therapies. Total RNA was extracted from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation (Ambion). Statistical analysis was performed with SPSS 14.0. Clinical outcomes analyzed were relapse rate and disease-free survival (DFS). Of 89 patients, 75 (84.3%) achieved CR, 7 (7.8%) PR, while 7 (7.8%) were chemoresistant. After a median follow-up of 43 months (range, 1–128), overall survival was 83% and DFS 74%. Results showed that miR-135a was underexpressed in the cHL L-428 cell line and in cHL samples compared to reactive lymph nodes. After pre-miR-135 transfection, the miR-135a levels in L-428 cells increased 12 Ct at 24h. Analysis at 24 and 48 hours post-transfection showed a proportion of apoptotic cells 20% and 30% higher than in control cells (pre-miRNA precursor Negative control). The analysis of Jak2 protein levels at 48 and 72 hours post-transfection showed a dose-dependent reduction of the protein expression of 28%(100nM-48h) and 58%(100nM-72h). The proliferation assay showed that pre-miR-135-transfected cells grew less than control cells transfected with pre-miRNA precursor negative control. All these results are along the same line as those found in primary tumor. Patients could be divided into two groups according to miR-135a expression: high and low expression. Patients with low miR-135a expression had a higher probability of relapse than those with high miR-135a expression (p=0.045). In the multivariate analysis, only miR-135a expression emerged as a prognostic factor for relapse (RR=6.533, p=0.021). In conclusion, miR-135a downregulation seems to play a role in the rescue of Hodgkin/Reed Sternberg cells from apoptosis and influence the transformation event through the regulation of Jak2 levels. In addition, expression levels of miR-135a may be a prognostic marker for risk of relapse in cHL patients.

Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin lymphoma

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 2945-2951 ◽  
Author(s):  
Alfons Navarro ◽  
Tania Diaz ◽  
Antonio Martinez ◽  
Anna Gaya ◽  
Aina Pons ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Critical Role ◽  
Disease Free Survival ◽  
Prognostic Impact ◽  
Free Survival ◽  
Protein Levels ◽  
Specific Subset ◽  
Classic Hodgkin Lymphoma

The behavior of classic Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the tumor cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. We examined the influence of our previously reported 25-microRNA signature for cHL on clinical outcome in 89 homogeneously treated cHL patients with a median follow-up of 80 months. Patients with low miR-135a expression had a higher probability of relapse (P = .04) and a shorter disease-free survival (P = .02). Functional analysis of cHL cell lines showed that mature miR-135a levels increased after pre–miR-135a transfection, causing apoptosis and decreased cell growth. Target analysis showed a direct regulation by miR-135a of JAK2, a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. miR-135a–mediated JAK2 down-regulation led to decreased mRNA and protein levels of the antiapoptotic gene Bcl-xL, suggesting a role for Bcl-xL in miR-135a/JAK2–mediated apoptosis. Our findings confirm the critical role of miR-135a in the survival of cHL cells and in the prognosis of cHL patients, indicating that novel treatment approaches targeting miR-135a may potentially benefit these patients.

Overexpression of GATA1 Confers Chemotherapy Resistance in Pediatric Acute Megakaryocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2039-2039
Author(s):  
Holly Edwards ◽  
Chengzhi Xie ◽  
Alan Dombkowski ◽  
Maggie Keller ◽  
Mark Stout ◽  
...  
Keyword(s):  
Cell Line ◽  
Histone Deacetylase Inhibitors ◽  
Target Genes ◽  
Negative Control ◽  
Pi3 Kinase ◽  
Luciferase Reporter ◽  
Toxic Dose ◽  
Protein Levels ◽  
Acute Megakaryocytic Leukemia ◽  
Shrna Knockdown

Abstract Abstract 2039 Poster Board II-16 Acute megakaryocytic leukemia (AMkL; M7) is a biologically heterogeneous form of AML, representing ∼10% of pediatric and 1-2% of adult AML cases. AMkL is the most common AML subtype of children with Down syndrome (DS). DS children with AMkL have an excellent prognosis with EFS rates of 80-100% when treated with ara-C/anthracycline-based protocols, in contrast to the <30% EFS rates of non-DS children with AMkL. This also contrasts to the ∼50% EFS rates of non-DS children with AML overall, indicating that AMkL is an extremely poor risk group amongst non-DS children with AML despite the use of intensive chemotherapy-based protocols. These clinical data make a compelling argument that new therapies are essential to improve the treatment outcome of this aggressive disease. Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected uniformly in nearly all DS AMkL cases, but not in non-DS AML and non-AMkL DS leukemia cases. The net effect of GATA1 mutations is an introduction of early stop codons and synthesis of a shorter GATA1 protein (designated GATA1s) that has altered transactivation activity, potentially contributing to the uncontrolled proliferation of immature megakaryocytes. It is conceivable that the altered GATA1 function between DS and non-DS AMkL may account for differential expression of GATA1 target genes in these two groups of patients. On the other hand, overexpression of GATA1 in megakaryoblasts from non-DS children with AMkL compared to myeloblasts from non-DS children with other subtypes of AML may contribute to differences in chemotherapy sensitivity via regulation of GATA1 target genes. We previously reported that GATA1 mutations in DS AMkL are associated with decreased expression of cytidine deaminase (encodes an enzyme which can convert ara-C to ara-U, the inactive form of the drug), thus contributing to the enhanced ara-C sensitivity of DS AMkL blasts. Further, when GATA1 was ectopically expressed in a DS AMkL cell line, CMK, it caused significantly increased resistance to ara-C. In the present study, we confirmed overexpression of GATA1 in non-DS AMkL blasts compared to non-DS AML blasts by real-time RT-PCR quantitation of GATA1 transcripts in our cohort of patient samples. shRNA knockdown of GATA1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivities to ara-C and daunorubicin, the two main drugs used for AML treatment, and significantly increased basal level apoptosis. This was accompanied by significantly decreased Bcl-xL transcript and protein levels in the GATA1 shRNA knockdown clones compared to a shRNA negative control. Binding of GATA1 to the two GATA elements in Bcl-x promoter and transactivation of Bcl-x promoter activity by GATA1 was demonstrated by ChIP assays and luciferase reporter assays, respectively, in Meg-01 cells. In our cohort of non-DS AMkL and AML patient samples, significant overexpression of Bcl-xL in non-DS AMkL compared to non-DS AML cases and a significant correlation between Bcl-xL and GATA1 transcripts were detected. Besides Bcl-xL, additional GATA1 targets (e.g. TNF) related to apoptosis were also identified by gene expression and ChIP-on-ChIP microarray analyses. Interestingly, our microarray data also suggest that GATA1 may have an impact on PI3-kinase/Akt pathway through modulating directly or indirectly a group of genes within the pathway. Western blotting revealed increased phosphorylation of Akt in the GATA1 knockdown clones compared to the negative control cells. Previous studies reported that histone deacetylase inhibitors (HDACIs) treatment causes hyperacetylation and subsequent degradation of GATA1, suggesting that these agents may be effective in targeting GATA1 in AMkL. Treatment of Meg-01 cells with an HDACI, valproic acid (VPA), resulted in decreased protein levels for GATA1 and Bcl-xL and increased phosphorylation of Akt. Co-treatment of Meg-01 cells with VPA and ara-C resulted in synergistic induction of apoptosis and activation of caspase-3. This drug synergy was amplified when a non-toxic dose of the PI3-kinase inhibitor LY294002 was added. Our results demonstrate that GATA1 causes resistance to chemotherapy in non-DS AMkL by promoting AMkL blast survival through regulating its target genes. Treatment of AMkL may be improved by integrating HDACI and PI3-kinase or Akt inhibitors into the chemotherapy of this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nodular Lymphocyte Predominant Hodgkin Lymphoma - A Retrospective Immunohistochemical Study of Patients in Bangalore, Karnataka

2021 ◽  
Vol 8 (23) ◽  
pp. 1966-1969
Author(s):  
Shankar Anand ◽  
Akshatha C ◽  
Libin Babu Cherian ◽  
Ramachandra C
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Classical Hodgkin Lymphoma ◽  
Double Positive ◽  
Cervical Lymph Nodes ◽  
Histiocytic Cell ◽  
Immunohistochemical Markers ◽  
Lp Cells

BACKGROUND The term Hodgkin’s lymphoma includes classical Hodgkin lymphoma (CHL) and the less common nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). NLPHL is a B cell neoplasm usually characterised by nodular or follicular and diffuse proliferation of small lymphocytes with single scattered large neoplastic cells (LP/L&H/Popcorn cells). NLPHL accounts for 10 % of all Hodgkin lymphoma. METHODS This is a retrospective study. Histopathology slides and blocks of 24 cases of nodular lymphocyte predominant Hodgkin lymphoma were collected from the archives of histopathology from 2011 to 2015. The immunohistochemistry slides of the corresponding histopathology cases were also assembled. Both the slides were reviewed by three expert onco-pathologists and IHC markers were studied and compared. RESULTS Patients were mostly young between 20 and 40 years (16 / 24, 66.67 %). There was a distinct male preponderance (20 / 24, 83.3 %). Most cases involved cervical, axillary or inguinal lymph nodes, with cervical lymph nodes being the most common (13 / 24, 54 %). It was found that CD45, CD20, CD79a and PAX5 staining highlighted the LP cells in all twenty-four cases, while OCT - 2 and BOB - 1 were highlighted in twenty-three cases (95.8 %), which was statistically significant. CD3 and CD5 IHC staining on T cell rosettes and background reactive T cells were examined, and it was seen that CD3 expression was far more consistent than CD5 expression in T cell rosettes and reactive T cells. Also, it was seen that, those cases which were double positive for CD3 and CD5 constitutes only eight cases (8 / 24, 33.3 %). CONCLUSIONS CD3 is a more consistent marker than CD5 in demonstrating surrounding reactive T cells in NLPHL. CD45, PAX5, CD20, BOB - 1 and OCT - 2 are consistent immunohistochemical markers of LP cells. KEYWORDS Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL), Classical Hodgkin Lymphoma (CHL), Cluster Differentiation (CD), Lymphocyte Predominant Cells (LP Cells), Lymphocyte and Histiocytic Cell (L & H Cell)

Immunohistochemical Analysis of the Expression of the B- Cell Markers BCL-6, MUM1/IRF4, CD79A and Transcription Factors BOB.1 and OCT.2 in Classical Hodgkin Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 974-974
Author(s):  
S. Valsami ◽  
D. Rontogianni ◽  
V. Pappa ◽  
T. Economopoulos ◽  
F. Kontsioti ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Expression Pattern ◽  
Disease Free Survival ◽  
Classical Hodgkin Lymphoma ◽  
Time To Progression ◽  
Free Survival ◽  
Cell Markers ◽  
Disease Free

Abstract INTRODUCTION: Classical hodgkin’s lymphoma can be considered in most cases as a B-cell lymphoma by the presence of potentially functional immunoglobulin gene rearrangements in Hodgkin/Reed Sternberg (H-RS) cells However the expression of B cell markers in classical Hodgkin lymphoma is rare and the light and heavy chain mRNA is lacking. The exact mechanism for this discrepancy is not known, and some studies have suggested a transcription machinery deficiency. The aim of our study was the detection of B cell markers like CD79a, bcl6 and MUM.1/IRF4 in relation to B cell transcription factors BOB.1 and OCT.2 in classical Hodgkin lymphoma in order to define subgroups with different histogenesis and prognosis. Patients and methods: The analysis included 107 cases of classical Hodgkin lymphoma 55 males and 52 females with a median age of 37 (13–79). They were staged as: 17 stage I, 55 stage II, 13 stage III and 22 stage IV. Advanced stage patients were risk stratified according to the IPI, and early according to EORTC. The histological subtype was: Nodular sclerosis 76, Mixed cellularity 19, Lymphocyte depleted 5 and lymphocyte rich 7 cases. Extranodal disease was present in 24/107 (22.4%) cases. Diagnostic biopsies were used for histochemical detection of bcl/6, CD79a, MUM-IRF4 and B cell transcription factors BOB.1, OCT.2. Expression was considered as low if detected in 1–25%, medium in 26–49% and high in &gt; 50% of H/RS cells. RESULTS Bcl6 was expressed in 22/101 cases, MUM.1 in 81/107 cases, BOB.1 in 89/100, OCT.2 in 17/100 and CD79a in 6/101 cases. In positive cases, bcl6, CD79a and OCT.2 have shown low expression, while MUM.1 and BOB.1 had a high expression in the majority of cases. There was not any difference in the expression pattern between different histologic subtypes for all these markers. Bcl6, MUM.1, OCT.2 and CD79a expression were not significantly different for different risk group categories and did not influence overall survival, disease free survival or time to progression. Cases positive for MUM expression had a significantly lower Hb (p=0.03), lower albumin (p=0.02), and higher IgG value (p=0.01). BOB.1 was expressed in 25% of early favourable, in 35% of intermediate and 40% of unfavourable early stage disease (p=0.06).There was a positive correlation between B symptoms and BOB.1 expression (p=0.01). There was a positive correlation between the expression of bcl6 and OCT.2 and between OCT.2 and CD79a (p=0.05, p=0.01) respectively. CONCLUSION MUM is expressed in the majority of classical Hodgkin lymphoma cases confirming its histogenesis from a late centrocyte and post/germinal centre B cell. B cell markers and transcription factors were not significant for survival, disease free survival and time to progression and their expression pattern was not different between different histological subtypes and risk groups of classical Hodgkin lymphoma.

A Novel Hodgkin Lymphoma Cell Line (KHL) Established from An Untreated Patient.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1448-1448
Author(s):  
Ta-Chih Liu ◽  
Yuan-Shiang Kao ◽  
Rachael Demuth ◽  
Nina Mathews ◽  
Carmen Espinoza ◽  
...  
Keyword(s):  
Lymph Node ◽  
Cell Line ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Chromosomal Aberration ◽  
Epstein Barr Virus ◽  
Early Stage ◽  
Classical Hodgkin Lymphoma ◽  
Barr Virus ◽  
Epstein Barr

Abstract The paucity of Hodgkin cell/Reed-Sternberg cell in classical Hodgkin Lymphoma (HL) represents a general problem for molecular and cytogenetic studies. The established HL cell lines could be used for such studies. However, only about 10 cell lines have been established and all of them were isolated from patients in late stage of illness when the tumor had recurred. Only one of these cell lines is known to be positive for Epstein- Barr virus antigen. In addition, the pattern of chromosomal aberration in these cell lines is highly complex. Therefore, it is necessary to have a cell line established from the early stage of disease, without prior treatment and with less chromosomal aberration for research. A sample of left axillary lymph node from a 27-year-old male with early stage of HL, and without prior chemotherapy, was cultured in RPMI 1640 media supplemented with fetal calf serum for routine cytogenetic study. The culture, passed weekly, now in its 60st week, 57th passes is growing autonomously without supplement of any growth factor. The original lymph node biopsy showed a classical Hodgkin lymphoma, mixed cellularity (WHO Classification) and the Hodgkin cells/Reed-Sternberg cells were positive for CD30, 4+(100%), CD20, 2+(33%) and negative for CD3, CD15, CD43, ALK-1 antigen and epithelial membrane antigen. EBV nuclear antigen 1 DNA and RNA are detected by PCR method. The cell line is positive for CD30, CD20, and Epstein-Barr virus (EBV) latent membrane protein, but negative for CD3, CD79a, and EBV early antigen. By florescent insitu hybridization the cell line is negative for p53 deletion, ALK gene rearrangement, MLL gene deletion, and t(12;21). A ploidy analysis by flow cytometry shows 80.22% diploid, and 19.78% hyperploids. The cells have doubling time of 30 to 36 hours. The initial karyotypes were: 45~46, XY, i(3)(q10), der(6)t(3;6)(p11;q22), t(6;13) (p21;q32), del(7)(q32), i(14) (q10)[cp3]/46, XY, del(3)(p10), der(6)t(3;6), der(6)t(6;13), del(7)(q32), add(10)(p13), der(13)add(13)(p11.1)t(6;13), i(14)(q10)[3]/46, XY[16]. Metaphase preparations from the cell line showed 46, XY, absence of the above mentioned chromosomal abnormalities, but 97% (1422/1466 cells) of cells were diploid; 2.5% (36/1466 cells) of cells were tetraploid/near tetraploid, and 0.5% (8/1466 cells) of cells showed endore-duplication. Chromosomal comparative genomic hybridization of the cell line showed microdeletion on chromosome 5q34 and 13q22~31 region, and gain on 12q12.1. Single nucleotide polymorphism array showed no abnormalities. This newly established cell line is unique in: it is the second cell line known to be positive for EBV antigen, it shows no complex chromosomal aberration by conventional karyotyping or molecular genotyping, and since the cell line showed mostly in diploid, and only 3% of the cells are tetraploid/near tetraploid and endoreduplication by conventional cytogenetic method, the cell line is ideal for the study of formation of hyperploid cells (i.e. Reed-Sternberg cells) from diploid cells.

Prognostic Significance Of a 4-Microrna Signature Targeting JAK2 In Classical Hodgkin Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 629-629 ◽  
Author(s):  
Alfons Navarro ◽  
Anna Gaya ◽  
Anna Cordeiro ◽  
Blanca Gonzalez-Farre ◽  
Marina Díaz-Beyá ◽  
...  
Keyword(s):  
Western Blot ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Renilla Luciferase ◽  
Luciferase Assay ◽  
Prognostic Impact ◽  
Classical Hodgkin Lymphoma ◽  
Expression Levels ◽  
Low Expression

Abstract Introduction The behavior of classical Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the Hodgkin/Reed Sternberg (HRS) cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. One of the genetic lesions most frequently found in HRS cells involves members of the JAK/STAT signaling pathway. However, few studies have identified microRNAs (miRNAs) that target JAK2 in cHL. In a previous study, our group identified miR-135a as a JAK2 regulator and showed the prognostic impact of this miRNA in a small set (n=89) of cHL patients (Navarro A. et al. Blood 2009). In the present study, we have examined novel miRNAs targeting JAK2 and evaluated the prognostic impact of their expression levels in 170 lymph nodes of cHL patients. Methods TargetScan and miRbase were used to identify JAK2-related miRNAs. The conserved miRNAs with higher scores were selected and further validated by Renilla-Luciferase assay and Western Blot. We performed a Renilla-Luciferase assay with a modified expression vector psiChecK-2 containing the complete 3’UTR region of JAK2 cloned in the 3’UTR region of Renilla luciferase gene. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in the HDMYZ cHL cell line together with 1μM of modified psicheck2 vector using Lipofectamine 2000, and luminescence was read at 24 hours. Further validation of the miRNA target was performed by Western Blot in three cHL cell lines (L428, L-1236 and HDMYZ). The expression levels of the miRNAs identified as targeting JAK2 were then assessed in lymph nodes from 170 cHL patients (median age, 33 years; male, 51%; EBV, 43.8%; HIV, 12%) diagnosed and treated in a single institution. Moreover, 15 reactive lymph nodes (RLN) were used as normal controls. RNA was purified from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation kit. The miRNA expression was analyzed using TaqMan microRNA assays. Statistical analyses were performed using R v2.13 and PASW Statistics 18. Results The bioinformatic analysis identified seven microRNAs as possible regulators of JAK2 (miR-34a, miR-101, miR-135a, miR-204, miR-216a, miR-216b and miR-375). The Renilla-Luciferase assay confirmed that in addition to miR-135a, miR-101 (61%; p=0.01), miR-204 (46%; p=0.003) and miR-216a (64.8%; p=0.02) also targeted JAK2. These findings were confirmed by Western Blot analysis of JAK2 levels after transfection with pre-miRNAs (median % of protein reduction in the 3 cell lines of 21.1%, 46.7% and 24%, respectively). The analysis of these miRNAs in RLN showed that miR-101, miR-135a and miR-204 were significantly downregulated in lymph nodes of cHL patients (p<0.001, p<0.001 and p=0.02 respectively) in comparison with RLN used as control. In contrast, miR-216 did not show significant differences in comparison with RLN. Interestingly, the analysis of the expression levels of these 4 miRNA in the patient lymph nodes had an impact on prognosis. First, low expression levels of miR-135a was associated with lower rate of complete response to first line treatment (76% vs 89%, p=0.012) and a trend was observed for miR-216(p=0.077). Secondly, patients with low expression levels of miR-101 or miR-204 had shorter disease-free survival (DFS) (151 vs 109 months, p=0.020; and 149.2 vs 105.5 months, p=0.055). Finally, patients with low levels of miR-135a, miR-204 or miR-216 had shorter overall survival (OS) (158 vs 136 months; p=0.039; and 157.5 vs 110 months, p=0.025; and 165 vs 140 moths, p=0.011 respectively). Since all four miRNAs showed prognostic impact (DFS, OS or treatment response), we decide to analyze the prognostic impact of the combination of the 4 miRNAs. Patients were then classified in two subgroups according to the expression levels of all four miRNAs. Patients with low expression of more than 2 miRNAs had shorter OS (163 vs 132 months; p=0.012). The multivariate analysis identified the combination of the four miRNAs as an independent prognostic marker of OS (OR, 7.6; 95% CI, 1.1-57.2; p=0.048) together with Age≥45years (OR, 5.9; 95% CI, 2.4-14.4; p<0.001) and B symptoms (OR, 2.6; 95% CI, 1.09-6.3; p=0.03). Conclusions MiR-101, miR-135a, miR-204 and miR-216 regulate JAK2 and are independent prognostic factors in cHL. Acknowledgments SDCSD of School of Medicine of UB. AC is an APIF-UB fellow. Disclosures: No relevant conflicts of interest to declare.

scholarly journals STAT6 and STAT1 are essential antagonistic regulators of cell survival in classical Hodgkin lymphoma cell line

Leukemia ◽  
2009 ◽  
Vol 23 (10) ◽  
pp. 1885-1893 ◽  
Author(s):  
D Baus ◽  
F Nonnenmacher ◽  
S Jankowski ◽  
C Döring ◽  
C Bräutigam ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Line ◽  
Hodgkin Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cell Line ◽  
Classical Hodgkin Lymphoma ◽  
Hodgkin Lymphoma Cell

Identification and Purification of Hodgkin Cells from Lymph Nodes Involved by Classical Hodgkin Lymphoma by Flow Cytometry and Flow Cytometric Cell Sorting.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2262-2262
Author(s):  
Jonathan R. Fromm ◽  
Steven J. Kussick ◽  
Brent L. Wood
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Cell Sorting ◽  
Classical Hodgkin Lymphoma ◽  
Tissue Sections ◽  
Hla Dr ◽  
Blocking Antibodies

Abstract The diagnosis of classical Hodgkin lymphoma (CHL) has historically been made in tissue sections, as attempts to identify the neoplastic Hodgkin and Reed-Sternberg (HRS) cells of CHL by flow cytometry (FC) have been largely unsuccessful. As HRS cells are known to be ringed (“rosetted”) by benign/reactive T cells, we hypothesized that in cell suspensions the HRS will be bound to T cells (forming T cell rosettes), and that consequently the rosettes would have a composite T-cell/HRS immunophenotype by FC (CD3+/CD15+/CD20−/CD30+/CD45+). We further hypothesized that specific antibodies to the adhesion molecules known to be involved in T cell/HRS cell binding (CD2 and LFA-1 on the T cell, and CD54 and CD58 on the HRS cell) might result in “naked” (unbound) HRS cells, enabling us to use FC to identify HRS cells with the expected immunophenotype (CD3−/CD15+/CD20−/CD30+/CD45−). Our initial FC studies of the HRS cell line L1236 demonstrated that CD15, CD30, CD40, CD71, CD86, CD95, and HLA-DR, but not CD3 or CD20, were brightly expressed on these cells and may be useful in identification of HRS in authentic cases of CHL involving lymph nodes. In mixing experiments, L1236 cells spontaneously bound normal T cells, analogous to T cell rosetting of HRS cells in CHL; these interactions could be blocked specifically using a cocktail of unlabeled antibodies to CD2, LFA-1, CD54, and CD58. Among 27 lymph nodes involved by CHL, this novel FC method, in which 250,000 to 500,000 total lymph node cells were evaluated, and in which up to ten cellular antigens were assessed simultaneously, enabled HRS cells to be identified in 89% of cases. 82% of these cases demonstrated interactions between HRS cells and T cells that could be disrupted with blocking antibodies. None of 29 non-CHL neoplasms, and none of 23 reactive lymph nodes, demonstrated HRS populations by FC. The proportions of cases where the HRS population showed expression of CD15, CD30, CD40, CD71, CD86, CD95, and HLA-DR, and absence of CD3 and CD20 was similar to that described previously in tissue sections by immunohistochemistry. Interestingly, in contrast to the findings in tissue sections, by FC the non-rosetted HRS cells of most CHL cases (73%) demonstrated detectable expression of CD45, usually at a low level. Finally, cell sorting experiments confirmed that (1) populations identified by FC have the cytomorphology of HRS cells, (2) HRS/T cell rosettes can be detected by FC, and (3) these rosettes can disrupted by the blocking antibody cocktail. This FC technique offers a potential alternative to immunohistochemistry in confirming the diagnosis of CHL and, through cell sorting, provides a means of rapidly purifying HRS cells.

Sign in / Sign up

Export Citation Format

Share Document