scholarly journals High CXCR3 on Leukemic Cells Distinguishes IgHVmut from IgHVunmut in Chronic Lymphocytic Leukemia: Evidence from CD5high and CD5low Clones

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Gayane Manukyan ◽  
Tomas Papajik ◽  
Zuzana Mikulkova ◽  
Renata Urbanova ◽  
Veronika Smotkova Kraiczova ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Chemokine Receptors ◽  
Prognostic Value ◽  
Surface Antigens ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Neoplastic Cells ◽  
Cxcr3 Expression ◽  
Mutational Status ◽  
Proliferative Pool

Despite the shared pattern of surface antigens, neoplastic cells in chronic lymphocytic leukemia (CLL) are highly heterogeneous in CD5 expression, a marker linked to a proliferative pool of neoplastic cells. To further characterize CD5high and CD5low neoplastic cells, we assessed the chemokine receptors (CCR5, CCR7, CCR10, CXCR3, CXCR4, CXCR5) and adhesion molecules (CD54, CD62L, CD49d) on the CD5high and CD5low subpopulations, defined by CD5/CD19 coexpression, in peripheral blood of CLL patients (n=60) subgrouped according to the IgHV mutational status (IgHVmut, n=24; IgHVunmut, n=36). CD5high subpopulation showed a high percentage of CXCR3 (P<0.001), CCR10 (P=0.001), and CD62L (P=0.031) and high levels of CXCR5 (P=0.005), CCR7 (P=0.013) compared to CD5low cells expressing high CXCR4 (P<0.001). Comparing IgHVmut and IgHVunmut patients, high levels of CXCR3 on CD5high and CD5low subpopulations were detected in the IgHVmut patients, with better discrimination in CD5low subpopulation. Levels of CXCR3 on CD5low subpopulation were associated with time to the next treatment, thus further confirming its prognostic value. Taken together, our analysis revealed higher CXCR3 expression on both CD5high and CD5low neoplastic cells in IgHVmut with a better prognosis compared to IgHVunmut patients. Contribution of CXCR3 to CLL pathophysiology and its suitability for prognostication and therapeutic exploitation deserves future investigations.

Mutational Status of the TP53 Gene As a Predictor of Response and Survival in Patients With Chronic Lymphocytic Leukemia: Results From the LRF CLL4 Trial

2011 ◽  
Vol 29 (16) ◽  
pp. 2223-2229 ◽  
Author(s):  
David Gonzalez ◽  
Pilar Martinez ◽  
Rachel Wade ◽  
Sarah Hockley ◽  
David Oscier ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Survival Data ◽  
Prognostic Value ◽  
Gene Mutations ◽  
Progression Free Survival ◽  
Tp53 Gene ◽  
Lymphocytic Leukemia ◽  
Tp53 Mutations ◽  
Mutational Status ◽  
Gene Tp53

Purpose TP53 mutations have been described in chronic lymphocytic leukemia (CLL) and have been associated with poor prognosis in retrospective studies. We aimed to address the frequency and prognostic value of TP53 abnormalities in patients with CLL in the context of a prospective randomized trial. Patients and Methods We analyzed 529 CLL samples from the LRF CLL4 (Leukaemia Research Foundation Chronic Lymphocytic Leukemia 4) trial (chlorambucil v fludarabine with or without cyclophosphamide) at the time of random assignment for mutations in the TP53 gene. TP53 mutation status was correlated with response and survival data. Results Mutations of TP53 were found in 40 patients (7.6%), including 25 (76%) of 33 with 17p deletion and 13 (3%) of 487 without that deletion. There was no significant correlation between TP53 mutations and age, stage, IGHV gene mutations, CD38 and ZAP-70 expression, or any other chromosomal abnormality other than 17p deletion, in which concordance was high (96%). TP53 mutations were significantly associated with poorer overall response rates (27% v 83%; P < .001) and shorter progression-free survival (PFS) and overall survival (OS; 5-year PFS: 5% v 17%; 5-year OS: 20% v 59%; P < .001 for both). Multivariate analysis that included baseline clinical variables, treatment, and known adverse genetic factors confirmed that TP53 mutations have added prognostic value. Conclusion TP53 mutations are associated with impaired response and shorter survival in patients with CLL. Analysis of TP53 mutations should be performed in patients with CLL who have progressive disease before starting first-line treatment, and those with mutations should be selected for novel experimental therapies.

scholarly journals Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia

2016 ◽  
Vol 35 (2) ◽  
pp. 150-157 ◽  
Author(s):  
Ksenija Vucicevic ◽  
Vladimir Jakovljevic ◽  
Natasa Colovic ◽  
Natasa Tosic ◽  
Tatjana Kostic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Mononuclear Cells ◽  
Prognostic Markers ◽  
Statistical Significance ◽  
Genetic Alterations ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
Mutational Status

Summary Background: In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. Aim: The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. Methods: We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. Results: We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=<0.001). Regarding the association with prognostic markers, the Bcl2/Bax ratio showed a negative correlation to lymphocyte doubling time (r=−0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Conclusions: Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

scholarly journals The IGLV3-21R110 Defines a Subset of Chronic Lymphocytic Leukemia with Intermediate Epigenetic Subtype and Poor Outcome

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Ferran Nadeu ◽  
Romina Royo ◽  
Guillem Clot ◽  
Martí Duran-Ferrer ◽  
Alba Navarro ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Rna Sequencing ◽  
Prognostic Value ◽  
Lymphocytic Leukemia ◽  
Whole Genome ◽  
Biological Features ◽  
Mutational Status ◽  
Bcr Signaling

Introduction: B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of pre- and post-germinal center B-cells named naïve-like (n-CLL), intermediate (i-CLL) and memory-like CLL (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. The integration of these factors might translate into novel insights in CLL pathogenesis with implications on the proposed stratification of the patients. Aim: To determine the clinical and biological features of the IGLV3-21R110 in CLL in the light of the epigenetic subtypes and immunogenetic, genomic and transcriptomic landscapes of the tumors. Methods: We characterized the immunoglobulin (IG) gene of 584 CLL cases from whole-genome/exome and RNA sequencing using our recently developed algorithm IgCaller (Nadeu et al., Nat. Commun. 2020) and MiXCR, respectively. The genomic makeup of the tumors was obtained from whole-genome/exome sequencing while RNA sequencing data for 369 cases was used for gene expression analyses. Expression levels of WNT5A and WNT5B were verified by quantitative PCR with reverse transcriptase. Primary end points were time to first treatment (TTFT) and overall survival (OS) calculated from the date of diagnosis. All patients gave written informed consent. The study was approved by the Ethics Committee of the Hospital Clínic of Barcelona. Results: The IGLV3-21R110 was detected in 6.5% of cases being similarly distributed between mutated (6.5%) and unmutated (6.6%) IGHV cases (P=0.56). In contrast, the IGLV3-21R110 was found in 30/79 (38%) i-CLL compared to only 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL (P&lt;0.001). All stereotyped subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped IG genes. IGLV3-21R110 i-CLL had a borderline IGHV mutational status (median 97.7%) that was higher than i-CLL lacking the IGLV3-21R110 (median 96.2%, P=0.005). IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL case and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL and had a specific expression signature of 64 genes with overexpression of WNT5A and WNT5B as hallmarks. No differences were observed in the expression profile of subset #2 and non-subset #2 IGLV3-21R110 i-CLL tumors. On the other hand, i-CLL lacking the IGLV3-21R110 phenotypically mirrored memory-like/mutated IGHV cases. In relation to prognosis, IGLV3-21R110 i-CLL had a short TTFT and OS similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Therefore, i-CLL cases, which have been associated with an intermediate prognosis between m-CLL and n-CLL in previous studies, can be divided in two subgroups of cases with opposed clinical evolutions based on the IGLV3-21R110. Indeed, the IGLV3-21R110 and n-CLL subtype retained independent prognostic value in multivariate analyses while the i-CLL lost its prognostic prediction both for TTFT and OS. The prognostic value of the IGLV3-21R110 was also independent of the IGHV mutational status. In terms of applicability in the clinics, all n-CLL cases were classified as unmutated IGHV and 98% of m-CLL were mutated IGHV. Thus, either a complete IG characterization (IGHV mutational status and IGLV3-21R110) or the integration of the n-CLL subtype and IGLV3-21R110 identified virtually the same subset of patients with aggressive disease. Conclusions: The IGLV3-21R110 defines a CLL subset with borderline IGHV mutations, specific driver alterations, a gene expression signature including WNT5A/B overexpression, and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes. Our findings support the identification of IGLV3-21R110 CLL as a particular subgroup of the disease with relevance in the risk stratification of the patients. Disclosures Nadeu: Janssen: Honoraria. Campo:NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America..

ZAP-70 Expression in Chronic Lymphocytic Leukemia: Correlation with Clinical and Biological Features.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1915-1915 ◽  
Author(s):  
Ilaria Del Giudice ◽  
Nnenna Osuji ◽  
Estella Matutes ◽  
Alison Morilla ◽  
Ricardo Morilla ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Prognostic Value ◽  
Clinical Stage ◽  
Univariate Analysis ◽  
Surrogate Marker ◽  
Lymphocytic Leukemia ◽  
Over Expression ◽  
Biological Features ◽  
Free Interval ◽  
Mutational Status

Abstract One of the most intriguing features of chronic lymphocytic leukemia (CLL) is its clinical heterogeneity. The mutational status of immunoglobulin heavy chain variable region (IgVH) genes is one of the most powerful predictors of overall survival (OS) and progression-free survival (PFS). The expression of the tyrosine kinase ZAP-70 in CLL cells has been proposed as a surrogate marker for the mutational status of IgVH genes. Recent reports suggest that ZAP-70 over-expression is an independent prognostic factor for shorter OS and PFS. There is little information about correlation between ZAP-70 expression and other clinical and biological features in CLL. The aims of this study are 1) to evaluate prospectively ZAP-70 expression in a series of CLL patients; 2) to correlate this with clinical stage, lymphocyte morphology, CD38 expression and chromosomal abnormalities detected by FISH and 3) to analyze the independent prognostic value of ZAP-70 in predicting time to first treatment (treatment free interval, TFI). ZAP-70 expression was analyzed using four-colour flow cytometry (as described by Crespo et al, NEJM2003; 348:1764–75). 201 previously untreated CLL patients were evaluated at diagnosis (32%) or during the disease course. Amongst them, 143 patients were entered in the LRF CLL4 trial. Clinical stage at the time of the study was A in 92 patients (A progressive in 46), B in 63 and C in 46. 28% were ZAP-70 positive (+) (cut-off ≥ 20% of CD5/CD19+ cells). ZAP-70 positivity was associated with prevalence of stage B/C at diagnosis (p=0.004), CLL with >10% prolymphocytes (CLL/PL) or atypical morphology (p<0.001), CD38 positivity (regardless of the cut-off point) (p<0.001), trisomy12 (p=0.001), del(6)(q21) (p=0.05) or no detectable abnormalities (0.002). ZAP-70 negative (−) cases presented more often with del(13)(q14) (p<0.001). There were no significant differences in the frequency of del(11)(q23) or delp53 between ZAP-70(+) and ZAP-70(−) cases. Median time from diagnosis to first treatment (treatment free interval, TFI) was 17.7 months for ZAP-70(+) and 44.6 months for ZAP-70(−) cases (p<0.001). The presence of the following parameters was associated with short TFI in univariate analysis: male sex (p=0.02), atypical morphology (p=0.02), clinical stage B/C (p<0.001), ZAP-70(+) (p<0.001), CD38+ (p<0.001), absence of del(13)(q14) (p<0.001), presence of delp53 (p=0.05). Multivariate analysis is being performed. In conclusion, we document a significant correlation between ZAP-70 over-expression in CLL and other adverse prognostic factors (such as CD38 and advanced stage), provide new data concerning its association with cytogenetic abnormalities detected by FISH and demonstrate the prognostic value of ZAP-70 expression.

scholarly journals Human interleukin-7 induces proliferation of neoplastic cells from chronic lymphocytic leukemia and acute leukemias

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 753-759 ◽  
Author(s):  
W Digel ◽  
M Schmid ◽  
G Heil ◽  
P Conrad ◽  
S Gillis ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Binding Sites ◽  
Lymphocytic Leukemia ◽  
Membrane Receptors ◽  
Receptor Expression ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Neoplastic Cells ◽  
Acute Leukemias ◽  
Interleukin 7

The biologic effects of interleukin-7 (IL-7) and the expression of specific IL-7 membrane receptors on isolated neoplastic cells from previously untreated patients with chronic lymphocytic leukemia as well as acute leukemias were investigated in vitro. Leukemic cells were incubated for up to 6 days with various concentrations of IL-7 (0.01 to 2,000 U/mL). Neoplastic cells of the T- or B-phenotype from chronic as well as from acute leukemias proliferated in a dose-dependent manner. Cells from acute myeloid leukemias also proliferated in response to IL- 7. An optimal proliferative effect was achieved between 96 and 120 hours with 200 U/mL IL-7. Combinations of IL-7 with IL-2 and tumor necrosis factor-alpha showed an additive effect on [3H]TdR incorporation. IL-7 binding assays gave a value of approximately 33 to 180 high-affinity (kd approximately 20 pmol/L) binding sites/cell and approximately 241 to 3,280 low-affinity (kd approximately 600 pmol/L) binding sites/cell. Receptor expression correlated with the proliferation in response to IL-7. These data indicate that IL-7 can induce proliferation of relatively mature tumor cells, and that this effect is not restricted to the lymphoid lineage.

Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells through Activation of the Death Receptor Pathway and Potentiates TRAIL Response.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4949-4949
Author(s):  
Laurence Lagneaux ◽  
Nicolas Gillet ◽  
Alain Delforge ◽  
Marielle Dejeneffe ◽  
Basile Stamatopoulos ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Chronic Lymphocytic Leukemia ◽  
Mononuclear Cells ◽  
Single Agent ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Potent Inducer ◽  
Mutational Status ◽  
Induced Apoptosis

Abstract Background: The anti-leukemic in vitro activity of valproic acid (VPA), a commonly used antiepileptic agent, was tested on lymphocytes derived from 40 patients with chronic lymphocytic leukemia (CLL) (Binet stage A=34, B=3, C=3). These patients had not been previously treated or remained untreated for the previous 6 months. Combined analysis of ZAP-70, CD38 and IgVH mutational status was performed for each patient. Methods: Mononuclear cells were incubated with VPA at 1, 5 and 10 mM for 24 hours. Cell viability was assessed by trypan blue exclusion assay, apoptosis by annexin V/propidium iodide(PI) labelling and PI staining after cell permeabilisation. Caspase activation was studied by flow cytometry analysis after cell treatment with selective caspase inhibitors. Results: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in all CLL patients tested. VPA-treatment induced apoptotic changes in CLL cells including phosphatidylserine (PS) externalisation and DNA fragmentation. The mean apoptotic rate was similar between IgVH mutated and unmutated patients or ZAP-70+/ZAP-70- cases. VPA induced apoptosis by the extrinsic pathway involving engagement of the caspase-8 dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA increased significantly the sensitivity of leukemic cells to TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand). In addition, VPA overcomed the prosurvival effects of bone marrow stromal cells. Conclusions: These data indicate that VPA, at the pharmacological concentration of 1 mM, is a potent inducer of apoptosis in CLL and should be further explored as a single agent. Also the combination of VPA and TRAIL may be a promising approach in the treatment of CLL.

Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4460-4468 ◽  
Author(s):  
Lesley-Ann Sutton ◽  
Efterpi Kostareli ◽  
Anastasia Hadzidimitriou ◽  
Nikos Darzentas ◽  
Athanasios Tsaftaris ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Large Scale ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Strongly Correlated ◽  
Class Switch ◽  
Mutational Status ◽  
Ighv Gene ◽  
The Common ◽  
Low Levels

Abstract Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).

scholarly journals Human interleukin-7 induces proliferation of neoplastic cells from chronic lymphocytic leukemia and acute leukemias

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 753-759 ◽  
Author(s):  
W Digel ◽  
M Schmid ◽  
G Heil ◽  
P Conrad ◽  
S Gillis ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Binding Sites ◽  
Lymphocytic Leukemia ◽  
Membrane Receptors ◽  
Receptor Expression ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Neoplastic Cells ◽  
Acute Leukemias ◽  
Interleukin 7

Abstract The biologic effects of interleukin-7 (IL-7) and the expression of specific IL-7 membrane receptors on isolated neoplastic cells from previously untreated patients with chronic lymphocytic leukemia as well as acute leukemias were investigated in vitro. Leukemic cells were incubated for up to 6 days with various concentrations of IL-7 (0.01 to 2,000 U/mL). Neoplastic cells of the T- or B-phenotype from chronic as well as from acute leukemias proliferated in a dose-dependent manner. Cells from acute myeloid leukemias also proliferated in response to IL- 7. An optimal proliferative effect was achieved between 96 and 120 hours with 200 U/mL IL-7. Combinations of IL-7 with IL-2 and tumor necrosis factor-alpha showed an additive effect on [3H]TdR incorporation. IL-7 binding assays gave a value of approximately 33 to 180 high-affinity (kd approximately 20 pmol/L) binding sites/cell and approximately 241 to 3,280 low-affinity (kd approximately 600 pmol/L) binding sites/cell. Receptor expression correlated with the proliferation in response to IL-7. These data indicate that IL-7 can induce proliferation of relatively mature tumor cells, and that this effect is not restricted to the lymphoid lineage.

Plasma Is a Reliable Source of mRNA for Testing IgVH Mutation Status in Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2949-2949
Author(s):  
W. Ma ◽  
I. Jilani ◽  
M. Gorre ◽  
M. Keating ◽  
H. Chan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Lymph Nodes ◽  
Plasma Cells ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
High Turnover ◽  
Mutation Status ◽  
Mutational Status ◽  
Reliable Source

Abstract The mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene in the leukemic cells of patients with chronic lymphocytic leukemia (CLL) is an important prognostic marker. Lack of IgVH mutation is associated with rapid disease progression and shorter survival. The assay used to determine IgVH mutation status requires specific amplification of the mRNA of the expressed clonal IgVH gene in CLL. However, in some patients with CLL or lymphocytic lymphoma, the bulk of the disease resides in the lymph nodes or bone marrow, with few circulating leukemic cells. In addition, contamination of leukemic clonal cells by reactive polyclonal plasma cells can cause difficulty in obtaining homogenous IgVH mRNA for sequencing, and may therefore lead to failed sequencing or sequencing of the wrong IgVH mRNA. We have reported that plasma is enriched with leukemia-specific DNA, RNA, and protein because of the high turnover of leukemic cells relative to non-neoplastic cells. We thus reasoned that plasma might be a more reliable source of IgVH mRNA than cells from peripheral blood or bone marrow, where mixed populations of leukemic cells and non-neoplastic lymphocytes or plasma cells could hinder sequencing. We tested the plasma from 8 patients in whom routine (cell-based) testing for IgVH mutation status failed to yield results due to a paucity of leukemic cells (&lt;10% of total cells). The plasma of all the 8 samples showed unique, easily defined amplification products of the IgVH mRNA. Sequencing of these products showed the presence of mutated IgVH in 5 patients and unmutated IgVH in 3. Testing paired plasma and cell samples from 19 patients with CLL showed identical mutation rates and family types of the expressed IgVH gene. In contrast, plasma from 20 normal individuals showed no IgVH amplification products that could be sequenced. This data suggest that plasma can be used as an alternative to cells for testing IgVH mutation status. More importantly, plasma is more reliable when few leukemic cells are in circulation and the bulk of the tumor is in the bone marrow or lymph nodes.

High Expression of Haematopoietic Cell Specyfic Lyn Substrate-1 (HS1) Predicts Survival of B-Cell Chronic Lymphocytic Leukemia Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2853-2853
Author(s):  
Aleksandra Butrym ◽  
Miroslaw Majewski ◽  
Justyna Dzietczenia ◽  
Tomasz Wrobel ◽  
Kazimierz Kuliczkowski ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Patient Survival ◽  
Clinical Course ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Mutational Status ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 2853 B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults in western countries. It is characterized by B lymphocyte accumulation in peripheral blood, bone marrow, lymph nodes and other lymphatic organs. Leukemic cells derive most commonly from B lymphocytes, rarely from T or NK cells. B-CLL is known from its heterogeneous clinical course from indolent to very aggressive. In spite of many known prognostic factors (such as immunoglobulin heavy chain gene mutational status – IgVH, expression of ZAP70 and CD38), is still difficult to classify a single patient to particular risk group and to predict CLL clinical course. That is why new prognostic factors are still needed. HS1 (hematopoietic cell specific Lyn substrate-1) is an intracellular protein, which expression occurs mainly in hematopoietic cells. HS1 plays an important role in regulating T cell immune synapse and affects many functions of NK cells, including the lysis of target cells, adhesion, chemotaxis and clustering of actin in the lytic synapse. The role of HS1 in B cells is poorly understood. This protein was identified in B cells as the primary receptor substrate for phosphorylation by BCR after antigenic stimulation. Other studies have confirmed the role of HS1 in the process of clonal expansion and deletion induced by antigen-receptor interaction in B cells and T. HS1 is rapidly phosphorylated in B cells in the vicinity of tyrosine residues and is a substrate for tyrosine kinases: the Src family and Syk, including Lyn, FGR, Fyn and Lck. It has been shown that HS1 interacts with the cell cytoskeleton in both: normal and leukemic B cells. HS1 protein is an important regulator of motility, migration and adhesion of leukemic cells and is involved in cytoskeleton rearrangement. HS1 can have impact on homing and migration of CLL cells. It can indirectly promote disease progression and influence patient survival. The aim of this study was to evaluate HS1 expression in CLL patients in connection with other known prognostic factors and patient survival. Material and methods: 92 untreated CLL patients (45 women and 47 men), aged between 42 and 88 years (median age 67 years), were included into the study. Diagnosis was made basing on typical clinical, hematological and immunophenotypical picture. The control group was consist of 28 healthy matched people (11 men and 17 women), aged between 36 and 79 years (median age 59 years). HS1 protein expression was determined by western blot. Comparative semi-quantitative indication of the degree of saturation of the bands analyzed by densitometry using the gel documentation system Gel-Doc (Bio-Rad) and a computer program to analyze the 1-D Quantity One (Bio-Rad). Assuming conventional units [AU - arbitrary units], depending on the saturation band, patients were divided into four groups with the expression of HS1 protein expressed in value from 0 to 3. Lack of expression was expressed as 0 [AU], and expression of the strongest, with the highest saturation band measured as 3 [AU]. Mutational status of IgVH, as well as CD38 and ZAP70 expression were also analyzed. Results: HS1 expression was significantly higher in CLL patients comparing to controls. Positive correlation was shown between HS1 and: age (p=0.0454), Rai stage (p=0.0412), leukocytosis (p=0.0129) and β2-microglobulin (p=0.0342). There was negative correlation between HS1 and hemoglobin level (p=0.0464) and platelet count (p=0.0310). Patients with lymphocyte doubling time shorter or equal to 6 months had higher expression of HS1. Expression of HS1 significantly influenced survival of CLL patients. Patients with higher HS1 expression had shorter survival than those with lower HS1 expression (p=0.0329). Conclusions: 1. Higher HS1 expression is observed in more advanced CLL stages. 2. Expression of HS1 in CLL cells is matched with shorter patient survival The relationship between expression of HS1 and survival of patients with B-CLL. Disclosures: No relevant conflicts of interest to declare.

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