Karyotype Results From CpG Oligodeoxynucleotide Stimulated Chronic Lymphocytic Leukemia (CLL) Cultures Are Consistent Among Laboratories: a CLL Research Consortium (CRC) Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1614-1614
Author(s):  
Nyla A. Heerema ◽  
John C. Byrd ◽  
Paola Dal Cin ◽  
Marie L Dell' Aquila ◽  
Prasad Koduru ◽  
...  
Keyword(s):  
B Cell ◽  
Prospective Trial ◽  
Pokeweed Mitogen ◽  
Fish Analysis ◽  
Cytogenetic Abnormalities ◽  
Interphase Fish ◽  
Cpg Oligodeoxynucleotide ◽  
Research Consortium ◽  
Clonal Abnormalities

Abstract Abstract 1614 Poster Board I-640 Background Cytogenetic abnormalities in B-cell CLL are important prognostic indicators for predicting disease progression and treatment response. Until recently, only interphase cytogenetics has been readily applicable to clinical use in CLL due to the inability of traditional mitogens to promote effective metaphase cytogenetic identification. Recently, cytokine or CpG oligonucleotide stimulation has been utilized to promote efficient metaphase analysis. One concern with this approach is the actual clonality and reproducibility of such clones when examined by different cytogenetic laboratories. The CLL Research Consortium (CRC) conducted a prospective trial to test (1) whether CpG would enhance detection of abnormal clones and (2) whether the CRC cytogenetic laboratories would achieve similar results when testing the same patient samples. Methods Initially, one laboratory compared stimulation of CLL cells using CpG versus pokeweed mitogen (PWM) + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the same samples. To test the reproducibility of CpG stimulation, fresh blood samples from 1 normal control and 12 CLL patients were collected at one site and distributed (blinded to phenotype and prior cytogenetic results) to 5 CRC cytogenetic laboratories where they were cultured for 3 days using CpG stimulation, harvested, banded and analyzed utilizing standard laboratory procedures. When possible, each laboratory analyzed 20 metaphases per case. Interphase FISH analysis for CLL (including probes for chromosomes 6q, 11q, 12, 13q, 17p, and IGH/CCND1) was also done on each sample. Results In the PWM+TPA versus CpG comparison, more cases were found to have clonal abnormalities with CpG stimulation (147 of 229, 64%) than with PWM+TPA (110 of 229, 48%; P=0.0005). All clonal abnormalities detected in the PWM+TPA cultures were also observed in the CpG cultures. In the 5-laboratory CpG comparison, the results were compiled and compared to the concurrent FISH results. The CpG karyotypes were concordant with the FISH results. The control case had a normal CpG-stimulated karyotype in all 5 laboratories. Three cases exhibited a complex karyotype; one of these also exhibited 11q-, one had 17p-, and one had neither. The cytogenetic descriptions varied modestly among labs, but all identified the complex clones. Every case including the control exhibited some nonclonal abnormal cells, but no pattern was detected among the 5 labs. When an abnormal clone was found by only 1 or 2 labs, it was present in only a very small number of cells. A small 13q- clone was observed in 2 cases in 1 and 2 labs (3 of 20 and 5 of 50 cells, respectively), and was confirmed by interphase FISH. A single lab found 2 of 30 cells with a t(12;15) in one case. Conclusions Abnormal clones in CLL are more readily detected with CpG stimulation than with traditional B-cell mitogens. The clonal abnormalities revealed by CpG stimulation are consistent with the “gold standard” interphase FISH results, and are reproducible among different cytogenetic laboratories. We have no evidence that CpG oligonucleotides create new clonal B-cell populations in vitro during 3 days of cell culture. CpG stimulation in vitro reveals clonal cytogenetic abnormalities and complexity in CLL that should be evaluated as potential independent prognostic parameters. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Cytogenetic Analysis of Normal Human B Cells Following CpG Stimulation: Implications for Interpretation of CpG Induced CLL Metaphase Analysis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3124-3124
Author(s):  
Xiaosheng Wu ◽  
Grzegorz S. Nowakowski ◽  
Stephanie A. Smoley ◽  
Bonnie A. Arendt ◽  
Mark A. Peterson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Clinical Utility ◽  
Chromosomal Abnormalities ◽  
Karyotype Analysis ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Cytogenetic Abnormalities ◽  
Trisomy 12 ◽  
Clonal Abnormalities

Abstract BACKGROUND: Karyotype analysis of metaphase cells for discovering genome-wide cytogenetic abnormalities has played an important role in prognosis and counseling of patients with chronic lymphocytic leukemia (CLL). Due to the low proliferative rate of CLL B cells, karyotype analysis has been of limited clinical utility in this disease. Thus, fluorescence in situ hybridization (FISH) on interphase cells has replaced karyotype analysis for the assessment of recurrent genetic changes in CLL. Recently, immunostimulatory CpG-oligodeoxynucleotides (CpG) have been shown to induce primary CLL B cell proliferation via engagement of toll-like receptor 9 (TLR9), thereby rendering karyotype analysis feasible and providing for a useful and reliable method to discover new cytogenetic abnormalities in CLL B cells. However, we and others have shown that CpG stimulation of CLL and normal B cells also induces robust expression of activation induced cytidine deaminase (AID), a mutagenic enzyme that is linked to the development of cytogenetic abnormalities. It is therefore plausible that cytogenetic abnormalities observed following CpG stimulation of CLL B cells may not reflect de novo abnormalities but rather reflect CpG induced-AID mediated cytogenetic alterations. We began to address this possibility by assessing the effects of CpG stimulation on the karyotypes of normal blood B cells. HYPOTHESIS: Since CpG stimulation induces AID expression in CLL and normal B cells, we hypothesize that: robust B cell activation by CpG could result in cytogenetic abnormalities thereby contributing to lymphomagenesis; and if CpG induces cytogenetic abnormalities that are linked to disease initiation and/or progression, such information would be of great value in the interpretation of cytogenetic data obtained from CpG-stimulated CLL B cells. METHODS: We performed interphase FISH analysis on freshly isolated blood B cells, and metaphase karyotype analysis on CpG stimulated B cells from 17 healthy blood donors. Pure (>98%) B cells obtained by negative selection, were hybridized at isolation with a probe set to identify deletion 6q, 11q, 13q, and 17p, trisomy 12, and IGH gene rearrangements, and cells were also stimulated for 5 days with 10 μg/ml CpG (CpG 2006, synthesized in-house) plus IL-2 and IL-15 for metaphase analysis. At least 20 post-CpG metaphase spreads were analyzed for each sample and abnormalities were considered clonal using the accepted convention (gains or translocations are present in two or more cells and loss present in at least 3 cells). The expression of AID in the B cells pre- and post-CpG stimulation was assessed by RT-PCR. RESULTS: FISH analysis of freshly isolated B cells showed that all 17 subjects were normal. After CpG stimulation, all samples examined exhibited strong AID expression as revealed by RT-PCR. By karyotype analysis, none of the 17 CpG stimulated samples showed evidence of clonal abnormalities. However, one or more nonclonal chromosomal abnormalities were observed in 7 out of 17 (41.2%) samples. Among those 7 samples, 4 samples had 1 abnormal metaphase out of a minimum of 20 examined while the other 3 samples exhibited two or more cells, each with distinct abnormalities. Some of those nonclonal abnormalities have also been observed in CLL (i.e., trisomy 3, trisomy 12 and 14q32 rearrangements). This is a much higher incidence of nonclonal abnormalities than has been seen with peripheral blood cells cultured by other methods. CONCLUSION: Our data have significance for the biology of CLL and for clinical practice. First, our data suggest that signaling through TLR9, and perhaps other receptors that likewise induce robust B cell AID expression, may represent a natural mechanism by which B lineage cells acquire chromosomal abnormalities during normal immune responses that may predispose toward neoplastic transformation. Second, given that none of the CpG-induced metaphases in normal B cells resulted in clonal abnormalities in the 5 day timeframe, our data also validate the clinical utility of CpG as a valuable tool for the cytogenetic analysis of CLL B cells since only clonal abnormalities are currently scored. However, it remains possible that non-clonal abnormalities seen in CpG-stimulated CLL B cells may be relevant to the disease process if future work shows that CpG stimulation uncovers recurring non-clonal defects of chromosomes/genes associated with tumorigenic events.

Improved Detection of Chromosomal Abnormalities in CLL by Conventional Cytogenetics Using CpG Oligonucleotide and Interleukin-2 Stimulation. A Belgian Multicentric Study

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3118-3118
Author(s):  
Natalie Put ◽  
Katrina Rack ◽  
Nadine Van Roy ◽  
Jeanne-Marie Libouton ◽  
Pascal Vannuffel ◽  
...  
Keyword(s):  
B Cell ◽  
Mitotic Index ◽  
Cytogenetic Analysis ◽  
Detection Rate ◽  
Interleukin 2 ◽  
Fish Analysis ◽  
Specific Change ◽  
Interphase Fish ◽  
Cpg Oligonucleotide ◽  
Clonal Abnormalities

Abstract Numerous studies have shown that the presence, number and type of chromosomal aberrations represent an independent predictor of prognosis in B-cell chronic lymphocytic leukemia (CLL). Consequently, cytogenetic analysis is routinely performed in this disease. However, CLL lymphocytes have a poor mitotic index, generating only 40–50% of abnormal karyotypes. The rate of detection can be increased to 80% by interphase FISH analysis. Since some aberrations, i.e. those not involving the classical regions (13q, 12, 11q, 17p, and 6q), can escape FISH detection, there has been great interest in improved culturing methods with an immunostimulatory CpG oligonucleotide (CpG). We performed a multicentric cytogenetic study to assess the impact of 2 different culturing procedures on the detection of clonal abnormalities in 159 consecutive unselected cases with CLL referred to our centers for routine analysis from October 2007 to July 2008. Dual 72 hours cultures of bone marrow or peripheral blood were set up with the addition of either a conventional B cell mitogen (TPA) or CpG and interleukin-2 (IL2). Cytogenetic analysis was performed on both cultures. FISH analysis using a CLL probe panel analyzing 1–6 regions (13q, centromere 12, 11q, 17p, 6q and 14q32) was also applied on uncultured material, on CpG and/or on TPA culture in 146 cases. Quality of banding and proliferation capacity were assessed in 38 cases. The quality was good in 17 (CpG) and 8 (TPA), intermediate in 12 (CpG) and 18 (TPA), and poor in 9 (CpG) and 12 (TPA) cases. The mitotic index was slightly higher in CpG cultures: <15 mitoses/slide were seen in 12 (CpG) and 15 (TPA) cases, 15–20 mitoses/slide in 13 (CpG) and 14 (TPA) cases, and >20 mitoses/slide in 13 (CpG) and 9 (TPA) cases, respectively. Clonal abnormalities were identified in 89 cases (56%). In 56 cases, the aberrant clone was detected in both cultures. Of these, the percentage of aberrant metaphases was similar in both cultures in 13, higher in CpG culture in 32 and higher in TPA culture in 11 cases. In 29 and 4 additional cases, a clonal abnormality was detected only in CpG or TPA culture, respectively. Thus, the percentage of abnormal karyotypes with CpG and TPA was 53 and 38%, respectively (p=0,005). “Typical” aberrations, including del(13q), +12, del(11q), del(17p) and del(6q), were detected in 16, 26, 15, 9, and 7 (CpG) cases, and in 10, 20, 16, 8, and 3 (TPA) cases, respectively. Translocations (balanced and unbalanced) were observed in 46 (CpG) and 27 (TPA) cases, whereas aberrations involving 14q32 were seen in 5 (CpG) and 4 (TPA) cases, respectively. Interphase FISH detected del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 53, 1, 5, 0, 0 and 1 cases, respectively, in which cytogenetic analysis was either normal or abnormal but did not show the specific change. Conversely, FISH did not detect del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 1, 1, 4, 1, 2 and 0 cases in which the specific change was visible at karyotypic level. FISH was performed on both CpG and TPA cultures in 9 selected cases harbouring an aberration for which a specific commercial probe was available (+12, n=3; del(13q), n=4; del(11q), n=1; and del(17p), n=1) and showing different percentages of aberrant cells in both cultures. In 3 of these cases, fresh uncultured material was also analyzed. The highest percentage of abnormal nuclei was observed in TPA culture in all cases. In the 3 uncultured samples the percentage of aberrant nuclei was similar to the CpG culture. In conclusion, our results confirm an increased detection rate of abnormalities in CLL by using CpG/IL2 stimulation. Interphase FISH can further increase the detection rate of recurrent abnormalities. However, neither cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques and the necessity of performing both.

scholarly journals Participation of suppressor T cells in the immunosuppressive activity of a heteroantiserum to human Ia-like antigens (p23,30).

1980 ◽  
Vol 151 (1) ◽  
pp. 257-262 ◽  
Author(s):  
S Broder ◽  
D L Mann ◽  
T A Waldmann
Keyword(s):  
T Cells ◽  
B Cell ◽  
Inhibitory Effect ◽  
Pokeweed Mitogen ◽  
Suppressor T Cells ◽  
Immunosuppressive Activity ◽  
Immunoglobulin Production ◽  
Cells Cultured ◽  
Simple Interaction

We studied the effects of an antiserum to human Ia-like antigens (p23,30) upon the polyclonal activation of normal B cells (cultured with various combination of irradiated and unirradiated T cells) to become immunoglobulin-secreting cells after stimulation with pokeweed mitogen in vitro. We found that the antiserum suppressed immunoglobulin production. The inhibitory effect did not appear to result from a simple interaction at the B-cell/monocyte level alone. Rather, the inhibitory effect required the presence of a radiosensitive subset of autologous suppressor T cells.

Inhibition of Antibody Production in Vitro with Bispecific CD20 X CD95 Antibodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1114-1114
Author(s):  
Kristina Nalivaiko ◽  
Martin Hofmann ◽  
Ludger Grosse-Hovest ◽  
Peter H Krammer ◽  
Hans-Georg Rammensee ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
B Cell ◽  
Antibody Production ◽  
Conflicts Of Interest ◽  
Death Receptor ◽  
Pokeweed Mitogen ◽  
Resting Cells ◽  
Cd20 Antibodies

Abstract Abstract 1114 Antibodies directed against the B-cell associated CD20 surface antigen can target normal as well as malignant B cells. They are sucessfully used for the treatment of B-cell derived leukemia and lymphoma and antibody mediated autoimmune disease, respectively. We have previously described that bispecific antibodies with specificity for CD20 and the death receptor CD95 are capable of inducing CD95 mediated apoptosis selectively in CD20-positive lymphoma cells. We now show that CD20 X CD95 hybrid antibodies induce apoptosis in pokeweed mitogen (PWM) activated B cells expressing CD95, but not in resting cells lacking it. Antibody production induced by PWM in vitro is profoundly inhibited. These results indicate that bispecific CD20 X CD95 antibodies may be used for the treatment of antibody mediated autoimmune disease. Compared to monospecific CD20 antibodies these reagents offer a new effector principle and specificity for activated rather than resting B cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals GENETIC CONTROL OF THE IMMUNE RESPONSE

1974 ◽  
Vol 140 (4) ◽  
pp. 977-994 ◽  
Author(s):  
Peter Lonai ◽  
Hugh O. McDevitt
Keyword(s):  
T Cells ◽  
B Cell ◽  
Tritiated Thymidine ◽  
Lymphocyte Transformation ◽  
Cross Reactivity ◽  
Pokeweed Mitogen ◽  
Thymidine Uptake ◽  
Soluble Antigen

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4°C followed by washing and incubation at 37°C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti-H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition.

Deletions of Chromosomes 6q and 13q, and Trisomy 4 Are the Most Common Cytogenetic Abnormalities in Waldenstrom Macroglobulinemia. Preliminary Results of a Multicentric Study.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1771-1771
Author(s):  
Florence Nguyen-Khac ◽  
Elise Chapiro ◽  
Sarah Mould ◽  
Carole Barin ◽  
Agnes Daudignon ◽  
...  
Keyword(s):  
B Cell ◽  
Recent Observation ◽  
Partial Trisomy ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Multicentric Study ◽  
Cytogenetic Abnormalities ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Trisomy 12

Abstract The genetic bases of Waldenstrom Macroglobulinemia (WM) are poorly understood. We have studied a cohort of 139 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM), by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). The sex ratio was 2.2M/1F, the average age at inclusion was 66 years [40–85]. The mean percentage of lymphoplasmacytic cells was 53% [8–97]. CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 7 probes CEP4, CEP12, 13q14, 11q22 ( ATM), 17p13 (TP53), IGH Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. Out of 110/137 successful karyotypes, 37% showed clonal abnormalities (9 additional cases had numerical changes of sexual chromosomes). Among abnormal karyotypes, there were 14 (34%) 6q deletions, 5 (12%) trisomy 4/partial trisomy 4, 4 (10%) trisomy 18, 3 (7%) trisomy 12. Using FISH, deletions of 6q21 were observed in 23/86 cases (27%), 13q14 in 12/89 cases (13%), TP53 9/85 cases (11%), ATM 6/81 cases (7%). Trisomy 4 was present in 9/82 cases (11%), and trisomy 12 in 4/86 cases (5%). No rearrangement of IGH was observed in the first 27 analyzed cases. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 27% with deletions of 6q21 (FISH), a low percentage compared to the literature [39–54%]. This could be explained either by the difference in the probe used or by the absence of selection of lymphoplasmacytic cells before cytogenetic analyses. Interestingly, we confirmed our recent observation that trisomy 4 was frequent in WM (12% if partial trisomy 4 was included). Furthermore we observed in this large series a frequent 13q14 deletion (13%). In conclusion, cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. 6q deletion is frequent compared to CLL or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. We didn’t observe cytogenetic involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. Searches for correlations with clinical and other biological parameters are ongoing.

Clinical and Pathological Features of Non-Hodgkin Lymphomas Harboring Concurrent t(14;18) and 8q24 Anomalies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3134-3134
Author(s):  
Ash A. Alizadeh ◽  
Matthew Anderson ◽  
Holbrook E Kohrt ◽  
Ragini M Shyam ◽  
Charles D. Bangs ◽  
...  
Keyword(s):  
Overall Survival ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Small Sample ◽  
World Health ◽  
Fish Analysis ◽  
Low Grade ◽  
Cytogenetic Abnormalities

Abstract Abstract 3134 Background: Concurrent t(14;18) and 8q24 translocations involving BCL2 and MYC in non-Hodgkin lymphomas (NHL) are rare, but are associated with a inferior overall survival (OS) regardless of the presenting or antecedent histological features (Johnson N, et al. Blood 2009). We sought to confirm these observations in an independent cohort of patients with NHL. Methods: Metaphase karyotypes and/or fluorescence in-situ hybridization (FISH) were used to identify cases of NHL with cytogenetic abnormalities involving 18q21 and 8q24 (BCL2 and MYC). Clinical and cytogenetic characteristics of these patients were assessed for correlations with pathological and clinical variables including outcome. Histological diagnoses were determined according to the 2008 World Health Organization Classification. Overall survival (OS) was calculated from the date a biopsy demonstrated an abnormality involving MYC to the last follow up date or death, as some cases acquired these lesions during their clonal evolution. Result: Among ∼1700 NHL patients diagnosed and/or treated at Stanford University Medical Center on whom cytogenetic studies were routinely performed, we identified 26 patients with evidence for concurrent cytogenetic abnormalities involving BCL2 and MYC. The histological diagnoses at the time of BCL2 and MYC rearrangements were available for 25 of the patients and included follicular lymphoma (FL1-2, n=3; FL3A, n=4), diffuse large B-cell lymphoma (DLBCL, n=2), and B cell lymphoma, unclassifiable, with features intermediate between Burkitt Lymphoma and DLBCL (BCLU, n=16). Cytogenetic analysis revealed that 13/26 cases with both BCL2 and MYC rearrangements had MYC translocations involving the immunoglobulin (Ig) loci. However, in striking contrast to the <10% prevalence of IgL or IgK partners in NHL harboring Ig-MYC, without Ig-BCL2 rearrangements, 9 of these13 cases (69%) involved IgL t(8;22), and only 4 of these 13 cases (31%) involved IgH t(8;14). 9/26 MYC translocations involved various non-Ig loci (35%). One case (1/26) demonstrated amplification of ?both the BCL2 and the MYC loci by FISH, while one case showed trisomy 8 (1/26). Three cases had FISH analysis only, precluding assessment of the MYC rearrangement partner. No correlation between MYC partner and histology was observed, although this analysis was limited by small sample sizes. Non-FL histology correlated with significantly poorer than expected outcome, with a median OS of 4 months compared to 2 y for FL (p=0.003). Interestingly, we found cases with low-grade follicular lymphoma (FL1-2) histology which harbored both BCL2 and MYC rearrangements. To better define the frequency of 8q24 anomalies in an unselected cohort of follicular lymphoma cases, FISH for MYC and BCL2 rearrangements was performed on 192 independent low-grade follicular lymphoma (FL1-2) cases. While a subset of these cases (71) showed the expected frequency of BCL2 rearrangements (72%) by FISH, none showed concomitant 8q24 anomalies by FISH, suggesting that routine FISH analysis of low-grade follicular lymphoma for MYC rearrangements would have a low diagnostic yield. Conclusion: NHL with rearrangements of both BCL2 and MYC are under-recognized due to lack of routine karyotyping or FISH analysis. Comprehensive analysis of BCL2 and MYC status should be performed on all HGBCL in the new WHO (2008) classification, though the low frequency of these lesions among FL tumors precludes a similar conclusion for low grade NHL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human immune response to group A streptococcal carbohydrate (A-CHO). I. Quantitative and qualitative analysis of the A-CHO-specific B cell population responding in vitro to polyclonal and specific activation.

1985 ◽  
Vol 161 (3) ◽  
pp. 547-562 ◽  
Author(s):  
F Emmrich ◽  
B Schilling ◽  
K Eichmann
Keyword(s):  
Immune Response ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Pokeweed Mitogen ◽  
Specific Antibodies ◽  
Group A ◽  
Precursor B Cells

The immune response to the group-specific carbohydrate of group A streptococci (A-CHO) provides an informative in vitro model for the investigation of several aspects of human anticarbohydrate immune responses. A-CHO-specific B cells can be polyclonally activated by pokeweed mitogen (PWM), and, specifically, by in vitro immunization with streptococcal vaccine. High levels of A-CHO-specific antibodies, mainly directed to the immunodominant side chain N-acetyl-D-glucosamine (GlcNAc), occur in healthy adult individuals. Serum antibody levels are reflected in high frequencies of precursor B cells among peripheral blood lymphocytes. In one particular case, greater than 15% of all B cells activated by PWM for IgM production were found to produce IgM anti-A-CHO antibodies, as determined in limiting dilution experiments, as well as by analyzing Ig concentrations in bulk culture experiments. The case with the lowest proportion observed had 0.3% A-CHO-specific B cells among IgM-producing B cells. Preferential PWM activation of anti-A-CHO-producing B cells could be excluded. The comparison of the proportions of anti-A-CHO IgM produced in vivo, and of B cells producing antibodies of this specificity in peripheral blood, suggests a similar distribution of specific precursor B cells in the antibody-producing lymphoid tissue compartments and in peripheral blood. However, nearly all specific antibodies produced in vitro belong to the IgM isotype, whereas IgG anti-A-CHO in high amounts, mostly exceeding the specific IgM, was found only among anti-A-CHO antibodies produced in vivo. Low anti-A-CHO IgG production was seen in polyclonally activated as well as in antigen-activated cultures, whereas, in contrast, total IgG was produced in considerable amounts after polyclonal activation. This suggests a different distribution pattern, and/or diverse differentiation requirements for anti-A-CHO-producing B cells, compared with other B cell species.

Detection of chromosomal changes in chronic lymphocytic leukemia using classical cytogenetic methods and FISH: application of rich mitogen mixtures for lymphocyte cultures

2016 ◽  
Vol 64 (4) ◽  
pp. 894-898 ◽  
Author(s):  
Dorota Koczkodaj ◽  
Sylwia Popek ◽  
Szymon Zmorzyński ◽  
Ewa Wąsik-Szczepanek ◽  
Agata A Filip
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mitotic Index ◽  
Cell Cultures ◽  
Lymphocytic Leukemia ◽  
Pokeweed Mitogen ◽  
Structural Aberrations ◽  
Chromosomal Changes

One of the research methods of prognostic value in chronic lymphocytic leukemia (CLL) is cytogenetic analysis. This method requires the presence of appropriate B-cell mitogens in cultures in order to obtain a high mitotic index. The aim of our research was to determine the most effective methods of in vitro B-cell stimulation to maximize the number of metaphases from peripheral blood cells of patients with CLL for classical cytogenetic examination, and then to correlate the results with those obtained using fluorescence in situ hybridization (FISH). The study group involved 50 consecutive patients with CLL. Cell cultures were maintained with the basic composition of culture medium and addition of respective stimulators. We used the following stimulators: Pokeweed Mitogen (PWM), 12-O-tetradecanoylphorbol 13-acetate (TPA), ionophore, lipopolysaccharide (LPS), and CpG-oligonucleotide DSP30. We received the highest mitotic index when using the mixture of PWM+TPA+I+DSP30. With classical cytogenetic tests using banding techniques, numerical and structural aberrations of chromosomes were detected in 46 patients, and no change was found in only four patients. Test results clearly confirmed the legitimacy of using cell cultures enriched with the mixture of cell stimulators and combining classical cytogenetic techniques with the FISH technique in later patient diagnosing.

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