IgH Gene Rearrangement Research Articles

Dense Methylation of Types 1 and 2 Regulatory Regions of the CD10 Gene Promoter in Infant Acute Lymphoblastic Leukemia With MLL/AF4 Fusion Gene

Infant acute lymphoblastic leukemia (ALL) displays distinct biologic and clinical features with a poor prognosis. The CD10-negative immunophenotype of infant ALL is a hallmark and provides a predictable signature of mixed-lineage leukemia (MLL) rearrangement. Although CD10 negativity reflects an earlier stage of B-cell development, complete IgH gene rearrangements (VDJH), found in almost half of the patients, show more mature IgH status. Discordance between immunophenotype and genotype of infant ALL suggests an aberrant process in immunophenotypic steps of differentiation or a secondary down-regulation of CD10 expression. In this study, CD10-negative infant ALL with MLL/AF4, CD10-positive infant ALL with germline MLL, CD10-positive pre-B ALL cell line, infant acute myeloid leukemia (AML; M5) with MLL/AF9 and pediatric AML (M2) with AML1/ETO were analyzed for VDJH status and methylation of CD10 gene promoters. Three of the 4 infant ALL samples showed complete rearrangements of the VDJH gene with productive joints. Bisulfite sequencing of CD10 type 1 and 2 promoters showed that more than 84% of the cytosine-phosphate-guanine (CpG) dinucleotides identified were methylated in all 3 CD10-negative infant ALL samples with MLL/AF4. The CpG dinucleotides distributed in the clusters of putative Sp1-binding sites and functionally active regulatory regions of the promoters were fully methylated. In contrast, none of the CpG dinucleotides were methylated in the CD10-positive ALL samples. Structural evidence of dense methylation in the CD10 gene promoter suggested that methylated transcription factor binding sites contribute to CD10 silencing as an epigenetic mechanism.

Pheno- and Genotypic Features of Epstein-Barr Virus Associated B-Cell Lymphoproliferations in Peripheral T-Cell Lymphomas

Among the 300 peripheral T-cell lymphomas (PTCL) searched for EBV positive non-resting B-cells by EBER in situ hybridization 12 have been identified with various forms of EBV-driven B-cell proliferation. This could be categorized into three major forms. i. In the first form scattered immature, mononuclear B-cells of immuno-, centroblastic type with CD20+. CD30+ CD45+, LMP1+ phenotype, reactive appearance and polyclonal immunoglobulin heavy chains gene rearrangement (IgH-R) were admixed to the PTCL cells. ii. The second form mimicked diffuse large B-cell lymphoma as homogenous sheets, largely demarcated from the PTCL, of mononuclear, immature B-cell of CD20+, CD30+, CD45+, LMP1+, EBNA-2+ phenotype but with lack of monoclonal IgH-R were present. iii. In the third form scattered Hodgkin-Reed-Sternberg (HRS) type of cells were noticed which exhibited the CD15+/-, CD20-/+, CD30+, CD45-, LMP1+, EBNA-2- phenotype and in 50% showed clonal IgH gene rearrangement in whole tissue DNA extract. The IgH associated transcription factors' (OCT2, BOB.1/OBF.1, PU.1) expression patterns in these cells corresponded to those of HRS cells in cHL. Based on analysis of 65 PTCLs, we have identified in the positive cases a highly significant increase of EBV+ small, reactive, resting B-cell compartment (75.9 / 100 HPF in PTCL vs. 1.5 / 100 HPF in control lymph nodes) likely to be due to the decreased immune surveillance. This progressive accumulation of EBV+ by-stander B-cell population in PTCLs might be the source of various B-cell proliferations, which in any form represent major diagnostic pitfalls and require a careful differential diagnostic procedure.

Presence of immunoglobulin heavy chain rearrangement in so-called “plasma cell granuloma of the lung”

Inflammatory pseudotumor of the lung appears to be a set of heterogeneous disorders. Histologically, three subtypes of pulmonary IPTs have been delineated. Among these, plasma cell granuloma (PCG) is characterized by prominent lymphoplasmacytic infiltration, and PCG has been added to the list of differential diagnostic problems of mucosa-associated lymphoid tissue (MALT) type lymphoma. To investigate the presence or absence of monoclonal B-cell proliferation, we analyzed the immunohistological and genotypic findings in three cases of pulmonary PCGs. Histologically, the three lesions were characterized by severe infiltration of mature plasma cells, plasmacytoid cells, and small lymphocytes intermixed. Scattered Russell bodies (intracytoplasmic inclusions) were present in all three cases, but there were no Dutcher bodies (intranuclear inclusions) or centrocyte-like cells. Immunohistochemical studies of light chain determinants demonstrated the polytypic nature of B-cells. There was no CD5(+), CD43(+) or cyclin D1(+) B-lymphocytes in any of the three lesions. There were no lymphoepithelial lesions detected within any of the three lesions even by immunostaining for cytokeratin. However, polymerase chain assay for immunoglobulin heavy chain gene demonstrated a clonal band in one of the three cases. It currently remains unclear whether this one case, demonstrating IgH gene rearrangement in our series, could be a sign of the prelymphomatous stage (e.g. incipient MALT type lymphoma) or merely represents an exaggeration of normal B-cell clonal response.

Endothelial Progenitor Cells within the Bone Marrow Microenvironment of Multiple Myeloma Patients Display Stem Cell Traits.

Abstract 2816Poster Board II-792 Background:Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM). Tumor growth and progression are promoted in MM through paracrine interactions between tumor cells, stromal cells, and matricellular components of the BM microenvironment. Endothelial progenitor cells (EPCs) are a key component of this microenvironment that mediate neovascularization. EPCs in MM patients contribute to neoangiogenesis and enhance the growth of tumor cells through the secretion of VEGF and IL-6. We found that EPC levels are increased in MM patients and covary with disease severity. Further, EPCs from MM patients were found by X chromosome inactivation studies to be clonally restricted, and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells. Based on new data regarding clonal restriction and on maintenance of MM EPCs in long-term culture compared both to EPCs obtained from normal controls and to human umbilical vein endothelial cells, the present study explored the hypothesis that EPCs in the vascular MM niche may be a source of MM-initiating cells with stem cell traits. The existence of MM stem cells is consistent with the finding of inevitable relapse of MM patients following response to treatment. We now show that clonal MM EPCs can be maintained in long term culture, unlike EPCs from healthy controls. In addition, our gene expression data show that MM EPCs have a higher level of expression of genes associated with stem cell function (e.g., GREM1, RUNX2, HOXC6, FOXF2) compared to the same patients' MM tumor cells, as well as to control EPCs. Genes such as WNT5A, SOX9, and ADAM12, which are involved in differentiation, an integral stem cell process, were also highly expressed in MM EPCs (manuscript in preparation). Stem cells have the abilities of self-renewal and differentiation. Cancer stem cells (CSCs), such as those found in acute myeloid leukemia, share these characteristics. Similar to hematopoietic stem cells (HSCs), CSCs are known to engraft to spleen and BM, where they proliferate and are capable of forming spleen colonies. To determine whether MM EPCs were able to home and proliferate in a similar fashion, we intravenously injected them into non-lethally irradiated mice. Using flow cytometry we examined the prevalence of human EPCs that expressed differentiation markers. The formation of spleen colonies was also assessed. Methods:EPCs (>98% vWF/CD133/ KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. Nine irradiated NOD/SCID mice were injected (i.v.) with 106 EPCs derived from 6 patients newly diagnosed with advanced MM. Spleens and BM were harvested 2-4 weeks later. The cells were analyzed by immunofluorescence and flow cytometry to determine the presence of human cell differentiation markers, including CD45 (a hematopoietic differentiation marker) and CXCR4 (a differentiation/migration marker). Results:EPCs cultured from 4 newly diagnosed patients were maintained in culture up to 7 months. Flow cytometry showed the engraftment of human cells to the spleen and BM of all mice 2-4 weeks following injection. The mean number of CD45+ cells in the experimental group increased within the spleen by 54.1 fold and in the BM by 35.9 fold compared to vehicle-injected controls. Similarly, CXCR4+ cells in the experimental group increased within the spleen by 15.9 fold and in the BM by 12.5 fold. In addition, of the 9 spleens harvested, 1 spleen showed colony formation, while none of the vehicle-treated animals displayed colony formation. Conclusion:Results show a high degree of proliferation of CD45+ cells within the spleen and BM following injection of MM EPCs. Since CD45 is a marker of hematopoietic differentiation, the proliferation of cells expressing this marker suggests that human hematopoietic cells expanded after EPCs were injected. Furthermore, the proliferation of cells expressing CXCR4+, which is required for HSC quiescence and which also plays a role in EPC differentiation, indicates that EPCs are part of the stem cell niche in MM. In addition to the sizeable increase seen in the number of cells expressing these differentiation markers, 1 of 9 mice injected, formed spleen colonies. Taken together, these data suggest that a portion of the MM EPC population possesses traits similar to those of HSCs and CSCs, and this thesis warrants further investigation. Disclosures:No relevant conflicts of interest to declare.

Unremitting Presence of Marrow Immunoglobulin Heavy Chain Gene Rearrangement (IGH-GR) 3 Months Following Autotransplant Is Associated with Poor Outcome in Disease Control in Patients with Multiple Myeloma (MM).

Abstract 1807Poster Board I-833In the study, we sought to determine the role of serial monitoring of immunoglobulin heavy chain monoclonality by IGH-GR tests in symptomatic MM patients who have been uniformly treated with induction chemotherapy, autotransplant following high-dose melphalan-based ablative therapy and post-transplant maintenance. Between September 2006 and April 2009, 51 patients (median age 60, range 37 – 77 years) were evaluated for conventional cytogenetics, myeloma FISH panel and IGH-GR on bone marrow samples prospectively every 3 months (mo) starting before initiation of therapy. The myeloma FISH panel included probes for chromosomes 1, 4, 11, 13, 14, and 17. IGH-GR test was performed by the InVivoScribe Technologies IGH Gene Rearrangement Assay using PCR primers targeting the conserved framework within the V-J region and J regions. A segment of the HLA-DQA1 gene was amplified as a control for patient DNA quality. 22 patients had IgG, 12 IgA, 13 light chain only and 4 nonsecretory MM. 30 patients (59%) were previously treated before and 16 (31%) had prior relapse. 23 patients (45%) received a tandem transplant and all patients received a maintenance regimen that included bortezomib and lenalidomide. Of the 51 patients, 11 (22%) had pre-therapy cytogenetic abnormalities (CA), 22 (43%) FISH abnormalities (FA) and 22 (43%) positive IGH-GR for monoclonality. Detection of positive IGH-GR was not associated with either CA (Fisher's exact test, p = 0.32) or FA (p = 0.14). The 3-year overall survival was 92% with 4 deaths. Relapse occurred in 10 with a 3-year cumulative index (CI) of 20%. In 3 to 12 mo following the first transplant, CA were seen in 7 (14% - 3 mo), 4 (8% - 6 mo) and 3 patients (6% - 12 mo); for FA, 9 (18%), 5 (10%) and 4 (8%); for IGH-GR, 10 (20%), 6 (12%) and 8 (17%). Findings before therapy were prognostic in survival and disease control. The 3-yr Kaplan-Meier survival was 80% for 11 patients (22%) with pre-therapy CA compared to 96% for those (n = 40) without CA (Logrank p = 0.02), 83% for 22 patients with FA (43%) compared to 100% for those (n = 29) without FA (p = 0.03), and 87% for 22 positive for IGH-GR (43%) versus 96% for those (n = 29) negative for IGH-GR (p = 0.03). Pre-therapy CA but not FA nor IGH-GR was significant for the CI of relapse: 33% vs. 15%, p = 0.06. Of the post-transplant follow-up on the CI of relapse, only the 3-mo evaluation was prognostically significant (Table). Although the current findings require further study for confirmation, the data suggests a potential role of IGH-GR tests in monitoring treatment responses in patients with MM. Additional primers such as the ones used in the BIOMED-2 may increase the sensitivity of test.Cumulative Index of RelapsePre-therapy3-mo post-transplant6-mo post-transplant12-mo post-transplantCA + vs. -33% vs. 15% P=0.0643% vs. 18% P = 0.0250% vs. 23% P = 0.0333% vs. 13% P = 0.7FA + vs. -17% vs. 17% P = 0.5233% vs. 16% P = 0.0240% vs. 19% P = 0.6725% vs. 15% P = 0.16IGH-GR + vs. -28% vs. 9% P = 0.5730% vs. 19% P = 0.00129% vs. 17% P = 0.325% vs. 19% P = 0.37 DisclosuresMyint:Seattle Genetics, Inc.: Research Funding.

IGH and IGK gene rearrangements as PCR targets for pediatric Burkitt's lymphoma and mature B-ALL MRD analysis

We recently reported that minimal residual disease (MRD) and minimal disseminated disease (MDD), assessed by long-distance PCR (LD-PCR) for t(8;14), are negative prognostic factors in mature B-cell acute lymphoblastic leukemia (B-ALL) and in Burkitt's lymphoma (BL). However, t(8;14) is detectable in only about 70% of patients, thus preventing MRD studies by this approach in the remaining patients. At present, no molecular assays have been reported for MRD and MDD analysis in t(8;14)-negative patients. The aim of our study was to evaluate the characteristics of patient-specific immunoglobulin (Ig) gene rearrangements as RQ-PCR targets for MRD analysis, in order to extend MRD studies to those patients who are not eligible for the LD-PCR assay. The study was performed according to the guidelines of the European Study Group on MRD detection in ALL (ESG-MRD-ALL). Overall, 36 B-ALL and 19 BL cases were analyzed. Multiple PCR reactions were performed for each sample to identify heavy and kappa light-chain rearrangements. A total of 97 RQ-PCR targets (62 for B-ALL, 35 for BL) were analyzed for sensitivity. The rearrangement pattern identified was similar to that reported for normal peripheral blood lymphocytes. In 88% of the targets, a sensitivity of at least 10−4 was achieved. In 87% of patients (84% of B-ALLs, 95% of BLs) at least one sensitive target was available. All PCR targets identified at diagnosis were preserved at relapse. Our results suggest that MDD and MRD can be successfully studied using a single sensitive Ig target in the great majority of B-ALL and BL cases. The combination of LD-PCR and Ig-based assays will allow MRD analysis in virtually all of the patients.

High Frequency of Clonal Immunoglobulin Receptor Gene Rearrangements in Sporadic Histiocytic/Dendritic Cell Sarcomas

The diagnosis of histiocytic/dendritic cell (H/DC) sarcomas is currently based on morphology and the presence of immunophenotypic features of H/DC differentiation. The issue whether clonal immunoglobulin receptor gene rearrangements are present in H/DC sarcomas has been debated over decades until the recent data by Feldman et al, which provided compelling evidence that patients with follicular lymphoma and concurrent/synchronous H/DC sarcoma share identical genotypic features, suggested the possibility of transdifferentiation or dedifferentiation of 2 otherwise morphologically and immunophenotypically distinctive neoplasms. Here we investigated the molecular characteristics of 23 patients with sporadic H/DC sarcoma. Nine of the 23 cases (39%) showed clonal IGH (+/-IGK) gene rearrangements, whereas 2 (9%) cases showed only clonal IGK gene rearrangements, which were further validated and confirmed by direct DNA sequencing. One histiocytic sarcoma showed t(14;18) by quantitative-polymerase chain reaction, which was confirmed by fluorescence in situ hybridization analysis showing IGH/BCL2 fusions in neoplastic histiocytes. Notably, all IGH/IGK-positive H/DC sarcomas were negative for B-cell-associated transcription factors PAX5 and BOB.1, whereas 4 of 7 IGH/IGK-positive histiocytic sarcoma cases were positive for Oct2. In addition, no evidence of Epstein-Barr virus infection was detected in 8 of 11 IGH/IGK-positive H/DC sarcoma cases by in situ hybridization, suggesting that Epstein-Barr virus infection may not play an important role in the pathogenesis of these tumors. This study provides evidence that clonal immunoglobulin receptor gene rearrangements may be detected at a high frequency in sporadic H/DC sarcomas. The findings suggest that a large subset of H/DC sarcomas have inherited B-cell genotypes, thus providing new insights for the pathogenesis of these rare but aggressive neoplasms.

Promoter DNA methylation of CD10 in infant acute lymphoblastic leukemia with MLL/AF4 fusion gene

10045 Background: Infant ALL displays distinct biologic and clinical features with a poor prognosis. The CD10-negative immunophenotype of infant ALL is a hallmark and provides a predictable signature of MLL rearrangements. While CD10 negativity reflects an earlier stage of B-cell development, complete IgH gene rearrangements (VDJH) show more mature IgH status. Discordance between immunophenotype and genotype of infant ALL suggests an aberrant process in immunophenotypic steps of differentiation or a secondary down-regulation of CD 10 expression associated with MLL rearrangements. We performed methylation analysis of full promoter regions of the CD10 gene to investigate epigenetic mechanisms responsible for CD10 negativity. Methods: CD10-negative infant ALL with MLL/AF4, CD10-positive infant ALL with germ-line MLL, CD10-positive pre-B ALL cell line, infant AML (M5) with MLL/AF9 and pediatric AML (M2) with AML1/ETO were analyzed for VDJH status and methylation of CD10 gene promoters. Results: Three of 4 cases with infant ALL revealed complete rearrangements of VDJH gene with productive joints. Bisulfite sequencing of CD 10 type 1 and 2 promoters identified more than 84% of methylated CpG dinucleotides in all three CD10-negative infant ALL cases with MLL/AF4. The CpG dinucleotides distributed in the clusters of putative Sp 1 binding sites and functionally active regulatory regions of the promoters were fully methylated. In contrast, none or a few of the CpG dinucleotides were methylated in the CD10-positive ALL, AML (M5) with MLL/AF9 or AML (M2) with AML1/ETO. Conclusions: Structural evidence of dense methylation in the CD 10 gene promoter suggested that methylated transcription factor binding sites contribute to CD10 silencing as an epigenetic mechanism. [Table: see text] No significant financial relationships to disclose.

PCR clonality detection in Hodgkin lymphoma

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.

Chromosome 14 copy number-dependent IGH gene rearrangement patterns in high hyperdiploid childhood B-cell precursor ALL: implications for leukemia biology and minimal residual disease analysis

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is generally a clonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor three rearrangements, a pattern that otherwise may be misinterpreted to be oligoclonal. Oligoclonal IGH rearrangements, on the other hand, may be instable at relapse and should therefore not be used for minimal residual disease analysis. We thus investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 HeH BCP ALL with either two (13%) or three copies (87%) of chromosome 14. HeH cases (44%) had an oligoclonal IGH rearrangement pattern, but true oligoclonality--after correction for the respective copy number of IGH alleles--was only 16%. Monoclonal and oligoclonal HeH cases had predominantly V(H) to preexisting DJ(H) recombinations, a finding that contrasts with oligoclonal cases of other major genetic BCP ALL subgroups in which V(H) replacements prevail. We conclude that for the precise assessment and correct interpretation of clonality patterns in BCP ALL, the IGH allele copy number has to be taken into consideration.

Very early onset of an acute myeloid leukemia in an adult patient with B‐cell lymphoblastic leukemia

We report on a case of a 30-year-old male with acute B-lymphoblastic leukemia (B-ALL) with immunophenotype CD19(+), CD22(+), CD20(+), CD10(+), with aberrant expression of CD13 and CD117, and IgH gene rearrangements. Three months after treatment with GMALL-2003 and Ida/FLAG protocols bone marrow showed predominance of blasts with myeloid morphology and phenotype MPO(+), CD13(+), CD33(+), CD64(+), CD15(+), CD56(+), EVI-1 gene overexpression and lack of IgH rearrangements. The case is the first report of a very early emergence of myeloid leukemia during the induction treatment for B-ALL in an adult patient. Different pathogenetic mechanisms are discussed - clonal evolution or selection, lineage switch or development of a de novo or therapy-induced leukemia.

Early onset of acute lymphoblastic leukemia with MLL rearrangement after autologous stem cell transplantation for multiple myeloma

Dear Editor, We would like to present our experience with a patient with multiple myeloma (MM) who developed acute lymphoblastic leukemia (ALL) with mixed lineage leukemia gene (MLL) rearrangement only 1 year after administration of alkylating agents. A 61-year-old woman presented with fatigue and was introduced to our hospital in January 2007. Serum immunoelectrophoresis showed increased monoclonal IgD (115.0 mg/dL) and λ light chain, and urine immunoelectrophoresis showed increased monoclonal Bence–Jones protein (4,360 mg/g creatinine). The bone marrow (BM) was infiltrated by malignant plasma cells (50% of nucleated cells) and conventional G-banding cytogenetics revealed normal karyotype (46,XX [20/20]). A diagnosis of MM was made. After four cycles of high-dose dexamethasone, we mobilized and collected her peripheral blood stem cells using cyclophosphamide (4 g/m) and granulocyte-colonystimulating factor in July 2007. She underwent autologous stem cell transplantation (ASCT) in August 2007 with highdose melphalan (200 mg/m) as a conditioning regimen. She made recovery without severe complication. We evaluated the response of treatment as very good partial remission. In July 2008, although she had no symptom, investigation revealed pancytopenia. The BM was heavily infiltrated by blasts (94%) with the immunophenotype as B cell lymphoblasts: CD19+, CD79a+, κ−, l−. G-banding chromosomal cytogenetics revealed chromosomal abnormalities: 46,XX,t(1;11)(p32–34;q23) [4/6]. Fluorescence in situ hybridization (FISH) examination showed split signals of MLL gene. A diagnosis of B-ALL with MLL rearrangement was made. She was commenced on the JALSG 202-O protocol (Clinicaltrials.gov; study ID:NCT00131027) and achieved hematological complete remission. We performed IgH gene rearrangement studies on genomic DNA extracted from the BM aspirates at the time of diagnosis of MM and that of ALL. Southern blotting (SRL inc., Nakagyo-ku, Kyoto, Japan) test showed different monoclonal bands on DNA extracted from both BM aspirates (Fig. 1). From these findings, we concluded that her MM and ALL were derived from different clones. Lau et al. [1] reported a patient with MM that terminated in ALL. Lau indicated that two diseases arose from different clones by IgH gene rearrangement analysis. Cases of secondary ALL supposed to be Topoisomerase II inhibitor-related were previously reported [2], and Lau’s case was administered doxorubicin. Although we did not administer Topoisomerase II inhibitor, we detected chromosomal abnormality of 11q23 that is typically determined from cases of Topoisomerase II inhibitor-related leukemia [2]. To our knowledge, this is the first report describing alkylating-agent-related ALL with MLL rearrangement. Our hypothesis is that her lymphoid progenitor cells had initial instability or a mutation that cannot be detected by conventional G-banding test, and different types of DNA modification separately led to MM and ALL. We suppose modification leading to ALL was MLL rearrangement. This is consistent with the reports that MLL rearrangement is Ann Hematol (2009) 88:813–814 DOI 10.1007/s00277-008-0680-8

Detection of immunoglobulin heavy chain gene rearrangement in b lymphoid malignancies in a teaching hospital in Northeastern Malaysia

<h3>Aims</h3> The aim of our study was to evaluate the incidence of clonal immunoglobulin heavy chain gene rearrangement in B lymphoid malignancies. <h3>Methods</h3> This prospective study was conducted at Hospital Universiti Sains Malaysia from May 2007 to May 2008. All cases of B lymphoid malignancies were included. Peripheral blood samples were collected and analysed by PCR for detection of clonal IGH gene rearrangement. Analyses were performed using IGH gene rearrangement assay with three different primers from InVivoScribe Technologies, USA. <h3>Results</h3> A total of 17 samples were collected from newly diagnosed B lymphoid malignancies which were B acute lymphocytic leukaemia (B ALL), B chronic lymphocytic leukaemia (B CLL) and non-Hodgkin lymphoma in leukaemic phase. We detected clonal IGH gene rearrangement in 82.4% (14/17) which was consistent with the presence of a clonal cell population. Results cannot be reported on three samples because there were DNA of insufficient quantity and quality for analysis. <h3>Discussion</h3> PCR analysis of IGH gene rearrangement was found to be an efficient technique for the initial determination of clonality in lymphoid proliferations. It is one of the most sensitive and valuable clonal markers in the diagnosis of B lymphoid malignancy and an important supplement to morphological diagnosis and immunophenotyping.

Immunoglobulin genes and T-cell receptors as molecular markers in children with acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is a malignant clonal disease, one of the most common malignancies in childhood. Contemporary protocols ensure high remission rate and long term free survival. The ability of molecular genetic methods help to establish submicroscopic classification and minimal residual disease (MRD) follow up, in major percent responsible for relapse. The aim of the study was to detect the frequency of IgH and TCR gene rearrangements and their correlation with clinical parameters. Forty-one children with ALL were enrolled in the study group, with initial diagnosis of IgH and TCR gene rearrangements by polimerase chain reaction (PCR). MRD follow-up was performed in induction phase when morphological remission was expected, and after intensive chemiotherapy. In the study group IgH rearrangement was detected in 82.9% of children at the diagnosis, while TCR rearrangement was seen in 56.1%. On induction day 33, clonal IgH rearrangements persisted in 39% and TCR rearrangements in 36.5% of children. Molecular analysis of genetic alterations and their correlation with standard prognostic parameters show the importance of risk stratification revision which leads to new therapy intensification approach. MRD stands out as a precise predictive factor for the relapse of disease.

Cytokine and Molecular Analyses of Intraocular Lymphoma

Purpose: The authors investigate the efficacy of using the cytokine levels and clonal heavy-chain immunoglobulin (IgH) gene rearrangements in the vitreous as adjunctive tools to diagnose intraocular lymphoma (IOL). Methods: The IL-10 and IL-6 levels and IgH gene rearrangements were analyzed in vitreous samples from 8 cases of IOL and in 14 uveitis patients. Results: The level of IL-10 with an IL-10/IL-6 ratio > 1 was significantly higher in all eyes with IOL. B-cell monoclonality was detected in only 5 of 8 eyes with IOL. Conclusions: The measurements of the levels of cytokines are valuable as a reliable biomarker.

Primary Cutaneous CD4+ Small/Medium-sized Pleomorphic T-cell Lymphoma Expresses Follicular T-cell Markers

Cutaneous CD4 small/medium-sized pleomorphic T-cell lymphoma (CSTCL) is a cutaneous T-cell lymphoma defined by a predominance of small-to-medium-sized CD4 pleomorphic T cells, with a favorable clinical course. Cases are also characterized by the presence of a rich infiltrate of reactive B cells. Recently, it has been reported that follicular helper T cells (TFH cells) display a distinct gene expression profile, positive for PD-1, CXCL13, and BCL-6. We report for the first time the expression of PD-1 and other TFH cell markers in CSTCLs and discuss its biologic significance. Sixteen CSTCLs were included in this study, and also 20 reactive inflammatory conditions, 10 primary cutaneous marginal zone, 10 follicular center lymphomas, and 5 primary CD30 cutaneous lymphomas. They were immunohistochemically analyzed for a large panel of markers. Double immunoperoxidase labeling of paraffin sections was performed for PD-1, OCT-2, and BCL-6. Clonal Ig and T-cell receptor rearrangements and Epstein-Barr virus-encoded RNA expression were also evaluated. Morphologic and clinical data were reviewed. Histologic examination showed a dense polymorphic lymphoid infiltrate throughout the dermis. Atypical large CD4 cells were positive for PD-1, CXCL13, and BCL-6 in all cases, and were attached in small clusters, or formed rosettes around CD30/OCT-2+ B blast cells. Epstein-Barr virus was not apparent in any of the cases. A dominant T-cell clone was identified in 14 cases, whereas polymerase chain reaction IgH gene rearrangement studies showed that all cases were polyclonal. None of the patients had lymphadenopathy or showed any evidence of systemic disease, nor did they have any previous history of mycosis fungoides or drug reactions. FTH cell markers are not exclusive to angioimmunoblastic lymphadenopathy but may also be seen in neoplastic cells of CSTCLs. Moreover, these findings suggest that B-cell stimulation by FTH could also take place in some cutaneous T-cell lymphomas.

Lymphocyte depleted Hodgkin lymphoma: an evaluation with immunophenotyping and genetic analysis

Lymphocyte depleted classical Hodgkin lymphoma (LDHL) is a vanishing category of classical Hodgkin lymphoma (CHL); many cases previously placed in this category are now recognised as diffuse large B-cell lymphoma (DLBCL), anaplastic large-cell lymphoma (ALCL), or nodular sclerosis CHL with lymphocyte depletion. In addition, the recent recognition of high grade B-cell lymphomas intermediate between DLBCL and CHL (grey-zone lymphomas) raises the question of whether LDHL exists at all as a category of CHL. We studied eight cases that fulfilled diagnostic criteria of LDHL according to the 2008 WHO Classification. The cases involved lymph nodes (7 cases) and pleura (1 case) from four males and four females (age 30–71 years; median 62 years). All tumors contained numerous Hodgkin-Reed-Sternberg (HRS) cells, fibroblasts and histiocytes and scattered lymphocytes. In three cases the tumors had a more diffuse fibrotic appearance, while in five cases they appeared reticular and anaplastic. Neoplastic cells in all cases expressed CD30, CD15, fascin, weak PAX5 and MUM-1 and lacked CD45, Alk-1, EMA, CD3, CD68, Mart-1 and cytokeratin. Oct.2 and/or Bob-1 were expressed in all cases. Two cases variably expressed CD20 but were CD79a negative. Four cases were positive for EBV. All the four cases with adequate DNA had clonally rearranged IGH genes. The combined morphologic, immunophenotypic and molecular genetic features of this group of cases distinguish LDHL from other disease entities, including grey-zone lymphomas.

Different Subclones of Adult Acute Lymphoblastic Leukemia Detected by Clonal Igh and TCR Markers Behave Differently during Remission and at Relapse

INTRODUCTION. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient–specific targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). However they might be unstable during the disease course. False-negative results due to clonal evolution are the major disadvantage of using Ig/TCR gene rearrangements as PCR targets for MRD detection. In order to minimize false-negative results, it is necessary to use more PCR targets for MRD monitoring.PATIENTS AND METHODS. Bone marrow samples of 34 ALL patients (27 B-ALL, 7 T-ALL) were analyzed for IgH and TCR gene rearrangement by means of PCR method at diagnosis, after induction and consolidation of remission, before and after stem cell transplantation and during maintenance therapy.RESULTS. Clonal IgH and TCR gene targets for MRD detection were identified in 97% of the patients (33/34). In 70% of the patients (23/33) two or more specific markers for MRD monitoring were detected. Three clonal IgH gene rearrangements were discovered in 2 patients. We wished to investigate whether it was important to monitor two or more targets, because of the dynamics of various markers (subclones) may be different). Prolonged MRD monitoring with 2–3 markers was carried out in 9 patients on different stages of treatment. In 4 of 9 patients (3 precursor B-ALL, 1 pre-T-ALL) the results of MRD detection by 2–3 clonal markers at different time points were similar. And in 5 of 9 patients (3 precursor-B-ALL, 1 precursor T-ALL, 1 B-mature ALL) marker detectability during treatment was dissimilar and not parallel. Of note, 4 of those 5 patients with varying dynamics of different subclones relapsed. Relapse was not observed in the B-mature ALL patient. No relapses were detected in 4 patients with similar dynamics of MRD markers. Bone marrow samples of 8 adult ALL patients were subjected to a detailed analysis of IgH and TCR gene rearrangements by PCR method at diagnosis and relapse. In most patients (7/8) rearrangements identified at diagnosis were present also at relapse. In one patient with precursor-B-ALL one clonal incomplete IgH gene rearrangement (DH-JH) detected at diagnosis was lost at relapse and another one DH-JH rearrangement was preserved. Of note, acute myeloid leukemia was diagnosed at relapse in this patient.CONCLUSIONS. Comparative PCR analyses of IgH and TCR gene rearrangements did not detect differences between newly diagnosed and relapsed ALL although in a small group of adult patients (unlike childhood ALL). Our data indicate the necessity to monitor MRD in ALL patients with more than one PCR target in order to minimize false-negative results and to predict the course of the disease. The detectability of various MRD markers (subclones) during treatment may differ and this fact could predict a higher probability of relapse.

Validation of CLL FISH Panel Scoring by Members of the Chronic Lymphocytic Leukemia Research Consortium.

Fluorescence in situ hybridization (FISH) probes and analysis methods for B-cell Chronic Lymphocytic Leukemia (CLL) vary extensively among cytogenetic laboratories. This is not unexpected, as neither national nor international standards have been established for most FISH studies. Lack of standardization is problematic when data collected at multiple institutions are used for clinical correlative studies. To circumvent such problems, the five participating laboratories in the CLL Research Consortium (CRC) designed and executed a joint CLL FISH validation study.Methods: Initially a survey was sent to assess equipment, methods and experience with FISH for CLL. In a pilot study to compare laboratory performance in scoring patient samples, slides from ten patients were prepared and sent to each participating lab to be hybridized with five probe sets (= 50 hybridizations) and analyzed according to their local protocol. In a second pilot study, slides from two patient samples and identical probe sets were sent to the participating labs where hybridization and analysis were carried out according to their local protocol. Next, technologists and directors from all participating labs attended a workshop where technologists working in pairs scored nuclei together, techniques and scoring criteria were established, and consensus reached on other concerns. In a proficiency test nine months after the workshop, slides from two patient samples (10 hybridizations) were hybridized and scored according to each lab's protocol and results shared using a common reporting form.Results: Survey results indicated that four labs used the same commercially available CLL FISH panel, and one used a combination of probes from the same vendor plus several home-brew probes. Each lab scored between 100 and 200 nuclei per hybridization site, and each independently set normal cutoff values. The FISH panel included probes to detect 11q, 13q, and 17p deletions, trisomy 12, and IGH gene rearrangement. One lab included probes to detect 6q deletion. In the first pilot study each lab used their hybridization methods, probe sets, and scoring criteria. Differences among labs were observed due to variations in probe strategy, reporting of anomalies, and perhaps most important, scoring criteria. Probe strategy differences resulted in variable reporting of 11q- vs monosomy 11 and 12q duplication vs trisomy 12. Some participants reported 13q-x1 and 13q-x2 as subclones and some reported only 13q-. One lab reported an IGH rearrangement whereas the others scored IGH as normal. In the second pilot study each lab used the same methods and probe sets to facilitate comparison of scoring by the technologists. All labs correctly identified the abnormalities, and there were no false positive results. Minor scoring differences were attributed to variation in scoring criteria or inexperience with an unfamiliar FISH probe strategy. The proficiency test that followed the workshop demonstrated 100% concordance in identification of abnormalities. Inter-lab scoring was much improved compared to the first pilot study. The only exceptions were a 13q- range of 72–90% in one case, and a 17p- range of 38–67% in another case.Conclusion: The pilot studies identified a need to develop common scoring criteria. The subsequent workshop and proficiency test demonstrated that the collaborative effort resulted in more standardized scoring among the CRC laboratories. Our collaborative study emphasizes the need to establish rigorous standards and guidelines for FISH procedures and scoring criteria. Standardization of FISH methods among participating laboratories will enhance the confidence in FISH studies for both clinical applications and cooperative intergroup clinical research.

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

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; andif 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.