Clonal immunoglobulin gene rearrangements in primary mediastinal clear cell lymphomas

Cells from two patients with primary mediastinal tumors of clear cell type were characterized by immunological and molecular biological techniques. In both cases a B cell immunophenotype was suggested by the positive staining for the B1 (CD20), B4 (CD19), Leu 14 (CD22), as well as staining with other B monoclonal antibodies by immunohistochemistry. However, no definitive evidence for the expression of Ig light or heavy chains at the protein level was found. Southern blot analysis of Ig heavy and light chain gene rearrangements revealed clonal B cell populations in both cases. There was no indication of a somatic joining of T cell receptor (TCR) genes using probes for TCR beta and or TCR gamma genes. Thus, our results suggest a clonal B cell origin of clear cell lymphomas in the mediastinum.     

Clonal immunoglobulin gene rearrangements and normal T-cell receptor, bcl-2, and c-myc genes in primary cutaneous B-cell lymphomas

Fourteen cases of primary cutaneous B-cell lymphomas were investigated at the immunohistochemical and molecular level to further characterize this newly defined entity. Neoplastic cells from all cases, phenotyped with a panel of monoclonal antibodies, were positive for HLA-DR, for the B-cell markers CD19, CD22, but not CD23 (except one case), and negative for the T-cell marker CD2. Monoclonal immunoglobulin light chains were demonstrated in six cases. The reactivity with the Ki-67 monoclonal antibody indicated that the neoplastic cells are proliferating. In five biopsies the presence of dendritic cells infiltrating the neoplastic areas was revealed using the monoclonal antibody Kim4b. By Southern blot analysis, clonal rearrangement of the immunoglobulin heavy chain gene (involving one or both alleles) was shown in 12 of 14 cases and of the light chain genes in 13 cases. The bcl-2 oncogene, normally involved in nodal follicular lymphomas, was in germ-line configuration. The c-myc and the beta and gamma chain genes of the T-cell receptor were also in the germ-line configuration. None of the cases presented Epstein-Barr virus sequences. These data indicate that primary cutaneous lymphomas of B-cell origin share morphological and phenotypic similarities with the nodal B-cell lymphomas of follicular histotype, are proliferating, and express in 45% of cases clear monoclonal immunoglobulin light chain; the molecular analysis confirms the B-cell derivation and the monoclonal nature of this neoplasia; it also shows that neither bcl-2 nor c-myc oncogenes are involved and that no inappropriate rearrangements of the T-cell receptor genes are found in this lymphoma.     

Detection of immunoglobulin gene rearrangement in B-cell lymphomas by polymerase chain reaction gene amplification.

This is a report on our attempt to use polymerase chain reaction (PCR) to detect rearrangement of the immunoglobulin gene in the tissue specimens obtained from 30 patients with non-Hodgkin's lymphomas. There were 20 B-cell lymphomas and 10 T-cell. All 20 B-cell lymphomas but none of the 10 T-cell lymphomas had JH rearrangement by Southern analysis. Two pairs of primers (V670/OL-4 and VH26/OL-4) were designed to amplify the CDR3 region of the immunoglobulin gene heavy chain. The PCR analysis was positive using either one or both pairs of primers in 11 of the the 20 cases (55 per cent) of B-cell lymphomas which all had positive rearrangement by Southern analysis. The two pairs of primers seemed to produce complementary results as the specimens may be positive to one pair but negative to the other. The false negative rate of 45 per cent is however much higher than the respective figures of 18 per cent and 0 per cent observed in our patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia in a previous study. Peripheral blood and bone marrow biopsy specimens obtained at the time of initial diagnosis were available from 10 patients with B-cell lymphomas whose lymph node biopsy specimens at the time of diagnosis were positive by both Southern analysis and PCR. All these peripheral blood and marrow specimens had no microscopic evidence of involvement by lymphoma cells and JH rearrangement was not detected by Southern analysis. However, rearranged bands identical to that of the lymph node biopsy specimen were detected by PCR in the peripheral and marrow blood of one of them. This PCR technique has been shown to have a sensitivity of 0.1 per cent in our previous report and may be more useful than morphology alone or Southern analysis in detecting minimal lymphomatous involvement in the peripheral blood and bone marrow at the time of initial diagnosis. Further clinical correlation is required to confirm the finding.     

Prognostic significance of clonal diversity of immunoglobulin gene rearrangements in patients with diffuse large B-cell lymphoma

Clonal diversity of the immunoglobulin (Ig) gene rearrangement represents the oligoclonality of B-cell neoplasm, and has been shown to be a marker for poor prognosis in acute lymphoblastic leukemia. However, no previous report has addressed its prognostic impact in diffuse large B-cell lymphoma (DLBCL). We investigated the clinical significance of clonal diversity in DLBCL patients. Lymph node samples from 98 DLBCL patients were examined for Ig heavy and light chain gene rearrangements using Southern blot analysis. Clonal diversity was defined as oligoclonality detected on Southern blotting as previously described, and PCR analysis for IgH oligoclonality was performed on parts of DLBCL samples with clonal diversity for confirming the Southern blot analysis results. We found that clonal diversity could be detected in 36 (36.7%) of DLBCL patients, and PCR analysis showed concordant results. Regarding the clinical relevance, clonal diversity was significantly associated with relapse or refractory disease. Survival analysis showed that clonal diversity is an independent prognostic factor in DLBCL (p=0.05, Cox's proportional hazard method), and stratified analyses found the most significant subgroup is the high-intermediate risk category (p=0.01, log-rank test). We conclude that clonal diversity of Ig gene rearrangements is associated with a high risk of relapse or refractory disease in DLBCL patients. It is also a factor of poor prognosis in DLBCL, especially for high-intermediate risk category.     

Detection of immunoglobulin heavy chain genes rearrangements in B-cell leukemias, lymphomas, multiple myelomas, monoclonal and polyclonal gammopathies

Polymerase chain reaction (PCR) based assays were found to be a realistic alternative to Southern blot hybridization for the assessment of clonal immunoglobulin heavy chain gene rearrangements. However, a comparison of the different PCR based studies reveals considerable variation in experimental design and marked differences in the reported results. This study compared different single- and double-step PCR assays relying on various FR3, FR2, FR1 and JH based primers for the detection of B cell clonality in acute lymphoblastic leukemias (ALL), non-Hodgkin's-lymphoma (NHL), multiple myeloma (MM), monoclonal gammopathies of unknown significance (MGUS) and three polyclonal gammopathies (PG). The highest monoclonality rate was observed using seminested CDR-III region amplification. This method achieved a monoclonal product in 6 of 13 pro-B ALL 21 of 29 c-ALL, 7 of 8 pre-B-ALL, 18 of 21 B-ALL, 14 of 17 B-NHL (intermediate or high grade) with bone marrow involvement, 0 of 9 B-NHL without bone marrow involvement, 9 of 9 low grade B-NHL (immunocytoma and including chronic lymphocytic leucemia), 13 of 19 MM, 2 of 9 MGUS, and 0 of 3 PG. Additional monoclonality was detected with nested CDR I PCR in 1 pro-B-ALL, 1 c-ALL, and 2 MM. CDR III IgH PCR has been confirmed as an efficient method for determining clonality in B-cell neoplasias. Some additional monoclonal products can be seen with CDR I-based PCR. Detection of monoclonality depends on the maturation grade of the neoplastic B-cell population.     

Chromosomal translocations and gene rearrangements in non-Hodgkin lymphomas

Unlike other types of malignancy, non-Hodgkin lymphomas have a specific reciprocal chromosomal translocation as a genomic hallmark in most patients. Among the more prevalent B-cell lymphomas, 65% of patients show a 14q + chromosome with rearrangement of the heavy-chain immunoglobulin genes at band 14q32.3, whereas the less common T-cell lymphomas typically have a selective rearrangement of the alpha T-cell receptor genes involving band 14q11.2. The presence of additional recurrent chromosomal defects in several subgroups of lymphomas is associated with tumor phenotype, evolution, and response to therapy.     

Characterization of IGH rearrangements in non-Hodgkin's B-cell lymphomas by fluorescence in situ hybridization

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin's B-cell lymphomas (NHL) and correlated to clinical relevant subgroups. However, the detection rate varied greatly with the technique used. The incidence of IGH rearrangements was analyzed using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 57 patients with nodal NHL. An IGH rearrangement was identified in 42 cases (73.7%). A t(14;18)(q32;q21) was found in 17 of the 20 follicular lymphomas (85%) studied and a t(11;14)(q13;q32) in 10 of the 11 mantle cell lymphomas (91%). IGH rearrangements were identified in 12 of the 26 diffuse large B-cell lymphomas (46%), including 5 t(14;18)(q32;q21) and 2 t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using Multi-FISH and/or chromosomal whole paint enabled the characterization of complex IGH translocations in follicular lymphomas and mantle cell lymphomas and the identification of all the chromosomal partners involved in the IGH rearrangement in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe could be the first approach in NHL, after which chromosome painting and M-FISH could be used to identify the chromosomal partner involved in the IGH rearrangement.     

Chemiluminescent detection of clonal immunoglobulin and T cell receptor gene rearrangements in Tunisian lymphoid malignancies, leukemias and lymphomas

Clonal rearrangement of antigen receptor genes is commonly used to characterize the lymphoproliferative diseases. In order to perform molecular characterization in the diagnostics and monitoring of lymphoid malignancies, leukemias and lymphomas in Tunisia, we have introduced the use of chemiluminescent probes for immunoglobulin (IG) and T cell receptor (TR) gene rearrangement detection employing the Southern blot method. The chemiluminescent and radioactive detection methods tested with alkaline phosphatase and 32P labelled probes, respectively, were used for the IG and TR gene rearrangement characterization. Our results show the same pattern of rearrangement. Moreover, the chemiluminescent signal is detected faster and it is as sensitive as the radioactive one. We report the optimized conditions for using IGH, IGK, IGL, TRB and TRG probes in non radioactive detection. We have applied the chemiluminescent Southern blot method to analyze examples of Tunisian leukemias and lymphomas. The results allowed the assessment of clonality and the T or B cell lineage of these cases. The use of non radioactive probes makes chemiluminescent Southern blot detection reliable, safe and sensitive. As the use of radioactivity is not common in our laboratories and the licensing requirements needed for its use prohibitive, the chemiluminescent technique will be of great help for detection and characterization of molecular markers in lymphoid malignancies in Tunisia.     

Prognostic significance of molecular staging by PCR-amplification of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma (DLBCL).

The prognostic value of the detection of peripheral blood (PB) and/or bone marrow (BM) involvement by polymerase chain reaction (PCR) amplification of rearranged immunoglobulin heavy chain (IgH) and immunoglobulin kappa light chain (Igkappa) genes was evaluated in 155 patients with diffuse large B-cell lymphomas (DLBCL). Immunoglobulin gene rearrangements (IgR) were detected in 35/155 (23%) patients. The presence of IgR in PB/BM was related to clinical stage (CS I-III vs CS IV; P<0.001), histopathological detection of BM involvement (P<0.001), and the International Prognostic Index (P<0.001). IgR-positive cases had a significantly lower complete remission (CR) rate (18/35, 51%) than IgR-negative patients (85/120, 71%; P=0.042), and a significantly poorer overall survival (OAS) at 5 years (25 vs 66%; P<0.001). There was a significant difference in the estimated OAS at 5 years between patients with negative BM histology and negative PCR results (66%), patients with negative BM histology but positive IgR (37%), and patients with positive BM histology (12%). Our results indicate that molecular methods improve the accuracy of staging in patients with DLBCL and define a group of patients with normal bone marrow histology who have a significantly poorer OAS due to molecular detection of PB/BM involvement.     

Cytomorphologic findings of B-cell lymphomas with concurrent IGH/BCL2 and MYC rearrangements (dual-translocation lymphomas)

BACKGROUND:

B‐cell lymphomas with concurrent IGH/BCL2 and MYC gene rearrangements, termed dual‐translocation or double‐hit lymphomas (DTLs), rarely are identified. They usually are characterized by highly aggressive behavior, a poor prognosis, and complex karyotypes. The objective of this study was to review and describe the cytomorphologic findings in different types of cytologic preparations and clinicopathologic characteristics of patients with DTLs.

METHODS:

Patient samples with IGH/BCL2 and MYC rearrangements that were detected by fluorescence in situ hybridization during the period from October 2003 to September 2009 were selected for morphology review. Clinical data and results from additional studies were collected from patient reports.

RESULTS:

Cytologic samples from 14 patients (5 men and 9 women) were reviewed. The most common cytomorphologic pattern was a mixed cell population consisting predominantly of large cells (88.2%), mainly centroblasts (94.1%), with dark blue cytoplasm (76.4%) accompanied by apoptotic bodies (64.7%), with marked cellular pleomorphism (94.1%). Nuclear segmentation was present in 64.7% of samples, conferring a “coffee bean” nucleus, and cytoplasmic vacuoles were observed in 46.6% of samples. Immunophenotyping revealed the expression of CD20, CD19, surface immunoglobulin, and CD10 in 13 samples. Other chromosomal aberrations were also identified. Seven patients died of their disease, and the time from progression to death ranged from 1 month to 16 months.

CONCLUSIONS:

Large cells with deeply basophilic cytoplasm, cytoplasmic vacuoles, and frequent segmented nuclei, particularly in fine needle aspirate smears and especially in patients with clinically aggressive and/or unusual clinical features, should trigger a fluorescence in situ hybridization analysis for IGH/BCL2 and MYC translocation to identify this entity. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.     

Development of consensus fluorogenically labeled probes of the immunoglobulin heavy-chain gene for detecting minimal residual disease in B-cell non-Hodgkin lymphomas

We have examined 72 patients with B-cell non-Hodgkin lymphoma (B-NHL) in order to search for consensus sequences of the immunoglobulin heavy chain (IgH) gene, and developed consensus fluorogenically labeled probes for use in an allele-specific oligonucleotide (ASO) real-time quantitative polymerase chain reaction (RQ-PCR) assay of minimal residual disease (MRD). We detected a clonal IgH variable region (VH) sequence in 51 (70.8%) of the 72 B-NHLs, the most frequent VH gene usages being VH3 and VH4 (45/51, 88.2%). It was possible to design three consensus fluorogenic probes for the VH3 gene and one for the VH4 gene avoiding these hypermutations. Our sequencing results suggested that consensus fluorogenic probes would be best based on the 5'-side of the framework region 3 (FR3) because the frequency of somatic hypermutations was significantly lower in the regions on which the probes were based than in the remaining parts of FR3 ( P <0.05). Nineteen (54.3%) of 35 B-NHLs with the VH3 gene and 5 (50%) of 10 with the VH4 gene had sequences identical to at least one of these probes. We found that probes containing one base substitution were still applicable for a MRD study, whereas those containing two or more were not. Therefore, our four probes were applicable for 37 (82.2%) of the 45 patients with VH3 or VH4. This limited number of probes makes a large-scale study of MRD in B-NHL more feasible.     

DNA rearrangements in non-Hodgkin's lymphomas

The configuration of DNA at genetic loci that undergo somatic recombination in lymphocytes provides genetic markers that are useful in the diagnosis and characterization of non-Hodgkin's lymphomas. The loci containing rearranged DNA fall into three groups: the immunoglobulin genes, the T cell receptor genes and sites of chromosomal rearrangements, such as chromosomal translocations. DNA fragments cloned from these loci can be used as hybridization probes to characterize the rearranged DNA sequences in biopsy tissues. Only probes for chromosomal rearrangements are capable of directly diagnosing neoplasia and histological subtypes; however, probes for rearrangements of immunoglobulin and T cell receptor genes are valuable for detecting clonal proliferations, determining B or T cell derivation of tumours and distinguishing individual clones of lymphocytes from each other. Applications of DNA rearrangements have already yielded a number of important findings concerning the biology of human lymphoma. These discoveries have included the existence of multiclonal B cell cancers, the widespread dissemination of occult tumour to lymph nodes in mycosis fungoides, the high incidence of circulating tumour cells in patients with low grade lymphomas and the molecular heterogeneity of chromosomal break-points in follicular lymphomas.     

弥漫性大B细胞淋巴瘤C-MYC基因异常分析

  目的   探讨弥漫性大B细胞淋巴瘤(DLBCL) MYC基因异常情况及其与BCL-2、BCL-6基因异常的关系。   方法   应用组织芯片和FISH技术对194例DLBCL的MYC、BCL-2、BCL-6基因异常情况进行检测, 并用免疫组织化学法检测CD10、BCL-6、MUM-1及Ki-67等蛋白标记物, 分析其相互关系。   结果   在164例MYC基因异常为38例(23.17%), 其中基因易位9例(5.49%), 基因扩增29例(17.68%); 同时存在MYC和BCL-6基因易位有2例, 未发现MYC和BCL-2同时易位或三者同时易位的病例; 资料完整(同时获得三种基因FISH结果) 的159例病例中, MYC基因扩增28例(17.61%) 与BCL-2基因扩增38例(23.90%) 呈显著正相关(r=0.291 6, P=0.000 4);MYC基因易位病例Ki-67高表达率(5/8, 62.50%) 明显高于非MYC基因易位病例(33/149, 22.15%, P=0.027 7), 2例MYC和BCL-6同时易位的病例均为Ki-67高表达, MYC基因扩增与Ki-67高表达无显著相关性。   结论   有关MYC基因在弥漫性大B细胞淋巴瘤中的异常改变除基因重排外, 还有基因扩增等活化方式, 目前对其作用机制尚缺乏了解, 值得进行深入研究。      

B-cell lymphomas morphologically resembling T-cell lymphomas

Seven cases of B-cell lymphoma that morphologically resembled T-cell lymphoma are described. These cases are of four morphologic types: atypical poorly differentiated lymphocytic lymphoma (PDLL) with convoluted nuclei, "Lennert's" lymphoma, mixed lymphocytic-"histiocytic" lymphoma with large variation in size of abnormal cells, and "histiocytic" lymphoma with large multilobed nuclei. These cases add further support to the belief that morphologic criteria alone are not sufficient for accurate immunologic classification of the malignant lymphomas since they may represent a distinct clinicopathologic entity.     

Oligoclonal immunoglobulin gene rearrangements in Philadelphia chromosome-positive common acute lymphoblastic leukemia

The occurrence of more than two rearranged bands of immunoglobulin heavy chain (IgH) genes in B precursor acute lymphoblastic leukemia (ALL) has recently been documented. To elucidate the nature of such leukemias, we studied 30 patients with common ALL, including 6 patients with Philadelphia chromosome (Ph1)-positive ALL, by immunophenotyping and genotyping. In 10 of the 30, Southern blotting showed oligoclonal patterns of IgH gene arrangements, which were frequently detected in Ph1-positive ALL. In one patient of the 10, three rearranged bands of Ig kappa chain genes were detected. Ph1 abnormality and co-expression of myeloid associated antigens were found in 5 and 5 of the 10, respectively. Detection of multiple fragments of IgH genes would be suggestive of multipotent progenitor origin of these ALL.     

Specificity of the rearrangements of the T-cell receptor gamma gene in human lymphomas

The structure and function of the human T-cell rearranging gamma gene are not completely understood. Several reports have suggested that this gene rearranges specifically in normal T cells, but the pattern of rearrangement in human lymphoid neoplasms is not clear. Some authors have described the rearrangements of this gene in unmanipulated leukemias as relatively specific for T-derived tumors, whereas others were unable to observe such specificity in malignant lymphomas. The present paper reports the analysis of the structure of the gamma gene in 32 lymphoid samples of different origin, with emphasis on non-T lymphomas. Four out of four T-cell lymphomas had this gene rearranged, whereas none of the 17 cases of B-cell lymphomas, 5 of Hodgkin's disease or 6 of nonneoplastic lesions showed any alterations of the gamma gene. Therefore, our data support the relative specificity of the gamma gene rearrangements in human T-cell malignant proliferations.     

Analysis of somatic hypermutations of immunoglobulin gene rearrangements in childhood acute lymphoblastic leukemia

The current study investigated the presence, frequency, and status of somatic hypermutations as well as their role in children with B lineage ALL. The obtained sequences were analyzed using IMGT/V-QUEST. Totally, 150 IGH sequences were evaluated; 139 from the 111 patients at the time of diagnosis and 11 from 8/111 patients at the time of relapse. The findings of the current report revealed the presence of somatically mutated V genes in childhood B lineage ALL. A higher frequency of somatic hypermutations was noted in unproductive rearrangements and was generally attributed to nucleotide mutation type, region, and IGHV gene subgroup biases.