Phenotypic and genotypic lineage switch of a lymphoma with shared chromosome translocation and T-cell receptor gamma gene rearrangement.

A case of non-Hodgkin's lymphoma showed a phenotypic and genotypic cell lineage switch twice during nine years of his clinical history; first, T-cell type, pleomorphic small cell lymphoma developed, followed by B-cell type, diffuse centroblastic/centrocytic lymphoma, and finally T-zone lymphoma without follicles again developed, from which AST-1 cultured cell line was established. Karyotype analysis demonstrated a shared abnormal chromosome, der(1)t(1;?)(p36;?), among the first relapsed B-cell tumor, the second relapsed T-cell tumor and AST-1 cell line. Furthermore, T-cell receptor (TCR) gamma gene rearrangement bands of the same size were observed in the first relapsed B-cell tumor and the second relapsed T-cell tumor as well as AST-1 cell line. These results suggested that both relapsed tumors of different cell lineages are derived from a common malignant clone, presumably a committed lymphoid stem cell. A unique translocation, t(2;14)(q37;q11.2), which may involve TCR delta/alpha gene complex, was observed in the second relapsed tumor and AST-1 cells. To attempt to isolate the breakpoint of this translocation, the configuration of TCR delta/alpha gene complex was studied. The result showed that two rearrangements of TCR alpha gene detected with J alpha probes were the products of the normal TCR rearrangement process, and were not involved in the translocation at this region. This patient, together with the AST-1 cell line, provided us a unique opportunity to study the development and clonal evolution of malignant lymphoma.     

Establishment of a human cell line (SKI-DLCL-1) with a t(1;14)(q21;q32) translocation from the ascites of a patient with diffuse large cell lymphoma.

Cytogenetic abnormalities at chromosome 1q21 are among the most common second genetic events observed in Non-Hodgkin's Lymphomas and have prognostic significance. Recently, BCL9 has been cloned from a pre-B-cell lymphoblastic leukemia cell line, which carried a t(1:14)(q21;q32). However, among a panel of 39 B-cell malignancies with 1q21 translocation, only two cases showed rearrangement for the BCL9 gene. We report the establishment of a new lymphoma cell line from a patient with relapsed diffuse large cell lymphoma. This cell line SKI-DLCL-1 showed cell surface antigens identical to the original tumor and demonstrated the profile of a mature B-cell phenotype: CD19 and CD20 positive, CD5 and C10 negative. It carried a t(1;14)(q21;q32) translocation identical to the original tumor. Although the clinical presentation was an isolated effusion lymphoma, studies for HIV-1, HHV8 and EBV were all negative. Southern blot analysis demonstrated that BCL9 was not rearranged in the SKI-DLCL-1 cell line. In addition, the BCL9 gene was not over-expressed in SKI-DLCL-1 cell line. The identification of a new locus at 1q21 will help clarify the pathogenesis of B-cell malignancies with a translocation involving this locus.     

Rearrangement of immunoglobulin, T-cell receptor, and bcl-2 genes in malignant lymphomas in Hong Kong

The pattern of malignant lymphomas in the Hong Kong Chinese population is characterized by a low incidence of Hodgkin's disease and follicular lymphomas. The authors studied the immunoglobulin (Ig), T-cell receptor (TCR), and bcl-2 gene rearrangement in 62 cases of malignant lymphoma in this population by Southern blot hybridization. Two cases of Hodgkin's disease showed no rearrangement of the Ig and TCR genes. All 42 cases of B-cell lymphoma had Ig heavy chain (JH) rearrangement with or without additional rearrangement of the light chains (C kappa and C lambda). One case of diffuse B-cell lymphoma had additional T-cell receptor beta-chain (C beta) rearrangement. Sixteen of 18 cases of T-cell lymphoma had C beta rearrangement, and one case of T-lymphoblastic lymphoma had additional JH rearrangement. Two of eight (25%) cases of follicular lymphoma but only one of the 34 (2.9%) cases of diffuse B-cell lymphoma had bcl-2 rearrangement that was detected by pFL-1 probe. None of the 62 cases showed bcl-2 rearrangement using the pFL-2 probe. In conclusion, the Ig and TCR gene rearrangement pattern of the lymphomas found in Hong Kong correlates well with the T-cell and B-cell lineage, which is similar to reports in the white population. However, the incidence of bcl-2 gene rearrangement in follicular B-cell lymphoma is lower than that reported in the US but comparable with that in Japan.     

Molecular analysis of an ataxia telangiectasia T-cell clone with a chromosomal translocation t(14;18)--evidence for a breakpoint in the T-cell receptor delta-chain gene.

We established a clonal T-cell line with a reciprocal chromosomal translocation t(14;18)(q11;q23) from a patient with ataxia telangiectasia (AT) and T-cell chronic lymphocytic leukemia (T-CLL). The tumor cells and the derived T-cell line were compared with respect to phenotype, karyotype, and rearrangement pattern. Restriction fragment analyses of the T-cell receptor (TCR)-delta gene, which is located within the TCR-alpha gene on chromosome 14q11, indicated that the breakpoint is located within the TCR-delta locus, splitting the TCR-delta gene between the variable and joining segments. This specific chromosomal translocation was only detected in the derived T-cell line and may be involved in the genesis of T-cell malignancies in AT.     

Cytogenetic and molecular characterization of a hepatosplenic T-cell lymphoma: report of a novel chromosomal aberration

Hepatosplenic T-cell lymphomas (HSTCL) are rare cancers and comprise 5% of peripheral T-cell lymphomas. These well-characterized extranodal lymphomas have a disguised onset, secondary to intrasinusoidal infiltration of the spleen, liver, and bone marrow, with a rapidly progressive course that is poorly responsive to chemotherapy and often ensues in the setting of immune system suppression. We describe the clinical, immunophenotypic, cytogenetic, fluorescence in situ hybridization, and molecular analyses for T cell receptor gene rearrangement in a 21-year-old man diagnosed with HSTCL. Immunophenotypic analysis revealed negativity for CD5 as well as double negativity for CD4/CD8 mature T-cell immunophenotype, which suggested the diagnosis of hepatosplenic T-cell lymphoma. Molecular analysis confirmed a TCR gene rearrangement, thereby verifying the common T-cell origin of the present HSTCL case. Furthermore, cytogenetic analysis revealed a novel chromosomal rearrangement, t(7;15)(p22;q21). Metaphase fluorescence in situ hybridization analysis confirmed the translocation of a chromosomal segment from 15q21 to 7p22.     

TEL-AML1 fusion precedes differentiation to pre-B cells in childhood acute lymphoblastic leukemia

The TEL-AML1 gene fusion results from a karyotypically cryptic t(12;21) translocation, the most common genetic abnormality in pediatric acute lymphoblastic leukemia (ALL). The presence of the TEL-AML1 fusion in utero, its protracted latency to overt leukemia, and secondary loss of the untranslocated TEL suggest it is an initiating event. Sequences of the TEL-AML1 genomic breakpoint and the immunoglobulin heavy chain (IgH) and/or T-cell receptor (TCR) gene rearrangements were characterized in four pediatric pre-B ALL patients. Analysis of these markers in relapsed patients revealed that immunophenotypically and cytogenetically distinct, and clonally unrelated antigen receptor leukemic cell populations harbored the same initiating TEL-AML1 molecular abnormality. Furthermore, TEL-AML1-positive cells persisted during remission even in the absence of detectable clone-specific IgH and TCR markers. We demonstrate that the TEL-AML1 translocation can occur in vivo during B-cell development before rearrangement of the IgH and TCR genes. We propose, in some cases, that the TEL-AML1 translocation occurs in a stem or B progenitor cell, and that recurrent TEL-AML1-positive pre-B ALL represents a de novo-transformed population that retains the same diagnostic initiating event.     

Establishment of a human herpes virus-8-negative malignant effusion lymphoma cell line (STR-428) carrying concurrent translocations of BCL2 and c-MYC genes

A new cell line, STR-428 was established from ascites tumor cells of a malignant effusion lymphoma patient without human herpes virus-8 (HHV-8) infection. STR-428 cells showed an immunophenotype of mature B-cells and produced few cytokines related to lymphomatous effusion. Karyotypic and genetic analysis revealed complex translocations including t(14;18)(q32;q21) effecting IgH/BCL2 and der(8)t(3;8)(q27;q24) involving c-MYC. STR-428 represents a unique, B-cell lymphoma cell line carrying concurrent rearrangement of BCL2 and c-MYC genes with features distinct from those of HHV-8-related primary effusion lymphoma. This cell line may be a valuable tool, other than HHV-8, to investigate the pathogenesis of primary lymphomatous effusion.     

Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11;14)(q13;q32) translocation.

The proto-oncogene PRAD1 (parathyroid adenoma 1) on chromosome 11q13 was found to be overexpressed in all five B-cell lines with t(11;14)(q13;q32) translocation tested. One B-cell lymphoma and four myeloma cell lines with this translocation demonstrated more than 10-fold overexpression as determined by Northern blot analysis, when compared with normal lymphoid tissues such as thymus, spleen and lymph node. Hematopoietic cell lines without the translocation were also examined, but none of these demonstrated the overexpression, confirming that overexpression of the PRAD1 gene is associated with t(11;14) translocation. A truncated form of mRNA was seen in one of five cell lines with the translocation, SP-49. Hybridization with different regions of the PRAD1 cDNA revealed that the truncated form of mRNA retained the coding region but had lost the 3' untranslated region. Southern blot analysis demonstrated a gene rearrangement in this SP-49 cell line. To study the genetic alteration responsible for the truncated form of mRNA in this cell line, the rearranged allele as well as the germline allele were cloned. The restriction map revealed that the rearranged portion was at the 3' end of the PRAD1 gene, eliminating the mRNA-destabilizing signal AUUUA. Human-rodent hybrid cell analysis demonstrated that the region introduced 3' of PRAD1 was derived from chromosome 11, suggesting that the PRAD1 gene region is deleted at the 3' end. Over-expression of the PRAD1 gene in association with t(11;14)(q13;q32) translocation suggested that in these cases the regulation of PRAD1 was altered by the juxtaposed gene, most likely the immunoglobulin heavy-chain gene from chromosome 14.     

B-cell marker negative (CD7+, CD19-) Epstein-Barr virus-related pyothorax-associated lymphoma with rearrangement in the JH gene

Pyothorax-associated lymphoma (PAL) develops decades after receiving artificial pneumothorax for pulmonary tuberculosis. The lymphomas, develop in tissue affected by long-standing severe inflammatory process. Most cases demonstrate diffuse large B-cell lymphoma. We present a patient with T-cell phenotype-positive and B-cell phenotype-negative (CD7+, CD43+, CD19-, and CD20-) PAL. Southern blot hybridization using immunglobulin heavy chain J region (IgH) gene probe revealed a monoclonal rearrangement, and hybridization using T-cell receptor beta chain (TCR) gene probe revealed a germline configuration. This indicates that the tumor origin was of B-lymphocytes. Chromosomal abnormality of the lymphoma was complicated. It suggested that many transformations occurred. In the transformation process, probably B-cell antigens were lost, and T-cell antigens were aberrantly expressed.     

Growth of human lymphoma cells in SCID mice.

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin. Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of TCL1 expression in B- and T-cell lymphomas and reactive lymphoid tissues

Chromosomal rearrangements observed in T-cell prolymphocytic leukemia involve the translocation of one T-cell receptor gene to either chromosome 14q32 or Xq28, deregulating the expression of cellular protooncogenes of unknown function, such as TCL1 or its homologue, MTCP1. In the human hematopoietic system, TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells causes mature T-cell proliferation in transgenic mice. In this study, using a newly generated monoclonal antibody against recombinant TCL1 protein, we extended our analysis mainly by immunohistochemistry and also by fluorescence-activated cell sorting and Western blot to a large tumor lymphoma data bank including 194 cases of lymphoproliferative disorders of B- and T-cell origin as well as reactive lymphoid tissues. The results obtained show that in reactive lymphoid tissues, TCL1 is strongly expressed by a subset of mantle zone B lymphocytes and is expressed to a lesser extent by follicle center cells and by scattered interfollicular small lymphocytes. In B-cell neoplasia, TCL1 was expressed in the majority of the cases, including lymphoblastic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (60%), and primary cutaneous B cell lymphoma (55%). TCL1 expression was observed in both the cytoplasmic and nuclear compartments, as confirmed by Western blot analysis. Conversely, TCL1 was not expressed in Hodgkin/Reed-Sternberg cells, multiple myelomas, marginal zone B-cell lymphomas, CD30+ anaplastic large cell lymphoma, lymphoblastic T-cell lymphoma, peripheral T-cell lymphoma, and mycosis fungoides. These data indicate that TCL1 is expressed in more differentiated B cells, under both reactive and neoplastic conditions, from antigen committed B cells and in germinal center B cells and is down-regulated in the latest stage of B-cell differentiation.     

Establishment of Epstein-Barr virus-negative diffuse large cell lymphoma cell line with an 8;22 chromosomal translocation

A new human B-cell lymphoma cell line was established from a pleural effusion of a patient with a diffuse large cell lymphoma which originated from an ileocecal tumor. The cell line, designated KAL-1, has been passaged 280 times over a period of 22 months. This cell line was successfully maintained in a chemically defined serum-free medium; its doubling time is approximately 24 h. Immunologically, the cells were demonstrated to express IgM lambda on the cell surface and to react with monoclonal antibodies to B-cell antigen including B1, B4, HLA-DR, and common acute lymphoblastic leukemic antigen but not with B2 and all the T-cell markers. Immunoglobulin gene analysis revealed rearrangements of both JH and C lambda. These data indicate that this cell line represents the B-cell lineage at the immature B-cell stage. This cell line was negative for Epstein-Barr virus nuclear antigen and had no detectable Epstein-Barr virus genome in cellular DNA. Chromosome analyses revealed that the cells carried an 8;22 chromosome translocation, reminiscent of variant type Burkitt's lymphoma. However, there was no histological evidence for Burkitt's lymphoma. Molecular studies showed that KAL-1 had deregulated high constitutive expression of c-myc. This cell line was demonstrated to be highly tumorigenic when injected into athymic nude mice. This tumor model should provide clues about the molecular mechanism involved in the pathogenesis of B-cell malignancy and appears to be a useful in vivo model for the study of molecular events during B-cell differentiation and therapeutic investigations.     

Molecular cloning of the chromosomal breakpoint of a B-cell lymphoma with the t(11;14)(q23;q32) translocation

The breakpoint of t(11;14)(q23;q32) chromosome translocation in a B-cell lymphoma line, RC-K8, was cloned. Immunoglobulin heavy chain (IGH) constant gene, C gamma 2 at the 5' end, was involved in this translocation, and the DNA segment juxtaposed to the C gamma 2 was proved to be derived from chromosome 11 by somatic cell hybrid study. The normal counterpart of chromosome 11 was also isolated. With a DNA probe near the breakpoint of chromosome 11, Southern blot analysis of RC-K8 and 10 other cases with translocation involving the 11q23 region was conducted, but no rearrangement bands have been observed thus far except for RC-K8.     

Application of Immunophenotyping and Heteroduplex Polymerase Chain Reaction (hPARR) for Diagnosis of Canine Lymphomas

Background: Canine malignant lymphoma is classified into B- or T-cell origin, as in the human case. Due to differences in prognosis, a suitable method needs to be developed for lineage identification. Aims: To determine the accuracy of immunophenotypic and molecular information between three methods: immunocytochemistry (ICC), immunohistochemistry (IHC) and heteroduplex polymerase chain reaction for antigen receptor rearrangements (hPARR) in spontaneous canine lymphomas. Materials and Methods: Peripheral blood, fine needle aspiration and tissue biopsies from enlarged peripheral lymph nodes prior to treatment of 28 multicentric lymphoma patients were collected. Cytopathology and histopathology were examined and classified using the updated Kiel and WHO classifications, respectively. Anti-Pax5 and anti-CD3 antibodies as B- and T-cell markers were applied for immunophenotyping by ICC and IHC. Neoplastic lymphocytes from lymph node and white blood cell pellets from peripheral blood were evaluated by hPARR. Results: In this study, low grade B-cell lymphoma accounted for 25% (7/28), high grade B-cell lymphoma for 64.3% (18/28) and high grade T-cell lymphoma for 10.7% (3/28). According to the WHO classification, 50% of all cases were classified as diffuse large B-cell lymphoma. In addition, ICC showed concordant results with IHC; all B-cell lymphomas showed Pax5+/CD3, and all T-cell lymphomas exhibited Pax5-/CD3+. In contrast to hPARR, 12 B-cell lymphomas featured the IgH gene; seven presented the TCRγ gene; five cases showed both IgH and TCRγ genes, and one case were indeterminate. Three T-cell lymphomas showed the TCRγ gene. The percentage agreement between hPARR and ICC/IHC was 60%. Conclusions: Immunophenotyping should not rely on a single method. ICC or IHC with hPARR should be used concurrently for immunophenotypic diagnosis in canine lymphomas.     

PATTERN OF T-CELL RECEPTOR DELTA GENE REARRANGEMENT BY SOUTHERN BLOTTING AND POLYMERASE CHAIN REACTION TECHNIQUE IN HONG KONG CHINESE PATIENTS WITH NON-T-CELL HEMATOLOGICAL MALIGNANCIES

It has been recognized that clonal T-cell receptor delta (TCRδ) gene rearrangement is present in both T- and B-cell malignancies. The highly sensitive polymerase chain reaction (PCR) technique may be applicable to cases of leukemia and lymphoma of non-T-cell origin for detection of minimal residual disease (MRD). A PCR technique was used in this study to investigate the pattern of clonal TCRδ gene rearrangement in Hong Kong Chinese patients with non-T-cell hematological malignancies. Seventy-three patients with the diagnosis of acute leukemia and non-Hodgkin's lymphoma of non-T-cell origin were included in this study. There were 20 patients with common ALL (cALL), seven precursal B-cell ALL (PreB-ALL), 23 acute myeloid leukemia (AML), and 23 non-Hodgkin's lymphoma of B-lineage (B-NHL). Clonal rearrangement was detectable by Southern analysis using a Jδ1 probe in 41 per cent of ALL of B-lineage but in none of the B-NHL or AML. The samples were also studied further by monoclonal PCR amplification for TCRδ gene rearrangement. Three different sets of primers were employed to detect clonal rearrangement of the TCRδ gene. The Vδ1(D)Jδ1 recombination typically seen in T-cell malignancies were not seen in any of the of the non-T-cell malignancies. The Vδ2(D)Dδ3 recombination was found exclusively in ALL of B-lineage and was seen in 73 per cent of the Southern positive cases. Although clonal TCRδ gene rearrangement was undetectable by Southern analysis in our AML cases, 26 per cent had a Vδ2(D)Jδ1 recombination found by the PCR technique. Sensitivity of the PCR technique was determined by serial mixing and was up to 5–10 leukemic cells per 10⁴ nucleated cells. It was apparent from this study that it was feasible to detect clonal TCRδ gene rearrangement by the PCR technique in a proportion of the cases of non-T-cell hematological malignancies. The PCR technique can be applied to detect residual leukemic cells in marrow of patients in an apparent morphological complete remission. The value of this application requires further clinical evaluation and correlation.     

Chromosome abnormalities in malignant lymphoma in patients from Kurashiki: histological and immunophenotypic correlations

Clonal chromosomal abnormalities were found in tumor tissue of 43 (84%) of 51 patients with non-Hodgkin's lymphoma (B-cell, 32; T-cell, 15) from an adult T-cell leukemia/lymphoma-nonendemic area in western mainland Japan. Four tumors were tetraploid, and the other 39 had a chromosome number in the diploid range. Trisomies 3, 5, 7, 18, and X, monosomy 13, and loss of an X in female and a Y in male were found in more than three patients each. Structural abnormalities in arms 1p, 1q, 2q, 3q, 13q, 14q, and 18q were found in eight or more patients each. Clustering of breaks occurred in 3q25-29 (ten patients, nine of whom with a B-cell tumor), 11q13 (five patients), dir dup(12)(q13-15----q21-24) (four patients), 14q32 (12 patients), and 18q21 (seven patients). The 14q32 translocations were all associated with B-cell tumors. t(8;14) was seen in a small noncleaved cell lymphoma, t(11;14)(q13;q32) in one follicular and three intermediately differentiated lymphocytic lymphomas, and t(14;18)(q32;q21) in two follicular lymphomas and one diffuse small cleaved cell tumor. The translocation partner of 14q could not be determined in the other four patients, three of whom had der(18)t(18;?)(q21;?). The seven 18q21 abnormalities, including a novel translocation t(2;18)(p11;q21.3), all occurred in B-cell tumors; even in the absence of t(14;18), they were closely associated with lymphomas of follicular center cell origin (six of seven).     

Chromosome 14 translocation in african and north american burkitt's lymphoma

Two established North American Burkitt lymphoma cell lines were studied by chromosomal banding techniques. The SU-AmB-1 line previously shown to be negative for the Epstein-Barr virus (EBV) was found to have, among other changes, a translocation from the long arm (q) of chromosome 8 onto 14q. The SU-AmB-2 line, which contains the EBV genome, also displayed the same 8/14 translocation. These results were compared with data from three EBV-positive tumor cell lines derived from patients with African Burkitt's lymphoma. Our findings indicate that a translocation from 8q onto 14q occurs in both African and North American Burkitt lymphomas, and that this abnormality apparently is not related directly to EBV. This chromosome translocation therefore may be an important event in the development of human lymphocytic malignancy, analogous to the occurrence of the Philadelphia chromosome rearrangement in chronic myelogenous leukemia.     

BCL-6 and other genomic alterations in non-Hodgkin's lymphoma (NHL).

This study reports on the frequency and disease association pattern of a number of gene rearrangements in a large panel of lymphoid tumours (n = 94). We detected the t(11;14) translocation, involving rearrangement of the BCL-1 locus, in 60% of mantle cell lymphomas. The BCL-2 gene, located at band 18q21, was rearranged in 42% of follicle centre lymphomas (FCL) and in 15% of diffuse large B-cell (DLBC) lymphomas. In this study, 80% of the c-MYC rearrangements were detected in aggressive diffuse lymphoma subsets but, interestingly, 9% of FCL showed involvement of t(8q24) translocation. In our study, rearrangements of the BCL-6 gene at band 3q27 were found in 31% of DLBC lymphomas. Interestingly, 50% of the BCL-6 rearrangement positive lymphoma cases had coexisting gene rearrangements involving all of the aforementioned gene loci. The molecular dissection of these genes will improve our understanding of the genesis of the diverse clinicopathological subtypes.