The clinical significance of antigen receptor gene rearrangements in lymphoid neoplasia.
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Blotting, Southern↗
Biomedical subjects
J Sklar
Publications and source records attributed to J Sklar.
Interallelic V(D)J trans-rearrangement within the beta T cell receptor gene is infrequent and occurs preferentially during attempted D beta to J beta joining.
Previous work has demonstrated that intergenic V(D)J rearrangement, a process referred to as trans-rearrangement, occurs at an unexpectedly high frequency. These rearrangements generate novel V(D)J combinations which could conceivably have some role in the normal immune system, and since they probably arise through chromosomal rearrangements akin to those associated with lymphoid neoplasia, they may also serve as a model for investigating recombinational events which underlie oncogenesis. In view of the existence of a mechanism that permits relatively frequent intergenic trans-rearrangements, it seems reasonable that interallelic trans-rearrangements involving segments belonging to each of the two alleles of a single antigen receptor gene might also occur. To determine the frequency of such rearrangements, we examined thymocytes of F1 progeny of a cross between SWR mice, which have a deletion spanning 10 of the known V beta segments, and NZW mice, which have a deletion involving all J beta 2 segments. Rearranged TCR-beta genes containing V beta segments from the NZW chromosome and J beta segments from the SWR chromosome were amplified from the DNA of F1 thymocytes with the polymerase chain reaction. Using this approach, we found that such rearrangements are relatively uncommon, being present in about 1 in 10(5) thymocytes, a frequency lower than that of V gamma/J beta intergenic trans-rearrangements. The ratio of conventional cis-rearrangement to interallelic trans-rearrangement for any particular V beta segment appears to be about 10(4):1. The structure of the junctions in all trans-rearrangements analyzed closely resembles conventional cis-rearrangements, indicating involvement of V(D)J recombinase in the ultimate joining event. However, in contrast to cis-rearrangements, a strong bias for inclusion of D beta 1 segments over D beta 2 segments was noted, suggesting that interallelic trans-rearrangement may occur preferentially during attempted D-J joining. J beta 2 segment usage in trans-rearrangements also appeared to differ from that expected from previously studied cis-rearrangements. The results have implications with respect to the events and timing of conventional cis-rearrangement during thymocyte differentiation, and the prevalence of various types of trans-rearrangements.
Alleles↗
Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease.
A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.
Adolescent↗
Acute lymphoblastic leukemia occurring as a second malignant neoplasm in childhood: report of three cases and review of the literature.
PURPOSE: The long-term effects of childhood cancer and its therapy are a problem of increasing concern. One of the most important of these late effects is the development of second malignant neoplasms (SMNs), which occur in approximately 8% of children within 20 years of diagnosis of a malignancy. These secondary cancers may result (individually or in combination) from increased genetic susceptibility, the mutagenic effects of chemotherapy and/or radiation therapy, or chance. Whereas the development of acute nonlymphocytic leukemia (ANLL) as an SMN is a well-recognized phenomenon, acute lymphoblastic leukemia (ALL) has been infrequently described as an SMN in either adults or children. PATIENTS AND METHODS: We report three patients treated at our institution in whom ALL developed as an SMN after treatment for neuroblastoma, Wilms' tumor, and Hodgkin's disease. These cases prompted us to review the published literature for cases of secondary ALL in childhood. Patients whose initial malignancy was diagnosed at age less than 16 years were classified as pediatric patients. SMNs were defined as cancers of clearly distinct histologic type occurring 6 or more months after diagnosis of the first malignant neoplasm. RESULTS: Including the three index cases, a total of 18 children with secondary ALL are reviewed, and the clinical features are discussed and compared with those of secondary ANLL. CONCLUSIONS: This review summarizes the published case histories of secondary ALL. The data suggest that ALL represents approximately 5% to 10% of the cases of acute leukemia that arise as SMNs in both adults and children.
Adolescent↗
AIDS and non-Hodgkin's lymphoma: a pre-pre-B-cell monoclonal lymphoma versus a novel mechanism of polyclonality?
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Clone Cells↗
Detection of the t(14;18) at similar frequencies in hyperplastic lymphoid tissues from American and Japanese patients.
Follicular lymphoma shows a wide geographic variation in incidence, occurring more frequently in the U.S. than in Japan. A translocation involving the bcl-2 gene on chromosome 18 and the immunoglobulin heavy chain gene on chromosome 14 is frequently found in follicular lymphomas and is believed to play a critical role in the pathogenesis of these tumors. Recently, bcl-2/IgH rearrangements have been detected in reactive lymphoid tissue obtained from European patients, indicating that such rearrangements occur at some low but measurable background rate. In non-malignant tissues, the polymerase chain reaction was used to study the frequency of bcl-2/IgH rearrangements in reactive lymphoid tissue obtained from American and Japanese patients to find out whether geographic variation in the incidence of follicular lymphoma was caused by differences in sporadic occurrence of the t(14;18). We found such rearrangements in 5 of 15 American hyperplastic tonsils and lymph nodes and 5 of 10 Japanese tonsils, an incidence close to that previously seen in European patients. These data suggest that the background incidence of such rearrangements is similar in all populations, regardless of the incidence of follicular lymphoma.
Adult↗
Transrearrangements between antigen receptor genes in normal human lymphoid tissues and in ataxia telangiectasia.
The polymerase chain reaction (PCR) was used on DNA obtained from various normal lymphoid tissues to amplify chimeric TCR gene rearrangements involving J segments of the beta gene and V segments of the gamma or delta genes. As found previously for the transrearrangements between the gamma and delta genes, transrearrangements involving the beta gene were more abundant in DNA of the thymus than in DNA of the spleen, lymph node, bone marrow, or PBL. In addition, transrearrangements between Ig H chain V region segment and J segment of TCR delta chain were also found in DNA of normal thymus. Sequence analysis of the trans-rearrangement PCR products revealed structures closely resembling normal intragenic rearrangements, with N insertions and often D segments at the junctions between segments. The sequences analyzed suggest that transrearrangements arise through the action of normal lymphocyte recombinase, involve trans recognition of heptamer/nonamer recombination signals, and follow the 12 + 23 spacer rule. To test whether transrearrangements result from chromosomal rearrangements with breakpoints at the sites of Ag receptor genes, PCR was performed on the DNA of PBL from patients with ataxia telangiectasia, a disorder in which circulating lymphocytes often have numerous karyotypic abnormalities with breakpoints at the cytogenetic positions of these genes. Comparison of the results of PCR on this DNA and that of normal tissues demonstrated a substantially increased frequency for most types of transrearrangements investigated. These results support the interpretation that transrearrangement among TCR genes may occur by chromosomal rearrangement.
Ataxia Telangiectasia↗
Chromosomal translocations joining LCK and TCRB loci in human T cell leukemia.
A case of T lymphoblastic leukemia (T-ALL) showing t(1;7)(p34;q34) as the sole karyotypic abnormality was investigated at the molecular level. Screening of a phage library of tumor DNA with a probe for the beta T cell receptor gene (TCRB), which maps to chromosomal band 7q34, resulted in the isolation of a clone containing DNA spanning the translocation breakpoint of the der(1) chromosome. This clone contained chromosome 1 DNA juxtaposed upstream of a D beta-J beta joint. Cloning of the corresponding germline region of chromosome 1 resulted in the isolation of a phage containing the breakpoint from the reciprocal, der(7), product, which showed chromosome 1 DNA joined downstream to a V beta segment. Comparison of germline and translocation clones demonstrated that breakage of chromosome 1 had occurred at the border of a tandem repeat of Alu sequences. To search for transcripts from DNA near the breakpoint, a chromosomal walk was initiated along chromosome 1. A probe consisting of chromosome 1 DNA from 24-30 kb upstream of the breakpoint hybridized to a transcript derived from the gene encoding the lymphocyte-specific tyrosine kinase p56lck, previously mapped to chromosomal band 1p34. The nonrandom nature of the breakpoints in this case was confirmed by the analysis of a second independent case of T-ALL containing a t(1;7) translocation, which was also found to show breakage within the LCK locus. The chromosomal breakpoint in the first case was localized 2 kb upstream of the lck upstream promoter and first nontranslated exon, while the breakpoint of the second case lay between the two alternative lck promoters, upstream of the second exon. Relative to normal thymus and activated T cells, levels of lck mRNA were greatly elevated in the first case and moderately elevated in the second. The existence of these translocations raises the possibility that alterations in the promoter region of the LCK locus may play a role in human cancer.
Base Sequence↗
Sensitive detection of clonal antigen receptor gene rearrangements for the diagnosis and monitoring of lymphoid neoplasms by a polymerase chain reaction-mediated ribonuclease protection assay.
This report describes a novel assay involving the polymerase chain reaction (PCR) and RNase protection for the rapid and sensitive detection of malignant lymphoid cells by nucleotide sequences within their individual rearranged gamma T-cell receptor (TCRG) genes. In this assay, clonal rearrangements are amplified from the DNA of diagnostic tumor specimens using a consensus V segment primer and a consensus J segment primer to which the promoter for T7 RNA polymerase has been appended. The PCR product from this amplification is transcribed into a radiolabeled RNA probe. Test RNA transcribed from the opposite DNA strand is synthesized by similar methods from TCRG genes of a subsequent biopsy specimen. The test RNA is hybridized with the probe, and mismatched nucleotide sequences in the RNA hybrids are digested by RNase A. Detection of fully protected probe by means of polyacrylamide gel electrophoresis and autoradiography indicates the presence of malignant cells in the test specimen. Dilution experiments with DNA of cell lines from acute lymphoblastic leukemias (ALLs) show that detection of one tumor cell among 10(5) normal bone marrow cells is usually possible. Residual disease was also successfully detected in several cases of ALL during clinical remission, including detection in one case at the 10(-5) level. The procedure described here may provide a simplified and rapid method for the sensitive diagnosis and monitoring of lymphoid malignancies. This procedure should be applicable to most antigen receptor genes, and unlike most comparable methods, requires neither analysis of nucleotide sequence nor synthesis of tumor-specific oligonucleotide probes or primers.
Autoradiography↗
TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms.
Previously we described joining of DNA in the beta T cell receptor gene to DNA of an uncharacterized locus in a t(7;9)(q34;q34.3) chromosomal translocation from a case of human T lymphoblastic leukemia (T-ALL). We now show that the locus on chromosome 9 contains a gene highly homologous to the Drosophila gene Notch. Transcripts of the human gene, for which we propose the name TAN-1, and its murine counterpart are present in many normal human fetal and adult mouse tissues, but are most abundant in lymphoid tissues. In t(7;9)(q34;q34.3) translocations from three cases of T-ALL, the breakpoints occur within 100 bp of an intron in TAN-1, resulting in truncation of TAN-1 transcripts. These observations suggest that TAN-1 may be important for normal lymphocyte function and that alteration of TAN-1 may play a role in the pathogenesis of some T cell neoplasms.
Amino Acid Sequence↗
Chimeric gamma-delta signal joints. Implications for the mechanism and regulation of T cell receptor gene rearrangement.
Rearrangement of Ag receptor genes requires recognition by the lymphocyte recombinase of heptamer-nonamer signal sequences followed by two endonucleolytic cleavages and two DNA ligations to form the coding and signal joints. The phenomenon of trans-rearrangement, in which Ag receptor gene segments located on different chromosomes recombine to yield chimeric products, provides an in vivo system in which to investigate the ability of the recombinase to carry out each of these functions in trans. Trans-rearrangements between TCRG and TCRD loci, similar in structure and frequency to those observed previously in human lymphoid tissues, were demonstrated in normal mouse thymus by PCR with crossed V gamma/J delta and V delta/J gamma primer pairs. A simple mechanistic model for trans-rearrangement was then tested. This model posits an ability of the recombinase to catalyze the formation of both coding and signal joints in trans and therefore predicts that trans-rearrangements will generate chimeric signal joints. In adult thymus, chimeric D delta 2-J gamma 1 and D delta 2-J gamma 2 signal joints, containing fused heptamer-nonamer sequences, could be detected by PCR and were each present at frequencies sufficient to account for a large proportion of the corresponding TCRG/TCRD trans-rearrangements. In agreement with the predictions of the model, chimeric signal joints were found as both linear chromosomal and circular episomal DNA. The results provide a framework for understanding the formation of chromosomal translocations in normal and neoplastic lymphoid cells and support the possibility of a looping mechanism for standard gene rearrangement. To test the form of regulation of TCRG rearrangement, the frequencies of specific signal joints from standard and trans-rearrangements were compared. Although J gamma 1 and J gamma 2 segments participated with equal frequency in trans-rearrangement with D delta 2, only the J gamma 1 segment participated in standard rearrangement with V gamma 5. The results suggest that V-J recombination in the TCRG locus is regulated directly at the DNA level by cis-acting constraints which do not affect the accessibility of individual TCRG gene segments to recombination in trans.
Animals↗
Polymerase chain reaction: the molecular microscope of residual disease.
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DNA, Neoplasm↗
Quantitation of T-cell DNA in cutaneous lymphoid infiltrates.
A method is described that allows the quantitation of T-cell DNA within a tissue biopsy specimen. This method is based on the densitometric evaluation of the multiband pattern of TCR-gamma gene rearrangements detectable in Southern blot analyses of polyclonal T-cell DNA digested with HindIII. These rearrangements can be detected down to a limit of approximately 5% T-cell DNA. The information derived from this type of analysis can be used to roughly assess the total T-cell content of lesional tissue specimens expressed as a percentage of total tissue DNA, and can be correlated with the pattern of T-cell infiltration visualized by immunohistology. This assessment can be used also to determine the threshold, expressed as a percentage of total lesional T-cell DNA instead of total tissue DNA, required for the detection of a monoclonal T-cell population by Southern blot analysis. These quantitative relationships may prove useful for studying cutaneous T-cell lymphoma and associated precursor conditions.
DNA↗
Nasal lymphoma. A clinicopathologic study with immunophenotypic and genotypic analysis.
We studied 13 cases of malignant lymphoma involving the nasal cavity, in six men and seven women, from 27 to 92 years of age (mean, 56 years; median, 55 years). All lymphomas had a diffuse pattern, with 10 of large-cell type (six immunoblastic polymorphous, one immunoblastic, three large cleaved cell), one of mixed small- and large-cell type and one of small cleaved-cell type. One case could not be subclassified. Angioinvasion and prominent necrosis were seen in 10 cases. Pseudoepitheliomatous hyperplasia of the overlying epithelium was present in five cases. Immunohistochemical studies on frozen or paraffin sections in nine cases revealed that the atypical cells were T cells in four cases (CD8+ in two cases) and B cells with monotypic immunoglobulin in two cases. In three cases, the findings were suggestive but not diagnostic of T lineage. Genotypic analysis in one of two cases of T-cell lymphoma revealed clonal rearrangement of the genes for beta and gamma chains of the T-cell receptor. Patients were treated initially with local radiation therapy (10 cases) or with radiation and chemotherapy (three cases). Eight patients (62%) had no relapse and were free of disease between 9 months and 23 years (mean, 6 years 5 months; median 2 years 1 month) after diagnosis. Five patients developed recurrent disease, three of whom were successfully salvaged. One patient was alive with tumor at the time of last follow-up and one died with tumor. Among cases of malignant lymphoma presenting with involvement of the nasal cavity, we find a high proportion of angioinvasive, diffuse large-cell lymphomas, with a predominance of T-cell type, and a relatively good prognosis when treated with radiation therapy.
Adult↗
Maintenance treatment of patients with myelodysplastic syndromes using recombinant human granulocyte colony-stimulating factor.
Myelodysplastic syndromes (MDS) are characterized by chronic refractory cytopenias resulting in increased risk of infection, bleeding, and conversion to acute leukemia. In an effort to improve these cytopenias we have treated 18 patients over a 6- to 8-week period with increasing daily subcutaneous doses of recombinant human granulocyte colony-stimulating factor (G-CSF). Sixteen patients responded with improvement in neutrophil counts. On cessation of treatment these counts returned to baseline values over a 2- to 4-week period. To maintain these improved blood counts 11 patients were treated with G-CSF for more prolonged periods. Ten patients again responded with an increase in total leukocyte counts (1.6- to 6.4-fold) and absolute neutrophil counts (ANC) (3.6- to 16.3-fold), with responses persisting for 3 to 16 months. A significantly decreased risk of developing bacterial infections was noted during periods with ANC greater than 1,500/mm3 as compared with periods of time with ANC less than 1,500/mm3. Two anemic patients had a greater than 20% rise in hematocrit over the study period, and 2 additional patients had a decrease in red blood cell transfusion requirements during G-CSF treatment. Bone marrow myeloid maturation improved in 7 of 9 maintenance phase patients. Three patients progressed to acute myeloid leukemia during treatment. The drug was generally well-tolerated and no severe toxicities were noted. These data demonstrated that G-CSF administered to MDS patients by daily subcutaneous administration was well-tolerated and effective in causing persistent improvement of the neutrophil levels and marrow myeloid maturation. These effects were associated with a decreased risk of infection and, in some patients, with decreased red blood cell transfusion requirements.
Colony-Stimulating Factors↗
Detection of non-Hodgkin's lymphoma in the peripheral blood by analysis of antigen receptor gene rearrangements: results of a prospective study.
Analysis of immunoglobulin (Ig) and T-cell receptor gene rearrangements, using Southern blot hybridization, has been applied to peripheral blood lymphocytes (PBL) in 335 samples from patients with non-Hodgkin's lymphoma. The incidence of circulating lymphoma cells detected by gene rearrangement analyses is related to the histologic subtype, clinical stage of disease, and clinical status. Among 104 patients studied at diagnosis, the incidence of positive analyses was 34% in low-grade lymphoma and only 8% in intermediate-grade lymphoma. Clonal Ig gene rearrangements were detected nearly universally in the small lymphocytic histologic subtype. PBL studies were related to the initial stage of disease: positive studies were seen in 35% of patients with stage IV disease, 29% of patients with stage III disease, and 12% of patients with stages I-II disease. The incidence of PBL rearrangements at the time of disease recurrence in 32 patients requiring cytoreductive therapy was 48%, somewhat greater than at initial diagnosis. A group of patients with low-grade lymphoma, who had treatment deferred after diagnosis or recurrence, was also studied; the incidence of PBL rearrangements was 38% in this population. Among 157 patients clinically free of disease, DNA analyses of the PBL were positive in only 10%. Subsequent relapse of disease in 26 patients was antedated by PBL rearrangement in only one patient. Clonal rearrangements detected in 15 patients have been followed by recurrence of clinical disease in only one patient over a median of 24 months from the time of analysis. The lack of detectable rearrangements in the peripheral blood in the majority of patients may be due to methodology or the biology of the disease. These issues may be further addressed with alternative methods for assessment of minimal disease. However, rigorous testing of any new molecular tool requires an adequate patient population in which disease status is closely monitored over a sufficient period of time.
Blotting, Southern↗
Rapid, nonradioactive detection of clonal T-cell receptor gene rearrangements in lymphoid neoplasms.
Southern blot hybridization analysis of clonal antigen receptor gene rearrangements has proved to be a valuable adjunct to conventional methods for diagnosing lymphoid neoplasia. However, Southern blot analysis suffers from a number of technical disadvantages, including the time necessary to obtain results, the use of radioactivity, and the susceptibility of the method to various artifacts. We have investigated an alternative approach for assessing the clonality of antigen receptor gene rearrangements in lymphoid tissue biopsy specimens. This approach involves the amplification of rearranged gamma T-cell receptor genes by the polymerase chain reaction and analysis of the polymerase chain reaction products by denaturing gradient gel electrophoresis. By use of this approach, clonal rearrangements from neoplastic lymphocytes constituting as little as 0.1-1% of the total cells in the tissue are detected as discrete bands in the denaturing gel after the gel is stained with ethidium bromide and viewed under ultraviolet light. In contrast, polyclonal rearrangements from reactive lymphocytes appear as a diffuse smear along the length of the gel. Our findings suggest that polymerase chain reaction combined with denaturing gradient gel electrophoresis may offer a rapid, nonradioactive, and sensitive alternative to Southern blot analysis for the diagnostic evaluation of lymphoid tissue biopsy specimens.
Base Sequence↗