scholarly journals Single cell origin of bigenotypic and biphenotypic B cell proliferations in human follicular lymphomas.

1988 ◽  
Vol 167 (2) ◽  
pp. 582-597 ◽  
Author(s):  
M L Cleary ◽  
N Galili ◽  
M Trela ◽  
R Levy ◽  
J Sklar

To investigate the possible relatedness of the subpopulations that make up so-called biclonal lymphomas, we examined five bigenotypic and biphenotypic follicular lymphomas using DNA probes specific for the t(14;18) chromosomal translocation, which is a characteristic feature of these neoplasms. On Southern blot analysis, both subpopulations from four of five lymphomas contained comigrating t(14;18) DNA rearrangements, confirming the single cell origins for these neoplasms. No comigrating t(14;18) DNA rearrangements were observed in the fifth lymphoma, but nucleotide sequence analysis of cloned, breakpoint DNA showed identical t(14;18) crossovers in the two subpopulations. The migration differences of both the Ig and chromosome 18 DNA rearrangements were shown to result from somatically acquired mutations of the Ig genes from the fifth lymphoma. These studies indicate that Ig gene rearrangements and idiotope expression are not consistently stable clonal markers since they are subject to variability as a result of somatic mutation. Although translocated chromosome 18 DNA rearrangements are more reliable, they may also vary among cells of some tumors since somatic mutation can affect, as well, DNA of translocated alleles in follicular lymphomas.

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 90-95
Author(s):  
MS Lee ◽  
MB Blick ◽  
S Pathak ◽  
JM Trujillo ◽  
JJ Butler ◽  
...  

The karyotypic abnormality t(14;18)(q32;q21) is reported to occur in 75% of follicular lymphomas. This translocation results in the rearrangement of a putative oncogene bcl-2, which resides at chromosome 18 band q21 (the 18q21 gene). Using two human genomic DNA fragments cloned from the chromosome 18 band q21 as probes, we analyzed 65 uncultured human lymphoma samples by the Southern blot technique. The 18q21 gene was rearranged in 18 of 26 (69%) follicular lymphomas, 3 of 5 (60%) follicular lymphomas transformed to large cell lymphomas, 8 of 20 (40%) diffuse large cell lymphomas (DLCLs), and 2 of 7 (29%) small noncleaved cell lymphomas (SNCs). Our analysis detected rearrangement of the 18q21 gene in 10 of 13 (77%) cases in which the t(14;18)(q32;q21) translocation was found by cytogenetic techniques. Our analysis also proved helpful in difficult karyotyping situations: (a) identifying the donor chromosome fragment as chromosome 18 band q21 in 4 of 9 (44%) cases that cytogenetically displayed a 14q+ chromosome of unknown origin, and (b) identifying a rearrangement of chromosome 18 band q21 in 12 of 18 (67%) cases that cytogenetically yielded no cells in metaphase. We also demonstrated three cases of submicroscopic rearrangement of the 18q21 gene. In our studies, patients with DLCLs and rearrangement of the 18q21 gene had a significantly higher incidence of extranodal involvement when compared with patients with DLCLs and no 18q21 gene rearrangement (P = 0.03).


eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Gilad D Evrony ◽  
Eunjung Lee ◽  
Peter J Park ◽  
Christopher A Walsh

Whether somatic mutations contribute functional diversity to brain cells is a long-standing question. Single-neuron genomics enables direct measurement of somatic mutation rates in human brain and promises to answer this question. A recent study (<xref ref-type="bibr" rid="bib65">Upton et al., 2015</xref>) reported high rates of somatic LINE-1 element (L1) retrotransposition in the hippocampus and cerebral cortex that would have major implications for normal brain function, and suggested that these events preferentially impact genes important for neuronal function. We identify aspects of the single-cell sequencing approach, bioinformatic analysis, and validation methods that led to thousands of artifacts being interpreted as somatic mutation events. Our reanalysis supports a mutation frequency of approximately 0.2 events per cell, which is about fifty-fold lower than reported, confirming that L1 elements mobilize in some human neurons but indicating that L1 mosaicism is not ubiquitous. Through consideration of the challenges identified, we provide a foundation and framework for designing single-cell genomics studies.


Blood ◽  
1991 ◽  
Vol 78 (6) ◽  
pp. 1552-1560 ◽  
Author(s):  
AD Zelenetz ◽  
G Chu ◽  
N Galili ◽  
CD Bangs ◽  
SJ Horning ◽  
...  

Abstract The t(14;18) chromosomal translocation that results in the juxtaposition of the bcl-2 proto-oncogene with the heavy chain JH locus is a common cytogenetic abnormality in human lymphoma. In particular, it is seen in about 85% of follicular lymphoma (FL) and up to one-third of diffuse lymphomas (DL). The chromosome 18 breakpoints have been shown to cluster into two regions. The major breakpoint region (mbr) within the 3′ untranslated region of the bcl-2 proto-oncogene accounts for approximately 60% of the cases and the minor cluster region (mcr) 30 kb 3′ of bcl-2 accounts for approximately 25% of the breakpoints. Because of variability in the position of the breakpoint, detection of the t(14;18) by Southern blot analysis provides an important clonal marker for the tumor. However, conventional electrophoresis (CE) fails to detect the translocation in 15% to 25% of cases. We have applied pulsed-field gel electrophoresis (PFGE) to the detection of the t(14;18) in a series of lymphoma prospectively analyzed by CE, polymerase chain reaction (PCR), and cytogenetic analysis. PFGE readily detected t(14;18) rearrangements as indicated by comigration of bands detected with probes for the mbr region (chromosome 18) and the JH locus (chromosome 14). In a series of 40 patients with FL, this method proved to be the most comprehensive for detection of the translocation compared with standard methods; in fact, in one case only PFGE was able to detect the chromosomal rearrangement. Ten percent of the FL cases were negative by all methods tested. In a separate analysis of matched tissue specimens from cases of tumor progression of FL to diffuse lymphoma, PFGE detected a common t(14;18) rearrangement confirming a clonal origin in seven of seven cases, whereas CE detected a rearrangement in only three of seven cases. Overall, PFGE was able to detect a translocation in 8 of 12 cases that were negative by CE and four of eight negative by cytogenetic analysis. In conclusion, PFGE analysis is more comprehensive than CE, PCR, and cytogenetic analysis for the detection of the t(14;18) breakpoint in tissue biopsies of malignant lymphoma.


Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2899-2907 ◽  
Author(s):  
Martina Vockerodt ◽  
Marta Soares ◽  
Holger Kanzler ◽  
Ralf Küppers ◽  
Dieter Kube ◽  
...  

Abstract Hodgkin’s disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VHgene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VHrearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 105 mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34+ stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation. © 1998 by The American Society of Hematology.


Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 90-95 ◽  
Author(s):  
MS Lee ◽  
MB Blick ◽  
S Pathak ◽  
JM Trujillo ◽  
JJ Butler ◽  
...  

Abstract The karyotypic abnormality t(14;18)(q32;q21) is reported to occur in 75% of follicular lymphomas. This translocation results in the rearrangement of a putative oncogene bcl-2, which resides at chromosome 18 band q21 (the 18q21 gene). Using two human genomic DNA fragments cloned from the chromosome 18 band q21 as probes, we analyzed 65 uncultured human lymphoma samples by the Southern blot technique. The 18q21 gene was rearranged in 18 of 26 (69%) follicular lymphomas, 3 of 5 (60%) follicular lymphomas transformed to large cell lymphomas, 8 of 20 (40%) diffuse large cell lymphomas (DLCLs), and 2 of 7 (29%) small noncleaved cell lymphomas (SNCs). Our analysis detected rearrangement of the 18q21 gene in 10 of 13 (77%) cases in which the t(14;18)(q32;q21) translocation was found by cytogenetic techniques. Our analysis also proved helpful in difficult karyotyping situations: (a) identifying the donor chromosome fragment as chromosome 18 band q21 in 4 of 9 (44%) cases that cytogenetically displayed a 14q+ chromosome of unknown origin, and (b) identifying a rearrangement of chromosome 18 band q21 in 12 of 18 (67%) cases that cytogenetically yielded no cells in metaphase. We also demonstrated three cases of submicroscopic rearrangement of the 18q21 gene. In our studies, patients with DLCLs and rearrangement of the 18q21 gene had a significantly higher incidence of extranodal involvement when compared with patients with DLCLs and no 18q21 gene rearrangement (P = 0.03).


Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 609-621 ◽  
Author(s):  
F Davi ◽  
C Gocke ◽  
S Smith ◽  
J Sklar

At presentation, bone marrow specimens from over 25% of B-lineage acute lymphoblastic leukemias (ALL) display more than two clonal rearrangements of immunoglobulin heavy chain (IgH) genes in Southern blot analyses. Nucleotide sequence analysis has shown predominantly different V(H)DJ(H) junctions among these genes, leading to the frequent description of such cases as oligoclonal leukemias. In the present study, we have analyzed the lgH genes from four patients whose leukemic cells contained different patterns of lgH gene rearrangements between presentation and relapse. Nucleotide sequence analysis of the lgH genes showed that three mechanisms could account for these differences: de novo V(H)DJ(H) rearrangement, V(H) to DJ(H) recombination, and V(H) replacement. In all cases, more than two totally different V(H)DJ(H) rearrangements appeared during evolution of the disease, formally consistent with the conclusion that these tumors were composed of apparently unrelated clones. However, the retention of some of the antigen receptor gene rearrangements, as well as the persistence of a chromosomal marker in two cases, indicated that these leukemias had a monoclonal origin. These findings support the hypothesis that some ALLs arise from a lymphoid progenitor cell at a stage of lymphocyte development before the onset of IgH gene rearrangement. These leukemic lymphocyte progenitors generate malignant daughter cells capable of an in vivo maturation that involves the completion of multiple different lgH rearrangements as well as the modification of preexisting rearrangements by V(H) to DJ(H) recombination or by a V(H) replacement.


Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 787-791 ◽  
Author(s):  
R Amakawa ◽  
S Fukuhara ◽  
H Ohno ◽  
S Doi ◽  
S Oguma ◽  
...  

Abstract A t(14;18) (q32;q21) chromosome translocation is closely associated with the follicular lymphoma, which is prevalent in the United States, and the t(14;18) causes the juxtaposition of a bcl-2 gene on chromosome 18 with an immunoglobulin heavy-chain gene locus on chromosome 14. Genomic DNAs from 30 Japanese patients with follicular lymphoma were examined for the molecular features by Southern blot hybridization. Using probe b for the major breakpoint cluster region of a bcl-2 gene, the rearrangements were detected in eight patients. Six of the eight patients had breakpoints located within the major breakpoint region, while two had breakpoints outside this cluster region but within the region of the 7.5-kb SstI fragment containing the probe b sequence. In two patients, pFL-2 probe detected the bcl-2 gene rearrangements that occurred near or within the minor breakpoint cluster region. These ten patients had a rearranged JH-containing fragment that migrated with the rearranged bcl-2 fragment. In the other 20 patients, these two chromosome 18-specific DNA probes did not detect the bcl-2 rearrangements. Compared with studies performed in the United States, the statistical analysis indicates a significant difference in frequency of the bcl-2 gene rearrangements near or within the major breakpoint cluster region (P = 0.0027) and the minor breakpoint cluster region (P = 0.029). However, the distribution difference of these events was not significant.


Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3520-3529 ◽  
Author(s):  
Ulrich Jäger ◽  
Silke Böcskör ◽  
Trang Le ◽  
Gerlinde Mitterbauer ◽  
Ingrid Bolz ◽  
...  

The human t(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between BCL-2 and DH/JH gene segments during V(D)J recombination in early B cells. De novo nucleotides are found inserted in most breakpoints and have been thus far interpreted as nontemplated N region additions. In this report, we have analyzed both direct (BCL-2/JH) and reciprocal (DH/BCL-2) breakpoints derived from 40 patients with follicular lymphoma with t(14;18). Surprisingly, we found that more than 30% of the breakpoint junctions contain a novel type of templated nucleotide insertions, consisting of short copies of the surrounding BCL-2, DH, and JH sequences. The features of these templated nucleotides, including multiplicity of copies for 1 template and the occurrence of mismatches in the copies, suggest the presence of a short-patch DNA synthesis, templated and error-prone. In addition, our analysis clearly shows that t(14;18) occurs during a very restricted window of B-cell differentiation and involves 2 distinct mechanisms: V(D)J recombination, mediating the breaks on chromosome 14 during an attempted secondary DH to JH rearrangement, and an additional unidentified mechanism creating the initial breaks on chromosome 18. Altogether, these data suggest that the t(14;18) translocation is a more complex process than previously thought, involving the interaction and/or subversion of V(D)J recombination with multiple enzymatic machineries.


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