Immunoglobulin Heavy Chain Translocations Research Articles

Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma

To detect immunoglobulin heavy chain ( IGH) gene translocations with specific oncogene loci, we established an interphase cytogenetic approach using double-color fluorescence in situ hybridization (DC-FISH), which we used to analyze 173 patients with B-cell lymphoma. DC-FISH using the IGH gene (14q32.3) in combination with c-MYC (8q24.1), BCL1 (11q13.3), BCL2 (18q21.3), BCL6 (3q27), and PAX-5 (9p13) gene probes detected IGH translocations in 70 (40.5%) of 173 patients. The partner genes involved in IGH translocations were identified in 56 (80%) of 70 patients, and fusion of the IGH gene with specific oncogenes was detected in 53 of 56 patients, particularly in interphase nuclei of 28 patients for whom cytogenetic analysis was not informative. The most common partner gene was BCL2 (19 patients; 27% of IGH translocation-positive patients), followed by BCL6 (16; 23%), BCL1 (11; 16%), c-MYC (7; 10%), and PAX-5 (2; 3%). These oncogenes were closely associated with subtypes of B-cell lymphoma. The other partners were 19q13 ( BCL3), 6p25 ( MUM1/IRF4), 1q36, and chromosome 8 identified in one patient each. Six of the nine patients with add(14)(q32) showed a BCL6/ IGH translocation. Double translocations of the IGH gene were found in three patients; c-MYC+ BCL1, c-MYC+ BCL2, and c-MYC+ BCL6 in each one. Interphase FISH using specific IGH-translocation probes is valuable for defining clinically meaningful subgroups of B-cell lymphoma.

Long-term clinical and molecular remission after allogeneic stem cell transplantation (SCT) in patients with poor prognosis non-Hodgkin's lymphoma.

From 1987 to 1999 35 patients with poor prognosis non-Hodgkin's lymphoma (NHL) underwent allogeneic stem cell transplantation (SCT) at the University Hospitals of Vienna and Graz. Initial biopsy specimens were reclassified according to the Revised European-American Classification of Lymphoid Neoplasms (REAL). All patients surviving 28 days engrafted. Twenty-eight of them (93%) attained clinical remission. At the last follow-up 14 patients were alive and disease-free at a median of 5.0 (range, 2.3-12.9) years after allogeneic SCT. The actuarial overall survival is 35%. Five patients relapsed 1.8 to 27.6 months after transplant, the probability of relapse is 23%. Of the 21 deaths following SCT, seven were due to relapse/refractory disease and 14 due to transplant-related causes. The probability of treatment-related mortality is 48%. After SCT, minimal residual disease (MRD) was monitored by polymerase chain reaction (PCR) in seven patients with a BCL-2/IgH translocation and in 13 with a clonal immunoglobulin heavy chain (IgH) rearrangement. All 20 patients attained clinical remission rapidly and converted to PCR negativity. In the follow-up nine of these patients are in long-term clinical and molecular remission, six PCR-negative patients died of transplant-related causes and five patients relapsed. In summary, allogeneic stem cell transplantation has a curative potential for patients with refractory and recurrent non-Hodgkin's lymphoma. In our series long-term disease-free survival was associated with molecular disease eradication after SCT. Treatment-related mortality rate was high, thus earlier referral of selected patients to allogeneic SCT should be considered.

Detection of Immunoglobulin κ Light Chain Rearrangements by Polymerase Chain Reaction: An Improved Method for Detecting Clonal B-Cell Lymphoproliferative Disorders

The clonal determination of B-cell lymphoproliferative disorders by immunoglobulin heavy chain (IgH) rearrangement by polymerase chain reaction (PCR) is widely used. However, few attempts have been made to detect immunoglobulin kappa light chain (Igkappa) gene rearrangement using PCR. We studied 145 cases of B-cell neoplasms, along with 58 atypical and 18 reactive lymphoproliferative disorders, using newly designed degenerate oligoprimers recognizing the framework 3 (FR3kappa) and the joint (Jkappa) regions of the Igkappa gene. PCR products were analyzed on nondenaturing polyacrylamide gel (ndPAGE). Clonal B-cell determination was further investigated using IgH rearrangement and t(11:14) or t(14:18). By combining these methods, we detected either clonality or translocation in 117 of 137 cases (85%) in mature B-cell neoplasms. The additional analysis of Igkappa rearrangement improved sensitivity from 66% to 85%. To investigate whether the Ig gene configuration could be characterized using Igkappa PCR in B-cell neoplasms showing severe breakdown of genomic DNA, 18 selected cases were analyzed. Successful amplification was detected in 72% of the cases using either FR3/2-JH and/or FR3Jkappa oligoprimers. Finally, clonality was detected in 21 of 58 atypical B-cell proliferations, and among them, the atypical marginal cell (54%) and atypical large cell (50%) proliferations showed the highest frequency of clonal immunoglobulin gene products. We concluded that PCR/ndPAGE analysis of Igkappa is a sensitive, rapid, and efficient method for assessing clonality in conjunction with IgH and specific translocation analysis. This approach is particularly useful in the characterization of B-cell lymphoproliferative disorders in archival material with poor preservation of the genomic DNA.

Lack of t(14; 18) Polymerase Chain Reaction-Positive Cells in Highly Purified CD34+ Cells and Their CD19 Subsets in Patients With Follicular Lymphoma

AbstractFollicular lymphoma (FL) is characterized in a significant proportion of cases by the t(14; 18) chromosomal translocation, which results in the juxtaposition of the oncogene bcl-2 to the joining region of the immunoglobulin heavy chain (IgH) gene. Molecular sequence analysis indicates that the t(14; 18) rearrangement occurs in a B-lymphoid progenitor cell at the time of IgH rearrangement. We were interested whether hematopoietic stem and progenitor cells as characterized by CD34 expression bear the translocation. Bone marrow (BM)-CD34+ cells were enriched from 14 patients with FL whose BM was known to be positive for bcl-2/IgH (major breakpoint region [MBR]). Six patients were in complete remission (CR), two patients were in partial remission (PR), and six patients had active disease. Six patients had histological BM involvement when the samples were obtained. Using an immunomagnetic selection device (MINIMACS), a mean purity of 88.7% ± 4% CD34+ cells was achieved. The CD34+ cells were further enriched by fluorescence activated cell sorting (FACS) using CD34 fluorescein isothiocyanate (FITC)- and CD19 phycoerythrin (PE)-conjugated antibodies. The IgH gene was rearranged in the CD34+/CD19+ cell subset of all patients assessed by polymerase chain reaction (PCR). This population is thought to represent the progenitor stage at which the bcl-2/IgH translocation occurs. The unseparated BM mononuclear cell fraction from all 14 patients was positive for bcl-2/IgH using a nested PCR, but the BM-CD34+ cell fraction and the respective CD34+/CD19+ subset were negative in 13 of these 14 patients. The one patient with a positive PCR signal in the CD34+ cell subset had a relapse with BM involvement. We conclude that CD34+ progenitor cells including CD34+/CD19+ B-cell progenitors are not involved in the malignant cell clone. These data are in agreement with a transgenic mouse model, which indicates that the malignant phenotype in FL is sustained by mature B cells.

Lack of t(14; 18) Polymerase Chain Reaction-Positive Cells in Highly Purified CD34+ Cells and Their CD19 Subsets in Patients With Follicular Lymphoma

Follicular lymphoma (FL) is characterized in a significant proportion of cases by the t(14; 18) chromosomal translocation, which results in the juxtaposition of the oncogene bcl-2 to the joining region of the immunoglobulin heavy chain (IgH) gene. Molecular sequence analysis indicates that the t(14; 18) rearrangement occurs in a B-lymphoid progenitor cell at the time of IgH rearrangement. We were interested whether hematopoietic stem and progenitor cells as characterized by CD34 expression bear the translocation. Bone marrow (BM)-CD34+ cells were enriched from 14 patients with FL whose BM was known to be positive for bcl-2/IgH (major breakpoint region [MBR]). Six patients were in complete remission (CR), two patients were in partial remission (PR), and six patients had active disease. Six patients had histological BM involvement when the samples were obtained. Using an immunomagnetic selection device (MINIMACS), a mean purity of 88.7% ± 4% CD34+ cells was achieved. The CD34+ cells were further enriched by fluorescence activated cell sorting (FACS) using CD34 fluorescein isothiocyanate (FITC)- and CD19 phycoerythrin (PE)-conjugated antibodies. The IgH gene was rearranged in the CD34+/CD19+ cell subset of all patients assessed by polymerase chain reaction (PCR). This population is thought to represent the progenitor stage at which the bcl-2/IgH translocation occurs. The unseparated BM mononuclear cell fraction from all 14 patients was positive for bcl-2/IgH using a nested PCR, but the BM-CD34+ cell fraction and the respective CD34+/CD19+ subset were negative in 13 of these 14 patients. The one patient with a positive PCR signal in the CD34+ cell subset had a relapse with BM involvement. We conclude that CD34+ progenitor cells including CD34+/CD19+ B-cell progenitors are not involved in the malignant cell clone. These data are in agreement with a transgenic mouse model, which indicates that the malignant phenotype in FL is sustained by mature B cells.

Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma.

In multiple myeloma, karyotopic 14q32 translocations have been identified at a variable frequency (10-60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q+), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus (i) are frequent (karyotypic 14q32 translocations and/or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); (ii) occur mainly in switch regions; and (iii) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.