Clinical significance of t(14; 18)-positive cells in the circulation of patients with stage III or IV follicular non-Hodgkin's lymphoma during first remission.

PURPOSE: To evaluate polymerase chain reaction (PCR) analysis as a method for the detection of circulating lymphoma cells in patients with stage III and IV t(14; 18)-positive follicular Non-Hodgkin's lymphoma (NHL) in first remission in a longitudinal prospective study. PATIENTS AND METHODS: Peripheral blood or bone marrow from eight patients with stage III and IV t(14; 18)-positive NHL was studied using PCR to detect the presence of t(14; 18)-positive cells in the circulation at different times during first remission. RESULTS: In four of six patients with no clinical evidence of disease (NCED), t(14; 18)-positive cells were detectable in the circulation. In one of two patients with clinical evidence of disease (CED), no t(14; 18)-positive cells were found at the four different occasions tested during first remission. First-remission duration ranged from 17 to 81+ months. The duration from the first PCR determination in remission until first relapse or the end of the observation period ranged from 10 to 37+ months. CONCLUSION: In patients with t(14; 18)-positive follicular NHL stage III and IV, treated with conventional remission induction therapy, the presence or absence of t(14; 18)-positive cells in the circulation shows no obvious correlation with the clinical remission status and the remission duration.

Detection of minimal disease using rearranged immunoglobulin heavy chain genes from intermediate- and high-grade malignant B cell non-Hodgkins lymphoma.

Rearranged immunoglobulin heavy chain (IgH) genes provide unique clonal markers for B cells. Since amplification of the rearranged gene by polymerase chain reaction (PCR) and demonstrating that the amplified sequence is indeed derived from tumor cells is more problematic in non-Hodgkin's lymphoma (NHL) than in other B cell malignancies, we used a comprehensive PCR primer set and formulated stringent selection criteria to identify tumor-specific rearranged IgH genes. Rearranged IgH genes amplified from lymphoma DNA were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was amplified with at least two independent VH-specific primers. From 11 of 13 (85%) intermediate- and high-grade malignant NHL, IgH rearrangements were isolated. Intraclonal IgH sequence heterogeneity was studied in four lymphomas, and detected in two of them. PCR using a lymphoma-specific primer followed by Southern hybridization of PCR product with a specific probe allowed detection of lymphoma DNA after 10,000-fold dilution. Circulating lymphoma cells were detected in patient blood and bone marrow samples which were negative by morphological and immunological criteria. Thus, also in intermediate- and high-grade malignant lymphoma, sensitive minimal disease detection using the rearranged IgH gene as a marker appears feasible.

Genomic organization of the translocations (8;14) and (14;18) in a new lymphoma cell line.

We generated a new lymphoma cell line carrying the translocations (8;14) and (14;18) and studied the genomic organization and expression of the BCL-2 and MYC genes. Polymerase chain reaction (PCR) and Southern analysis showed that the breakpoints of t(14;18) were located in the major breakpoint region (mbr) of the BCL-2 gene and just 5' of JH6 in the IgH locus. The breakpoints of the t(8;14) were located upstream of exon 2 in the non-coding region of the MYC gene and near the switch region of the IgH locus. Both IgH loci were involved in chromosomal translocations resulting in the absence of a functional B-cell receptor. Normal BCL-2 and truncated MYC transcripts were detected in these cells. The BCL-2 protein was expressed.

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin's lymphoma.

Minimal residual disease (MRD) detection in B cell non-Hodgkin's lymphoma (NHL) patients has been shown to be possible using the rearranged heavy (IgH) chain gene as a tumor marker. To explore a second independent tumor marker, we used specific PCR primer sets to identify tumor-specific rearranged Ig light chain (IgL) genes. Rearranged IgL genes were amplified from lymphoma DNA by multiplex PCR using separate primer sets for the Igkappa and the Iglambda genes. They were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was isolated from at least two independent PCR products. From 12 out of 13 intermediate- and high-grade malignant NHL, PCR products could be obtained with IgL specific primers. PCR products from five NHL were studied in detail by cloning and sequencing. The rearranged IgL genes showed 85-100% homology with their closest germ line counterparts. Intraclonal IgL sequence heterogeneity was studied in five lymphomas and detected in only one. Minimal disease was studied in three patients by PCR, followed by Southern hybridization of the PCR product with a lymphoma-specific oligonucleotide probe, which allowed for detection of lymphoma DNA following 1000-fold dilution. Blood samples from one patient, who is in long-term clinical remission, were negative for the lymphoma-specific rearranged Igkappa gene. In the second patient the rearranged Iglambda gene was detected during the first clinical remission, that was followed by a nodal relapse, but not during the second remission, that has been stable for almost 3 years now. The third patient was negative for the rearranged Iglambda gene in blood samples up to 102 months after diagnosis. Circulating lymphoma cells were detected in blood and bone marrow samples which were negative by morphological and immunological criteria. Our studies show that the rearranged IgL gene can be used as a second independent tumor marker in intermediate- and high-grade malignant NHL.

New polymorphism and a new chromosome breakpoint establish the physical and genetic mapping of DXS369 in the DXS98-FRAXA interval.

Recently some of us cloned a new probe RN1 (DXS369), which appears a close marker for the fragile X locus (FRAXA) [Oostra et al.: Genomics 1990]. We present here new evidence for its physical and genetic mapping in the DXS98--FRAXA interval. We used 2 different somatic cell hybrid lines with breakpoints in the Xq27-q28 region: L10B Rea and PeCHN, and we established the order: (DXS105, DXS98)-L10B Rea-DXS369-PeCHN- (DXS304, DXS52). We detected an additional TaqI RFLP at the DXS369 locus which increases its informativeness up to 57%. Two point linkage analysis in a large set of families gave high lod scores for the FRAXA-DXS369 linkage (z(theta) = 10.1 at theta = 0.044) and for DXS369-DXS304, a marker distal to FRAXA (z = 19.2 at theta = 0.070). By multipoint analyses we established the localization of DXS369 in the DXS98-FRAXA interval. DXS369 is a much closer proximal marker for FRAXA than DXS105 or DXS98 and any new probe mapping between the breakpoints in L10B Rea and PeCHN will be of potential interest as a marker for FRAXA.

Translocation (14;18)-positive cells are present in the circulation of the majority of patients with localized (stage I and II) follicular non-Hodgkin's lymphoma.

Stage I and II follicular non-Hodgkin's lymphoma (NHL) is clinically defined as a localized disease. To study the possibility that this disease is in fact disseminated, we used the sensitive polymerase chain reaction (PCR) method using translocation (14;18) as marker. Samples from 21 patients who were clinically diagnosed with stage I or II follicular NHL were analyzed for the presence of t(14;18)-positive cells using PCR. We analyzed (1) the diagnostic lymph node biopsy and (2) the peripheral blood or bone marrow samples from these patients. Translocation (14;18) cells were detected in the diagnostic lymph node biopsies of 12 patients. In 9 of these patients, t(14;18)-positive cells were detected in peripheral blood and/or bone marrow samples at diagnosis and/or after therapy. Thus, in 75% of the follicular NHL patients carrying the t(14;18) as a marker for lymphoma cells, t(14;18)-positive cells were detected in peripheral blood and bone marrow at diagnosis and after therapy. Our results show that t(14;18)-positive cells can be detected in the circulation of patients with stage I and II follicular NHL, indicating that, although diagnosed as localized, the disease is disseminated.

New polymorphic DNA marker close to the fragile site FRAXA.

DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.