Molecular characteristics and gene segment usage in IGH gene rearrangements in multiple myeloma.

BACKGROUND AND OBJECTIVES: Analysis of IgH rearrangements in B-cell malignancies has provided clinical researchers with a wide range of information during the last few years. However, only a few studies have contributed to the characterization of these features in multiple myeloma (MM), and they have been focused on the analysis of the expressed IgH allele only. Comparison between the expressed and the non-functional IgH alleles allows further characterizion of the selection processes to which pre-myeloma cells are submitted. DESIGN AND METHODS: We analyzed a cohort of 84 untreated MM patients in order to characterize their functional VDJH and non-functional DJH rearrangements. The pattern of mutations and gene segment usage for both types of rearrangements was analyzed by polymerase chain reaction and sequencing. RESULTS: VH3 and VH1 family members were over- and under-represented, respectively. VH3-30 and VH3-15 segments were the most frequently used, whereas VH4-34 was found only in non-functional or heavily mutated VDJH rearrangements. DH2 and DH3 family members were over-represented in both VDJH and DJH repertoires, while the DH1 family was under-represented only in the productive VDJH rearrangements. Finally, DH3-22 and DH2-21 gene segments were found to be over-represented in the functional repertoire while segments commonly used by less mature B-cell malignancies, such as DH6-19 or DH3-3, were under-represented. INTERPRETATION AND CONCLUSIONS: Data reported here help to identify the clonogenic MM cell as a post-germinal center B cell that has undergone selection processes during the germinal center reaction.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Two new 3' PML breakpoints in t(15;17)(q22;q21)-positive acute promyelocytic leukemia.

In the present article, two new types of PML/RARA junctions are described. Both were identified in diagnostic samples from two t(15;17)(q22;q21)-positive acute promyelocytic leukemia (APL) patients who failed to achieve complete remission. By using different sets of primers, reverse transcriptase polymerase chain reaction (RT-PCR) of PML/RARA junctions showed atypical larger bands compared with those generated from the three classical PML breakpoints already described. Sequence analysis of the fusion region of the amplified cDNAs allowed us to determine the specificity of these fragments in both patients. This analysis showed two new hybrid transcripts that were 53 and 306 base pairs (bp) longer than that expressed by the NB4 cell line (PML breakpoint within intron 6), and are the result of the direct joining of RARA exon 3 with PML exon 7a (patient 2) or the 5' portion of PML exon 7b (patient 1), respectively. In patient 1, RT-PCR analysis of the reciprocal RARA/PML junction showed a smaller transcript than that expected in bcr1 cases, while in patient 2 no amplified fragment was obtained. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) showed that both patients had the t(15;17) translocation. The clinical and hematological profiles expressed by the two patients carrying these unexpected types of PML/RARA rearrangement did not differ significantly from that commonly seen in other APLs with the exception of the poor outcome. Genes Chromosomes Cancer 27:35-43, 2000.