NOD SCID indicates nonobese diabetic severe combined immunodeficiency; PCR, polymerase chain reaction; Ig, immunoglobulin; lt chain, light chain.

Single-stage polymerase chain reaction (PCR) used strategy A and nested PCR used strategy B. The single-stage CDR2/CDR3 primed reaction provides a control to confirm the presence of clonotypic messenger RNA in the patient peripheral blood mononuclear cells (PBMCs) used for xenografting to NOD SCID mice, using intracardiac injection, as previously described. Nonclinical clonotypic isotypes were detected only by nested reverse transcriptase (RT)-PCR (strategy B) in the patient PBMCs, suggesting they were quite infrequent cells. Results indicating the presence of nonclinical clonotypic transcripts are underlined. The majority of positive PCR reactions were for murine bone marrow (BM) and vertebral tumors, but spleen cells were also positive. A variety of evidence has suggested that strategies A and B, which are highly specific for the isotype being amplified, do not efficiently amplify clinical or nonclinical clonotypic isotypes in patients and in xenografted mice. This evidence suggests that the results in Table 4are likely to underestimate the true frequency of clonotypic isotypes. This suggestion is exemplified by the results for patient 4, where a single-stage CDR2/CDR3 (strategy D) strongly detected clonotypic transcripts (column 3), but none of the CDR3/nonclinical isotype combinations were able to detect clonotypic transcripts in these 4 mice, nor in all tissues from each mouse that were positive after PCR with CDR2/CDR3 primers (line 4). In contrast, use of a different strategy (PCR stage 1, Vh leader/constant region; stage 2, CDR2/CDR3) detected clonotypic IgM but not any other isotype in BM and spleen of these same mice (not shown). All 4 clonotypic isotypes were detectable in the peripheral cells harvested from each of the 5 patients and used for xenografting. For patient 8, pleural effusion cells that had exuded from a thoracic BM lesion were xenografted. The high clonogenicity of these cells, including detection of clonotypic IgM and nonclinical postswitch IgG using strategy A, suggests that the clonogenic multiple myeloma cells might be of BM origin. No Epstein-Barr virus (EBV) transcripts were detectable in tissues from any of these xenografted mice, eliminating the possibility that clonotypic IgM or any other clonotypic isotypes were derived from EBV transformants.