Second Clones In Molecularly-Defined Biclonal Multiple Myeloma Are Derived From Unrelated B Cells, Can Be Present at High Frequency and Exhibit Chromosomal Abnormalities

Abstract 2981

Typically MM involves a single monoclonal protein, predicting a single CDR3 peak. We have shown that a sizeable number of patients do not fit this pattern. RT-PCR and genomic PCR (gPCR) were performed using universal primers that bind to FR3/JHc or FR3/CH1 to identify the monoclonal CDR3. In a cohort of 49 MM patients, 10 (20%) exhibited two CDR3 peaks, one of the clinically identified MM clone and another “second” clone. With one exception, a biclonal gammopathy, these second clones were not detected serologically by serum protein electrophoresis or immunofixation. Molecular biclonality is categorized into 3 groups based on the isotype of second clones: group I (1 IgD and 4 IgG MM) where second clone has a pre-switch isotype; group II (3 IgG MM) where the second clone has a post-switch isotype and group III (2 IgA MM) where the second clone exhibits ongoing class switch recombination. All MM clones undergo somatic hypermutation (2.44-15.03%, median = 8.55%). For second clones, 2/10 are unmutated (0-1.05%, below 2% cut off) and 8/10 are mutated (2.44-13.54%, median = 5.90%). The VH3 gene family was utilized in 6/10 of MM clones and 5/10 of second clones. For 10/10 patients, the clinical MM clones are unrelated to second clones as shown by different combinatorial VDJ, different length of CDR3 and different VDJ junction. Biclonal distribution was compared by grading of CDR3 peak heights generated by gPCR. For 9/10 patients, the MM clone is most frequent in BM, 3 of which also predominated in blood. For 6/10 patients, second clones predominated in blood. Q-PCR of mononuclear cells (MNC) showed that second clones ranged from 0.06–24% (median = 1%, n=7) in BM and 0.1–31% (median = 1.45%, n=6) in blood. Chromosomal abnormalities in plasma cells (PC) were seen in 8/8 patients analysed; among these, 4 had t(11;14). Other genetic abnormalities included deletion 13q14, amplification 1q21, trisomy and tetrasomy of chromosome 1. For 4/5 patients where second clones could be distinguished from the MM clone by IgH immuno-FISH, chromosomal abnormalities were detected in the second clone, which in some cases were different from those in the MM clone. Longitudinal analysis was studied in 3 patients. For case 1, a biclonal gammopathy, the IgGλ clone exhibited deletion 13q14 and amplification 1q21, and was dominant in a sternal biopsy (IgGλ/IgGκ = 3.6%/<0.01% MNC; PC<5%MNC); the IgGκ clone exhibited t(11;14) and was the dominant clone in sequential iliac biopsies (IgGλ/IgGκ = 0.3%/3.3% MNC; PC=5-10%MNC). Both clones responded to treatment as measured by Q-PCR, and CDR3 analysis. Case 2 had an IgG MM clone and an IgM second clone; the IgG clone had del13q14 and t(11;14) while the second clone had no detected chromosomal abnormalities. Both clones persisted for at least 2 years during which the MM clone comprised 26–41% of blood MNC while the second clone comprised 12–31% of MNC. The abnormally high abundance suggested a pathological role for the second clone in this patient. Case 3 had an IgD MM clone with an IgM second clone utilizing unmutated VH1-69/DH3-3/JH6 and a long CDR3. Several chromosomal abnormalities including t(11;14), trisomy 11, 14 and 17 were observed in IgD⁺ and IgM⁺ cells. The second clone is 0.06% in BM and 1.2% in pre-treatment blood, higher than the expected frequency of antigen-specific B cells. The second clone persisted over 7 months although both MM and second clones responded to treatment. Overall, our studies show that molecular biclonality is frequent in MM, and some second clones have a high frequency in blood and/or BM. Biclonal detection in multiple specimens suggests the persistence of partner clones. The unrelated VDJ origin and distinct chromosomal abnormalities between MM and second clones suggests that they may arise from different transformation events. The particularly high abundance of the second clone in some patients predicts a direct contribution to pathogenesis and disease outcome. Although the clinical significance of second clones remains unclear, the high frequency for some, the presence of chromosomal abnormalities and their persistence during therapy suggest that they may be clinically significant. The prevalence of MM patients having genetically abnormal and frequent second clones suggests that MM may be characterized by more than one transformation event, with clonal dominance defining the clinically identified MM clone.