Near-Tetraploidy Can Mask the Presence of Chromosome 13q Deletions in Plasma Cells of Patients with Multiple Myeloma.

Ploidy status and chromosomal aberrations are important prognostic features in multiple myeloma (MM). In particular, non-hyperdiploidy i.e. hypodiploid, pseudodiploid and near-tetraploid (NT), and chromosome 13q aberrations are associated with a worse outcome. We have used combined I-FISH and cell morphology analysis with the Duet™ system for the diagnosis of del 13q and IgH aberrations in bone marrow (BM) samples from MM patients (pts). Using this method we suspected that del 13q can be masked in plasma cell populations due to NT chromosome number.

We studied 125 consecutive BM samples from MM pts for the presence of NT. Mean pts’ age was 56±11 years, sixty percent were men, sixty-seven percent had stage III disease and forty-eight percent had IgG myeloma.

Two commercial probes were used in all FISH experiments: LSI 13 (RB1) and LSI IGH dual-color, break-apart rearrangement probe. For further clarification other probes of Vysis were used.

The measurement of plasma cells DNA content (DI) was done by determining the DAPI intensity using the Volocity Grid Confocal system controlled by the Volocity Acquisition software. To examine the accuracy of measurements DI of 2 MM cell lines: U266 and NCI-H929 were determined. DI in the two cell lines deviated only by 3.2% and 3.6%, respectively, therefore supporting accuracy of ploidy measurements. The criteria for the diagnosis of NT included:

1. Duplication of hybridization patterns in both 13q and IgH

2. Normal hybridization pattern in 13q and duplication of hybridization patterns in IgH and another probe

3. Normal hybridization pattern in IgH and duplication of hybridization patterns in 13q and another probe

4. If normal hybridization signals were seen in both 13q and IgH probes but plasma cells had large nuclei, at least two other probes with duplication of hybridization patterns were required for NT determination.

NT was diagnosed in 45/125 BM samples (36%), median plasma cells with NT 12.5%, range 2–100% of the plasma cells population.

DI was determined in 10 BM samples suspected for NT. All the samples studied were verified as NT with a mean DI of 1.7 ± 0.14 (range 1.58–1.90).

Chromosome 13q status was normal in only 8 NT samples (18%), i.e. four hybridization signals were detected. In 32/45 (71%) NT samples there were two hybridization signals in a portion of plasma cells population, representing a duplication of del 13q in the presence of NT. Importantly, in 5 samples (11%) all plasma cells had two hybridization signals for del 13q. These cases could actually be overlooked unless NT was specifically searched for and ruled out, therefore potentially loosing important prognostic information.

In conclusion, we found a high prevalence of NT in MM pts using a sensitive novel assay. The determination of NT is important as del 13q can be masked in BM samples harboring NT DNA content.