Phenotypic, Transcriptomic and Genomic Characterization Of Clonal Plasma Cells (PCs) From Newly Diagnosed Patients With Light Chain Amyloidosis (AL)

In AL, a small PC clone synthesizes a misfolded light-chain that forms amyloid fibrils causing organ dysfunction. Significant progress was made regarding the characterization of the amyloid fibrils, but little attention has been paid to the molecular features of clonal PCs; this is most likely explained by the low tumor burden in AL, often masked by a polyclonal PC background. Here, we investigated the phenotypic, transcriptomic and genomic profile of clonal PCs from a total of 22 patients with newly-diagnosed AL. Through multidimensional (12-color) flow cytometry (MFC) combining the evaluation of 10 antigens plus cyκ/cyλ (thus confirming clonality of aberrant phenotypes), we detected clonal PC in all 22 (100%) patients (median 0.76%; range: 0.01% - 30%). Clonal PCs mainly differed from normal PCs by down-regulation of CD19 (100% of cases), CD27 (50%), CD38 (41%), CD45 (50%) and CD81 (50%); CD117, CD28 and CD56 were aberrantly bright positive in 32%, 50% and 64% of patients, respectively. Principal component analysis showed overlapping immunophenotypic expression profiles between clonal PC from AL vs multiple myeloma (MM) and MGUS patients.

Using patient-specific aberrant phenotypes, we then sorted clonal PCs (purity ≥97%) by MFC for subsequent molecular studies. Gene expression profiling (GEP) was performed (HumanGene1.0ST) on extracted RNA from clonal PCs of 10 of the 22 AL patients, and compared to FACS-purified PCs from 7 healthy donors. Overall, clonal PCs showed deregulation (SAM Excel add-in with a FDR q-value<10^-5) of only 74 genes (3 up- and 71-downregulated); thus, clonal PC from AL patients showed a GEP that mainly overlapped to that of normal PCs, with only a few relevant functional categories potentially altered (increased cell survival and impaired cell migration) by down-regulation of CD9, CD99, CDH1, FAS, FOSL2, GLIPR1, HIF1A, KCNMA1, TUBA1A, PIK3R5, and TRIO, with over-expression of PRDM5. Noteworthy, gene expression of CD27 and CD81 was also significantly decreased in clonal PCs, consistent with their aberrant phenotype.

Afterwards, we investigated the genomic profile of clonal PC from 8/22 AL patients through high density Cytoscan750K array. Interestingly, copy number abnormalities (CNA; defined by >25 consecutive imbalanced markers per segment, >100Kb length and <50% overlapping variants with patient-paired control DNA) were detected in 100% of patients with AL, with a median of 10 CNA/patient (range, 4-24). Whole chromosomal gains were observed for chromosomes 3, 5, 9, 11 and 19, with 2 patients (20%) showing a hyperdiploid genomic profile. Whole chromosomal arm gains were noted at 1q and 15q, while losses were detected at 13q, 16q, 17p and 22q. A total of 30 interstitial losses and 41 interstitial gains were also found. Further investigation of recurrent chromosomal imbalances unraveled that gains at 1q21.1 was present in 63% of AL patients, while gains at 11q13.3 and del(13q12.2-13q14.2) in 50%. Copy number neutral loss of heterozygosity (CNN-LOH) larger than 3Mb were detected in 6/8 (75%) of AL patients (median of 2.5 LOH/patient); interestingly, two of the CNN-LOH were located on the 1q21 and 11q13 loci (the latter encoding for cyclin D1), which overlapped with previously identified recurrent CNA.

In this integrated phenotypic, molecular and genomic characterization of purified clonal PCs from AL patients, we showed that these display aberrant phenotypes similar to MM, but that the GEP of AL clonal PCs is far less deregulated. Conversely, CNA are present in virtually all patients and with frequencies similar to MM. However, while hyperdiploidy was a less frequent phenomenon in AL as compared to MM, our results show that in highly purified clonal PCs, gains at 1q21.1 can be detected in approximately two-thirds of AL patients.