Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: Next-generation sequencing of immunoglobulin genes: toward personalized medicine in plasma cell disorders.

Immunoglobulin light chain (AL) amyloidosis is a hematologic disorder caused by a generally small, poorly proliferating plasma cell clone which resides in the bone marrow and produces immunoglobulin light chains that circulate in the bloodstream, misfold and aggregate as extracellular amyloid fibrils. The process of amyloid formation and deposition can lead to cell death, subversion of tissue architecture, organ dysfunction and death. The primary sequence of the disease- causing light chain is patient-unique and dictates the protein’s propensity to aggregate and possibly its organ tropism and toxicity. Since the PC clone is small and difficult to detect, the advent of efficient next- generation sequencing (NGS) techniques to sequence Ig genes may improve our understanding of the molecular mechanisms of the disease as well as the disease management. Research’s laboratory of Italian Amyloid Center – Fondazione IRCCS Policlinico San Matteo in Pavia has recently established an NGS-based technique to sequence the complete variable (V) region of Ig genes from multiple bulk samples in parallel. Here, we established a pipeline of bioinformatics analyses to identify and characterize the V region of both the clonal Ig gene produced by a bone marrow PC clone and of by standing polyclonal Ig genes present in the same biological sample. As a proof of concept, we have applied it first in the context of a validation data set exploring the accuracy, sensitivity and reproducibility of this approach. Sensitivity was evaluated by serial dilutions of RNA extracted from two human cell lines (one - and one -expressing light chains) in the RNA extracted from a healthy donor lacking PC clones. Reproducibility was evaluated by the realization of five replicates of two AL patients (one  and one ) and duplicates from three additional AL λ patients. These studies demonstrated that this newly established NGS-based technique coupled with bioinformatics analyses can accurately identify the complete sequence of the V region of dominant, clonal Ig genes, with the obtained sequences showing 100% identity with sequences identified by conventional methods, with a sensitivity that is dictated by the number of reads achieved during sequencing and with high reproducibility also in terms of defining the molecular clonal size, as a measure of the relative frequency of clonal reads in a given sample. Next, we applied the NGS technique to a cohort of 86 patients affected by AL amyloidosis. We determined the molecular clonal size and the dominant immunoglobulin light chain sequence in these patients and assigned the variable germline gene used through immunogenetics analyses. These analyses confirmed published knowledge on the relative frequency of specific immunoglobulin germline genes in AL amyloidosis. We confirmed known associations of specific light chain sequence features with clinical parameters in AL (including association of IGLV1-44 with heart involvement). Furthermore, we exploited light chain sequence information from previously published amyloidogenic and control clonal light chains and from a series of 220 patients with AL amyloidosis or multiple myeloma to define sequence and spatial features of N-glycosylation. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic k light chains. In conclusion, this novel NGS approach may facilitate obtaining clonal immunoglobulin gene sequence(s) at diagnosis even from small, but dangerous B cell clones. This will significantly expand our currently limited knowledge of molecular determinants of light chain pathogenicity. Additionally, knowledge of the underlying clonal immunoglobulin gene sequence may facilitate the sensitive detection of residual tumoral plasma cells resisting chemotherapy during minimal residual disease assessment. Besides AL amyloidosis, this strategy may be applicable to sequence and detect immunoglobulin involved in other plasma cell dyscrasias or B cell disorders.