Defining the utility and suitability of Immune Diversity Genotyping (IDG) in multiple myeloma: Early steps in immune biomarker development

High-throughput sequencing of B- and T-cell receptors may reveal insights into adaptive immunity in cancer, yet its clinical utility in multiple myeloma (MM) remains unclear. This thesis first develops, then critiques parallel bulk-repertoire sequencing assays for these receptors, then utilizes them in two large trial cohorts from the NCRI Myeloma X trial.

Chapter 2 outlines the development of multiplex PCR assays for IGH and TCRβ, exploring the role of starting material, primer choice and mechanisms designed for error correction. Through the creation of a bioinformatics pipeline, comparable repertoires were able to be generated. Incoporating FastQC, PRESTO, MiXCR and Immunarch, the pipeline was able to demonstrate that sequencing depth profoundly skews diversity metrics and that extensive bioinformatic processing was unable to reproducibly correct artefacts in library preparation and sequencing.

Chapter 3 interrogates 425 peripheral-blood samples from the Myeloma X trial, with samples from multiple timepoints available for each patient. However, the age of the samples, their quality and the advent of the pandemic severely impacted the sequencing results. Only 105 libraries from 52 patients meet quality thresholds, and primer bias, variable sequencing depth and absent cell counts obscured any independent link between T-cell diversity and outcome.

Chapter 4 sequences IGH in 94 libraries produced from the same cohort. The technical confines of B-cell sequencing in MM were even more pronounced than in T-cell sequencing and sample quality limited conclusions further. Within the assessable samples, lower B-cell diversity trended towards male sex and age but there was no reproducible association with progression-free or overall survival.

Collectively, the thesis outlines the technical confines within which bulk repertoire profiling can inform MM biology, particularly when using imperfect samples. It highlights prerequisites for future studies and repertoire sequencing techniques, positioning similar methods as complementary tools for monitoring immune competence in plasma-cell disorders, an area of increasing importance in an era of evolving immunotherapies.