Proliferative CD8 T-Cells: A Novel Immune Microenvironmental Determinant of Outcomes in Multiple Myeloma with Translocation (4;14)

Background

High risk (HR) Multiple Myeloma (MM) remains an unmet clinical need. Translocation 4;14 is a HR cytogenetic abnormality occurring in 15% of MM, and is associated with an adverse prognosis. We sought to identify factors that correlated with progression free survival (PFS) in a cohort of t(4;14) patients through high dimensional profiling of the malignant plasma cells (PCs) and tumor micro-environment (TME) through single cell multi-omics.

Methods

We analysed diagnostic bone marrow (BM) samples from 13 patients with t(4;14) MM using the ESCAPE™ platform from Singleron Biotechnologies which simultaneously measures gene and cell surface protein expression of 64 proteins in single cells. Cryopreserved BM samples were stained with antibodies and subsequently sorted on CD138 expression. The CD138 positive and negative fractions were recombined at a 1:1 ratio for analysis using the 10x Genomics 3’ RNAseq kit. Resulting data were analyzed with Singleron Biotechnology’s MapSuite™ single cell analytics platform. Subsequent validation of our findings was performed using the 5’ chemistry version of ESCAPE™ with the 10x Genomics RNAseq platform.

Results

We analysed 107,382 cells from 13 patients and 2 controls. All patients received novel agent-based induction and had a median age of 63 years. Median PFS and overall survival(OS) were 22 and 34 months respectively. No gene or protein expression patterns within the PCs were identified that correlated with PFS or OS. We detected a population of proliferative, effector CD8 T-cells (cluster 26) whose identity was confirmed by independent bioinformatics tools “MapCell” and “DISCO”. For each patient, we derived the proportion of Cluster 26 cells among TME cells and defined the patients in the top 50% as “Clus26 high” and the bottom 50% as “Clus26 low” samples. Survival analysis revealed that Clus26 high patients had significantly shorter PFS (P=0.04, log rank test).This result was independent of R-ISS stage and treatment characteristics. Notably, Cluster 26 expressed LAG3 and TGIT mRNA, suggesting an exhausted phenotype, akin to that reported in dysfunctional CD8 T-cells from solid tumors.

We next derived a gene signature from Cluster 26 cells, comprising CD8A+ and MKI67+ which we applied to an independent cohort of 15 MM patients. Using the gene signature to stratify patients into “Gene-sig high” and “Gene-sig low” groups similarly to what we did for cluster 26 cells, the gene signature also correlated significantly with adverse PFS in this cohort (P=0.03). We subsequently validated the presence of Cluster 26 cells using multiplexed immunofluorescence in a tissue microarray from a separate cohort of 86 MM patients including those without t(4;14). We derived the proportion of cells co-expressing CD8 and Ki67(double-positive cells), among the CD138-negative cells and selected a cut-off of 3.0% double positive (DP) cells to split the patients into “DP high”, (n=49), and “DP low” (n=37) groups. "DP high” patients had an inferior PFS (P=0.04) in keeping with the results from the ESCAPEseq cohorts. Lastly, we performed multiplexed immunohistochemistry on whole slide BM trephine biopsies, preliminary analyses show that proliferative CD8 T-cells are spatially related to PCs, suggesting intercellular interactions.

Conclusions

We present the first application of single cell multi-omic immune profiling in a genomically defined subset of MM and have identified a proliferative T-cell subset of prognostic significance. This population also retains its negative correlation with PFS in non t(4;14) patients, suggesting a broader significance across cytogenetic subtypes of MM. Further work is ongoing to delineate the functional characteristics and therapeutic implications of cluster 26.