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The bone marrow microenvironment interacts with malignant cells and regulates cancer survival and immune evasion in multiple myeloma (MM). We investigated the immune profiles of longitudinal bone marrow samples from patients with newly diagnosed MM (n = 18) using cytometry by time-of-flight. The results before and during treatment were compared between patients with good (GR, n = 11) and bad (BR, n = 7) responses to lenalidomide/bortezomib/dexamethasone-based treatment. Before treatment, the GR group had a lower tumor cell burden and a higher number of T cells with a phenotype shifted toward CD8+ T cells expressing markers attributed to cytotoxicity (CD45RA and CD57), a higher abundance of CD8+ terminal effector cells, and a lower abundance of CD8+ naïve T cells. On natural killer (NK) cells, increased expression of CD56 (NCAM), CD57, and CD16 was seen at baseline in the GR group, indicating their maturation and cytotoxic potential. During lenalidomide-based treatment, the GR patients showed an increase in effector memory CD4+ and CD8+ T-cell subsets. These findings support distinct immune patterns in different clinical contexts, suggesting that deep immune profiling could be used for treatment guidance and warrants further exploration.

Background In this phase 2 study patients received 3 cycles of RVD (lenalidomide, bortezomib and dexamethasone) followed by autologous stem cell transplantation (ASCT) and lenalidomide maintenance. We evaluated the bone marrow (BM) immune profile with CyTOF (cytometry by time-of-flight) at treatment start and during lenalidomide maintenance focusing on two different response groups: good and poor responders at pre- and post-treatment phases. Our hypothesis was that there could be distinct differences in immune cell profiles between these groups, especially in T and NK cell subsets and exhausted T-cells. Methods Twenty-six patients were included in this study, 18 from this trial. BM samples were collected from all 18 patients at diagnosis, from 11 the 1st sample when achieved good response during maintenance after a median of 21 (6-46) months and the 2nd sample if good response was maintained after a median of 56 (45-67) months and from 5 patients at relapse after a median of 6 (2-23) months. Patients in good response cohort (n=11) had progression-free survival (PFS) > 5 years. For comparison we included 4 BM samples, taken at good response after ASCT from MM patients not exposed to lenalidomide and 4 BM samples collected from age-matched, healthy donors. Results With a median follow-up of 81 (13-97) months the median PFS was not reached in the good response cohort and was less than 18 months in the poor response cohort. CyTOF analysis revealed distinct good (GR) and poor responder’s (PR) immune signatures at baseline level. GR, baseline group has shifted phenotype of T cells toward the CD8 T cells, expressing markers, attributed to the cytotoxicity (CD45RA, CD57), as well as having slightly higher abundance of CD8 TE and lower abundance of CD8 naive T cells. Total T cell amounts were significantly higher in good responders. Increased expression of CD56, CD57, and CD16 were also seen on NK cells in good responders at the baseline, indicating both maturation and cytotoxic potential of NKs. In contrast, a significant decrease of CD56 and CD16 expression suggesting reduced cytotoxic potential and increase of CD57 were seen on NKs in poor responders, baseline indicating senescence status a phenotype associated with exhaustion. Treatment stimulates the expression of cytotoxic/effector-like phenotype on T cells which is confirmed by the significantly increased amounts of CD4 and CD8 effector memory cells, with the respective decrease of naive cells. Conclusions Patients responded to the treatment, have higher effector/cytotoxic cells, expressing higher levels of CD57 and/or CD45RA for T cells, and CD57, CD16, and CD56 for NK cells. Additionally, those patients have less degree of tumor burden as well as decreased expression of chemokine receptors (CCR7, CCR6, CXCR4, CXCR3, and CXCR5). Good responders showed the increase in effector memory CD4 and CD8 subsets of T cells abundance, indicating even the higher cytotoxic effect of immune system.

Introduction Here we report the results of one of the secondary endpoints of the Finnish Myeloma Group-MM02 study; composition of bone marrow (BM) immune cell subsets at treatment start and during lenalidomide (Len) maintenance focusing on 2 different response groups: good responders (GRs) and poor responders (PRs) at pre- and post-treatment stages. We evaluated the BM immune profile with CyTOF (cytometry by time-of-flight). Our hypothesis was that there would be distinct differences in immune cell profiles between patients with good and poor response, especially in the T and NK cell subsets. Patients and methods Twenty-two patients were included in this CyTOF study. Eighteen were NDMM patients from FMG-MM02 study who received 3 RVD cycles followed by ASCT. Len started 3 months after ASCT 10 mg/day in 21/28-day cycles until progression or toxicity. BM samples were collected to the Finnish Hematology Registry Clinical Biobank (FHRB Biobank) at several timepoints; from 18 patients at diagnosis, from 11 patients 1 st sample at good response during Len and 2 nd sample if this good response was maintained and from 5 patients at relapse during Len. The patients in the good response cohort (n=11) had progression-free survival (PFS) > 5 years. For comparison, we included 4 BM samples from the FHRB Biobank, taken at a good response after ASCT from MM patients without exposure to Len. Results With a median follow-up of 81 months (13-97) the median PFS was not reached in the good response (GR) cohort and was 18 months in the poor response (PR) cohort. The 1 st GR samples were collected after a median of 21 (6-46) months of Len. The 2 nd samples in GR cohort were collected after a median of 56 (45-67) months of Len. The PR samples were taken after a median of 6 (2-23) months of Len. CyTOF analysis revealed distinct good and poor responder's immune signatures at baseline level. GR, baseline group has shifted phenotype of T cells toward the CD8 T cells, expressing markers, attributed to the cytotoxicity (CD45RA, CD57), as well as having slightly higher abundance of CD8 TE and lower abundance of CD8 naïve T cells. Total T cells amounts were significantly higher in GR. Increased expression of CD56, CD57, and CD16 were also seen on NK cells in GR at the baseline, indicating both maturation and cytotoxic potential of NKs. In contrast, a significant decrease of CD56 and CD16 expression suggesting reduced cytotoxic potential and increase of CD57 were seen on NKs in PR, baseline indicating senescence status a phenotype associated with exhaustion. By using viSNE we discovered two novel populations - MAC1, and MAC2, - associated with disease pathogenesis. We further investigated the composition of these populations, and it appears that MAC1 expressed CD38+CD56+CD45-CD19- presumably malignant plasma cells and MAC2 expressed CD45- CD14+CD38+CD56+TCRgd+CCR7+CCR6+CXCR4+ CXCR3+CXCR5+CD294+, which might be the novel populations, having the features of myeloid suppressor cells. These two immune cells were higher in PR (9.2% and 8.2%, respectively) at baseline as compared to the GR (2.3% and 1.7%). NKs, T cells, and B cells showed a high expression of monocytic marker CD14 and chemokine receptors (CCR7, CCR6, CXCR4, CXCR3, and CXCR5) in PR both at baseline and relapse. This might be attributed to either biological features of MM environment, or the high level of interaction between these populations and monocytes, which left traces of their membranes on the non-monocytic populations. Conclusions Patients, responded to the treatment, have higher abundances of effector/cytotoxic cells, expressing higher levels of CD57 and/or CD45RA for T cells, and CD57, CD16, and CD56 for NK cells indicating proper differentiation and maturation of T, and NK cells to effector and cytotoxic subsets. Additionally, those patients have less degree of tumor burden as well as decreased expression of chemokine receptors. During the therapy administration, good responders show the increase in effector memory CD4 and CD8 subsets of T cells abundance, indicating even the higher cytotoxic effect of immune system. Disclosures Silvennoinen: Amgen: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding. Luoma: Amgen: Honoraria; Janssen: Honoraria; Incyte: Honoraria. Anttila: Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Takeda: Honoraria. Säily: Takeda: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria. Partanen: Takeda: Honoraria; Abbvie: Honoraria; Behring: Honoraria. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding.