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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.

The role of allogeneic hematopoietic stem cell transplantation (allo-SCT) in multiple myeloma is controversial. We analyzed the results of 205 patients transplanted in one center during 2000–2017. Transplantation was performed on 75 patients without a previous autologous SCT (upfront-allo), on 74 as tandem transplant (auto-allo), and on 56 patients after relapse. Median overall survival (OS) was 9.9 years for upfront-allo, 11.2 years for auto-allo, and 3.9 years for the relapse group (p = 0.015). Progression-free survival (PFS) was 2.4, 2.4, and 0.9 years, respectively (p < 0.001). Non-relapse mortality at 5 years was 8% overall, with no significant difference between the groups. Post-relapse survival was 4.1 years for upfront-allo and auto-allo, and 2.6 years for the relapse group (p = 0.066). Survival of high-risk patients was reduced. In multivariate analysis, the auto-allo group had improved OS and chronic graft-versus-host disease was advantageous in terms of PFS, OS, and relapse incidence. Late relapses occurred in all groups. Allo-SCT resulted in long-term survival in a small subgroup of patients. Our results indicate that auto-allo-SCT is feasible and could be considered for younger patients in the upfront setting.

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.

Autologous stem cell transplantation (ASCT) combined with novel agents is the standard treatment for transplant-eligible, newly diagnosed myeloma (NDMM) patients. Lenalidomide is approved for maintenance after ASCT until progression, although the optimal duration of maintenance is unknown. In this trial, 80 patients with NDMM received three cycles of lenalidomide, bortezomib, and dexamethasone followed by ASCT and lenalidomide maintenance until progression or toxicity. The primary endpoint was the proportion of flow-negative patients. Molecular response was assessed if patients were flow-negative or in stringent complete response (sCR). By intention to treat, the overall response rate was 89%. Neither median progression-free survival nor overall survival (OS) has been reached. The OS at 3 years was 83%. Flow-negativity was reached in 53% and PCR-negativity in 28% of the patients. With a median follow-up of 27 months, 29 (36%) patients are still on lenalidomide and 66% of them have sustained flow-negativity. Lenalidomide maintenance phase was reached in 8/16 high-risk patients but seven of them have progressed after a median of only 6 months. In low- or standard-risk patients, the outcome was promising, but high-risk patients need more effective treatment approach. Flow-negativity with the conventional flow was an independent predictor for longer PFS. Electronic supplementary material The online version of this article (10.1007/s00277-019-03815-7) contains supplementary material, which is available to authorized users.

Introduction: The median progression free survival (PFS) and overall survival (OS) of multiple myeloma (MM) patients have been prolonged due to novel agents combined with ASCT but the median OS in MM is still 7-8 years. Thus, the feasibility of new combinations and dosing of available agents must be investigated. The first proteasome inhibitor (PI), bortezomib (B), combined with elotuzumab and dexamethasone (d) showed superiority to Bd with PFS of 9.7 vs. 6.9 months, respectively, without excessive toxicity (Jakubowiak et al. Blood 2016;127:2833-40). In our study we investigate the safety, feasibility and initial efficacy of a second generation PI carfilzomib (K), SLAMF7 antibody elotuzumab (E) and dexamethasone (D) combination (KED) in relapsed or refractory MM (RRMM) patients. Patients and methods: Forty RRMM patients after 1-3 prior lines will be included in this phase 2 study after written informed consent. The primary endpoint is overall response rate (ORR). In patients achieving at least very good partial remission (VGPR) the quality of response will be assessed with high-sensitivity multicolour flow cytometry (MFC) according to the 8-colour EuroFlow protocol. Carfilzomib is given once weekly 20 mg/m2 on D1C1 and thereafter 70 mg/m2 in 28 day cycles on days 1, 8 and 15 in cycles 1-8 and on days 1 and 15 thereafter combined with weekly elotuzumab 10 mg/kg on days 1, 8 and 15 in cycles 1-2, thereafter on days 1 and 15; dexamethasone 40 mg on days 1, 8, 15 and 22 on cycles 1-8, thereafter on days 1 and 15. Treatment will continue until progression or excessive toxicity. Carfilzomib dose was 20/56 mg/m2 for the first two cycles for the first five patients to evaluate the safety. Additionally, patient samples collected prior to treatment will be comprehensively profiled by whole exome and RNA sequencing and evaluated for ex vivo response to the agents. Together, the study addresses clinical response, ex vivo-in vivo translation, identifies molecular biomarkers for the KED combination and facilitates precision guided clinical trials for RRMM. Results: By the end of July 2018, 11 patients have been enrolled. Median number of prior lines was 2 (1-3). Seven IgG-κ, two IgA-κ and one kappa and lambda light-chain patient are included. After a median of 6 (2-12) cycles ORR is 91% with 3 patients in VGPR (median MFC-MRD of 0,002%), 7 patients in PR, one in MR. Initial molecular characterization highlighted diverse subclonal backgrounds among the treated patients (Figure 1), but interestingly, the best responding VGPR patients displayed mutations to RAS genes in the dominant clones (NRAS 828, 2662; KRAS 733). At least PR was achieved after a median of 1 (1-4) cycle. Three patients have progressed and one patient withdrawn due to suspected thrombotic microangiopathy with manifestation of convulsions and pulmonary embolism. We noticed only one grade 2 infusion reaction after premedication. One patient developed autoimmune hemolytic anemia (AIHA), without red cell antibodies, suspected to be related to elotuzumab. She recovered with steroids and elotuzumab discontinuation and continued Kd without reappearance of AIHA. Another patient had grade 3 liver transaminase elevation but was able to continue treatment with dose reduction of carfilzomib and dexamethasone. Conclusion: To the best of our knowledge this is the first study evaluating the carfilzomib, elotuzumab and dexamethasone combination in RRMM with comprehensive molecular annotations. Among this small group of patients we noticed two unexpected severe adverse events; atypical AIHA and suspected thrombotic microangiopathy. AIHA should be excluded if unexpected anemia will appear during elotuzumab treatment. Preliminary results of this KED combination showed efficacy in patients with clonal RAS mutations and ORR of 91% after the median of two prior treatment lines using weekly carfilzomib dose of 70 mg/m2. Figure 1: Subclonal diversity in patient derived plasma cells. Clonal and subclonal fractions were evaluated by assessing peaks of variant allele frequencies of somatic mutations present in the myeloma plasma cells using kernel density estimation. For each plot, the fraction of cells carrying the mutation is represented on the x axis (1 = 100% of cells), and the probability density on the y axis. The mutated cell fraction (x axis) was calculated by adjusting mutant allele burden by copy number of the mutated loci. Disclosures Silvennoinen: Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Tsallos:Novartis: Research Funding. Anttila:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Räsänen:Amgen: Honoraria. Luoma:Amgen: Honoraria. Jantunen:Amgen: Honoraria; Genzyme/Sanofi: Honoraria; Takeda: Honoraria. Heckman:Celgene: Research Funding; Orion Pharma: Research Funding; Novartis: Research Funding.