PMC5804097_01
M-protein levels over time. After initial presentation in 2010, the patient received five rounds of treatment and relapsed each time. The patient received the first dose of daratumumab (16 mg/kg) in October 2013 when he enrolled in the SIRIUS study. M-protein levels were measured at the local laboratory and then centrally after the first dose of daratumumab
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Fig1
The patient's M-protein levels over time and the response to treatment are shown in Fig. 1. 
Change in immune cell populations and properties. A diagram of the T-cell receptor sequencing protocol is shown in a. DNA was extracted from peripheral blood mononuclear cells and subjected to 2 rounds of PCR amplification. During the first round, primers specific for the variable (forward) and joining (reverse) were used to amplify somatically recombined T-cell receptors. During the second round, each amplicon was amplified using primers containing barcode and adapter sequences to facilitate T-cell receptor sequencing via the Illumina platform in the next step of the protocol. The sequencing data were used to evaluate T-cell receptor clonality, diversity, and changes from baseline. Percent changes in CD8 T cells from baseline among patients in the SIRIUS study are shown in b. The patient, indicated by the red line, showed a rapid expansion of CD8+ T cells that was maintained over time. c The percent change from baseline of regulatory T cells over time. d T-cell receptor clonality at baseline versus on-treatment time points. For all patients in black, the on-treatment time point was 3 months. For the patient, the sample time points were 3 and 32 months, in orange and red, respectively. e The T-cell receptor clonality in the case study patient at baseline, 3, and 32 months. PBMCs peripheral blood mononuclear cells, V variable region, D diversity region, J joining region, PCR polymerase chain reaction, TCR T-cell receptor
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Fig2
T-cell receptor sequences with the ImmunoSEQ  assay (Adaptive Biotechnologies, Seattle, Washington, USA) (Fig. 2a). The assay used prequalified multiplex polymerase chain reaction (PCR) assays (TR2015CRO-V-019) composed of forward and reverse primers that directly targeted the family of variable (V) genes (forward primers) and joining (J) genes (reverse primers). T cells. The expansion of CD8+ T cells in this patient was among the largest in the study population, resulting in an increase of approximately 800% from baseline by 3 months following the first daratumumab dose (Fig. 2b). The expansion of the CD8+ T-cell population in this patient was accompanied by a decrease in regulatory T cells of 67% at 3 months (Fig. 2c). T-cell receptor clonality from baseline to 3 months of daratumumab treatment in 16 different patients enrolled in the SIRIUS study are shown in Fig. 2d. The patient had the greatest change in clonal T cells from baseline after 3 months. This clonal T-cell expansion was sustained for 32 months (Fig. 2e).
Minimal residual disease. A diagram of the protocol used to assess minimal residual disease shown in a. Bone marrow aspirates subjected to bulk red blood cell lysis and separated into 2 tubes. Each tube was incubated with an 8-marker antibody combination. Tube 1 was stained for surface markers only and tube 2 surface and intracellular markers. Minimal residual disease was then measured by flow cytometry merging data from each tube into one analysis. As shown in b plasma cells (PC) displayed a CD38high CD138int phenotype with low CD19 and CD45 expression indicative of an immature plasmablast (PC) population. By applying a cutoff value of 1 abnormal/clonal plasma cell per million nucleated event for MRD positivity (i.e., 10-6 sensitivity threshold of MRD positivity), the bone marrow aspirate tested MRD negative. BM bone marrow, RBCs red blood cells, MRD minimal residual disease
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Fig3
Briefly, bone marrow aspirates were incubated post red cell lysis (Pharm Lyse  Buffer, BD Biosciences, San Jose, California, USA) in two separate tubes containing 10-marker antibody combinations against CD138, CD38, CD45, CD19, CD56, CD81, CD117, CD27, and immunoglobulin kappa and/or lambda (see Additional file 1: Table S1) (Fig. 3a). The patient was MRD negative at the 10-5 sensitivity threshold (one cancer cell in 100,000 normal cells; Fig. 3b). The plasma cell distribution represented 0.0035% of total nucleated cells, with no phenotypic abnormalities or kappa-to-lambda ratio bias.