Biomarker assessments
BMBx
PROLIFERATION
For biomarker and correlative studies, peripheral blood (PB), BM aspirates (BMA) and BM core biopsies (BMBx) were obtained at screening, on treatment at cycle (C) 2 day (D) 15 or prior to C4, and at end of study from patients receiving Atezo monotherapy (Cohort A) or Atezo+Dara (Cohorts D1, D2 and D3) (Flow cytometry was performed using longitudinal PB and BMA to characterize CD8+ cytotoxic T cells using 8 color flow panels at the central laboratory (Covance, NC, USA) according to the laboratory protocol. CD8+ T-cell activation and proliferation were analyzed using %CD8+HLA-DR+Ki-67+.23Formalin-fixed and decalcified paraffin-embedded tissue sections (4 mm thickness) taken at baseline were used for dual-plex immunohistochemistry (IHC) assay (CD138/tartrate-resistant acid phosphatase [TRAP]) at Histogenics Corp (MA, USA). Osteoclasts were enumerated based on TRAP positivity and morphology (RNA sequencing (RNAseq) was performed using longitudinal CD138+ and CD138 (–) enriched BM fractions. RNA isolation, library preparation using the Illumina (CA, USA) TruSeq RNA Access Kit and sequencing using the Illumina HiSeq System (2X50 bp paired-end and target (Mass cytometry immunophenotyping (39 marker CyTOF panel; Fluidigm, CA, USA) and proteomic profiling (multiplex immuno-oncology assay [Olink]; Olink Proteomics, Uppsala, Sweden) were performed on BM mononuclear cells (BMMC) and BM plasma (For the unbiased integrative analysis, a similarity network fusion (SNF) algorithm (Gene set enrichment analysis was implemented to further understand the underlying biological difference between clusters using the gene expression data. One vs all comparison was performed using the hallmark gene set (MSigDB v5.2) (