Overall, 228 MM diagnostic (n=66) and follow-up (n=162) BM samples (n=110 in VGPR or CR/sCR) were evaluated in parallel with the NGF MRD approach vs local routine flow-MRD methods (that is, conventional 8-color flow-MRD technique).24 (link) Detailed description of these BM samples, related patient clinical data, disease status and time points at evaluation is provided in Supplementary Tables 1, 3 and 4 . Briefly, conventional flow-MRD was based on staining of 300 μl of whole BM with a single 8-color antibody combination (CD45-PacB CD138-OC515 CD38-FITC CD56-PE CD27-PerCPCy5.5 CD19-PECy7 CD117-APC CD81-APCH7), as previously described.24 (link) In turn, for the NGF approach a median volume of 1.5±1.3 ml (range: 0.1–5.3 ml) was employed adding up to a median total sample volume of 1.8 ml (maximum of 5.6 ml). PC populations that coexisted in individual BM samples were identified based on a combination of the CD38, CD138, CD45 PC-associated markers and light scatter characteristics, the presence vs absence of myeloma-associated phenotypes, plus CyIg light chain restriction in case of NGF, as described elsewhere.48 (link) According to consensus recommendations,42 (link) the limit of quantitation and the LOD of the NGF MRD method was calculated at <5 × 10−6 and <2 × 10−6 aPC, based on the identification of ⩾50 and ⩾20 aPC among 107 events, respectively. More detailed information about instrument conditions, data acquisition and analysis, and the specific reagents used in the present study is provided as Supplementary Material .
Automatic identification and enumeration of aPC was performed in 110 MM BM follow-up (VGPR or CR/sCR) samples using the automatic gating function of the Infinicyt software and previously described procedures,49 , 50 and the results were compared against the conventional expert-guided PC-identification/gating approach. For automatic gating, a database consisting of a subset of 14 normal BM samples stained with Version 5 of the antibody panel was constructed and used.50 In a subset of 31 MM follow-up BM samples with low MRD levels (for example, ⩽10−4) in which enough DNA was available, MRD was also evaluated by NGS. For this purpose, patient-specific VDJH rearrangements were amplified and directly sequenced from DNA extracted from diagnostic samples using the DNAzol reagent (MRC, Cincinnati, OH, USA) and IGHV family-specific primers that covered framework regions 1 (FR1) and FR2, plus a JH consensus primer, as described elsewhere.26 (link), 32 (link)VDJH rearrangements identified at diagnosis were used as MRD-targeted sequences for subsequent follow-up samples. Follow-up DNA samples were amplified using the LymphoTrack IGH Assay (InVivoScribe Technologies, San Diego, CA, USA) and sequenced in an Illumina MiSeq platform (Illumina, San Diego, CA, USA). To all reactions, a known amount of DNA from the MWCL-1 cell line was added as reference control for cell enumeration. The Fastq files generated were analyzed with the LymphoTrack/MiSeq Software (InVivoScribe/Illumina). The number of MRD cells was calculated from the number of reads for the diagnostic VDJH target rearrangements and the number of reads of the reference rearrangement; percentage MRD was calculated upon dividing the number (× 100) of MRD cells by the total number of cells in the reaction.
Automatic identification and enumeration of aPC was performed in 110 MM BM follow-up (VGPR or CR/sCR) samples using the automatic gating function of the Infinicyt software and previously described procedures,49 , 50 and the results were compared against the conventional expert-guided PC-identification/gating approach. For automatic gating, a database consisting of a subset of 14 normal BM samples stained with Version 5 of the antibody panel was constructed and used.50 In a subset of 31 MM follow-up BM samples with low MRD levels (for example, ⩽10−4) in which enough DNA was available, MRD was also evaluated by NGS. For this purpose, patient-specific VDJH rearrangements were amplified and directly sequenced from DNA extracted from diagnostic samples using the DNAzol reagent (MRC, Cincinnati, OH, USA) and IGHV family-specific primers that covered framework regions 1 (FR1) and FR2, plus a JH consensus primer, as described elsewhere.26 (link), 32 (link)VDJH rearrangements identified at diagnosis were used as MRD-targeted sequences for subsequent follow-up samples. Follow-up DNA samples were amplified using the LymphoTrack IGH Assay (InVivoScribe Technologies, San Diego, CA, USA) and sequenced in an Illumina MiSeq platform (Illumina, San Diego, CA, USA). To all reactions, a known amount of DNA from the MWCL-1 cell line was added as reference control for cell enumeration. The Fastq files generated were analyzed with the LymphoTrack/MiSeq Software (InVivoScribe/Illumina). The number of MRD cells was calculated from the number of reads for the diagnostic VDJH target rearrangements and the number of reads of the reference rearrangement; percentage MRD was calculated upon dividing the number (× 100) of MRD cells by the total number of cells in the reaction.
