P020

Comparing Minimal Residual Disease Detection in Multiple Myeloma
using NGS-Based LymphoTrack® Assays and Flow Cytometry
Ying Huang1, Austin Jacobsen1, Jeff Panganiban1, Edgar Vigil1, Kasey Hutt1, Fuensanta W. Martinez1, Alejandro Medina2, Cristina Jiménez2, Ramón García-Sanz2,Jeffrey E. Miller1
1Invivoscribe, Inc, San Diego, CA, and 2Hospital Universitario de Salamanca-IBSAL, IBMCC-CSIC, Salamanca, Spain

Results: MRD Samples by LymphoTrack Assays – MiSeq®

Introduction
Multiple myeloma (MM), which can be identified by the presence of >10% clonal plasma cells inbone morrow (BM), is the second most common hematological malignancy. Multiparameter
flow cytometry (MFC) is a standard tool used to detect and monitor MM patients. Next
generation sequencing (NGS) based methods have demonstrated advantages with improved
sensitivity, and international organizations (NCCN, IMWG and ESMO) have recently included
NGS for minimal residual disease (MRD) assessment in MM. Here we report results of a pilot
study comparing the detection of MRD using NGS-based LymphoTrack® Assays and MFC bytesting 101 specimens from MM subjects.

MRD Samples for FR1/FR2

LymphoTrack®
FR1/FR2MiSeq® Assay

N=82

Detected

Not Detected

Detected

3511

Not Detected1125

ConcordanceMaterials and Methods
LymphoTrack (% Clonal cell equiv.)

The workflow for the LymphoTrack IGH FR1, IGH FR2, IGH FR3, and IGK Assays is depicted
below:
PCR

Quantify

Amplicons

Amplicons

Pool
LibraryRun
MiSeq®

AnalyzeData

101 paired BM samples from MM patients were tested in this study. The MFC methods utilized
an 8-color direct immunofluorescence technique and tested 1-5 million cells. Our NGS based
MRD assessment tested less than ~1/10 as many cell equivalents of genomic DNA (0.7µg,~107,000 cell equivalents) from the collected specimens which were blinded prior to testingwith four LymphoTrack Assays (IGH FR1, FR2, FR3 and IGK) on a MiSeq®. LymphoTrack Software
was used, which automatically sorted data by target and index. The primary clonalrearrangement (clonotype) identified by LymphoTrack FR1 or FR2 Assay was then used to trackdisease in the 91 subsequent samples. LymphoQuant® Internal Control was added to each PCR
reaction at 100 cell equivalents to estimate the cell equivalents within each sample.
LymphoTrack Software and LymphoTrack MRD software were used to analyze the sequencing
results from baseline and follow up samples, respectively.

Results: Baseline Samples by LymphoTrack Assays – MiSeq®
Individual Assays

FR1

FR2

73.2%

Detection Comparison:
LymphoTrack vs Flow Cytometry

The LymphoTrack® IGH and IGK Assays for the MiSeq were manufactured under cGMP
standards per ISO 13485. Master Mix with 24 different indices to allow the simultaneous
testing of multiple samples and targets on the same MiSeq® flow cell.

Purify

MFC

FR311
97

R² = 0.9303

531-1-1

1

35
7Flow Cytometry (% Tumor cells)

9Samples (N=11) are LymphoTrack - / MFC+Sample

LymphoTrack
DNA input (Cell Equiv. )%Tumor Cells
by MFC

SampleLymphoTrack
DNA input (Cell Equiv. )%Tumor Cells
by MFC

9700ng ( ~1.1x105 )

0.0005159289ng ( ~4.4x104 )

0.001

41700ng ( ~1.1x105 )0.0628880225ng ( ~3.5x104 )0.03204

44700ng ( ~1.1x105 )0.003336

83138ng ( ~2.1x104 )0.086148

47486ng ( ~7.5x104 )0.036504

90243ng ( ~3.7x104 )

0.789004

49430ng ( ~6.6x104 )0.04552101

237ng ( ~3.6x104 )0.1922151

263ng ( ~4.0x104 )

0.0008MRD Detection Comparing DNA input levels
by LymphoTrack vs Flow Cytometry

IGK

60%
Clonal(C)

84/101(83.2%)

80/101(79.2%)

63/101(62.4%)

87/101(86.1%)

Non-Clonal(NC)

15/101(14.9%)

21/101(20.8%)

38/101(37.6%)

14/101(13.9%)

Invalid(I)

2/101(2.0%)

0/101
(0%)

0/101
(0%)

0/101
(0%)

50%

50%

40%
30%

43%
30%

20%
10%

Combined AssayResults

FR1/FR2

Clonal(C)

95/101(94%)

99/101(98%)

98/101(97%)

101/101(100%)

Non-Clonal
(NC)

6/101
(6%)

2/101
(2%)

2/101
(2%)

0/101
(0%)

Invalid
(I)

0/101
(0%)

0/101
(0%)

1/101
(1%)

0/101
(0%)

FR1/FR2/FR3

FR1/FR2/FR3/IGK

FR1/IGK

Clonal Detection Rate in Baseline Samples
100%
94%
80%
60%

83%

98%

86%
79%
62%

40%

97%

100%

35%

13%

13%

8%

0%
FR1/FR2 MRD

EXCLUDES SAMPLES <700 NG OF DNA

LymphoTrack - / Flow Cytometry LymphoTrack + / Flow Cytometry -LymphoTrack + / Flow Cytometry +LymphoTrack - / Flow Cytometry +MRD Samples (Excluding <700 ng DNA)

LymphoTrack
IGH FR1 MiSeq® AssayAlone

6%

MFC

N=48

Detected

Not Detected

Detected

234

Not Detected318

Concordance

85.4%

Conclusions• LymphoTrack assays were able to detect clonotype sequences in 100% ofbaseline samples from MM subjects.
• LymphoTrack IGH FR1 Assay achieved 85.4% agreement with MFC in detecting
MRD - despite testing only 1/10th cell equivalents used in MFC assessment.

20%• LymphoTrack assays when spiked with LymphoQuant Internal Control were able
to report clonal cell percentage in MRD samples and have a good correlation(R2= 0.93) with MFC measured % Tumor cells.

0%• Unlike MFC assays, the LymphoTrack Assays and accompanying bioinformatics
software can be validated for submission to regulatory authorities worldwide.

AMP Global May 16-18 2019 Hong Kong