H045

Detection of Clonal Rearrangements in Multiple Myeloma Samples using LymphoTrack® Assays
Ying Huang1, Austin Jacobsen1, Jeff Panganiban1, Edgar Vigil1, Kasey Hutt 1, Joshua Heilman1, Martin Blankfard2, Even H Rustad2, Malin Hultcrantz2, Ola Landgren2 andJeffrey E. Miller1
1Invivoscribe, Inc., San Diego, USA, 2Memorial Sloan Kettering Cancer Center, New York, USA

Introduction
Total N=193

IGH FR1

IGH FR2

IGH FR3

IGK

Overall(IGH FR1/FR2/FR3 + IGK)C (Clonal)

88/193(45.6%)

81/193(42.0%)

90/193(46.6%)

106/193(54.9%)

152/193(78.8%)NC (Non-clonal)

83/193(43.0%)

109/193(56.5%)

101/193(52.3%)

78/193(40.4%)

34/193(17.6%)I (Invalid)

22/193(11.4%)

3/193(1.6%)

2/193(1.0%)

9/193(4.7%)

7/193(3.6%)• Despite testing baseline specimens which were generally from “last pull” aspirates
with low tumor cell content (50ng ~= 7,700 cell equivalents), LymphoTrack Assays wereshown to detect clonality in about 80% of MM baseline research specimens.
• Despite testing only 200 ng DNA (~ 30,000 cells), typically from “last pull” aspirates
with low tumor cell content, the same LymphoTrack Assays and testing procedures
were able to track the clonal sequences in 87% of MM follow up specimens.
• The presence of LymphoQuant IC within each PCR reaction allowed the % clonal cells
within each specimen to be estimated.
• LymphoTrack Assays can be potentially useful tools to identify and monitor diseasestatus in MM samples at both diagnosis and subsequent time points throughout thecourse of treatment.
• Unlike MFC, the LymphoTrack Assays and accompanying bioinformatics software canbe easily standardized between laboratories and submitted for approval to regulatory
authorities worldwide.
MiSeq® is a registered trademark of Illumina, Inc..
Experiments were performed with CE-IVD products not available in North America

133

C (clonal)

NC (Non-clonal)

I (invalid)

MRD Results% Clonal cells insample

LymphoQuant
Frequency

Sample1_TP1
Sample1_TP2
Sample1_TP3
Sample1_TP4
Smaple2_TP1
Smaple2_TP2
Sample3_TP1
Sample3_TP2
Sample4_TP1
Sample5_TP1
Sample5_TP2
Sample6_TP1
Sample6_TP2
Sample6_TP3
Sample6_TP4Sample6_TP5
Sample7_TP1
Sample7_TP2
Sample8_TP1
Sample8_TP2
Sample8_TP3
Sample8_TP4
Sample8_TP5
Sample8_TP6
Sample9_TP1
Sample9_TP2
Sample10_TP1
Sample10_TP2
Sample11_TP1
Sample12_TP1

FR2
FR2
FR2
FR2
FR2
FR2
IGK
IGK
FR3
IGK
IGK
IGK
IGK
IGK
IGK
IGK
FR3FR3
FR3
FR3
FR3
FR3
FR3
FR3
FR2
FR2
FR3
FR3
FR1
FR3

Detected
Detected
Detected
Detected
Detected
Detected
Detected
Detected
Not Detected
Detected
Detected
Detected
Detected
Detected
Detected
Detected
Detected
Detected
Detected
Detected
DetectedDetected
Detected
Detected
Detected
Detected
Not Detected
Detected
Not Detected
Not Detected

0.144
1.876
1.628
0.001
0.004
0.003
0.002
0.001
0.000
1.717
0.093
0.001
0.003
0.004
0.019
0.002
2.773
0.346
0.011
0.044
0.070
0.862
0.081
24.223
1.180
0.035
0.000
0.002
0.000
0.000

1.63E-01
2.83E-02
2.89E-01
2.16E-02
1.22E-01
1.16E-01
2.63E-01
2.71E-02
7.12E-02
1.17E-01
1.44E-01
2.15E-01
7.35E-02
9.63E-02
1.05E-01
1.25E-01
5.38E-02
1.55E-02
4.14E-03
9.97E-03
1.77E-02
1.55E-02
2.38E-02
7.46E-03
1.19E-01
2.57E-01
2.93E-01
1.51E-02
8.15E-01
2.46E-01

IGH FR2 Assay: Follow-up Sample 1
2.0

2.0

1.6

1.5

1.2

1.0

0.8

0.5

0.4
0.0

0.00

1324

5

Frequency for LymphoQuant

Assay

Timepoint
SampleLymphoQuant

IGH FR3 Assay: Follow-up Sample 8
100.0

0.4

10.0

0.3

1.0

0.2

0.1

0.1

0.0

0.00

1234567

Frequency for LymphoQuant

Conclusions

Follow-up Sample%clonal cells in sample• Five (5) LymphoTrack® Assays (IGHV Leader, IGH FR1, IGH FR2, IGH FR3 and IGK) for theMiSeq®, each with available with 24 indices, were designed, developed, andmanufactured per cGMP, then QC tested and validated under a QSR-compliantregulatory system.
• 50 ng of genomic DNA (only ~7,700 cell equivalents) from 193 MM bone marrow(BM) baseline specimens were procured, anonymized and blinded prior to testingwith the four LymphoTrack Assays (IGH FR1, FR2, FR3 and IGK) using the MiSeq®
platform.
• Libraries generated from all 4 assays were purified, harmonized, pooled, andsequenced in a single MiSeq run. Fourteen (14) samples that tested negative by theabove 4 assays were reflex tested with the 5th LymphoTrack Assay, IGHV Leader.
• The most prominent clonal rearrangement sequence identified by one of theLymphoTrack Assays was then tracked using the single assay.
• 200 ng DNA (~ 30,000 cells), typically from “last pull” aspirates with low tumor cell
content were tested from 30 subsequent residual follow up specimens.
• In order to generate an estimation of lymphoid cell equivalents within each specimen
LymphoQuant® Internal control (IC; 100 cell equivalents) was added to each PCR
reaction in follow up specimens.
• LymphoTrack® Software (MiSeq®) and LymphoTrack MRD software were used toanalyze the sequencing results from baseline and follow up samples, respectively.

Test 7 Invalid Samples by IGHV Leader

Results: Using LymphoTrack® Assays - MiSeq ® to Track Sequences in Follow up MM Samples

Materials and Methods%clonal cells in sample

Multiple myeloma (MM) is a malignancy of plasma cells. Currently, multiparameter flow
cytometry (MFC) is the tool most often used to detect and monitor MM in patients.
However, MFC requires fresh specimens and is difficult to standardize between testcenters. Since genomic DNA is stable and the assays, platforms, and the accompanying
software can be easily standardized between test centers, we developed next generation
sequencing (NGS)-based assays with bioinformatics software to detect the clonal Ig
rearrangements associated with MM and track Minimal Residual Disease (MRD). Here wereport the results of a pilot study of clonal rearrangement detection and tracking bytesting 193 anonymized/blinded MM specimens using LymphoTrack Assays. These assays
identify B-cell populations by targeting the IGHV Leader, IGH FR1, FR2, FR3, and IGK.
Thirty (30) follow-up samples were also tested to demonstrate MRD utility.

Results: Using LymphoTrack® Assays - MiSeq® to Detect Clonality in Baseline MM Samples

TimepointSample

LymphoQuant

Overall detecting samples: 26/30 (87%)

AMP, November 1-3 2018, San Antonio, TX, USA