Validation and clinical implementation of next generation sequencing for routine
IGH and TCRG clonality assessment
Maria E. Arcila, Tessara Baldi, Mustafa Syed, Ivelise Rijo, Jake Bakas, Zeineen Momin, Khedoudja Nafa

H44Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NYResults

Introduction
IGH and TCR gamma clonality testing is an important component
in the diagnosis of lymphoproliferative disorders. Capillaryelectrophoresis (CE) of multiplexed PCR products is a common
method of analysis. Although robust, simple and highly
reproducible, it does not provide a full characterization of clonalsequences and lacks sensitivity and specificity to track clones insubsequent samples. We describe our clinical validation andimplementation of an NGS based assay for initial clonal
characterization and minimal residual disease assessment
through patient specific clone tracking.

Lymphotrack read summary for polyclonal sample
Total countRank
123
456789
10Sequence
LengthRaw countV-geneJ-gene% total reads
Cumulative %TGGGTAAGACAAGCAACAAAGTGGAGGCAAGAAAGAATTCTCAAACTCTCACTTCAATCCTTACCATCAAGTCCGTAGAGAAAGAAGACATGGCCGTTTACTACTGTGCCGCTCCCCACTTTGGCAGTG1291257
Vg10
Jg1/2
0.2768912
0.2768912
GGACTCAGTCCAGGAAAGTATTATACTCATACACCCAGGAGGTGGAGCTGGATATTGAGACTGCAAAATCTAATTGAAAATGATTCTGGGGTCTATTACTGTGCCACCTGGCTTGGTTCAAGATATTTGCTG1061110
Vg3
JgP10.2445101
0.5214012
GGACTCAGTCCAGGAAAGTATTATACTCATACACCCAGGAGGTGGAGCTGGATATTGAGACTGCAAAATCTAATTGAAAATGATTCTGGGGTCTATTACTGTGCCACCTGGGACAGGCCTAGCCTTTTGGATCAAGACGTTTGCAA
121940Vg3
JgP2
0.2070626
0.7284638
GGAATCAGCCCAGGGAAGTATGATACTTACGGAAGCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGATGTAGGATTATTATAAGAAACTCTTTGGCAGTG
146898Vg4
Jg1/2
0.1978109
0.9262747
GGAATCAGTCGAGAAAAGTATCATACTTATGCAAGCACAGGGAAGAGCCTTAAATTTATACTGGAAAATCTAATTGAACGTGACTCTGGGGTCTATTACTGTGCCACCTCCGAATTATTATAAGAAACTCTTTGGCAGTG
140835
Vg8
Jg1/20.1839333
1.1102080
TGGGTAAGACAAGCAACAAAGTGGAGGCAAGAAAGAATTCTCAAACTCTCACTTCAATCCTTACCATCAAGTCCGTAGAGAAAGAAGACATGGCCGTTTACTACTGTGCTGCGTGGGATACCACTGGTTGGTTCAAGATATTTGCTG
121799Vg10
JgP1
0.1760032
1.2862112
GGAGTCAGTCCAGGGAAGTATTATACTTACGCAAGCACAAGGAACAACTTGAGATTGATACTGCGAAATCTAATTGAAAATGACTCTGGGGTCTATTACTGTGCCACCTGGGACGTCTTGATTGGATCAAGACGTTTGCAA
116733Vg2
JgP2
0.1614648
1.4476759
GGACTCAGTCCAGGAAAGTATTATACTCATACACCCAGGAGGTGGAGCTGGATATTGAGACTGCAAAATCTAATTGAAAATGATTCTGGGGTCTATTACTGTGCCACCTGGGACCGAGTGGTTCAAGATATTTGCTG
111677Vg3
JgP1
0.1491291
1.5968051
GGAATCAGTCGAGAAAAGTATCATACTTATGCAAGCACAGGGAAGAGCCTTAAATTTATACTGGAAAATCTAATTGAACGTGACTCTGGGGTCTATTACTGTGCCACCTGGAATAAGAAACTCTTTGGCAGTG
133629Vg8
Jg1/2
0.1385557
1.7353608
GAAGACTAAGAAACTTGAGGTAAGTAAAAATGCTCACACTTCCACTTCCACTTTGAAAATAAAGTTCTTAGAGAAAGAAGATGAGGTGGTGTACCACTGTGCCTGCTGGCTAAAGGCCTACTAATAGTAGTGATTGGATCAAGACGTTTGCAA
128629Vg11
JgP2
0.1385557
1.8739165

5%

1%

Methods
Blood, bone marrow and FFPE tissue samples submitted forroutine clonality assessment were selected. DNA was extracted
and tested using both standard CE and LymphoTrack® IGH +TRG
– MiSeq assays (InVivoScribe). Positive, negative and notemplate controls were run with all assays. Sensitivity and LOD
were assessed based on dilution studies for detection of initialclonal population and subsequently for tracking of a precharacterized clone at minimal residual disease levels.

Results
A total of 160 samples were analyzed including 126 clonal(defined by initial CE testing, 92 IGH, 34 TCRG) and 34
non-clonal samples with 94% concordance between the 2
methods. Discordant results (clonal by CE, non clonal by NGS)
were attributable to pseudoclonality in the post treatment setting.
Based on a minimum input of 50ng of high quality DNA, analyticalsensitivity was 5% for diagnostic samples (un-characterized clone)with good Inter and intra-assay reproducibility. Further dilution
studies to establish LOD for tracking a previously characterized
clone showed accurate detection at 1x10-5 with 1-2ug DNA input.
The mean number of reads per sample was approximately
500,000.

0.1%Acknowledgements
The authors would like to thank the Diagnostic Molecular Pathology
Laboratory members and Invivoscribe for their technical support.

Comparison of lymphotrack and CE
method in the initial diagnosis of clonality.
For a previously uncharacterized clone, theNGS method shows higher sensitivity withan LOD of approximately 1% (vs 5% forthe CE method)

0.01%

Lymphotrack read summary for clonal sample (biclonal/biallelic)
Total countRank
1234
5678910572,268

IGH dilution study (cell line - 1ug input)

SequenceLengthRaw countV-geneJ-gene% total reads
Cumulative %GGAATCAGCCCAGGGAAGTATGATACTTACGGAAGCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGAAACAGATACCACTGGTTGGTTCAAGATATTTGCTG
122241712
Vg4
JgP1
42.2375530
42.2375530
GGACTCAGTCCAGGAAAGTATTATACTCATACACCCAGGAGGTGGAGCTGGATATTGAGACTGCAAAATCTAATTGAAAATGATTCTGGGGTCTATTACTGTGCCACCTGGGCCGATGGGGGAAACTCTTTGGCAGTG
138141811
Vg3
Jg1/2
24.7805224
67.0180754
targetTargetTotalDilution
Total reads Total target Total reads
GGAATCAGCCCAGGGAAGTATGATACTTACGGAATCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGAAACAGATACCACTGGTTGGTTCAAGATATTTGCTG
1228247
Vg4
JgP1
1.4411080
68.4591835
sequence Sequencereads(IVS-0013 into IVS 0000)
run 2
sequence(run 1+2)
GGACTCAGTCCAGGAAAGTATTATACTCATACACCCAGGAGGTGGAGCTGGATATTGATACTGCAAAATCTAATTGAAAATGATTCTGGGGTCTATTACTGTGCCACCTGGGCCGATGGGGGAAACTCTTTGGCAGTG
1385451Vg3
Jg1/2
0.9525257
69.4117092
run 1
run 1
run 2
GGAATCAGCCCAGGGAAGTATGATACTTACGGAAGCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGAAACAGATACCACTGGTTGGTTCAAGATATTTGCT
1213590Vg4
JgP1
0.6273285
70.0390377
1% (1/100)
51525898
498,330 565,180
11050
1,063,510
GGACTCAGTCCAGGAAAGTATTATACTCATACACCCAGGAGGTGGAGCTGGATATTGAGACTGCAAAATCTAATTGAAAATGATTCTGGGGTCTATTACTGTGCCACCTGGGCCGATGGGGGAAACTCTTTGGCAGT
137
2399
Vg3
Jg1/2
0.4192092
70.4582468
0.1% (1/1000)
381
447
396,417 470,979
828
867,396GAATCAGCCCAGGGAAGTATGATACTTACGGAAGCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGAAACAGATACCACTGGTTGGTTCAAGATATTTGCTG
121
1669
Vg4
JgP1
0.2916466
70.7498934
0.01% (1/10,000)
6427
637,782 422,769
GGAATCAGCCCAGGGAAGTATGATACTTACGGAAGCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGAAACAGATACCACTGGTTGGTTCAAGATATTTGCTGA
123
1308
Vg4
JgP1
0.2285642
70.9784576
91
1,060,551
GGAATCAGCCCAGGGAAGTATGATACTTACGGAAGCACAAGGAAGAACTTGAGAATGATACTGCGAAATCTTATTGAAAATGACTCTGGAGTCTATTACTGTGCCACCTGGGAGGGATTATTATAAGAAACTCTTTGGCAGTG
143
1104
Vg4
Jg1/2
0.1929166
71.1713743
0.001% (1/100,000)
174
431,755 356,569
21788,324
GGAATCAGTCGAGAAAAGTATCATACTTATGCAAGCACAGGGAAGAGCCTTAAATTTATACTGGAAAATCTAATTGAACGTGACTCTGGGGTCTATTACTGTGCCACCTGGGGAATTATTATAAGAAACTCTTTGGCAGTG
141
997
Vg8
Jg1/2
0.1742191
71.3455933
0.0001% (1/1,000,000)
03425,060 387,118
3812,178

Calculated% targetreads
1.039012
0.095458
0.008580
0.002664
0.000369

0.5
0012

345

6-0.5-1

SAMPLE

-1.5-2-2.5-3

Conclusion
Assessment of clonality by NGS methods provides significant
improvement over existing clonality assays using fragment
analysis by CE. Sensitivity for detection of a diagnostic clone issimilar to the CE assays but provides full characterization of theclone to enable tracking in subsequent samples at the MRDlevel. NGS testing readily resolved pseudoclonality calls in
post-treatment samples by differentiating clonal products ofsame size but different sequences interpreted as clonal by theCE method. However, the LymphoTrack® assays remain
expensive and with higher TAT compared to CE.

MRD case study #2
57 yo male with growing subcutaneous nodule in the arm diagnosed as
typical T cell proliferation most consistent with angioimmunoblastic T
cell lymphoma. Bone marrow submitted for evaluation of disease,
pretransplant

453,969-3.5

R² = 0.9864

DIAGNOSTICPRE TRANS RUN-1
PRE TRANS RUN-2
PRE TRANS RUN-3
PRE TRANS RUN-4
PRE TRANS RUN-5

RAW COUNTCLONALSEQUENCE
CLONE 1
6230

5139
5271
10732
17583
6268

TOTAL
READS

% TOTAL
READS

42580557118779130711486492020955872680

14.6
0.9
0.7
0.9
0.9
0.7

-4

References

MRD case study #1 - 51 yo male with newly diagnosed plasma cell neoplasm
1.
2.

3.

4.

Diagnostic sample
Bone marrow: Plasma cell myeloma, involving 80-90%
marrow cellularitySmall monotypic B-cell population
detected by flow cytometry

Post treatment sampleBone marrow: Morphology: no evidence . Flow cytometry: Minuteabnormal plasma cell population detected by flow cytometry (0.035%)
Lymphotrack: 0.12% (744/632030, 99% identity)

5.

Emerson RO et al. Defining the alloreactive T cell repertoire using high-throughput
sequencing of mixed lymphocyte reaction culture PLoS One. 2014;9(11):e111943
Logan AC. Et al. Immunoglobulin and T cell receptor gene high-throughput sequencing
quantifies minimal residual disease in acute lymphoblastic leukemia and predicts
post-transplantation relapse and survival. Biol Blood Marrow Transplant
2014;20:1307-13.
Miller, J.E. (2013). Principle of Immunoglobulin and T Cell Receptor Gene
Rearrangement. In Cheng L, Zhang D, Eble J.N. (Eds), Molecular Genetic Pathology(2nd Ed). New York, USA: Springer Science & Business Media.
Schumacher JA et al. A comparison of deep sequencing of TCRG rearrangements vstraditional capillary electrophoresis for assessment of clonality in T-Celllymphoproliferative disorders. Am J Clin Pathol. 2014;141(3):348-59
Wu D et al. Detection of minimal residual disease in B lymphoblastic leukemia byhigh-throughput sequencing of IGH. Clin Cancer Res. 2014;20(17):4540-8

AMP meeting , Austin, Texas 2015