Minimal Residual Disease Detection of Lymphoid and Plasma Cell Neoplasms
Using a Next-Generation Sequencing (NGS)-Based Assay

H44Caleb Ho, MD; Juan Gomez-Gelvez, MD; Mustafa H Syed, MS; Kseniya Petrova-Drus, MD, PhD; Ahmet Zehir, PhD; Wayne Yu, BS;Tessara Baldi, BS; Marc Ladanyi, MD; Ahmet Dogan, MD, PhD; Jinjuan Yao, MD, PhD; Khedoudja Nafa, PharmD, PhD; Maria E. Arcila, MD

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

Introduction
Lymphoid and plasma cell neoplasms are characterized by clonally-restricted T-A. Diagnostic Sample with two distinct clones with TRG Primers

C. 6 Months Post 2nd Allogeneic Stem Cell Transplant

cell receptor (TCR) or immunoglobulin (Ig) rearrangements. Across clinicallaboratories, this is generally demonstrated with standardized multiplex
polymerase chain reaction (PCR) assays, in which V-J or D-J products areseparated by fragment sizes on capillary electrophoresis (CE). However, thisapproach has relatively low sensitivity and does not provide the specific clonalsequence information required for tracking a clone at low level or in minimal
residual disease (MRD) setting. In this study, we assessed the performance of aNGS-based assay, LymphoTrack®(LT) (Invivoscribe, San Diego, CA), for detectionof low level and MRD among various lymphoid and plasma cell neoplasms incomparison to CE and flow cytometry (FC) assays.

Flow: 0.0026% Abnormal T-Lymphoblasts; 1% Blasts on Aspirate Smears

D. 8 Months Post 2nd Allogeneic Stem Cell Transplant

Material and Methods
DNA was extracted from bone marrow, blood, and formalin-fixed paraffinFlow: 8% Abnormal T-Lymphoblasts; 12% Blasts on Aspirate Smears

embedded tissue from 48 patients with diagnostic and post-therapy (PT) samples.
For clonal Ig rearrangement, PCR primers flanking the IGH conserved framework

B. 1 Month Post 2nd Allogeneic Stem Cell Transplantregion 1 (FR1) in VH and conserved JH region were used. For clonal TCR
rearrangement, primers flanking the TRG conserved Vγ and Jγ regions were used.
The amplified products were sequenced on the Illumina MiSeq platform, andanalyzed with the proprietary LymphoTrack® analysis software, which provided
the quantitation and V-J gene family usages of all unique sequences. With the aidof an in-house developed software, MSK-LymphoClone, the patient-specificdiagnostic clonal sequences were used to detect residual disease involvement in

Flow: No Abnormal T cells; Morphologic Remission

Fig. 1: Residual Disease Detection in a Patient with Relapsed T-Lymphoblastic Leukemia/Lymphoma, using TRG Primers on LymphoTrack®.
The patient shows evidence of subsequent relapse by PET scan, and is currently on palliative therapy.

subsequent samples, and compared to concurrent CE and 10-color FC results

Results

available at MSKCC.

AFlow: No Abnormal T cells; Morphologic Remission

Conclusion

BResults

Compared to capillary electrophoresis and flow cytometry, LymphoTrack® provides
comparable or better MRD detection sensitivity of lymphoid neoplasms, and withincreased diagnostic certainty by utilizing patient-specific clonal sequences for MRD
detection.

Table 1: Lymphoid and Plasma
Cell Neoplasm cases used for
Residual Disease Detection byLymphoTrack®

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

References
1.

Fig. 2: Comparison of Residual Disease Detection by Lymphotrack (LT) vs. (A) Capillary Electrophoresis (CE) and (B) Flow Cytometry (FC)
Table 2: Summary ofTotal Sequencing Reads
and Percentage of Reads
supporting ResidualDisease• In 2/14 Plasma Cell Neoplasm PT samples, FC detected suspicious atypical plasma cells (0.0007% and 0.0028% of total WBC), but noclonal sequence detected by LT.
 In one sample, total sequencing reads was suboptimal for MRD detection (65,960 total reads).
 In the other sample, two subsequent samples from the same patient showed no evidence of disease by all detection methods.
• In 12 PT samples from 10 patients, LT detected residual disease, while neither FC nor CE detected disease.
 2/10 patients showed subsequent overt evidence of persistent/recurrent diseases, with median follow-up time of 3 months.
• In 18 PT samples from 17 patients, there is no evidence of residual disease by all detection methods.
 16/17 patients showed no subsequent evidence of disease, with median follow-up time of 2.7 months.

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