Validation of Next Generation Sequencing-Based Clonality Assays for Diagnosis and Minimal Residual Disease Tracking in Lymphoid Malignancies
Authors: Wu Haiyan , Li Feng , Cai Jiacheng , Zhang Ke , Zheng Xin , Yao Jicheng , Wang Weifeng , Wang Kai 
Published: November 2021

Abstract
Introduction:
B or T cell receptor (BCR/TCR) clonal rearrangements have served as important diagnostic markers and minimal residual disease (MRD) tracking markers to guide treatment decisions in lymphoid malignancies. Next generation sequencing (NGS)-based BCR/TCR clonality assessment allows easier sample preparation, higher sensitivity and simpler standardization than flow cytometry and polymerase chain reaction (PCR)-based assays. Here, we developed NGS-based BCR/TCR clonality assays to identify and track disease-associated clonotypes of IGH, IGK, IGL, TCRB and TCRG rearrangements and BCL1/2-IGH translocations in lymphoid malignant cells. Our studies validated the analytical performance of the assays using genomic DNA (gDNA) from cell lines and patient samples diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), lymphoma and lymphoid blast phase chronic myeloid leukemia (BP-CML), as well as peripheral blood gDNA and circulating tumor DNA (ctDNA) from lymphoma patients.
Methods:
Our BCR/TCR clonality assays were based on two rounds of multiplex PCR followed by NGS. In the first-round PCR, the sequences of complementary determining region 3 (CDR3) in rearranged immune receptor genes were amplified by multiplex primers, and sample-specific index and NGS adapters were then added in the second-round PCR. Sequencing was performed on NovaSeq 6000 System and processed with customized bioinformatics pipelines. BCR clonality assays could identify IGH (V H-D H-J H or D H-J H), IGK (V κ-J κ, V κ-Kde and intronRSS-Kde) and IGL (V λ-J λ) rearrangements, as well as BCL1-IGH and BCL2-IGH translocations. TCR clonality assays could detect TCRB (V β-D β-J β) and TCRG (V γ-J γ) rearrangements. To evaluate the performance of our assays, we detected BCR or TCR clonality in gDNA from 4 cell lines and 40 clinical samples with ALL, MM, CLL and lymphoma, as well as paired chronic- and blast-phase samples of CML. Limit-of-detection (LOD) was estimated by clinical samples and cell lines with a background of peripheral blood gDNA from healthy donors. Linearity of detection was established with gDNA of cell lines spiked into normal gDNA to generate across orders of magnitude of clonal frequencies. To ensure consistent performance of the assays, we tested separate reactions and a single mixed reaction for IGH VDJ, IGH DJ, IGK and IGL. In addition, peripheral blood gDNA matching aforementioned lymphoma cases and ctDNA samples were also tested by our assays.
Results:
Both BCR clonality of B-cell lymphoid malignancies (B-ALL, MM, CLL and lymphoma) and TCR clonality of T-cell lymphoid malignancies (T-ALL and lymphoma) showed above 90% positive detection rate and mostly positive in more than one receptor gene. Chronic and blast phase samples from the same CML patient showed an identical dominant clonotype. The assays had high sensitivity, with LoD defined between 1 to 2 malignant cells in both BCR and TCR clonality assessment. Linearity was observed with clonal frequencies from 1 to 10 -6, which indicated consistence between observed and expected frequencies. The sequencing results with a single adjusted mixed reaction for IGH VDJ, IGH DJ, IGK and IGL were comparable to that with separate reactions, suitable for both diagnosed samples and MRD samples, suggesting the robustness of the assays. Our testing results also showed that peripheral blood gDNA of lymphoma patients carried identical clonotypes found in malignant tissues and ctDNA.
Conclusion:
We characterized and validated the performance of our NGS -based BCR/TCR clonality assays. We also demonstrated its potential application as a highly sensitive tool for diagnosis and MRD tracking for lymphoid malignancies, including ALL, MM, CLL, lymphoma and even CML at risk of lymphoid blast transformation.

Disclosures
No relevant conflicts of interest to declare.