Next Generation Sequencing Demonstrates Clinical Utility and Increased Sensitivity in Detection ofIgVH Somatic Hypermutation in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL)

Isabel Hubbard, M.Sc., Alice Layton, Ph.D., Bevan Tandon, M.D.
Maryville, TN, United States

Introduction

Materials and Methods

Results

Conclusions

Chronic Lymphocytic Leukemia/Small lymphocytic
lymphoma (CLL/SLL) is the most common
leukemia diagnosed among adults in Westerncountries and is associated with heterogeneous
clinical outcomes. IgVH somatic hypermutation(SHM) status is a primary component of the CLL
International Prognostic Index (CLL-IPI) working
1group formulation for disease risk stratification.
Unmutated IgVH has been established as a strong
and independent predictor of adverse clinicalprognosis and reduced overall survival.

NGS: IGH framework 1 V-J amplicon libraries were prepared using
Invivoscribe panel primers, PGM adapters, and patient sample DNA.
Pooled libraries were quantified and sequenced on the Ion Torrent PGM™
instrument, with generated FASTQ files analyzed by LymphoTrack® PGM®
Software (Version 2.0). A ≥ 4-fold difference between first and third mostabundant IGH sequences defined clonal read data,5 and IgVH somatic
hypermutation status was resulted in accordance with recommended CAP
reporting guidelines.6• SS-PCR failed to detect a clonal population in 5/25
cases evaluated (20%), whereas, NGS detected clonal
populations in all 25 cases studied.

Evaluation of IgVH SHM status using NGS is feasible
for routine clinical testing in the private referencelaboratory setting and confers significant advantages
over traditional methodologies:

Clinical laboratory evaluation of IgVH SHM status
traditionally involves rt-PCR followed by Sanger
Sequencing using RNA extracted from patient
peripheral blood or bone marrow aspirate samples,
however RNA lability places significant burden on
the submitting physician to ensure specimen transit
time is minimized. Furthermore, Sanger Sequencing
is time and labor intensive, and sensitivity of IgVH
SHM detection may be limited for low abundance
CLL/SLL clones. ZAP-70 expression by flow
cytometry has therefore been widely utilized asa surrogate marker for IgVH SHM status. PositiveZAP-70 expression is usually associated with IgVH
unmutated disease and an unfavorable clinical2course. Unfortunately, standardization for thismarker is known to be poor; interpretation ofthe flow cytometry list mode data may be highly
subjective, contributing to significant variability in3clinical reporting. Significant discordance between
ZAP-70 expression patterns and expected IgVH SHM
results may also be seen and has been attributed
to pre-analytic sample processing factors in some
4studies.
Herein we report on the clinical utility of a nextgeneration sequencing (NGS) based approach toIgVH SHM testing using DNA derived from CLL/SLL
patient samples.

Sanger Sequencing (SS): Samples were previously tested at an outside
national reference laboratory. Patient sample RNA was extracted and reverse
transcribed into cDNA followed by rtPCR amplification using VH leader and
JH primers. Identified clonal VH sequences were searched against a known
database for homology. IgVH homologies were defined as follows:>98% = unmutated, 97%-98% = borderline, <97%=mutated. CLL clones wererequired to comprise at least 50% of total analyzed B-cells.
ZAP-70: Flow cytometry was performed on a Beckman Coulter Navios
instrument utilizing ZAP-70 Clone 1E7.2 (Invitrogen). T and NK-cells served
as internal control subpopulations. CD19+/CD5+ B-cell ZAP-70 results weredefined as follows: >25% = positive; 20-25% = borderline; <20% = negative.

• V3-21 gene utilization was detected by NGS in 1 of the 5cases for which SS-PCR failed to detect a B-cell clone.

• Accuracy: NGS demonstrated 100% concordance with SSPCR among evaluable cases where SS-PCR did not fail todetect a clonal population.
• NGS Limit of detection: ~3% clonal B-cells (data notshown)• NGS Precision: 100% (data not shown)• Strength of agreement between NGS and ZAP-70 assays
was 0.64 (Good), whereas, SS-PCR and ZAP-70 assays
showed moderate agreement (0.535) (Kappa statistic).

• NGS may show improved sensitivity in detection ofclonal populations compared to Sanger Sequencing.
• DNA is an acceptable specimen substrate for NGS
IgVH SHM detection, limiting practical constraints
typically associated with RNA based testing.
• NGS significantly streamlines laboratory workflow:• Three day clinical reporting is feasible.
• Assay hands on time is minimized (<5 hours).
• Batch multiplexing of patient samples facilitatessignificant reductions in cost and labor.
• NGS sample input requirements are minimized.
Clinically valid NGS results may be obtained using
1ml of peripheral blood or bone marrow, or 0.025 µgDNA.
• NGS data output is automated and nonsubjective, and may include relative frequencies ofclonal IGH reads, percentage homology to mostclosely matched germline IGH reference sequences,and VH gene utilization profiles of patient read data.

References
1. International CLL-IPI working group. An international prognostic index for patients with chronic
lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data. Lancet Oncol.
2016 Jun;17(6):779-90.
2. Rassenti et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status asa predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med. 2004 Aug26;351(9):893-901.
3. Wilhelm C, Neubauer A, Brendel C. 2006. Discordant results of flow cytometric ZAP-70
expression status in B-CLL samples if different gating strategies are applied. Cytometry B Clin
Cytom. 2006 Jul 15;70(4):242-50.
4. Sheikholeslami MR et al. Variations in the detection of ZAP-70 in chronic lymphocytic leukemia:
Comparison with IgV(H) mutation analysis. Cytometry B Clin Cytom. 2006 Jul 15;70(4):270-5.
5. Schumacher et al. A comparison of deep sequencing of TCRG rearrangements vs traditional
capillary electrophoresis for assessment of clonality in T-Cell lymphoproliferative disorders.
Am J Clin Pathol. 2014 Mar;141(3):348-59.
6. Duncavage et al, Template for Reporting Results of Biomarker Testing of Specimens FromPatients With Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma. Arch Pathol Lab
Med. 2016 Apr 15.

For potential collaboration or comments please contact:
btandon@mplnet.com