A Single Institution Experience Using Next Generation Sequencing (NGS) for Analysis of Immunoglobulin Heavy Chain
Variable Region (IGVH) Somatic Hypermutation in Chronic Lymphocytic Leukemia
Kurt Bessonen, Ming Mai MS, Lori Frederick, Rong He MD, David Viswanatha MD
Division of Hematopathology, Dept. of Laboratory Medicine and Pathology
Mayo Clinic, Rochester, Minnesota
Introduction

Materials and Methods

Somatic hypermutation (SHM) status of the immunoglobulin heavychain variable region (IGVH) gene is an important prognostic marker inB-cell chronic lymphocytic leukemia (CLL). SHM, defined as amutation load ≥2% (compared to germ line sequence), is associatedwith a better prognosis, whereas an unmutated status (mutation <2%)
is associated with adverse prognosis. SHM status is commonly
detected by PCR amplification of the monoclonal CLL IGVH-JH region
followed by Sanger sequencing, but this process is relatively laborintensive. Recently, Next Generation Sequencing (NGS) technology
has been used to detect clonal IGVH rearrangements and to determine
SHM status in CLL patients. NGS represents a significant improvement
over Sanger sequencing by simultaneously detecting andcharacterizing IGVH rearrangements using a single multiplex PCR
library, thereby allowing direct identification of the SHM status in CLLpatients. This study presents our experience using NGS for thedetection of IGVH SHM status in CLL.

Sequences were analyzed using Sequencher software version 5.0(Gene Codes Corporation, Ann Arbor, MI) and the consensus contig
sequence was analyzed using IMGT/V-QUEST software (IMGT,Montpellier, France).

Materials and Methods
Patient Samples and RNA Preparation
Peripheral blood and bone marrow aspirate samples with at least 5%
clonal B cells were obtained and RNA was isolated using the ZR WholeBlood RNA MiniPrep kit (Zymo Research Corp., Irvine, CA) for peripheral
blood and miRNeasy Mini Kit (Qiagen USA, Valencia, CA) for bonemarrow samples. Reverse transcription was performed using theMultiscribe Reverse Transcriptase and Random primer cDNA synthesis
kit (Life Technologies, Grand Island, NY).

Sanger Sequencing
Leader PCR was performed with 7 individual PCR primer sets for eachpatient (7 consensus V-family primers and JH primer). A rapid capillary
gel electrophoresis step was used to assess the quality and presence ofclonal bands using the QIAxcel DNA High Resolution Kit (Qiagen USA,Valencia, CA). If a distinct clonal leader band was not present, FR1 PCR
was then pursued using 6 individual PCR primer sets (6 consensus Vfamily primers and 1 JH primer) and assessed for the presence of aclonal band, following which the positive PCR product was treated withexoSAP-IT (Affymetrix, Santa Clara, CA) to remove excess nucleotides
and primers. The purified PCR product was sequenced using the BigDyeTerminator kit version 1.1 (Life Technologies, Carlsbad, CA) per
manufacturer’s instructions. Sequencing reactions were purified using
EdgeBio Performa DTR Ultra 96 well plates (EdgeBio, Gaithersburg,MD) and analyzed by capillary electrophoresis on the ABI 3130xL(Applied Biosystems, Foster City, CA).

ResultsResults• Discordant findings were present in 2/43 (5%) of comparison samples;

Figure 1. Representative data from a productive mutated NGS sample

additional minor discrepancies identified in 8 cases• Causes of discordant results:• NGS versus Sanger differences:• VH nomenclature (IMGT vs. IgBLAST)• Differing SHM status (M vs. UM), possibly from suboptimal
Sanger data and sequence contig with increased ambiguousbases• NGS result negative and Sanger result positive for clonal IGHV:
differences in multiplex versus single primer reaction PCR setup, or low clonal B-cell percentage• NGS leader versus FR1• Leader result negative and FR1 positive: leader primer site
mutation or PCR efficiency• Other: “N” base artifact (sequencing platform/chemistry/low complexity region)vs. true intraclonal heterogeneity

Next Generation Sequencing
Multiplex leader PCR was performed including adapter ligation andsample indexing using the LymphoTrack IGHV Leader SomaticHypermutation Assay Panel for Miseq (Invivoscribe, San Diego, CA).
A check gel was run to assess the quality and presence of clonal PCR
product using the QIAxcel DNA High Resolution Kit. If no band or an
indistinct band was present, FR1 multiplex PCR was performed using
the LymphoTrack IGH FR1 Panel for Miseq (Invivoscribe, San Diego,CA). PCR product libraries were purified using the Agencourt AMPureXP Purification kit (Beckman Coulter, Brea, CA) and subjected to next
generation sequencing (NGS) on the MiSeq platform (Illumina, SanDiego, CA), after normalization and sample pooling. NGS generated
data was evaluated for sequence quality and converted to FASTQC
files. After de-multiplexing, individual patient samples were analyzed
and graphically displayed using the Invivoscribe LymphoTrack IGHVSHM Software for MiSeq (Invivoscribe, San Diego, CA). A minimum
total read (coverage) depth of 100,000X and a clonal IGVH fraction ofat least 10% was required to adequately evaluate clonalrearrangements for SHM status. Recurrent patterns of clonal generearrangements and potential artifacts were determined. In somecases, additional assessment using IMGT/V-QUEST was performed.

Discussion

Figure 4. Representative data from a biclonal NGS sample

Conclusions

Figure 2. Representative data from a polyclonal NGS sample• NGS methodology is accurate and acceptable for clinical use in thedetermination of somatic hypermutation status in CLL. Results obtained in ourexperience are highly concordant (41/43, 95.3%) in comparison to the current
standard of PCR and Sanger Sequencing.

• Advantages of NGS include direct determination of the IGVH rearrangement andResults
Figure 5. Representative data showing intraclonal heterogeneity in an NGS sample. Inset
shows base differences between sequences 1-5.

• 12 normal donor samples and 46 CLL samples with known IGVH
status (by Sanger sequencing) were evaluated using the NGS
methodology• All normal samples were negative for clonal gene rearrangements• 3/46 CLL samples were excluded from the study due to Sanger
sequencing or NGS failure.
• Concordance with Sanger method was observed in 41/43 of thesamples (95.3%) using NGS.
• Additional findings: a) Different VH gene designation between
Sanger and NGS informatics; b) Different SHM status between
NGS and Sanger methods; c) Occasional biclonal rearrangements
variably detected between platforms• Patterns of artifact and intraclonal heterogeneity were identified• Successful PCR and NGS required a clonal B-cell population of atleast 5%percent of somatic hypermutation; however, a clear understanding of the various
rearrangement patterns, technical or informatics limitations, potential analytic
artifacts, and adequate quality parameters is central to ensuring high quality
results. Additional merits of NGS in this situation include the ability to batch
multiple samples simultaneously and to potentially enable easier minimal
residual disease monitoring.

References
Figure 3. Representative data from an unproductive unmutated Sanger sample

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computation from unselected RNA-seq reads in chronic lymphocytic leukemia.
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2. Szankasi P, Bahler D, Clinical Laboratory Analysis of Immunoglobulin Heavy
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3. McClure R, et al., High Throughput Sequencing Using the Ion Torrent Personal
Genome Machine for Clinical Evaluation of Somatic Hypermutation Status in
Chronic Lymphocytic Leukemia. JMD 2015; 17: 2: 145-154.
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