Table 1 Recommendations for the assessment of the somatic hypermutation status of the IGHV gene in clonotypic IGHV-IGHD-IGHJ gene rearrangements for standard (A) and difficult (B) cases in CLL.

From: Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia: the 2022 update of the recommendations by ERIC, the European Research Initiative on CLL

A. STANDARD CASES

Item

Recommendations

1. Methodology

Report type of: primers,a PCR product analysis, sequencing method, bioinformatics tools for SHM status assessment, and stereotypy analysis.

2. IGH gene and allele identification

IGHV, IGHDb, IGHJ genes and alleles.

3. Functionality

SHM status determined only for productive rearrangements; if the rearrangement is unproductive, mention reasons for that (e.g., IG pseudogene, out-of-frame junction, stop codon, large indel).

4. IGHV gene: % of nucleotide identity to the germline to 2 decimal points as reported by IMGT

Classification: U-CLL ≥ 98%; M-CLL < 98%; borderline CLL when 97–97.99%.

5. Subset identification/BcR IG stereotypy

For subsets with well-established prognostic value (currently, subsets #2 and #8).

B. CHALLENGING CASES

Item

Recommendations

1. Single unproductive rearrangement

Repeat the PCR with alternative primer sets and using cDNA. Perform NGS to get more detailed information regarding the clonal architecture.

SHM status disclosed as not determined only in case all different approaches fail.

2. Double rearrangements

2.1 One productive and one non-productive

Same as for standard cases: mutational status defined by the productive rearrangement, irrespective of the SHM status of the unproductive rearrangement.

2.2 Double productive

2.2.1 Concordant SHM status

Same as for standard cases i.e., consider as M-CLL or U-CLL, according to the SHM status.

2.2.2 Discordant SHM status

Check immunophenotype for the presence of 2 clonal populations.

Recommend to the physician that it is safer to consider as U-CLL; close follow-up.

3. Multiple (>2) productive rearrangements

Check immunophenotype for the presence of 2 or more clonal populations.

Perform NGS to assess the relative frequency of each clonotype and consider the predominant clonotype, if it is clearly identified (NOTE: specific guidelines are still to be provided/developed here).

4. Missing anchors (C104/W118)

Mutational status assessment is possible if evidence for IG expression on leukemic cells and/or preserved G-X-G motif within the VH FR4.

  1. aLeader primers are the only recommended option. That said, in rare cases when the application of a multiplex PCR with leader primers is unsuccessful VH FR1 primers can be used. The result should only be used to facilitate the application of a new round of PCR using IGHV subgroup-specific leader primers. Only if the result is suboptimal again, the report can be based on the VH FR1 PCR but it should be clearly stated that the use of VH FR1 primers might underestimate the total number of IGHV somatic hypermutations since a part of the VH domain is missing.
  2. bIn a percentage of cases, IGHD identification may be difficult due to: (i) excessive exonuclease trimming of the IGHD gene; and/or (ii) SHM within the VH CDR3, hindering the assignment to the closest germline IGHD gene and allele.
  3. CLL chronic lymphocytic leukemia, IG immunoglobulin, M-CLL mutated CLL, U-CLL unmutated CLL.
  4. Examples of lab report for both types of cases are provided in Supplementary Material.
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