IGHV gene mutation at heart of CLL treatment

Variable regions have seven amino acid segments: three hypervariable as well as complementarity determining regions (CDR) and four framework (FR) regions. The CDRs help constitute how antigens are recognized, whereas the framework regions provide the support for proper folding and orientation of the CDRs.

Many will have seen variations of VDJ (variable, diversity, joining) recombination maps that occur within the heavy chain of the immunoglobulin. (Fig. 2). That is what drives antigen specificity as we pull together the various combinations of variable, diverse, and joining region genes. The IGHV gene is found on chromosome 14 and is encoded in gene segments. In normal B-cell maturation, chromosomal recombination of the V, D, and J segments come together to form that variable region of the immunoglobulin heavy and light chains, which is a separate pathway but using this same kind of process. Each developing B-cell will assemble a unique Ig V region by this somatic V, D, and J gene segment recombination together with hypervariable changes.

Antigen affinity is increased by the somatic hypermutation that occurs after that B-cell comes into contact with an antigen in the follicular germinal center. Somatic hypermutation introduces random nucleotide changes into the V genes, which leads to B-cells that express immunoglobulins with a high degree of antigen specificity. Since CLL originates from a single lymphoid cell, each daughter cell that occurs off that original cell will reflect that same IGHV rearrangement of the V, D, and J regions together with these hypervariable, somatic mutation changes.

The IGHV mutation assay can be done by Sanger sequencing or next-generation sequencing. With Sanger, only one sample can be run at a time. NGS technology is the best approach and represents a significant improvement over Sanger. It allows for batch analysis and simultaneous identification of the clonal IGH rearrangement, the tumor-specific rearrangement sequence, and, importantly for this assay, determination of the somatic mutation percentage.

To determine IGHV mutation status, blood or bone marrow can be used. In our laboratory, EDTA is preferred by far but ACD is an acceptable specimen. RNA is extracted and converted to cDNA using reverse transcription. PCR amplifies the IGH gene rearrangements with multiplex primers that span the leader, all V and D segments, and a portion of the J segment. Those sequence data are then analyzed to identify the IGHV rearrangement and the unique sequence, and results are compared to a germline IGHV database. The percent identity of the tumor IGHV to the closest germline sequence is calculated.

According to National Comprehensive Cancer Network and International Working Group on CLL guidelines, rearrangements with a mutation frequency of greater than or equal to two percent are mutated and considered a good prognosis, and rearrangements with a mutation frequency of less than two percent are unmutated and considered as having a poor prognosis. This information should be in the report, of course, but we as pathologists need to think beyond that: What do our clinicians need to know in terms of prognosis and how can this information be translated into other information to inform treatment decisions?

The interpretation is dependent on having enough clonal cells to amplify the clonal IGH gene rearrangement. We use five percent of total lymphocytes, by flow, to make sure we have enough clonal cells for a reliable, specific, and sensitive assay, and the prognostic significance of IGHV mutation status can be determined only if we can find a single, functional IGH rearrangement—functional meaning the sequence implies that it can go on and form an intact immunoglobulin molecule.

One of the advantages of having a lot of experience is being able to recognize when there are problems in interpretation. Here are two such problems: You may find more than one functional rearrangement, or you may find a nonfunctional rearrangement. In both situations, these are findings of uncertain significance and thus the IGHV status cannot be determined.

When the mutation status is at or near the two percent cutoff, interpret with caution, particularly if the entire IGHV could not be sequenced because of the use of framework region 1 V region primers. While two percent sounds like a perfect objective number, we all know there are degrees of subjectivity when the results get close to the cutoff number.

The CAP now has a proficiency testing program for IGHV. Sequence analysis of the IGHV gene is used to determine the somatic hypermutation status. Any sequencing method can be used, and one would submit the V-gene allele, percent similarity, and mutation status.

Now for the clinical value of IGHV gene mutation status. CLL suffered for a long time because there were too many prognostic markers. Individually they were informative, but the large number of markers became confusing and probably slowed progress.

But we have learned a lot about all these prognostic markers in the past five or so years. Clinicians wanted a simpler prognostic risk categorization as the first step, which has led to a more appropriate understanding of these new therapies that came out at the same time. Along that line, ZAP70 and CD38 early on were thought to be possible surrogates for IGHV. The thinking was, “Everybody can do a flow assay. Doing the IGHV status is hard. Maybe we can use these flow-based assays instead.”

Various studies have looked at the concordance between CD38 and ZAP70 expression, using flow cytometry, and IGHV mutation status. And although there is a general concordance, there is not sufficient equivalence such that either marker can be used in place of IGHV analysis. And with these surrogate markers, there is too much variability in assigning consistent cutoff percentage expression. The conclusion is that neither marker can be used as a replacement for IGHV mutation status in determining prognosis and subsequent treatment. Studies done at Mayo Clinic have also confirmed these findings. It’s time to move away from these surrogate-type markers to IGHV, which has clear clinical value.

The International Working Group on CLL is the group that subsequently drove the CLL International Prognostic Index and, in my view, the development of prognostic markers and how to use them, and now how to use new CLL therapies. The IWCLL guidelines, updated in 2018, say that IGHV mutation status should always be required before treatment in the baseline evaluation of patients with CLL (Fig. 3).

The CLL-IPI was a study of more than 3,400 treatment-naive patients from world-renowned CLL cancer centers in five countries. A subsequent validation was done of 838 patients from Mayo Clinic. A large set of prognostic markers were looked at and only five were found to have any value for use in a composite score: (del)17p FISH, IGHV, β2-microglobulin, clinical stage, and age.

Based on these five parameters, the group came up with four risk groups: low, intermediate, high, and very high. The clinical application is not only improved staging, but also for testing novel therapeutics in high- or very high-risk groups. Fig. 4 is a summary of those parameters and the prognostic scoring system that was developed. The only way to get to a seven to 10 score—that is, the very high category—is to have a TP53 abnormality. Conversely, the only way to be in a low-risk group is to be age 65 or older with none of the above factors, or having a Rai stage zero and none of the above. The Mayo Clinic validation data from that CLL-IPI study separate patients into these four categories. You can also see that a score of two for the unmutated IGHV status is critical to have in order to move into high or very high status. As pathologists we need to understand what its role is, and thus its inclusion in the workup of patients with CLL, in particular those patients being considered for therapy or showing signs of progressive disease.

A clinical colleague of mine approaches the risk categories in this way: If a patient falls into a low-risk group, he would typically do nothing, and that’s an important clinical statement. He also would not typically treat those who are in the intermediate-risk category unless they’re symptomatic. He would likely treat those in the high-risk group unless they’re asymptomatic, and that’s where we get into the decision: chemoimmunotherapy with fludarabine and rituximab or targeted therapy for BTK with the small-molecule inhibitor ibrutinib. Most important, if the patient is in the very high-risk category, it’s important to treat in experimental protocols or with ibrutinib or other small-molecule inhibitors.

To sum up, CLL FISH testing for prognosis is well established and well understood. More important than FISH for prognostic determination at this time, however, with the exception of (del)17p, is IGHV status. IGHV needs to be included in the initial comprehensive workup and assessment of CLL patients because its status can help clinicians make better treatment decisions.

The full webinar is available at www.captodayonline.com.