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Molecular clonality testing for lymphoma

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William Check, PhD

May 2013—Most jobs in the kitchen or the home workshop can be done with a basic set of tools. But every once in a while you need something special—a zester, say, or a dremel—and in those situations it’s nice to have that special tool on hand. Even more important, it’s nice to know how to use it.

So, too, in hematopathology, where the special tool is clonality testing by PCR. Most cases of lymphoma can be diagnosed with a basic tool kit—morphology, with H&E staining in essentially all cases, plus some type of immunophenotyping, typically immunohistochemistry or flow cytometry. But in a small proportion of cases, molecular clonality testing is necessary.

“Molecular clonality testing is not needed for most cases [of lymphoma diagnosis],” Patricia J.T.A. Groenen, PhD, clinical molecular biologist in pathology at the Radboud University Nijmegen Medical Centre, the Netherlands, told CAP TODAY. Dr. Groenen is a member of the EuroClonality Group, which developed, validated, and generated guidelines for the most widely used PCR clonality kit, the EuroClonality/BIOMED-2 multiplex PCR kit, marketed in the U.S. by InVivoScribe. “Pathologists can diagnose leukemia or lymphoma and identify malignancy in most specimens,” Dr. Groenen notes. She estimates that only 10 percent to 15 percent of suspected lymphoma cases in her institution are submitted for clonality testing by PCR. Last year the EuroClonality Group published guidelines on the correct interpretation of results obtained with the EuroClonality/BIOMED-2 PCRs (Langerak AW, et al. Leukemia. 2012;26:2159–2171). Dr. Groenen and her coauthors wrote, “As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory.”

At the Association for Molecular Pathology 2012 annual meeting, Dr. Groenen spoke on the development of the EuroClonality/BIOMED-2 multiplex PCR kit and the recently published guidelines for its use. She was joined for a discussion of challenging cases by Rita Braziel, MD, professor and director of hematopathology in the Department of Pathology at Oregon Health and Science University, and James R. Cook, MD, molecular hematopathology section head in the Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic.

“For the vast majority of B-cell lymphoma cases that we diagnose, we are not going to do molecular clonality testing,” Dr. Braziel said in an interview. “We have flow cytometry, kappa and lambda analysis by in situ hybridization, and other immunophenotypic profiles. We will almost always do molecular clonality analysis in new peripheral T-cell lymphomas.”

Molecular clonality testing is utilized more for T-cell lymphoma, Dr. Cook agrees. He and colleagues do it more often for T-cell lymphomas because there is no flow method to assess clonality. “For B-cell lymphoma we usually look at clonality by flow cytometry,” Dr. Cook said in an interview. “We do PCR [for B-cell lymphoma] only when flow is not available or gives equivocal results.” Dr. Cook estimates that T-cell lymphomas make up only about 10 percent of non-Hodgkin lymphomas, and that he uses PCR clonality analysis in perhaps five percent to 10 percent of lymphoma diagnoses. “And we see unusual cases [in our referral practice],” he says. “In a community practice setting you may need it even less often. Most community practices are going to send these cases out rather than do it themselves.”

It’s no surprise that molecular analysis of clonality by PCR can be difficult, including interpretation of patterns obtained from the EuroClonality/BIOMED-2 multiplex primer sets, since clonality testing usually is performed on difficult cases. “It is one thing to have this test on the market,” Dr. Groenen says. “However, the other critical thing is that we want to make sure the PCR kit is used properly.” So the EuroClonality Group organizes workshops to teach users to interpret the reagents correctly. The first workshop was held in 2006, only three years after information about the BIOMED-2 primer set was published (van Dongen JJ, et al. Leukemia. 2003;17:2257–2317). “We were doing molecular clonality testing for diagnostics,” Dr. Groenen says. “Then within a short period a couple of groups asked whether they could come to our lab for one or two weeks. But that takes so much time. So we started thinking—apparently there is a need for this. That’s how the Dutch workshops started.” Now workshops are held in Nijmegen annually. “Each year these workshops are full,” Dr. Groenen says. “People come from Europe, but also Australia, Asia, and the U.S. So obviously there is still a need.”

In 2008 the AMP invited the EuroClonality team to do a daylong workshop on BIOMED-2 testing, sponsored jointly with InVivoScribe. The workshop was repeated in 2010. Last year Dr. Groenen again crossed the Atlantic where she joined Dr. Cook and Dr. Braziel in the workshop on how to apply the new guidelines. Dr. Groenen presented a synopsis of the guidelines, after which the three participants discussed five challenging cases. “In a workshop people can get the impression that interpretation can be so difficult,” Dr. Groenen said in an interview. “Because workshops should have educational value, we present cases that people can learn from.” They therefore present cases that are difficult from the pathology or molecular biology perspective. “People may forget that the test and the uniform scoring system [guidelines] works well for more than 95 percent of the cases. You can have 10 straightforward cases, but these will not be the cases that are discussed in the workshops.”

Difficult diagnostic case: follicular lymphoma? Polyclonal IGH-rearrangements and IGK-VJ rearrangements were detected; however, a clonal IGK-DE rearrangement was detected (arrow). The overall molecular interpretation of this case is: Monoclonality detected.

But cases that come to molecular clonality testing may be a bit more problematic than the ordinary run of specimens to start with. “From the pathologist’s viewpoint, these cases may already be more difficult,” she says. Having a specialized and accurate method can be of help.

“The problem I’ve been seeing and that is of concern to me,” Dr. Braziel says, “is that now with kits available from InVivoScribe with multiplex PCR for antigen receptor clonality, it’s very easy technically to do the test. So now lots of people are doing the test who have limited experience in molecular testing and even more limited experience in interpreting the results.” She has been doing PCR analysis of immunoglobulin (Ig) and T-cell receptor (TCR) genes, so-called antigen receptor genes, for about 15 years. “Even so, it was a really steep learning curve for us when we started using the IVS BIOMED-2 GeneScan kit. I will still send samples [to Dr. Groenen] for a second opinion.” Use of multiplex PCR reaction tubes in the EuroClonality/BIOMED-2 multiplex PCR kit creates complex patterns that require an understanding of the primers to resolve. In Dr. Braziel’s section there are six hematopathologists, all of whom sign out clonality tests. “Most of the time we will look at cases together and discuss them,” she says.

Where this level of expertise is not available, mistakes can be made. In their consultative practice, Dr. Braziel says, “We have seen cases where misinterpretation of clonality results has led to misinterpretation of the hemepath diagnosis.” Helping participants to achieve a more sophisticated understanding of the EuroClonality/BIOMED-2 primer sets and how to interpret them was the major objective of the AMP workshop. “Most people who attended the workshop wanted to increase their level of understanding of the kits and to increase their skills at doing the test and interpreting it,” Dr. Braziel says. “My goal in this workshop was not to say, ‘You should send your cases to me,’ but to help them understand the test better.”

Case 3 showing three clonal TCRB gene rearrangements and a clonal TCRG rearrangement (not shown here) as well. The technical interpretation for all shown PCR tubes is: clonal. The overall molecular interpretation is consistent with the presence of one T-cell clone (monoclonality), with on one allele two clonal TCRB gene rearrangements (VDJ and DB2-J) and on the other allele one clonal TCRB VDJ-rearrangement.

“The BIOMED-2 primers and protocols are used widely in the U.S.,” Dr. Cook says. Numbers in the CAP Molecular Hematologic Oncology Survey bear this out. “Many labs have been using their own in-house tests,” he says. “I think they are being phased out and replaced by InVivoScribe kits. We use the EuroClonality primers through IVS for both B- and T-cell PCR.”

Of course, as Dr. Braziel noted, molecular clonality testing was performed before the EuroClonality/BIOMED-2 primer sets were introduced in 2003. It was done by Southern blotting, which was the gold standard for many years, and even by PCR in some institutions. However, Southern blotting was cumbersome and neither method was sufficiently sensitive.

“When we started doing clonality testing by looking at genes for immunoglobulin heavy chain [IgH],” Dr. Braziel says, “some labs used only one primer set, for framework three. Others used primers for framework one or framework two. People found they had the best results if they used primers for all three framework regions.” By relating this piece of history, Dr. Braziel was making an important point about the composition of the EuroClonality/BIOMED-2 multiplex primer kit—the critical feature that makes it work and challenging to interpret in some situations: Single-primer PCR reactions were abandoned in favor of multiplexed primer combinations for Ig and TCR gene rearrangements. “Using multiplex PCRs has markedly improved this type of testing,” Dr. Braziel says. “It means that most people are using the same primers and it has standardized testing, so that most people will get the same results if they do the test right and interpret it correctly, which most of them do.”

One can get some idea of the need for complex testing in identifying Ig and TCR gene rearrangements by looking at the genetic events that give rise to these large and complex molecules. (A good explanation is provided in the preface to the group’s 2003 Leukemia paper.) Basically, during human development, recombination occurs among the genetic regions for three components of the Ig molecule—the V, D, and J regions. (TCRs share the basic structure of Ig molecules and are formed in the same way.) Joining of these three regions in various combinations creates a repertoire of Ig molecules. Because each region consists of a family of genetic variants, the number of possible combinations in the resulting immunoglobulin population in an individual is very large—estimated at 1012. Thus the need for multiplex PCR reactions in the test.

Interpretation of TCRG VJ-rearrangements (tube A), showing polyclonal TCRG rearrangements (case 2A) and a clonal TCRG rearrangement (arrow) in a polyclonal background (case 2B). The rearrangement patterns were detected by both GeneScanning and heteroduplex analysis.

In the 2003 Leukemia publication, the kit contained 95 different primers for the Ig/TCR targets in 14 multiplex PCR tubes. How was this amazing feat accomplished? “We made use of family-specific primers,” Dr. Groenen explains. Each region of the gene for Igs is composed of many variants, but variants are grouped in families that share sequence homology. In addition, tubes are selected based on complementarity among regions. For instance, Dr. Groenen calls the combination of TRG tube A and TRB tube A “my personal favorite” when there is limited amount of DNA from a diagnostic specimen. “We tested the primers and verified that all family members [for each genetic region] can be identified with the primer set. So the kit works quite well.”

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