Thursday, June 30, 2011

Searching for Surrogates for IGHV mutations in chronic lymphocytic leukemia

I posted this accidentally the other day. It is the draft for an article I am writing for Leukemia Research. The idea is that a lot of people are beavering away trying to find simple tests that will give the same information as IGHV mutations but are not so difficult to do. My conclusion is that these tests don't deliver the goods and that anyway we can get even more worthwhile information from IGHV mutations and in any case the tests has become at least as easy to do as flow cytometry tests.

Searching for Surrogates for IGHV mutations in chronic lymphocytic leukemia


Despite a startling separation of chronic lymphocytic leukemia (CLL) into two clinically different diseases with average survivals of 8 years and 25 years, the mutational status of immunoglobulin variable region (IGHV) genes has not entered routine clinical practice to assess prognosis, although their assessment is regarded as an essential for clinical trials. Instead, surrogates that may be measured by flow cytometry have been sought. Measurements of the expression of CD38 and ZAP-70 have been the most popular assays for prognosis although both are in their own ways unsatisfactory. Many other candidates have emerged, but none has been universally endorsed.
As the assay for IGHV mutations has been standardized the standard of difficulty has diminished and as greater numbers of cases have been assessed it has become clear that there is even more information to be gathered from the study of the sequence of IGHV genes. It has been recognised that stereotypy within CLL is associated with more specific clinical features than mere longevity and an even greater heterogeneity has been revealed. It seems clear that the genetic prognostic markers cannot be replaced by expression markers and the search for surrogacy and IGHV mutational status should become a routine investigation in CLL.


More than a decade ago the simultaneous publication of two papers seemed to explain the marked heterogeneity that clinicians had observed in the natural history of chronic lymphocytic leukemia (CLL). [1, 2] The rearrangement of immunoglobulin (Ig) genes is an essential process in the maturation of normal B cells, whereby each lymphocyte acquires a unique B cell receptor (BCR) formed by the selection and recombination of individual gene segments from a large repertoire.

For the heavy chain of Ig, selection and recombination takes place from one each from 51 VH genes, 27 D genes and 6 JH genes.[3] The junctions of VH to D and D to JH are imprecise, with the deletion by exonucleases of templated nucleotides or the insertion by terminal deoxytransferase (TdT) of non-templated nucleotides in a random manner [4]. This introduces a further huge diversity into the shape of the Ig molecule, especially as the D segment can be read in any of the three frames [5]. The consequence is that the third complementarity-determining region (CDR3) of any given lymphocyte is virtually unique, and provides a clonal signature for any tumor deriving from it. Rearrangement of the light chain variable region genes occurs in a similar manner, involving single-step recombinations of V/J gene segments but with no D segments.

On completion of this maturation the B-cell leaves the bone marrow for the periphery where it may encounter antigen. It then undergoes affinity maturation, usually in the germinal centers of the peripheral lymphoid organs. Here, somatic mutation is induced under the influence of CD40+ve T cells, cytokines and antigen-bearing follicular dendritic cells [6]. The rate of introduction of base pair changes is of the order of 10e4 – 10e3 per generation. The mutations tend to cluster in the CDRs, possibly for structural reasons and possibly because of antigenic selection.

Interest in immunoglobulin genes in CLL originally stemmed from attempts to recognize the normal cell counterpart to the CLL cell. Because CLL cells express CD5 many authorities had accepted that CLL cells derived from the minor population of CD5+ve naïve B cells. Early sequences of the IGHV genes of tumor cells from patients with CLL found them to be in germline configuration [7-9] tending to confirm their origin from a naïve B cell. However, reports began to appear in the literature detailing cases with evidence of somatic mutation culminating in 1994 with a review of the literature by Schroeder and Dighiero [10] which found that 36/75 reported cases had IGHV genes with less than 98% sequence homology to the appropriate germline gene. The figure of 98% was chosen because polymorphisms, which are quite common in IGHV genes, can account for that degree of disparity [11]. Subsequently, a multicenter study of 64 patients with undoubted and classical CLL also found two groups of roughly equal numbers with respectively mutated and unmutated IGHV genes [12].

IGHV genes as prognostic factors

The first suggestion that IGHV mutations might have prognostic significance came from Oscier et al [13] who examined 22 patients with classical CLL segregated according to karyotype. Tumors with trisomy 12 had unmutated IGHV genes but those with 13q14 abnormalities detected by conventional cytogenetics had evidence of somatic mutations. By 1998 this series had been extended and presented at several meetings, demonstrating a more aggressive disease and a shorter survival for patients with unmutated IGVH genes [14-16] before the definitive publication of the two papers that documented and mutually corroborated the finding that IGVH mutational status identifies two subsets of CLL, one with a median survival of 8 years and one with a median survival of approximately 25 years [1,2].

Searching for surrogates

From the beginning it was recognized that sequencing of IGVH genes would not be available to most laboratories, especially if the technique involved a post-doctoral student, a sheet of X-ray film, a ruler and a lot of patience. One of the original papers proposed the percentage of B-cells expressing CD38 measured by flow cytometry as a surrogate measurement [1]. Despite its initial attraction CD38 expression later proved to be discordant with IGHV mutational status in 30% of cases and vary during the course of the disease in up to 25% of cases [17]. In fact, CD38 expression is an independent prognostic variable that can be combined with IGHV mutational status to enhance prognostic predictability [17].

Once IGHV mutational status had so comprehensively separated CLL into two clinical types, the question was asked as to whether it was one or two diseases [18]. Gene expression profiling demonstrated a distinct pattern for CLL distinguishing it from other lymphoid tumors and any particular B-cell population, although the closest normal cell profile was that of a memory B-cell [19, 20]. However, the expression of a small number of genes in the mutated and unmutated subtypes was different, with ZAP-70 standing out as the gene that most stringently separated the subsets.

Flow cytometric assays for ZAP-70 expression have proved difficult, especially as it is an intracellular antigen so that the cells require permeabilization, but at least three different methods have been reported [21-23]. Seen as surrogate assays for IGHV gene mutations, the first two assays performed similarly with around 94% concordance, but the third, which used a different and directly conjugated antibody, had only a 77% concordance with IGHV gene mutations. On the other hand, in this study ZAP-70 expression performed better than VH mutations in predicting treatment-free survival. Patients who were ZAP-70 positive; IGHV mutated had a worse survival than those who were ZAP-70 negative; IGHV unmutated.

Nevertheless, establishing a standardized assay for ZAP-70 expression has proved difficult [24] and many of the most experienced laboratories have dropped it from their repertoire as being unreliable. The need for a dependable surrogate for IGHV mutational status remains. Although no other prognostic factor has been widely adopted, there has been no shortage of candidates. A recent review described nineteen new prognostic markers: TCL1 gene expression, CLLU1 expression, miRNA signature, mRNA signature, and expression of Lipoprotein lipase A, ADAM29, HEM1, Septin 10, DMD, and PEG 10, levels of VEGF and thrombopoietin; telomere length and activity; surface expression of CD49d, CD69 and FCRL, expression of anti-apoptotic genes such as MCL-1 and the Bcl-2/Bax ratio, MDR1/MDR-3 genes, and AID mRNA [25].

Combinations of prognostic factors might be more useful than individual factors. CD38 and IGHV mutations [17] or CD38 and ZAP70 [26, 27] both perform better than any one factor. A scoring system based on six surface molecules (CD62L, CD54, CD49c, CD49d, CD38, and CD79b) detectable by flow cytometry has been proposed [28].

Immature laminin receptor

In a recent issue of Leukemia Research, Friedrichs et al [29] proposed that as high expression of the immature laminin receptor (iLR) protein predicted a favorable prognosis for CLL correlating with mutated IGHV genes and being detectable by flow cytometry, it might be used as a surrogate. ILR is an oncofetal antigen overexpressed in several tumor tissues but absent in the normal differentiation process. It is the precursor molecule of the mature 67 kDa protein that plays a role in the cell adhesion-associated processes initiated by laminin binding.

Friedrichs et al studied 134 patients with CLL. The flow cytometry assay used direct staining with a FITC-conjugated mouse anti-ILR monoclonal antibody and cells were double stained with an anti-CD19-PE antibody or an isotope control in the conventional way. ILR scores over CD38 and ZAP-70 in not being present on T cells nor on normal B cells. A cut-off 30% cells staining was determined best to distinguish between positive and negative. 41% of CLLs in their series were judged to be positive. There was a correlation between the expression of iLR and low ZAP-70 expression, mutated IGHV status and low CD38 expression. There was no change in iLR expression over time as there sometimes is with CD38 and ZAP-70 expression [17, 30]. However, the correlation of high expression of iLR with mutated IGHV genes was no better than for ZAP-70 or CD38. Only 78.9% of patients with mutated IGHV genes were iLR positive and 81.7% of unmutated cases were iLR negative. ILR negativity therefore behaves with a similar degree of concordance to IGHV mutations as ZAP-70 or CD38 expression. In a multivariate analysis iLR expression retained some independent predictive value for prognosis.

In summary, iLR expression is not an exact surrogate for IGHV mutational status, but yet another prognostic factor with some independent value.


Perhaps the time has come to draw a line under the search for surrogates. Over time the technology for sequencing IGHV genes in CLL has improved and a recent study reported over 7500 sequences among patients worldwide [31]. The striking feature of these sequences was stereotypy, the finding that the IGHV sequences of large numbers of CLL patients were identical or very similar. The implication is that perhaps a third of all CLLs are derived from populations of B cells responding to a restricted range of antigens or superantigens. Originally at least 48 separate stereotypes could be identified, each subset comprising between 2 and 20 cases [32] but the wider collaboration [31] identified 952 ground-level clusters of cases with between 2 and 56 cases in each, though clearly most of these had very small numbers of cases. Common sequences among ground-level clusters made it possible to group them into considerably larger higher order clusters that displayed biased features with regard to IGHV usage, somatic hypermutation and VH CDR3 pattern composition. The largest higher order cluster with 213 sequences is clearly recognizable as subset 2 which uses IGHV3-21 with an acidic residue at VH CDR3 position 107. This well known subset has been identified as the major exception to the prognostic importance of IGHV gene mutations in which CLLs with up to 4% somatic mutations behave aggressively [33].

These stereotypes are often confined to a single IGHV gene, but not necessarily so and B cell receptors can derive their gene sequence from a number of VDJ combinations, all belonging to the same phylogenetic clan. For example, the second largest higher order cluster which included 184 sequences used 6 different IGHV genes of clan 1 (IGHV1-2, 1-3, 1-8, 1-18, 5a and 7-4-1) with a QWL motif at VH CDR3 positions 108-110.

Of relevance to our current topic is the fact that different stereotypic subtypes appear to be associated with distinct patterns of clinical behaviour. For example, subtype 8 which uses the VH4-39 gene in an unmutated form but class-switched to IgG production is associated with a 24-fold greater risk of Richter transformation and has a higher frequency than expected of trisomy 12 yet no significant association with progressive disease [34]. On the other hand, subtype 4 which uses VH4-34 in a mutated form tends to present in the fifth decade and be non-progressive [32]. As further cases are assembled it is to be expected that even greater heterogeneity will be recognised and assigned to specific subtypes.


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Anonymous said...

I understand that a cutoff needs to be made, but I think the 2% level is arbitrary. I 'd like to see a paper examining patients with borderline mutational status, say 1.5-2.5%. I'll bet you'd see a number of the so-called mutated patients (2.1-2.5%) with aggressive disease and many so-called unmutated ones (1.5-2.0%) with indolent disease.

Terry Hamblin said...

I have written such a paper in 2008. 3% mutations are a bit of a mixture of benign (mostly) and aggressive. 4% apart from the 3-21s are all benign. The new article tells us that the individual subtype is going to be more important.

Wayne said...

I am aware of subtype #4 and #16 from your writings and am curious when you commented "4% apart from the 3-21s are all benign."

I am VH4-34 6% mutated so does this mean I get a subtype of my own due to the aggressive behavior of my CLL?

Hoping to hear you are still progressing with feeling better.


Terry Hamblin said...

As yet we know little about the clinical expression of subset 16 but we do know that it is not as benign as subset 4. There are several genes involved in proliferation that are expressd at greater levels than in subset 4. Then the 4-34s that are in neither subset are also more aggressive than subset 4. It is only subset 4 that is extremely benign.

The 4% benign figure comes from my own series of 300+ cases but might not apply universally with 7500 sequences