How many mutations are you allowed?

Legend of figure.

Figure 1. Comparison of survival in months of patients with CLL with different degrees of sequence homology for IgVH genes.
For both panels.  represents < 97% homology,  between 97% and 97.9%,
 between 98% and 99.9% and  100%.
Panel A shows overall survival censored for deaths unrelated to CLL; panel B shows treatment-free survival.

Summary

The choice of 98% sequence homology for immunoglobulin heavy chains to distinguish between mutated and unmutated versions of chronic lymphocytic leukaemia (CLL) was arbitrary and was chose to account for supposed polymorphisms. Some authors chose 97% or even 95%. In this study we have examined survival curves for cohorts of patients with varying degrees of sequence homology. All patients with <97% homology behaved as if mutated. Those with 97-98% homology were more aggressive than the mutated cases, but less aggressive than those with >98% homology.

Introduction

Although the difference between chronic lymphocytic leukemia (CLL) with mutated and unmutated immunoglobulin variable region heavy chain (IgVH) genes is well established and this distinction is recognised as one of the most important prognostic variables (Damle et al 1999; Hamblin et al 1999), the choice of 98% sequence homology as the limit of the unmutated subset was arbitrary, based on possible polymorphisms that might produce that degree of variation (Matsuda et al, 1993). Some authors chose 97% (Krober et al, 2002) or even 95% (Lin et al, 2002) homology as a possibly more appropriate cut-off. When a comparison was made of IgVH gene homology in leukaemic cells and granulocytes from the same patient, it became apparent that even small numbers of mutations were caused by somatic hypermutation rather than polymorphisms (Davis et al 2003).

Individuals whose IgVH genes have only a few somatic mutations comprise a small proportion of CLL patients and until recently it has not been possible to study enough patients in this group to distinguish a different prognostic impact of choosing the threshold for unmutated status at >97% or >98% homology. The problem is further complicated by the fact this is an elderly population in which many deaths are unrelated to CLL, and this adds ‘noise’ to the system making small differences difficult to perceive. A final difficulty is the discovery that the use of the VH3-21 gene is associated with a poor prognosis whether of not there are somatic mutations (Tobin et al 2002). This is especially problematic because such cases frequently have between 96 and 99% sequence homology.

In order to resolve these difficulties we have made a retrospective survey of 310 patients with CLL who have passed through our hands in the past 30 years and who have had their IgVH genes sequenced. We have compared outcomes of patients with different degrees of sequence homology. There were only four whose tumor used the VH3-21 gene, none of which fell in the disputed area of 97-98% homology; two had 100% and two <97% sequence homology. Patients were observed until treatment was indicated because of symptoms or evidence of progression, and then treated according to best practice of the day; largely with chlorambucil prior to 1990 and increasingly with purine analogues or combinations including purine analogues since then.

Methods.

310 patients with CLL were studied. Of these 260 were local patients representing the normal referral pattern of a district hospital and 50 were patients referred from other hospitals for a second opinion. The diagnosis was based on standard morphological and immunophenotypic criteria. Local patients were followed closely by the authors; data on referred patients were assembled from the referring physician by letter or telephone call. In the cases of patients who have died, an assessment of whether or not the cause of death was related to the CLL was made independently by two of the authors (TJH and DGO) and any discrepancy resolved by discussion. Except in cases with bone marrow suppression, deaths from cardiac or cerebral events were regarded as unrelated to CLL unless an infective episode or thrombocytopenia was involved. Deaths from cancer were regarded as unrelated unless there was an obvious relationship to the CLL.

IgVH gene analysis

Prior to October 2004 IgVH genes were originally sequenced as previously described (Hamblin et al, 1999)1. The preferred source material was RNA. cDNA was synthesized and amplified by polymerase chain reaction (PCR) using a mixture of oligonucleotide 5’ primers specific for each leader sequence of the VH1 to VH6 families or a consensus 5’ FW1 region primer, together with either a consensus 3’ primer complementary to the germ line JH regions or a 3’ primer complimentary to the constant region. From 2004 onwards gDNA was extracted from whole blood using the QIAmp®DNA mini kits (Qiagen, Crawley, West Sussex, UK) according to the manufacturers instructions. gDNA was amplified in a single multiplexed PCR reaction consisting of 6VH framework 1 primers combined with one JH consensus primer (standardises BIOMED-2 primers) (van Dongen et al, 2003; Matthews et al, 2004). Clonal sequences were determined by sequencing amplicons from at least 2 independent PCR reactions. The majority of samples were sequenced directly using an automated DNA sequencer. Nucleotide sequences were aligned to EMBL/GenBank and current databases (V-BASE sequence directory IMGT/V-QUEST, using MacVector 4.0 sequence analysis software; International Biotecnologies, New Haven, CT, and Lasegene; DNASTAR, Madison, WI.). Percentage homology was calculated by counting the number of mutations between the 5’ end of FR1 and the 3’ end of FR3.

Statistical methods

Data were analysed using GraphPad Prism 4. Survival functions comparing patients have been estimated using the product limit method of Kaplan Meier.

Results

There were 99 patients with 100% sequence homology, 22 with between 98 and 99.9%, 22 with between 97 and 97.9%, 24 with between 96 and 96.9%, and 143 with <96% homology with germ line genes. There have been 139 deaths of which 79 were determined to be unrelated to CLL. Survival curves are shown in figure 1. The median survivals, censored for unrelated deaths, were 102 months for patients with 100% IgVH gene homology; 132 months for those with 98-99.9%, 184 months for those with 97-97.9% and not yet reached for those with 96% or <96%. The difference between 100% and 97-97.9% was statistically significant (p=0.002) as was the difference between 97-97.9% and <97% (p<0.0001). However, the differences between 100% and 98-99.9% and between 98-99.9% and 97-97.9% did not reach statistical significance. The survival curves for those with 96-96.9% homology and <96% were virtually identical.

Using treatment-free survival as an alternative end-point gave very similar results. Median times to treatment were 35 months for 100% homology, 36 months for 98-99.9%, 156 months for 97-97.9%, and 272 months for <97%. The difference between 100% and 97-97.9% was statistically significant (p=0.001) as was the difference between 97-97.9% and <97% (p=0.0003), but the differences between 100% and 98-99.9%, between 98-99.9% and 97-97.9%, and between 96-96.9% and <96% were not.

Discussion

The pattern revealed by this study is not a continuous gradation with survival increasing with increases in the number of mutations. Those with 97%-97.9% homology comprise a mixture of benign and malignant cases rather than a homogeneous group with moderate malignancy.

It is not understood how accumulations of somatic mutations affects prognosis in CLL. The initial explanation, that in those with mutated IgVH genes the cells had passed through the germinal centre while the cells of those with unmutated IgVH genes had not, seems unlikely to be true. Tumour cells from both types of patients most closely resemble memory B cells (Klein et al, 2001) and both express CD27 (Dong et al, 2002). Furthermore, among both mutated and unmutated CLLs there are now many examples of non-stochastic pairing of immunoglobulin heavy and light chains predicated by the sequences of the CDR3 of the Ig heavy chain, combining to form stereotypic antibody-combining-sites indicative of selection by as yet unknown extrinsic or self antigens (Widhopf et al, 2007). All these facts point to the clear conclusion that both types of CLL should be thought of as derived from antigen-experienced B cells.

Poor prognosis in patients with unmutated IgVH genes is particularly associated with those who acquire other poor prognostic factors such as CD38 (Hamblin et al, 2002), ZAP-70 (Rassenti et al, 2004) and chromosomal deletions at 11q23 and 17p13 (Dohner et al, 2000). It is not known why these abnormalities are preferentially acquired in patients with unmutated IgVH genes, nor whether those with between 97% and 97.9% homology are among those that preferentially acquire them.

One possible mechanism is that the mutator mechanism is impaired in CLLs with unmutated IgVH genes. The anomalously high expression of activation-induced cytosine deaminase (AID), an enzyme necessary for somatic hypermutation and Ig class switching, in cases with unmutated IgVH genes (Albesiano et al 2003; McCarthy et al 2003; Oppezzo et al, 2003) may be implicated. It has been suggested that high levels of AID may result in loss of substrate specificity and the development of mutations in c-MYC, PAX-5 and RhoH genes which are associated with more aggressive forms of the disease (Reiniger et al, 2006). It would be interesting to see whether the level of AID correlates with the degree of somatic hypermutation and whether this is a factor in the acquisition of other poor prognostic factors.



References
Albesiano E, Messmer BT, Damle RN, Allen SL, Rai KR, Chiorazzi N. (2003) Activation-induced cytidine deaminase in chronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone.
Blood, 102, 3333-3339.

Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N. (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood, 94, 1840-1847.

Davis ZA, Orchard JA, Corcoran MM, Oscier DG. (2003) Divergence from the germ-line sequence in unmutated chronic lymphocytic leukemia is due to somatic mutation rather than polymorphisms. Blood, 102:3075.

Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, Dohner K, Bentz M, Lichter P. (200) Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 343, 1910-1916.

Dong HY, Shahsafaei A, Dorfman DM. (2002) CD148 and CD27 are expressed in B cell lymphomas derived from both memory and naive B cells. Leuk Lymphoma. 43,1855-1858.

Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. (1999) Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood, 94,1848-1854.

Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK, Oscier DG. (2002) CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 99, 1023-1029.

Klein U, Tu Y, Stolovitzky GA, Mattioli M, Cattoretti G, Husson H, Freedman A, Inghirami G, Cro L, Baldini L, Neri A, Califano A, Dalla-Favera R. (2001) Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. Journal of Experimental Medicine, 194, 1625-1638.

Krober A, Seiler T, Benner A, Bullinger L, Bruckle E, Lichter P, Dohner H, Stilgenbauer S. (2002) V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood,100,1410-1416.

Lin K, Sherrington PD, Dennis M, Matrai Z, Cawley JC, Pettitt AR. (2002) Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia. Blood, 100,1404-1409.

McCarthy H, Wierda WG, Barron LL, Cromwell CC, Wang J, Coombes KR, Rangel R, Elenitoba-Johnson KS, Keating MJ, Abruzzo LV. (2003) High expression of activation-induced cytidine deaminase (AID) and splice variants is a distinctive feature of poor-prognosis chronic lymphocytic leukemia. Blood. 101, 4903-4908.

Matthews C, Catherwood M, Morris TC, Alexander HD. (2004) Routine analysis of IgVH mutational status in CLL patients using BIOMED-2 standardized primers and protocols. Leuk Lymphoma 45,1899-1904.

Matsuda F, Shin EK, Nagaoka H, Matsumura R, Haino M, Fukita Y, Taka-ishi S, Imai T, Riley JH, Anand R, Soeda E, Honjo T. (1993) Structure and physical map of 64 variable segments in the 3'0.8-megabase region of the human immunoglobulin heavy-chain locus. Nature Genetics, 3, 88-94.


Oppezzo P, Vuillier F, Vasconcelos Y, Dumas G, Magnac C, Payelle-Brogard B, Pritsch O, Dighiero G. (2003) Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation. Blood, 101, 4029-4032.


Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, Neuberg DS, Flinn IW, Rai KR, Byrd JC, Kay NE, Greaves A, Weiss A, Kipps TJ. (2004) ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia.
N Engl J Med. 351, 893-901.

Reiniger L, Bodor C, Bognar A, Balogh Z, Csomor J, Szepesi A, Kopper L, Matolcsy A. (2006) Richter's and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation. Leukemia, 20, 1089-1095.

Tobin G, Thunberg U, Johnson A, Thorn I, Soderberg O, Hultdin M, Botling J, Enblad G, Sallstrom J, Sundstrom C, Roos G, Rosenquist R. (2002) Somatically mutated Ig V(H)3-21 genes characterize a new subset of chronic lymphocytic leukemia. Blood, 99, 2262-2264.

Van Dongen JJ, Langerak AW, Bruggermann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, Garcia-Sanz R, Van Krieken JH, Droese J, Gonzalez D, Bastard C, White HE, Spaargaren M, Gonzalez M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA. (2003) Design and standardization of PCR primers ad protocols for detection of clonal immunoglobulina nd T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17, 2257-2317.

Widhopf GF, Goldberg CJ, Toy TL, Rassenti-LZ, Wierda WG, Byrd JC, Keating MJ, Gribben JG, Rai KR, Kipps TJ. (2007) Non-stochastic pairing of immunoglobulin heavy and light chains expressed by chronic lymphocytic leukemia B cells is predicated on the heavy chain CDR3. Blood (prepublished online August 3, 2007; DOI 10.1182/blood-2007-02-073130