1. ¹INSERM, U762, Paris, France
2. ²Université Pierre et Marie Curie-Paris6, Paris, France
3. ³Laboratoire d'Onco-Hematologie, CEA/iRCM, Fontenay aux Roses, France
4. ⁴Service d'Hématologie Biologique, Hôpital Pitié-Salpêtrière, Paris, France

Correspondence: J Delic, E-mail: jozo.delic@cea.fr; H Merle-Béral, E-mail: helene.merle-beral@psl.ap-hop-paris.fr

Tumours with microsatellite instability (MSI) exhibit a mutator phenotype and consequently disclose inactivating mutations or epigenetic silencing of post-replicative DNA mismatch repair (MMR) genes, predominantly MSH2 and MLH1.¹ Microsatellite (MS)-repeated motifs are highly prone to insertion or deletion during DNA replication. In MMR-deficient tumours, oncogenesis has been attributed to the accumulation of frame shift mutations in coding MS located in genes playing a role in cell proliferation, cell cycle checkpoints and apoptosis. MSI may also occur through an excess in the activity of 3-methyladenine DNA glycosylase and apurinic endonuclease (APE1), enzymes involved in base-excision repair.² Numerous MSI target genes are essential in DNA repair such as DNA-PKcs, ATR, RAD50 and MRE11,³ and some are involved in non-homologous end-joining repair (NHEJ), a DNA repair pathway that we previously showed to be altered in the aggressive subset of chronic lymphocytic leukaemia (CLL).⁴

CLL displays a general genome hypomethylation, reviewed in Bouley et al.⁵ and Raval et al.,⁶ shown as being both a hallmark and a promoter of human cancers.⁷ The bcl-2 proto-oncogene is overexpressed in at least 95% of CLL, due to cytosine hypomethylation of the gene promoter CpG sequences. In addition to disturbing apoptosis, overexpression of Bcl-2 may alter MMR by inhibiting the expression of MSH2, a key component of MMR, as well as recombination (reviewed in Bouley et al.⁵). Owing to the involvement of some MMR components in both apoptosis and DNA repair, and because MSI-driven mutations often target proapoptotic genes, we hypothesized that MSI could be involved in CLL, notably in those that are refractory to DNA damage-induced apoptosis. Bax, a major opposite partner of Bcl-2 in controlling apoptotic death balance, may be suspected as a potential candidate for loss of function in B-cell CLL (B-CLL). Yet, Bax, as well as Bcl-10, caspase 5, FAS or Apaf-1 genes all contain a coding MS targeted by MSI-driven mutations.¹ Conflicting results have been reported on the incidence of MSI in CLL (reviewed in Bouley et al.⁵ and Raval et al.⁶). While some studies claimed no appearance of MSI in CLL, MSI has been detected in rare cases of non-Hodgkin's lymphomas, developing in immunocompromised patients, predominantly diffuse large B-cell lymphoma.⁸ Other investigators reported that aberrant hypermethylation of the promoter region of MLH1 associated with MSI occurred in a number of CLL patients undergoing Richter's transformation (reviewed in Bouley et al.⁵ and Raval et al.⁶).We recently showed that the capacity of DNA repair through NHEJ is greater in a subset of CLL cells resistant to DNA damage-induced apoptosis as compared to sensitive subset, a feature fitting well with patient's response to therapy with alkylating agents.⁴ Here, we have addressed MSI in these two subsets of CLL cells using a highly sensitive and specific methodology recently recommended for MSI testing by the National Cancer Institute.⁹ Considering the heterogeneity of the response of B-CLL cells whether to activate apoptotic death or not following DNA damage in vitro as well as the clinical outcome of CLL patients (Chiorazzi et al.,¹⁰ Stilgenbauer and Dohner¹¹ and references within), we included a large series of 59 primary cell samples among which 42 were sensitive and 17 were resistant to DNA damage-induced apoptosis in vitro, obtained from 25 and 14 leukaemic donors, respectively. For three patients, the 'classification' based on apoptotic death susceptibility of their cells in vitro changed concomitantly with changes in clinical status during the time of samples' collection, evolving from indolent to aggressive form with the malignant cells becoming in parallel resistant to apoptosis in vitro. Two patients underwent Richter's transformation (patients 33 and 38). It is noteworthy that 80% (11/14) of patients having malignant B cells resistant to apoptosis relate to the aggressive form of the disease and had already received chemotherapy, with only one remaining in remission more than 6 months; the time of sampling for these patients was at least 3 months after the end of chemotherapy. Despite the fact that 8 of the 14 patients with resistant B cells disclosed mutations in immunoglobulin heavy-chain (IgVH) genes, a feature generally associated with a good clinical prognosis, all but one received chemotherapy arguing for the aggressiveness of their disease. Although unmutated genes were predominantly classified as resistant (P=0.05; that is only one patient with malignant cells sensitive to apoptosis was devoid of IgVH mutation, Table 1 and Figure 1), the association between the mutational status of IgVH genes and the sensitivity to DNA damage-induced apoptosis was not strong enough to be considered as a valuable marker for disease aggressiveness. The level of soluble CD23 (sCD23) that has also been proposed as a faithful prognostic marker, reached high values independently of the presence of IgVH mutations. Similarly, the sCD23 levels did not discriminate resistant from sensitive B-CLL cells to apoptosis, whatever was measured at the time of the diagnosis (P=0.46) or at the time of sampling (P=0.84; Figure 1); maximal sCD23 levels also failed to correlate with apoptosis (P=0.15). The most frequent chromosomal aberration consisting of 13q14 mono- or biallelic deletions was present in both the resistant and the sensitive groups. Three patients, all belonging to the resistant subset, disclosed 17p13 deletions in addition to 13q14 deletions, affecting p53 function. In addition, p53 mutations were detected in four resistant cases (patients 30, 33, 36 and 38), while no mutation could be detected in any of the sensitive cases. In parallel, among all CLL cases where the mutational status of p53 was determined, all sensitive cases and more than 50% of resistant cases had p53 wild type (not shown). According to these observations, B-CLL cells susceptibility to undergo DNA damage-induced apoptosis or not in vitro, combined with other already established biological parameters (that is type of chromosomal aberrations, mutational status of IgVH genes or sCD23 levels), should be considered as a reliable and faithful prognostic marker.

Figure 1.

Comparison of the sensitivity/resistance of B-cell chronic lymphocytic leukaemia (B-CLL) cells to undergo apoptosis following DNA damage with the status of immunoglobulin heavy-chain variable (IgVH) gene mutations and with the soluble CD23 (sCD23) PVT expression level. Among the 39 patients, 25 have malignant cells sensitive (S) and 14 resistant (R) to DNA damage-induced apoptosis. In the sensitive group, among the patients for whom the IgVH status was defined, all but one had mutated (M) IgVH genes, whereas all of the other unmutated (U) cases were in the resistant group (P=0.05); resistant cell subset disclosed eight cases with mutated and six cases with unmutated IgVH genes. The variations in the sCD23 levels defined at the moment of cells' sampling were comparable whatever cells underwent apoptosis following DNA damage or not (from 46 to 716 and 60 to 443, respectively, P=0.84). Similarly, among the CLL cases resistant to apoptosis, there were no significant differences in the sCD23 according to the IgVH status (P=0.85).

Full figure and legend (37K)

Table 1 - Peripheral blood samples from CLL donors were used for negative selection of B cells by RosetteSep procedure (StemCell Technology, USA).

Full table

MSI phenotyping could be adequately performed since germline DNA was successfully amplified with the five markers of the pentaplex assay: NR27, NR21, NR24, BAT25 and BAT26. Polymorphisms at these loci are rare and have been well-characterized among different populations worldwide in a study showing that tumour MSI can be determined for all human populations using this panel of markers without the need for matching normal DNA.¹² Because the five mononucleotide repeats are all located outside the coding sequences, being in the 5'UTR sequences (NR21 and NR27), 3'UTR sequences (NR24) or introns (BAT25 and BAT26), their mutations are unlikely to be selected in MSI tumours. Thus, these markers should display high levels of instability in tumours originating from other tissues, making this method appropriate to detect MSI in haematological tumours, as previously shown with the BAT25 and BAT26 markers.⁸ The fact that several genes targeted by MSI in haematological tumours are also inactivated in MSI gastrointestinal tumours, including BAX, further strengthens the possibility to use the pentaplex method to efficiently detect MSI in CLL. Inactivating mutations of BAX occurring in half of colorectal MSI tumours decrease apoptosis in these tumours, which frequently overexpress Bcl-2.¹³ Likewise, MSI-driven BAX mutations could have been expected in CLL overexpressing Bcl-2.

Since none of the markers displayed a single profile reminiscent of MSI in any of the B-CLL samples analysed, all tumours were classified as MS-stable; the variations in the sizes of each marker were all consistent with the known polymorphisms. Examples of amplification profiles of samples derived from four sensitive p53 wild-type tumours (patients 10, 13, 21 and 25), two resistant p53 wild-type tumours (29 and 39) and two resistant p53 mutated tumours (30 and 33) are shown in Figure 2.

Figure 2.

Pentaplex PCR method for microsatellite instability (MSI) determination in chronic lymphocytic leukaemia (CLL). Purification of total DNA from CLL B lymphocytes was carried out according to standard protocol edited by the manufacturer ('DNeasy Tissue kit', Qiagen, Hilden, Germany). MSI determination was carried out using the pentaplex PCR method, as previously described.¹² In this method, the germline DNA was successfully amplified with the five markers in a pentaplex assay: NR27 (inhibitor of apoptosis protein-1 5'UTR), NR21 (SLC7A8 5'UTR), NR24 (zinc-finger 2 3'UTR), BAT25 (c-kit intron 16) and BAT26 (MSH2 intron 5). For each marker, the antisense primer was labelled with a fluorescent dye: HEX for NR27 and NR24, 6-FAM for NR21 and BAT26, and NED for BAT25. PCR products were mixed with ROX-labelled HD400 size markers and analysed in denaturating conditions on an ABI PRISM 3100 Genetic Analyzer in 36-cm capillaries using POP-7 (Applied Biosystems, Foster City, CA, USA). Apparent sizes of the various alleles were determined using the GeneScan software (Applied Biosystems). The profiles shown correspond to four sensitive (#10, 13, 21 and 25) samples, all devoid of p53 mutation and four resistant samples with (#33 and 35) or without (#29 and 31) p53 mutations. The numbers in each graph correspond to the patients' numbers presented in Table 1.

Full figure and legend (182K)

In conclusion, the analysis of 59 CLL cell samples (42 sensitive and 17 resistant cell samples derived from 39 patients) showed no MSI irrespectively of the cell sensitivity to undergo DNA damage-induced apoptosis or not, and the clinical phenotype features of CLL patients. Thus, MMR does not appear as a safeguard mechanism potentially altered in the pathogenesis of CLL, opening the possibilities for other DNA repair pathways to drive this type of malignancy. Moreover, MMR defect could not be documented in the new emerging subset of patients (unpublished data) whose cells are resistant to DNA damage-induced apoptosis but display unaltered NHEJ, thus suggesting that other DNA repair pathways may be involved in their resistance.