In our previous study, we reported that ALL patients with the HLA-DP 1 and DP 3 supertypes had significantly worse outcomes than those with four other DP supertypes.16
Of the four key DP polymorphic antigen-binding residues lining pockets 1, 4, 6 and 9,17
DP 1 and DP 3 are the only two supertypes that share aspartic acid in pocket 1 at position 84 linked to lysine in pocket 4 at position 69 of DPβ, (though they differ for residues in pockets 6 and 9). We therefore wondered whether the antigen-binding molecular signature influenced by aspartic acid in pocket 1, and lysine in pocket 4 (Asp84-Lys69) of DPβ1, rather than the motif determined by pocket 1, 4, 6 and 9 might provide a more specific functional surrogate of outcome in UKALL XI.
Although the DP-typed cases represent only a proportion (38%) of total UKALL XI patients, lack of significant differences in clinical characteristics between typed and non-typed patients suggest that no bias was introduced by analysing the association of DP molecular signatures with outcome in two-fifths of UKALL XI cases. Our analysis confirmed that 5 and 10 year EFS in patients with Asp84-Lys69 was significantly (~10%) worse than non-Asp84-Lys69. Of the DP-typed patients, 44% of Asp84-Lys69 cases had events, while only 36% of non-Asp84-Lys69 cases had events. The difference in EFS between the Asp84-Lys69 and non-Asp84-Lys69 patients remained significant even after adjustment for other prognostic factors, strongly suggesting that Asp84-Lys69 is an independent predictor of adverse outcome in UKALL XI. A total of 294 of the 798 DP-typed patients presented in this paper survived relapse. Of the patients that typed for Asp84-Lys69, 60% had post-relapse events, compared with 54% of the patients with non-Asp84-Lys69, this difference being nonsignificant.
We previously clustered DP alleles into six structural DP supertypes (DP 1–4, 6, 8) defined by polymorphic amino-acid residues lining pockets 1, 4 and 6, at positions 84, 69 and 11 of the DPβ subunit.18
In the present study we included patients with two additional supertypes (DP 11, 15), and classified patients only by the presence of polymorphic amino-acid residues at positions 84 and 69. Position 84 of the DPβ subunit lines pocket 1 of the DP antigen-binding site, and is equivalent to position 86 of DRβ.17
In DPβ, residues 84–87 form a linked unit, which influences the size and charge of the amino-acid side chains of antigens preferred by pocket 1. Thus, the aspartic acid 84–glutamic acid 85–alanine 86–valine 87 (DEAV) peptide binding motif was present in 20 (64%) of the 31 DP alleles in the current patient series, while the remainder had either glycine 84–glycine 85–proline 86–methionine 87 (GGPM) in 10 (32%) alleles, or valine 84–glycine 85–proline 86–methionine 87 (VGPM) in one (3%) DP allele (DPB1*1501). Structure-function studies of DP molecules have shown that DEAV differs from GGPM by modifying the contact area between the DPα1 and DPβ1 subunits of the DPβ heterodimer, as well as influencing the binding of amino acids at the P1 and P2 positions of antigenic peptides by increasing the negative charge of the P1/P2 pockets.17
In HLA-DR alleles, DRβ86 is occupied by glycine or valine and peptides may bind to Gly86, but not to Val86 owing to steric effects.23, 24
Castelli et al.25
identified pockets 1 and 6 of DP as accommodating the main anchor residues of DP-binding peptides, but our results suggest that the outcome of UKALL XI may have been influenced by the peptide binding motif (DEAV or GGPM) of DP pocket 1 alone.
Previous studies of HLA and outcome in childhood ALL have generally involved small patient numbers, limited HLA allele resolution, inadequate diagnostic detail or different treatment regimens to enable clear overall conclusions to be drawn.26, 27, 28, 29, 30, 31
A typical example of the confusion that this has engendered is the reported association of HLA-DR5 with long remission,30
no impact on remission31
and an increased incidence of relapse.32
It is possible that the reason for this lack of agreement is that HLA associations with treatment outcome can only be evaluated in the context of specific trial regimens, due to differences in the effects of therapy on HLA-mediated functions. Despite indications of major histocompatibility complex loss33
and HLA class I downregulation by ALL cells34, 35
no recent attempt has been made to establish the baseline contribution of heritable major histocompatibility complex variation to the clinical outcome of childhood ALL. Although prospective HLA typing of children presenting with ALL using serological methods previously presented a significant technical challenge, the availability of DNA-based molecular techniques now affords a rapid and reliable means of determining HLA genotypes. Although a fully prospective study has yet to be carried out, the availability of remission samples from patients recruited by a large UK population-based case–control epidemiological study of childhood ALL aetiology (the UKCCS20
) most of whom were randomised to the UKALL XI trial, has provided a timely opportunity to redress this deficiency and to establish the contribution of HLA-DP supertypes to outcome, preparatory to an analysis of HLA association with outcome in more recent childhood ALL trials using different treatment regimens.
Although a functional explanation for the association of Asp84-Lys69 with adverse outcome in childhood ALL in UKALL XI is a matter for speculation, we favour a role for the activation of CD4+ T cells by Asp84-Lys69-restricted (that is, bound) peptides. Sidney et al.36
recently reported an extensive overlap in the peptide binding specificity of DP alleles, but peptide binding by Asp84 (DP 1) alleles could clearly be distinguished from Gly84 (DP 2) alleles by a preference for positively charged side-chain residues, notably arginine and lysine. The precise identity of any bound peptides is a matter for further investigation, but we note the presence of arginine at the P1 position of a 9-mer TEL-AML1 core junctional peptide, RIAECILGM, which elicited a DP 1-restricted CD4+ T response in vitro
, and that DP 1 frequency is reduced in patients with TEL-AML1 ALL.37, 38
Our results were obtained by analysing patients in the UKALL XI trial, which is known to have had a higher rate of relapse than concurrent childhood ALL trials. Extrapolation to more recent trials, such as ALL97, with significantly improved EFS39
thus requires caution, and further study. The higher relapse rate in UKALL XI may be partly attributable to the lower anti-leukaemic toxicity of prednislone (PRED) than dexamethasone,40
but this does not explain why this should selectively involve DP alleles with an Asp84 residue. Currently, there is only limited information on changes in HLA class II expression by relapsed ALL cells41, 42
but this seems to exclude loss of HLA class II expression at relapse as an explanation for escape from T-cell control. In contrast, expression of HLA class II molecules including DP by relapsed ALL cells might lead to the binding of leukaemia-associated peptides leading to the recruitment of regulatory CD4+ T (Treg) cells.43
This might lead to a loss of effector (CD8+) T-cell control of minimal residual disease. Although direct evidence of such an effect is currently lacking, the ability of established tumours to induce Treg-associated immune tolerance associated with tumour metastasis44
and reduced survival45
has been well documented in other tumours. In the context of the UKALL XI trial, a key point may be that the use of PRED was insufficiently toxic for leukaemia cells, but exacerbated a tolerogenic Treg response, as PRED is known to promote the differentiation of Tregs and to ameliorate autoimmune disease and allergy.46, 47
We recently reported that the DP 6 supertype, in which Asp84 is linked to Glu69, was strongly associated with susceptibility to childhood leukaemia. We found that this was due to a single infrequent DPB1 allele, DPB1*0601.28
In the present analysis, DP 6 was associated with a slightly worse post-relapse EFS (P
=0.07), but it was not associated with overall outcome. Of the 34 relapsed DP 6 patients included in the outcome analysis, one third typed for DPB1*0601, but there was no discernable difference in their clinical characteristics compared with non-relapsed DP 6 patients (age, sex, presenting white cell count, immunophenotype, karyotype of cells at diagnosis), indicating that DP 6 does not have independent prognostic value in UKALL XI.
In summary, we report that patients in UKALL XI with a DP Asp84-Lys69 antigen-binding molecular signature are more likely to relapse and probably have a worse post-relapse outcome than patients with a non-Asp84-Lys69 molecular signature. We suggest that this might be due to the pocket 1 residue, Asp84, a marker for the DEAV peptide binding motif of DPβ1. We propose a mechanistic explanation for this association in which recruitment of Tregs, possibly exacerbated by PRED (glucocorticoid) therapy, in response to the recognition of Asp84-bound auto- or leukaemia-associated peptides by CD4+ T cells, leads to loss of minimal residual disease control. As we find no association between HLA-A supertype and outcome in UKALL XI48
we suggest provisionally that Asp84 may be an independent prognostic factor at least in this trial. It will nonetheless be important to analyse the contribution of this and other HLA amino-acid signatures in the outcome of more recent childhood ALL trials, and specifically to determine how they might affect risk-adapted therapy. The technical simplicity of single HLA amino-acid genotyping suggests that this could be done at ALL diagnosis.