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Temporal changes in incidence and pattern of central nervous system relapses in children with acute lymphoblastic leukaemia treated on four consecutive Medical Research Council Trials, 1985–2001

Despite the success of contemporary treatment protocols in childhood acute lymphoblastic leukaemia (ALL), relapse within the central nervous system (CNS) remains a challenge. To better understand this phenomenon, we have analysed the changes in incidence and pattern of CNS relapses in 5564 children enrolled on four successive MRC-ALL trials between 1985 and 2001. Changes in the incidence and pattern of CNS relapses were examined and the relationship with patient characteristics assessed. Factors affecting post-relapse outcome were determined. Overall, relapses declined by 49%. Decreases occurred primarily in non-CNS and combined relapses with a progressive shift towards later (≥30 months from diagnosis) relapses (p<0·0001). Although isolated CNS relapses declined, the proportional incidence and timing of relapse remained unchanged. Age and presenting white cell count were risk factors for CNS relapse. On multivariate analysis, the time to relapse and the trial period influenced post-relapse outcomes. Relapse trends differed within biological subtypes. In ETV6-RUNX1 ALL, relapse patterns mirrored overall trends while in High Hyperdiploidy ALL, these appear to have plateaued over the latter two trial periods. Intensive systemic and intrathecal chemotherapy have decreased the overall CNS relapse rates and changed the patterns of recurrence. The heterogeneity of therapeutic response in the biological subtypes suggests room for further optimisation using currently available chemotherapy.


Introduction
Over the last three decades, survival of children with acute lymphoblastic leukaemia (ALL) has improved from around 50%1 to nearer 80%.2 During the same period the outcome for those who relapse has remained poor. [3][4][5][6][7][8][9] Relapses result when evolutionary pressures of frontline therapy favour emergence of a subclone from within the original blast population. 10 The incidence and pattern of relapses thus vary according to protocols used. Irrespective of frontline treatment, relapsed ALL is characterised by two recurring features. The first is the critical prognostic impact of the duration of first remission (CR1).3,7-9,11 Patients who relapse within 18 months of initial diagnosis have a significantly worse outcome when compared to those with later relapses. The second is the predilection for relapse in extramedullary sites, particularly the central nervous system (CNS). At initial diagnosis, <2% of children have disease within the CNS. This can rise to 40% at first relapse.3,7,9 CNS relapses may occur isolated or in conjunction with marrow disease (combined relapse) -therapy outcomes appear to differ in these two groups. In a Children's Oncology Group (COG) report of standard risk ALL relapses, overall survival was better in children with isolated extramedullary rather than combined relapse. 8 The advent of sensitive molecular investigations has complicated the distinction between these two relapse categories. For instance, the Berlin-Frankfurt-Münster (BFM) group observed that ~80% of those with apparent isolated CNS relapse had molecular evidence of marrow disease. The numbers may be small and not adjusted for the duration of CR1 but the BFM observations also indicate a poorer outcome in extramedullary relapses with concomitant higher marrow tumour burden. 12 We do not know why relapses occur in the CNS. Within the limitations of our understanding, modern ALL protocols are designed to limit CNS relapses. Over the last 3 decades in the UK and elsewhere, intrathecal chemotherapy has replaced cranial irradiation as CNS-directed prophylaxis for most children with ALL. 13 Along with progressive intensification of systemic therapy, these measures have in general been successful and the overall incidence of CNS relapses has steadily declined.2 Nevertheless, CNS relapse remains a significant preventable and therapeutic challenge. What then has been the impact of modern therapies on the pattern of CNS relapses in childhood ALL? Have we reached the end of optimisation with conventional drugs or is there room for further refinement of existing therapy? To answer these questions we have analysed the trends in incidence and outcome of CNS relapses among children treated for ALL in the UK over a 17-year period spanning 4 trial eras. The 5-year event free survival (EFS) improved from around 60%14 to nearly 80%15 during this period.

Patients
All patients, treated on national protocols for childhood ALL between 1985 and 2001 and who experienced a relapse in the CNS, were included in these analyses. Patients with a non-CNS relapse (n-CNSr) were included for comparison. For purposes of this study, an isolated CNS relapse (i-CNSr) was defined as the presence of ≥5 white blood cells (WBC) per μl in cerebrospinal fluid with blasts identified on cytospin, or a biopsy-proven recurrence in the CNS or eye, in the absence of morphological disease in the bone marrow. A combined relapse (c-CNSr) was defined as the presence of CNS disease with ≥5% blasts in the bone marrow aspirate. Patients aged 1-9·99 years at presentation with diagnostic WBC counts <50×10 9 /L were designated NCI (National Cancer Institute) Standard Risk; all other patients were classified as NCI High Risk. Relapses were categorised as very early, early or late based on time to relapse from first diagnosis, i.e. <18 months, 18-30 months or ≥30 months respectively.

Clinical Trials
The Medical Research Council (MRC) clinical trials that ran during this period have previously been reported and include chronologically, UKALLX,16 UKALLXI,17,18 and ALL97.15,19 A major amendment was made to ALL97 in 1999 and the second phase of this trial is analysed separately (ALL97/99).20 Salient relevant differences between the trials are outlined in Table 1 and in the Supplemental. For purposes of analyses, patients have been grouped according to category of relapse (i-CNSr, c-CNSr or n-CNSr), NCI risk group, time to relapse and immunophenotype.7 Analyses were censored to the annual follow-up of 30 th April 2007 for UKALLX and UKALLXI and to 31 st October 2007 for ALL97 and ALL97/99. The small numbers of patients lost to follow-up were censored at the date of last contact. Median follow-up from commencement of treatment for UKALLX is 18·6 (range 0·0-22·3) years, for UKALLXI is 13·0 (range 0·3-16·6) years, for ALL97 is 9·3 (range 3·5-10·8) years and for ALL97/99 is 6·6 (range 2·3-8·0) years.

Cytogenetic and molecular genetic characterisation
Diagnostic cytogenetics was performed at regional laboratories and karyotypes were confirmed centrally.21 Some UKALLXI patients treated after 1994 were screened for ETV6-RUNX1 fusion using reverse transcriptase polymerase chain reaction.18 Fluorescent in situ hybridization (FISH) analyses for ETV6-RUNX1 and other translocations became routine from the start of ALL97. High hyperdiploidy (HH) was characterised by karyotyping (51-65 chromosomes) or by centromere FISH (using the Multiprobe-I system or by detection of classic trisomies).22 Patients were classified as having an MLL rearrangement if an established 11q23/MLL translocation was seen on karyotyping or if a split signal pattern was observed using a breakapart MLL FISH probe.

Statistical Methods
Analyses of relapse excluded patients who died without attaining remission. Differences in clinical features, cytogenetics and proportions of relapses between patients enrolled on the distinct protocols were analysed by the chi-squared test. The long follow-up means that analyses using simple proportions are equivalent to competing risk cumulative incidence calculations. These proportions indicate the number of patients experiencing an event of interest given the level of competing events and, when added for the different relapse categories, provide the total relapse rate. Kaplan-Meier life tables were constructed for survival curves and trials were compared using the log-rank test. Patients were censored at events other than the one of interest. Secondary malignant neoplasms (SMNs) were included in EFS estimations (Table 1) and all post-SMN ALL relapses (overall, 3) were included in the analyses. Overall survival (OS) post relapse was defined as the time between first relapse and death from any cause. Univariate analyses using log-rank tests were performed to examine the significance of a number of variables in relation to risk of relapse and overall survival. Multivariate Cox regression analysis was used to determine factors independently associated with outcome. Both methods of analysis (proportions and Kaplan-Meier) provide different information and both have thus been presented.23 All p-values quoted are twosided. Analyses were carried out using SAS statistical software, version 9·1 (SAS Institute Inc, Cary, NC, USA), and in-house programs.

Change in incidence and pattern of CNS relapses with protocols
During this period, both EFS and OS have improved with significant declines in both CNS and non-CNS relapses ( Figure 1A-C and Table 2). Among those who relapsed in the CNS, the proportion with c-CNSr has fallen (p = 0·0001) while the proportion of i-CNSr has remained relatively unchanged (p = 0·8) ( Table 2).

Change in time to CNS relapse with protocols
Along with the decrease in relapse rates, the duration of CR1 prior to relapse has also changed ( Table 3). As the lowest relapse rates were seen with ALL97/99, a comparison has been made between ALL97/99 and all previous trials. Among relapsing patients, while the proportion of very early relapses is similar for pre-ALL97/99 and ALL97/99, there has been a drop in the proportion of early relapses, and an increase in late relapses (p<0·0001). This change in the timing of relapse with trials is also seen in c-CNSr (p=0·01) and n-CNSr (p=0·0004), but not in i-CNSr (p=0·5). Table 4 shows the influence of risk factors on recurrence. Age, WBC count and NCI risk group were significant risk factors across all relapse categories. Unlike n-CNSr, gender was not a risk factor for CNS relapse. Immunophenotype influenced i-CNSr and n-CNSr but not c-CNSr. When analysed by trial, there were no differences in the effect of risk factors, with two exceptions (data not shown). First, the effect of gender on n-CNSr differed significantly by trial protocol (p(heterogeneity)=0·0001), with the greatest effect seen in the earlier trials. Second, while there was a suggestion that the effect of the T-cell immunophenotype on i-CNSr differed with trial (p(heterogeneity)=0·02), this was no longer observed when the lessthan-robust data from UKALLX was excluded from the analyses (p=0·6).

Factors influencing risk of CNS relapse
We have sufficient data to analyse the pattern of relapses in the four main cytogenetic subtypes (Table 5). There was a significant decrease in relapse rates in ETV6-RUNX1 patients over successive protocols (p<0·0001). The change in pattern was similar to that observed for the whole group, i.e. a proportionate decrease in c-CNSr but not in i-CNSr with time. Patients with HH ALL also showed a significant decline in relapse rates (p<0·0001), primarily between UKALLXI and ALL97. Unlike in ETV6-RUNX1 patients, there was no apparent change in relapse pattern, with the proportion of i-CNSr and c-CNSr remaining essentially unchanged over the trials (Table 5). Although numbers were small, there were also suggestions of a decrease in relapse rates over time in those with MLL (p(trend)=0·009) and t(9;22) (p(trend)=0·05) rearrangements. A decline in i-CNSr was seen in t(9:22) disease, with none observed in the later trials. However increasingly with trials, patients with adverse cytogenetic subtypes received an allograft in CR1 and thus in ALL97 and ALL97/99 most patients with t(9;22) and MLL rearrangements would have been transplanted.24

Outcome following CNS relapse
Similar to the COG experience,8 the overall outcome was significantly better in patients with i-CNSr (p=0·04)(Supplemental). Supplementary Figure S1A-C shows differences in post-relapse OS in each relapse category by trial. There were excess relapses in UKALLXI ( Table 2) but many patients were subsequently salvaged. Excluding UKALLXI, there is no significant difference in post-relapse OS for UKALLX, ALL97 and ALL97/99. The 5-year OS post c-CNSr in UKALLX and ALL97/99 are comparable. While OS post n-CNSr appeared to be better, and for i-CNSr worse in ALL97/99, when compared to UKALLX these differences are not statistically significant.
The prognostic significance of a number of variables in relation to outcome post relapse is shown in Table 6. Univariate analyses showed that time to relapse, WBC count, adverse cytogenetics and NCI risk group were predictive of OS for all categories of relapse. As with n-CNSr, age and HH were predictive for OS in i-CNSr while immunophenotype and the ETV6-RUNX1 genotype significantly influenced post-relapse OS in c-CNSr. Multivariate analysis, after exclusion of cytogenetic subtypes due to small numbers and missing data, confirmed the independent prognostic impact of time to relapse on post-relapse OS in all three relapse categories. Additional factors independently and significantly associated with post-relapse OS were WBC count (i-CNSr and n-CNSr) and the blast immunophenotype (c-CNSr and n-CNSr).
Besides time to relapse, multivariate analysis indicated that the trial period significantly influenced post-relapse OS across all relapse categories. The choice of steroid in frontline therapy has been reported to influence outcome. 15 Overall, EFS was significantly higher with frontline dexamethasone treatment (Table 1), although within trials, this effect was observed in ALL97 and not in ALL97/99. However, frontline steroid therapy had no significant influence on OS in relapsed patients who were randomised to receive either prednisolone 40mg/m 2 (n=280) or dexamethasone 6·5mg/m 2 (n=120) in frontline protocols (p=0·4). This was equally true for post-relapse OS in each relapse category [p(heterogeneity)=0·2)] although numbers in these subgroups were small.

Transplantation
The proportion of patients treated with an allogeneic stem cell transplant (SCT) has decreased progressively with each trial (p<0·0001) as has the proportion of transplants carried out post relapse over time, p(trend)=0·0007 (Supplementary Table S1). There was no significant variation in the proportion of patients receiving a transplant post i-CNSr or n-CNSr over the study period. There was a decrease in SCT for those with c-CNSr (p=0·02), although numbers are small for ALL97/99. In patients transplanted post CNS relapse (i-CNSr or c-CNSr), two-year OS, defined from the date of transplant, was 46% (95% CI: 34%-58%) in UKALLX, 64% (56%-72%) in UKALLXI, 55% (40%-70%) in ALL97 and 65% (40%-90%) in ALL97/99. Comparison of outcome between patients treated with chemotherapy only and those treated with SCT has not been attempted due to the inherent bias in therapy selection.

Discussion
Our analyses confirm that current UK therapy for ALL is effective in preventing extramedullary relapses in most children. This does not adequately explain why the decline in CNS relapses is seen predominantly in combined relapses or the proportionately little change in the pattern of i-CNSr with progressive trials.
Though UKALLXI investigated different CNS-directed therapies, the highest incidence of all relapses, and notably c-CNSr, were seen during this era. In UKALLX all children received cranial irradiation and an anthracycline during induction. 16 In ALL97/99, anthracyclines were only given to NCI high risk patients or those with poor early response to therapy15,20 and radiotherapy was reserved for those with CNS disease at diagnosis. Nevertheless there is a significant decrease in relapse rates in all categories in ALL97/99. Thus CNS recurrence can largely be prevented without the use of high dose methotrexate or cranial irradiation. The more frequent use of intrathecal methotrexate is contributory, but as this was introduced in UKALLXI, it is not the sole factor. In ALL97 and ALL97/99, steroid therapy was randomised between prednisolone and dexamethasone. The latter is thought to have better penetration into the CNS. While significant improvements in EFS and in both CNS and non-CNS relapse rates were seen with dexamethasone, there was no difference in OS. 15 Additionally, there was no significant heterogeneity of effect of the randomised steroid on outcome by trial. Thus other chemotherapeutic changes in ALL97/99 are primarily responsible for the improvement in outcome between ALL97 and ALL97/99. In ALL97/99, UKALLX/ALL97 intensification phases were replaced with BFM-type consolidation blocks, therapy was risk stratified and intrathecal therapy extended. The duration of therapy for boys was extended to 3 years, so that from 1998, most boys received 3 instead of 2 years of therapy. The additional randomisation of 6-mercaptopurine with 6thioguanine (6-TG) found the latter to be protective against i-CNSr, but also hepatotoxic. 19 Although synergy with dexamethasone is a possibility, neither 6-TG nor its active metabolites cross the blood brain barrier. 19 Thus the evidence suggests that risk-stratified intensification of systemic therapy along with frequent intrathecal chemotherapy is the most successful approach to prevention of CNS relapse. A similar observation has been reported by COG in the CCG-1961 study. Children receiving early post induction intensification of therapy showed a significant decrease in n-CNSr and c-CNSr but not i-CNSr.25 Thus both intensification and more frequent intrathecal therapy appear to have played a role in the decline in CNS relapses. With multi-agent chemotherapy, it is difficult to identify the key responsible agent(s). The differential relapse trends in the cytogenetic subtypes offer room for speculation. In patients with ETV6-RUNX1, the incidence of relapses has progressively decreased with each successive protocol from UKALLXI onwards. Further decline in ETV6-RUNX1 associated relapses in ALL97/99 occurs primarily in the c-CNSr group. We have already commented on the fact that the improvement in outcome with ALL97/99 cannot be attributed to dexamethasone alone. ETV6-RUNX1 leukaemias are thought to benefit from intensive asparaginase therapy.26,27 UKALLXI and ALL97 used suboptimal doses of Erwinase. The improved outcome of ETV6-RUNX1 patients in ALL97/99 is probably related to the more effective use of E. coli asparaginase (Elspar®, Merck, USA) in this trial. The additional intensification of asparaginase therapy in ALL2003 is expected to further reduce ETV6-RUNX1 relapses. In HH ALL, a predilection for extramedullary relapses in patients treated on contemporary chemotherapy regimens has been reported from a single centre.28 Though relapses in HH patients halved in ALL97, ALL97/99 provided no apparent further benefit and overall, the proportion of i-CNSr and c-CNSr has remained the same over trials. HH ALLs are more likely to be responsive to intensive methotrexate regimens.28 In UKALLXI, high dose intravenous methotrexate (HDMTX) was found to be protective against i-CNSr. 13 The relapse rate for HH is also

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts lower in this protocol than that for ETV6-RUNX1. It is thus tempting to postulate that outcome in patients with HH may be further improved by the targeted reintroduction of HDMTX in future trials. Thus, there are still opportunities to biologically adapt current therapy to improve outcomes.
Though the incidence of relapse has decreased with time, post-relapse outcomes have not improved. This suggests that by optimising treatment, we are now preventing relapses in those who were earlier cured with salvage therapy. Given our incomplete understanding of why CNS relapses occur and the paucity of new agents, the best therapeutic strategy remains unclear. The results of transplantation in children with i-CNSr relapses have been variable. 7,8,11,30-32 In retrospect, a number of children who were transplanted for disease recurrence in earlier trials would have been cured by current chemotherapy. The standard approach for those who are not transplanted is chemoradiotherapy. However, there is little consensus on the dose, type and timing of CNS irradiation.2 Radiotherapy no longer has a role in preventing CNS relapses in frontline therapy. Is it really of benefit as a therapeutic adjunct to systemic chemotherapy in relapsed disease? This is a difficult question to answer as the small numbers and heterogeneity of disease preclude a randomised approach to this problem. At the moment the most effective strategy remains prevention of disease recurrence. Our analyses suggest that both the biological heterogeneity of the disease and combined systemic and intrathecal chemotherapy influence the incidence and pattern of CNS relapse. Thus further optimisation with currently available agents is possible and may further decrease CNS recurrence.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    Table 1 Details of MRC childhood ALL trials analysed in this study. Both five-year EFS and OS have improved with trials. The proportion of boys and girls, immunophenotype, CNS disease at diagnosis and NCI risk groups are comparable between trials. The age limit of eligibility increased from ALL97 onwards and as a result, a greater proportion of older children and slightly fewer younger children were recruited to ALL97 and ALL97/99 when compared with previous trials      Table 5 Changing trends in relapses in four cytogenetic subtypes over successive trials. The rise in incidence of translocation-associated leukaemias in the later trials reflects the use of FISH screening.   Table 6 Log-rank analyses of factors influencing outcome in each category of relapse (incomplete data for cytogenetic subtypes). O/E = Observed/Expected ratio