General Discussion
Summary
Recommendation
Samenvatting
Intisari
9
9
General Discussion
Summary
Recommendation
Samenvatting
Intisari
Leukemia in children is found mostly among the poor,
or they may become poor or very poor because of
suffering from leukemia, or any other malignancy. To
solve the problems in the management of children with
cancer, the government should play a primary role, with
the cooperation with doctors, nurses, psychologists
and social workers as well as the parents.
General Discussion
The main aim of this thesis was to improve diagnostics in childhood acute leukemia in Indonesia. Hence the research focused on cellular and molecular characteristics of childhood leukemia in Yogyakarta, Indonesia.
The first important step was to make an overview of the epidemiology of Indonesian children with acute leukemia from a hospital-based data set.
The second part of our study concerned the implementation of immunophenotyping by flowcytometry, which is the core of diagnostic improvement on childhood acute leukemia. We started the study with immunocytochemistry examination on cytospinned mononuclear slides. From the simple test then we developed immunophenotyping on single color method and finally, we used a-three color method.
The third part of this thesis concerns the possible improvement for treatment based upon a randomized study of corticosteroids. And finally the relevance of polymorphisms of certain gen of enzymes relevant for methotrexate (MTX) metabolism was studied.
Part 1. Epidemiology of childhood leukemia in Yogyakarta, Indonesia.
Few children with leukemia survived in Indonesia before 1990. The situation is comparable to that in Western countries before 1970. In the academic children’s clinic of the Dr Sardjito Hospital in Yogyakarta (DSH) a treatment protocol was used originating from the first Dutch protocols of the early 1970ies. Beginning with a twinning program between DSH and VUmc in Amsterdam, a new protocol was drafted, named COM-ALL-92. This was based upon the successful and simple Dutch ALL-6 protocol with elements from the German based BFM protocols [1, 2]. The results were still disappointing, mainly due to abandonment of treatment, and through complications, which were often fatal. But registration and follow-up were not yet fully developed; the real situation was virtually unknown. This is the case in many low-income countries. So we asked the questions as stated above.
We started to discuss and answer these questions with the occurrence of a global economic crisis in 1998. The crisis had intense impact on the treatment costs especially of childhood leukemia patients. As a result of devaluation of the Indonesian Rupiah, the costs of imported medicines increased about 5-fold almost overnight. Meanwhile there were no data on childhood cancer and childhood leukemia
available both on national or local/regional levels due to lack of infrastructure. There was no national protocol for childhood leukemia. Each center/teaching hospital in Indonesia used their ‘own protocol’ and in Yogyakarta we used the Dutch adapted childhood leukemia protocol since 1992. The Pediatric Leukemia Seminar and Workshop on improving leukemia diagnostics and patient care was then held in Yogyakarta in 1998. In this seminar the basis was laid for a cost effective protocol and data management was set up. A new treatment protocol was introduced as a result of this national Indonesian meeting and it was called Wijaya Kusuma 2000-ALL protocol, after the flower that is reputed to cure all illness [3].
The DSH is a tertiary care hospital, with a coverage area including the Yogyakarta Special Province (YSP) and the southern part of Central Java. The population within this area is estimated to be 5 million. The YSP has a known population of 3.3 million (population census of 2005). All leukemia cases are referred to the DSH as a tertiary hospital. Analysis of all children (0-14 years) with leukemia admitted between 1998 and 2009 showed that AML was almost twice as frequent as in most Western countries. So we already speculated upon environmental or genetic factors to explain this difference. These data were based on the official catchment area as determined by the DSH. However, because the catchment area of the Pediatric Cancer Unit (PCU) might not be the same as that for the hospital as a whole, a separate analysis was made of patients whose address was in the province of Yogyakarta. Here reliable population data are available, with a number of children below 15 years of age of 711,691 in the year 2005. To avoid the potential influence of population changes in the calculation of rates, we used mid census (2005) data, which are often used as representative data to calculate the rates [4].
In the United States, approximately 1 in 7,000 children between 0 to 14 years of age are diagnosed with cancer each year [5]. That means that 1 out of about 400 children will develop childhood cancer before 18 years of age. In Western countries it is predicted that in the near future about 1 out of every 600 adults will be a survivor of childhood cancer. So childhood cancer is fortunately quite rare. Cancers in children are the cause of more than 10% of all deaths in children below 15 years of age [6, 7]. The mortality rate in children under 5 years of age was varying between developed and developing countries. There is a big gap between developing and developed countries in the healthcare system [8].
Leukemia, a malignant disorder of hematological progenitor cells, is the most frequent type of cancer in children. Acute lymphoblastic leukemia (ALL) is the most common malignancy that affects children, representing nearly one-third of all pediatric cancers and 10% of cancers in adolescents (15 –19 years of age) [9].
The annual incidence of ALL is about 30-50 cases per million people, with a peak incidence in patients aged 2–5 years [10].
In our PCU the number of children with acute leukemia in the catchment area of the DSH increased from 45 per year in 1999, the second year of the study period, to 91 per year at 2009. This resulted in a significant increase in AIR of 34.6 in 1999 to 70.0 in 2009 (ANOVA, P=0.003). The increase probably reflects a higher referral rate, or expanding catchment area or both, and not a real increase in the incidence of childhood leukemia. The AIR in 2009 was far above the world’s incidence rate, which is 30-50 per million people per year [11, 12]. Therefore, a separate analysis was made of the patients referred from the YSP, where the number of inhabitants is reliably known. The Average Annual Incidence Rate (AAIR) in the YSP was 20.8 for ALL and 8.0 for AML. The AAIR of ALL in the YSP is relatively low compared to the Western countries; the lowest was Norway (22.4) and the highest was Australia (37.9) [11]. Our AIR of 25.7 for ALL in 2009 is also relatively low compared to AIR data for the USA (34.6) as well as for West Germany (36.4), but it is quite close to that cited for East Germany (24.0). A low incidence of ALL might be explained by the circumstance that in Indonesia the early mixing of children, and early exposure to infectious agents is very common, which is supposed to protect against the occurrence of ALL [13, 14]. Due to this relatively low incidence of ALL the proportion of AML is relatively high (25% vs. 15% in developed countries). We found in YSP that 27.7% of all referred acute leukemias concerned AML, while in Western Germany the relative incidence of AML is 13.1% and in the USA (Whites) is 15.6%.
The AAIR of AML (8.0) is comparable to the Western countries although in the high range. The lowest AAIR is in Canada Western provinces (4.6) and the highest is Norway (8.0). However, our AAIR data are lower compared to other Asian countries such as Japan (9.7) and China, Shanghai (12.1) [15]. ALL and AML showed a different age pattern, similar to that reported in The Netherlands [16]. The number of childhood leukemia cases in the YSP and surrounding South Central Java increased over the years 1998–2009. Part of the explanation may be that the pediatric care unit (PCU) of the Dr Sardjito Hospital (DSH) was first organized in 1998, and subsequently referrals increased. The overall AAIR of childhood leukemia was still lower compared to Western countries [17].
Part 2. Improving diagnosis for better treatment: the role of Immunophenotyping
The diagnosis of ALL in Indonesia is based on clinical findings and morphological analysis of peripheral blood and bone marrow. Immunophenotyping was not yet
available here. Immunophenotyping has become an important diagnostic tool for classification, prognosis and disease monitoring of acute leukemias [18-21]. Immunophenotyping data have been published with various results for the relative frequency of AML and ALL as well as for B- and T-lineage ALL [22-38]. We started to immunophenotype all patients suspected of leukemia in 2006. In the cohort of 498 patients, boys were slightly more frequent. This result was similar to what we have found previously [17] and similar with data for other developed countries [39]. The proportion of AML was relatively high compared to western countries 25% vs. 15%. As stated above, this is presumably not because AML incidence is higher than in most Western countries, but as an effect of a lower incidence of ALL in Indonesia [40]. Our result showed that the relative proportion of B-lineage ALL was 83%, and T-lineage ALL was 17%. For T ALL, this result is slightly higher than in most developed countries. Biphenotypic acute leukemia and mature-B ALL were lower compared to Western countries.
In the first period of the study, 136 samples were immunophenotyped using a single color method and a set of 9 monoclonal antibodies. The discordance between morphology and immunophenotyping of AML with this method was far too high. A new method was therefore developed. The new method applied a three-color Facs scan and a set of 15 antibodies. We expanded the old panel by adding antibodies directed against cytoplasmic antigens. Cytoplasmic CD3 was introduced, this is considered to be a highly specific marker for T-lineage ALL, cytoplasmic CD79a for B-lineage ALL and cytoplasmic MPO and CD117 for cells of myeloid lineage (AML). The new panel was applied to 318 samples, and resulted in less samples (4%) being labeled as ‘non conclusive’, compared to 18% in the old panel. There were only few samples (1%) that showed ‘low marker expression’ as compared to the old panel (14.7%). Concordance between morphology and immunophenotyping methods in the old method was 0.43 (moderate agreement), while in new method was 0.82 (almost perfect agreement) [41]. That is a compliment for our laboratory staff evaluating the morphology of blood and bone marrow. Of 318 patients tested, a relatively high percentage of acute leukemia was classified as AML (23%). Of the ALL samples 83% were B-lineage ALL and 17% were T-lineage ALL. From cases of morphological ALL, we found that 3.8 % was labeled AML by immunophenotyping. From cases of morphological AML, 15% were shown to be ALL. Nine out of 239 morphological ALL were labeled AML, and 12/79 morphological AML were in fact ALL. This means that a sizeable number of patients (21/318 = 6.6%) were shifted from ALL to AML protocols or vice versa.
Finally, we conclude that the incidence of ALL is relatively low in Indonesia [40], while the incidence of AML is similar to incidences reported from western countries
[11]. The relative proportion of B-lineage ALL was 83%, and T-lineage ALL was 17%, the latter is slightly higher than in most developed countries. The three-color method was much better compared to one three-color method for classification of leukemias. A set of 15 antibodies in three-color method proved to be ideal and can be implemented in Indonesia and other low-income countries [42]. By implementing immunophenotyping in the diagnostic procedure, the diagnosis of childhood acute leukemia has been improved in Indonesia, which was formerly based on the morphology and cytochemistry only. Immunophenotyping also facilitates the confirmation of a diagnosis ALL versus AML.
Immunophenotyping of abnormal hematological cells is important for the diagnosis, classification, and prognosis in patients with hematological malignancies [43-47]. It has been applied to diagnose and stratify patients with suspected leukemia in Yogyakarta. We also examined myeloid expression (CD13, CD33, CD117 and cMPO), the expression of CD10, CD34 and the combination of CD10 and CD34, both in B- and T-lineage ALL. Myeloid markers, as well as CD10 and CD34 (alone or in combination) are surface markers that have been reported to have prognostic relevance in childhood acute lymphoblastic leukemia (ALL) [48-57]. Two hundred and eleven patients met the inclusion criteria and were analyzed for the correlation of both markers with the clinical and biological features of those patients. We also analyzed whether the markers had any significance as prognostic factor.
Myeloid antigen expression
Improvement in immunophenotyping allows detecting mixed antigen expression in one cell, including myeloid antigen expression in ALL. The clinical significance of myeloid antigen co expression has remained controversial [48, 51, 53-55, 57-60]. We tried to find clinical significance of myeloid antigen co expression in 239 Indonesian children with ALL. Myeloid expression in ALL was defined if finding one or more of these markers: CD13, CD33, CD117 and MPO. In the Indonesian ALL population 25% of cases showed myeloid antigen co expression. This finding was slightly higher compared to Malaysia (23%) [53], but lower compared to European population (36%) [54]. A study conducted by Wiersma et al. 1991 and Kurec et al. 1991 found that myeloid antigen co expression was the most important predictor for a poor EFS outcome [51, 52]. These findings are consistent with a finding that leukemic cells from myeloid-positive ALL patients were more resistant to glucocorticoid-induced killing than cells from Myeloid-negative ALL patients [61]. In our population, the majority of patients with morphology ALL-L1 had no myeloid antigen expression (p=0.02). However univariate analysis for EFS, ages at diagnosis and risk group were found to be statistically significant predictors for event-free survival. Multivariate
analysis showed that age category was the only factor significant, HR 2.00 (95%CI: 1.23 – 3.23, p=0.006). Overall, in this patient cohort, patients with myeloid-negative ALL cells had a better prognosis. Kaplan Meier analysis of EFS showed no difference for myeloid expression. However, for LFS Kaplan Meier analysis showed that myeloid-negative patients had a better prognosis. Kaplan Meier analysis showed that LFS at 4 years was 80% ± 5% in the myeloid-negative group compared to 67% ± 8% in the myeloid-positive group.
The proportion of myeloid antigen expression in the B-lineage group was 27%, while 10 out of 56 patients (18%) were in T-ALL group (p=0.15). Moreover, the groups did not differ in relevant clinical and biological features. Since both groups have a distinct clinical prognosis we also stratified the data for the precursor B or T lineage.
CD10 and CD34 expression
The clinical significance of CD10 and CD34 expression has been debated. Studies on the expression of CD10 and CD34, alone or in combination, as a prognostic factor, found discrepancies. The expression of CD10 and CD34 also varies in different studies [49, 50, 56, 62-68]. In our total study cohort, 211 patients were analyzed for clinical and biological features. 59% were boys, the largest group was 1-9 years of age (78%), and most had FAB-L1 classification (83%). The most common subtype was B-lineage ALL (84%). High WBC (>50.000/mm3) was found in 21% of patients. Risk classification used NCI criteria plus blast count at day 8, and 55% were classified as high risk (HR). This is quite a high percentage and more than is usually seen in western countries. Apparently patients come to diagnosis rather later in our PCU than in western countries. The prognosis in our cases is heavily influenced by other factors than the leukemia itself. Death was the event was most frequently observed (n=50, 23.7%). Fourteen patients (6.6%) relapsed and 4 patients (1.9%) failed in induction. In the whole patients cohort, EFS at 4 years was 58% ± 4% while OS at 4 years was 84% ± 4%.
Studies elsewhere on the correlation of CD10 expression with outcome also showed various results. Two different studies found that in their patient cohort CD10+ expression in B- and T-lineage ALL had no independent prognostic significance [50, 65]. In our study CD10 antigen was found in 148 patients (70%) of the whole patient cohort. The relative frequency of CD10 expression was higher in SR patients (83%) than in HR patients (60%, p<0.001). In univariate analysis for OS and EFS, CD10 expression had a significant prognostic value. But in multivariate analysis of OS and EFS, this expression was no longer significant. Kaplan-Meier analysis of OS showed that expression of CD10 was associated a better prognosis. Patients with CD10+ had an OS at 4 years of 62% ± 6%, vs. CD10- of 40% ± 7% (p < 0.001).
CD34 expression was found in 112 patients (53%). This expression was also significantly associated with risk group stratification (p<0.001); the relative frequency of CD34 expression was higher in SR patients (67%) than in HR patients (42%). Pui et al., [49] found that the expression of CD34 was correlated with several favorable features at diagnosis such as age between 1-10 years, white race, absence CNS leukemia, low serum LDH, CD10 expression and hyperdiploidy. In concordance with our study, CD34 was associated with standard risk stratification but it had no significant impact on prognosis.
Combined CD10 and CD34 expression and risk stratification has a significant (p<0.001) association. The relative frequency of CD10 and CD34 co-expression was 56% in the SR group compared to 27% in the HR group. Co-expression of negative CD10 and negative CD34 occurred in 6.4% of patients in SR group and 24.8% of patients in the HR group. In Morocco, Dakka et al, found that the five years survival rate for the CD10−CD34− group was only 22% [56]. We also found that lack of both CD10 and CD34 expression was related to a worse prognosis; OS was 61% in the CD10+ and/or CD34+ group vs. 31% in the double negative group, p<0.001. This double negativity is also an independent prognostic factor in our population besides risk classification.
B-lineage ALL
Myeloid antigen expression
Univariate analyses for EFS showed that sex, age at diagnosis and risk classification were significant factors. On multivariate analysis (correcting for protocol used) nothing remained significant. For LFS, there was no factor significant in univariate analysis. In the Kaplan-Meier analysis for EFS, precursor B-ALL patients with and without myeloid antigen expression had a similar prognosis. EFS at 4 years of myeloid-positive B-lineage ALL was 53% ± 8% and of myeloid-negative it was 55% ± 6%, p=0.621). Kaplan Meier analysis for LFS also showed no association of survival with myeloid antigen expression (LFS at 4 years were 73% ±6% for myeloid-negative versus 70% ± 9% for myeloid positive, p = 0.420). No difference in prognosis between myeloid-positive and myeloid-negative cases was found in B-lineage ALL.
CD10 and CD34 expression
Studies on the significance of CD10 expression on outcome also showed various results in the literature. Some studies found that CD10+ expression in B- and T-lineage ALL had no independent prognostic significance [50, 65]. Our study showed that in the B-lineage ALL group, the majority of samples were CD10+, 78%.
Expression of CD10+ did differ slightly, but not significantly between SR patients and HR patients, (83% vs. 71%; p=0.07). In literature, Dakka found that the 5-year survival rate of B-lineage ALL with CD10+ is higher than in CD10- patients. Another study of CD10 expression in Mexico showed no significant prognostic value, EFS of CD10+ cases was 78% and CD10- was 71% (p=0.6) [56, 66]. This result is similar to our findings. EFS at 4 years was 61% ± 6% and CD10- was 49% ± 9%, p=0.084. Expression of CD34 was frequently found in B-lineage ALL and was a favorable prognostic factor [62]. In B-lineage ALL, expression of CD34 was associated with favorable presenting features: age between 1 and 10 years, white race, absence of CNS leukemia, low serum lactate dehydrogenase level, leukemic cell CD10 expression, hyperdiploidy [49]. In 1997 a study of CD34 expression conducted by Cascavilla et al., found that expression of CD34 antigen was frequently expressed in B-lineage ALL and was a positive prognostic factor in childhood ALL. In our study, expression of CD34 was 59% in B-lineage ALL. CD34 expression was higher in SR patients than in HR patients (67% vs. 50%; p=0.02). CD34+ alone was not found to be a predictive factor (log-rank test p = 0.223). Expression of CD34 thus has no prognostic impact. The survival rates of CD34+ and CD34- patients were similar. The already mentioned study in Morocco found in B-lineage ALL, that the co-expression of CD10 and CD34 was associated with 1-10 years of age at diagnosis, male gender and low WBC [56]. In our study, expression of CD10 and/or CD34 was associated with risk classification, p=0.037. CD10+ and/or CD34+ was found in 160/178 (90%). Expression of CD10 and/or CD34 was associated with risk classification, p=0.037. The minority (36%) of CD10+CD34+ patients was high risk, whereas the majority (67%) of CD10-CD34- patients were classified as high risk. Multivariate analysis for EFS showed only risk classification and CD10-CD34- to be a statistically significant prognostic factor. After correction for the protocol used hazard ratios were 1.76 (95% CI: 1.13 – 2.76, p = 0.013) for CD10-CD34- and 1.65 (95% CI: 1.03 – 2.65, p = 0.037) for risk classification. Multivariate analysis for OS showed that only CD10-CD34- and age category remained statistically significant. Hazard ratios were 2.57 (95% CI: 1.54 – 4.31, p < 0.001) for CD10-CD34- and 1.74 (95% CI: 1.02 – 2.96, p = 0.041) for age category. OS for CD10+ and/or CD34+ was 61% ± 5% vs. 31% ± 9% for CD10-CD34- (log-rank test p < 0.001). So lack of expression of CD10 and CD34 is an important negative prognostic factor in B-lineage ALL in our study.
T-lineage ALL
Myeloid antigen expression
significant adverse prognostic factor (p=0.04). It has to be emphasized that only a small number of patients were studied in the T-ALL group. However, LFS analyses also showed a worse prognosis for myeloid-positive patients; LFS at 4 years was 52%, while for myeloid-negative patients LFS at 3 years was 96% (p=0.001). In T-lineage ALL however, cases with myeloid antigen expression had a worse prognosis. In myeloid-negative T-ALL patients (n=46), there was only 1 relapse in the first year of treatment. Hence, therapy for this group might be less intense to prevent toxic deaths. Our results thus showed that a lack of myeloid antigen expression had a better prognosis in T-lineage ALL.
CD10 and CD34 expression
In published data on T-lineage ALL, CD10 expression was associated with lower WBC [69]. In addition, CD34- was correlated with age over 10 years, CNS disease at diagnosis, and higher WBC. In a different study, CD34 expression was associated with poor disease free survival and overall survival [70]. In our findings, a univariate analysis of T-lineage ALL showed that age at diagnosis, CD10 expression and combination of CD10 and CD34 were significantly associated with OS. In multivariate analysis, only CD10-CD34- found to be related to OS, with a hazard ratio after corrected to the protocol was 6.1 (95% CI: 1.7 – 21.3, p-value 0.005). Kaplan-Meier analysis showed that patients with CD10 expression in this T-lineage ALL group had a better survival. Survival for CD10 expression was 88% ± 12% compared to 26% ± 10% of CD10- (p=0.005). Expression of CD34 did not have a significant impact on outcome. The combination of CD10- and CD34- in T-lineage ALL was associated with a significantly worse prognosis of 13% vs. 77% in the cases that showed expression of either CD10+ or CD34+ or the combination of both markers (p=0.002). T-ALL has generally a worse prognosis than precursor B-ALL, and should be classified in the high-risk group and be treated with a high-risk protocol.
Prognosis and prognostic factors are heavily dependent upon the therapy protocol used. Differences in protocol can modify the effect of prognostic factors. Indeed, theoretically all prognostic factors loose their effect if the cure rate reaches 100%. In St Jude Children’s Research Hospital, cure rates over 90% are achieved. It is therefore understandable that the prognostic value of any marker, including myeloid antigen expression, CD10 and CD34 expression will be most obvious in situation with lower OS, EFS and LFS. Which is the case in Indonesia.
In T-lineage patients, even though they are HR in particular the CD10+ patients and the combined CD10 and CD34 expression have a relatively good prognosis. Hence, a special care should be taken for those who are in T-lineage group with no expression of CD10 and/or CD34.
Part 3. Treatment outcome: the role of Prednisone-Dexamethasone randomization, and molecular genetic study.
Prednisone-Dexamethasone randomization: treatment outcome.
Clinical trials and in vitro studies have shown that dexamethasone is superior over the standard corticosteroid prednisone in killing lymphoblasts and in lowering the incidence of CNS involvement and improving the cure rate of children with ALL [71-74]. Since dexamethasone is a very inexpensive drug, Indonesian protocols for childhood ALL were dexamethasone-based instead of prednisone-based since 1992 until present (Indonesia 2006 ALL protocol), but the toxicity was quite disturbing [75]. We noticed that in large randomized Western studies EFS was higher, but also toxic deaths were more frequent in the dexamethasone arm. Although the difference did not reach statistical significance, the clinical importance was potentially high, because the toxic death rate in a UK study was almost twice as high in the dexamethasone arm (4.6%) as in the prednisone arm (2.5%) [73].
Therefore the Indonesian ALL 2006 protocol randomized the use of dexamethasone and prednisone. The doses used were 4mg/m2 for dexamethasone and 40mg/m2 for prednisone. This was used in a 4-drug induction for both HR and SR patients, so including an anthracycline. Surprisingly, our randomized study comparing dexamethasone and prednisone showed up till now no major differences between both arms of the study. Abandonment rates of patients in the dexamethasone group and in the prednisone group were similar (24.5% vs. 25.5%, P = 0.91), as were death (17.7% vs. 14.9, p = 0.54), leukemic events (13.7% vs. 11.7%, P = 0.59) and continuous CR achievement (44.1% vs. 47.9%, P = 0.60). A separate analysis of SR patients revealed a trend for higher induction death in the dexamethasone compared to the prednisone arm (16.2% vs. 6.1%, P = 0.067) with equal leukemic events in both arms. In HR patients the dexamethasone versus prednisone arms did not show statistical significant difference in induction death rate (8.8% vs. 14.3%, P = 0.69) and leukemic events (17.6 vs. 7.2%, P = 0.43). The possible explanation could be a notable tendency towards higher septicemia and induction death in childhood ALL treated with dexamethasone than prednisone or prednisolone [76-79]. The major causes of treatment failures in our setting were abandonment of treatment and death during induction or in remission. These events were occurring in appreciably higher rates than in higher income countries where abandonment is almost unknown and death rates are reported to be between 1-5% [77, 80-84]. The high abandonment rate during induction in our study may have resulted from the higher incidence of behavioral changes during corticosteroid treatment. Sitaresmi et al. found that side effects were an important reason for abandonment. Another
explanation is related to social and economical aspects [75, 85]. High toxic death and abandonment rates during induction treatment and in remission represent problems typically seen in low or medium-income countries [75, 86-90]. Both adverse events are a major contributor to treatment failures responsible for low complete remission rates and lower EFS as compared with international studies [91-93]. The clinical outcomes overall were not significantly different in the dexamethasone and prednisone arms in our study. The 3 years EFS was lower in the dexamethasone group, although not statistically significant. The EFS in the dexamethasone arm was 31.5% ±6.6%, versus 41.5% ±5.9% (p = 0.51) in the prednisone arm.
The Indonesia-ALL-2006 protocol introduced 4 doses of anthracycline during induction for both the standard risk and high-risk protocols, while the previous protocol WK-ALL-2000 only used 2 doses in the HR group only. In retrospect, the addition of an anthracycline may have caused more pronounced life-threatening conditions in a setting of a low-income country like Indonesia with a high incidence of infections and limited access to supportive care. The use of an anthracycline during induction will augment the toxicity of dexamethasone as shown in a study by Belgaumi et al. [94] In the UKALL VIII Study those who received daunorubicin in induction experienced twice as many induction failures (non-remitters + deaths) compared with those who did not receive it (6% vs. 3%). Early remission death rates were also higher in those who received daunorubicin (8% vs. 4% of remitters) [95]. In the Netherlands, Veerman et al., found that the results of DCOG ALL-9 (dexamethasone-based) protocol are better than those of the previous Berlin– Frankfurt–Münster-based protocols ALL-7 and ALL-8. The EFS of the non high-risk group who were treated without anthracyclines, was 84% comparable to that seen in ALL-6 (83%). This protocol used high cumulative doses of dexamethasone and vincristine, but without the use of anthracyclines, etoposide, cyclophosphamide, or cranial irradiation. Therefore the risk of certain side effects was minimized [96]. A number of studies in Italy, USA and in the UK showed that SR patients treated with a 3-drug induction without anthracycline achieved complete remission rates around 95% with 5-years overall EFS 56% - 83% [95, 97, 98].
In conclusion, in 4-drug induction treatment, dexamethasone is dangerous for our patients. It leads to more deaths and abandonment during induction. Nevertheless, the treatment outcomes of childhood ALL patients in Indonesia has advanced during the last decade with increasing 3 years EFS almost doubled, from around 20% in WK- ALL-2000 protocol to 37% in this Indonesia-ALL-2006 protocol. A recommendation to reduce the 4-drug induction treatment for SR patients to become a 3-drug induction treatment is made. Administering prednisone in induction and reserve dexamethasone for re-induction and pulses in maintenance is probably the optimal
approach in SR patients. In a country like Indonesia where poor supportive care and few children are currently being cured, developing protocols should be performed with extreme caution regarding potential toxic and drug-related life-threatening complications [99].
MTHFR C677T and TSER polymorphisms in Indonesian children with and without leukemia
Differences in ethnicity may affect the results of treatment, since genetic differences can affect drug sensitivity. Several studies on ethnic variations in genetic polymorphisms have been published [93, 100-102]. The prevalence of the TSER and
MTHFR C677T polymorphisms may be different among populations [103, 104]. These polymorphisms may affect the efficacy of Methotrexate (MTX). The MTHFR C677T
might play a role in toxicity due to MTX treatment in ALL, and the variant allele associated with increased toxicity with MTX [105-109]. Therefore, identification of these polymorphisms may be an important pharmacogenetic determinant to predict both response to and toxicity of chemotherapy in childhood leukemia. We studied especially genetic polymorphisms relevant for MTX metabolism. We found variations in polymorphisms in the Thymidylate synthase enhancer region (TSER) and in Methylenetetrahydrofolate reductase (MTHFR) between European and Indonesian children with ALL as well as healthy controls from both countries.
TSER can have 2 or 3 repeats, resulting in 3 genotypes: 2R/2R, 2R/3R and 3R/3R. In Indonesian children a much higher proportion of the 3R allele, and especially of the TSER 3R/3R group was present. The TSER 3R/3R repeat was three-fold more frequent in the Indonesian children, while the 2R/2R repeat was only 1% compared to 21% in the European children. TSER is related to ALL prognosis [93, 110]. TSER 3R/3R results in a higher TS activity, faster metabolism of MTX, and consequently less sensitivity to MTX [111]. TSER 3R was also reported to be associated with poorer outcome compared to the presence of at least one 2R allele [110]. There are more genes involved in MTX sensitivity, however TSER may play a role in the decision of dosing [108, 110].
The role of MTHFR polymorphisms in development of leukemia and MTX toxicities were studied. The MTHFR gene encodes for an enzyme called methylenetetrahydrofolate reductase. This enzyme plays a role in processing amino acids. Methylenetetrahydrofolate reductase is important for a chemical reaction involving forms of the folic acid.
Development of ALL and CML is more dependent on folate status, and more susceptible to DNA instability than that of AML [112]. In a meta-analysis, MTHFR
C677T polymorphism was associated with increased risk of MTX-induced toxicities [113]. Caucasian people are homozygous for a C
→
T polymorphism located at nucleotide 677 (referred to as the TT genotype). The substitution of alanine to valine increases thermolability and reduces the activity of MTHFR. MTHFR C677Tpolymorphism may have an effect on the response to MTX, and recent studies have suggested that the TT genotype may be associated with an increased toxicity of MTX in leukemia patients [107, 114].
We found that for MTHFR, the frequency of the TT genotype was very low in Indonesia both for healthy and leukemia children, compared to the Netherlands. The frequencies of MTHFR TT and CT genotypes are twofold higher in European children compared to Indonesian children. Because of the lower frequency of the
MTHFR 677T allele, Indonesian children may have less toxicity with respect to Caucasian children. This may also mean that they are less responsive to MTX in a therapeutic sense.
A frequent homozygosity of TSER 3R/3R polymorphism was found in Indonesia. This may mean a risk of under-treatment for Indonesian children with leukemia. In addition, because of the lower frequency of the MTHFR 677T allele, they may experience less toxicity in comparison to Caucasian children. Therefore future research should focus on characterization of both side effects and clinical outcome in these patients in relation to genetic polymorphisms.
In Indonesia, the MTX dose that is adopted from Western protocol might be relatively low for many children with ALL. It is therefore important that the guidelines for dosage of MTX during maintenance are strictly followed: those patients who have consistently high WBC, and are taking their medicines, should have increased dosages. The protocol requires those patients who have WBC above 4,000/mm3 to take higher dosages of MTX and the other maintenance drug, 6-mercaptopurine. This is a very economical strategy, since white blood cells counts have to be done regularly anyway also to prevent overdosing with the associated risk of severe complications (infection, bleeding, toxic death). Since the use of the required higher dose of MTX in relatively MTX resistant patients in Indonesia might be limited by lack of financial resources, alternative, less expensive treatments might be considered. Individualized treatment for instance, the dose of MTX is based on pharmacogenetic differences may increase effectivity of the protocol. It would be possible to determine the genotype before maintenance treatment is started, and select dosage of MTX based upon the results of MTHFR and TSER determinations. This analysis would make it possible to improve cure rates and limit toxicity for childhood leukemia, besides the usual personalized clinical aspect (stratification into SR and HR) and initial response to therapy [115].
Summary
Problems on the management of childhood cancers particularly in childhood leukemia in Yogyakarta, Indonesia were more complicated since 1998 when global economic crisis occurred. But the existence of a twinning program between Gadjah Mada University and VU University Medical Center was highly beneficial. In 1998, a national meeting on childhood leukemia was held Yogyakarta. Koningin Wilhelmina Fonds (KWF) sponsored this meeting. All pediatric oncology centers including pediatric oncologists, technicians and nurses participated in that meeting. Separated workshops and seminar for oncologists, technicians, nurses as well as public and general practitioners were held. The participants had improved their knowledge during the workshop. At the end of the workshop, a cost- effective protocol for childhood ALL was set up by three international experts, Prof. AJP. Veerman from the Netherlands, Prof. Gunter Henze from Germany and Prof. James Nachman from the US. The protocol was called Wijaya Kusuma 2000-ALL protocol, after the flower that is reputed to cure all illness. A pediatric cancer registry was also set up in Yogyakarta. One dedicated person named Purwanto was hired for a data manager. The study began on March 2006 supported by KWF.
The DR. Sardjito Hospital (DSH) is a tertiary care hospital, with a coverage area including the Yogyakarta Special Province (YSP) and the southern part of Central Java. The population within this area is estimated to be 5 million. All leukemia cases are referred to the DSH as a tertiary hospital. Analysis of all children (0-14 years) with leukemia admitted between 1998 and 2009 showed that the number of children with acute leukemia in the catchment area of the DSH increased from 45 per year in 1999, the second year of the study period, to 91 per year at 2009. This resulted in a significant increase in AIR of 34.6 in 1999 to 70.0 in 2009. The Annual Incidence Rate (AIR) in 2009 was far above the world’s incidence rate, which is 30-50 per million people per year. Therefore, a separate analysis was made of the patients referred from the YSP, where the number of inhabitants is reliably known. The Average Annual Incidence Rate (AAIR) in the YSP was 20.8 for ALL and 8.0 for AML (Chapter 2). The incidence of ALL was low compared to developed countries. Due to this relatively low incidence of ALL resulting the proportion of AML is relatively high (25%) compared 15% in developed countries. The number of childhood leukemia in the YSP and surrounding South Central Java during 1998–2009 was increased. The increase possibly reflects a higher referral rate, or expanding catchment area. Before the study began, diagnosis of childhood leukemia was made by morphology and cytochemistry only. This method is not yet standard diagnosis based on WHO criteria. In 2006, immunophenotyping was set-up in Yogyakarta (Chapter 3). From
March 2006 until November 2007, 136 samples were immunophenotyped. In this first period we used a single color method and a set of 9 monoclonal antibodies. The result showed discordance between morphology and immunophenotyping of AML was too high, resulting kappa score 0.43 (moderate agreement). A new method then developed. Starting December 2007 the new method, introduced a three-color Facs scan and a set of 15 antibodies. We expanded the old panel by adding antibodies directed against cytoplasmic antigens. Cytoplasmic CD3 was introduced, a highly specific marker for T-lineage ALL, Cytoplasmic CD79a for B-lineage ALL and cytoplasmic MPO and CD117 for cells of myeloid lineage (AML). The new panel was applied to 318 samples, and resulted in few samples (1%) that showed ‘low marker expression’ as compared to the old panel (14.7%). Concordance between morphology and immunophenotyping was increased. Kappa score was 0.82 (almost perfect agreement). The quality control of this test was done in Cancer Center Amsterdam, validating our results. In our patient cohort a relatively high percentage of acute leukemia was classified as AML (23%). Of the ALL samples 83% were B-lineage ALL and 17% T- lineage ALL. Nine out of 239 morphological ALL were labeled AML, and 12/79 morphological AML were in fact ALL. This means a number of patients (6.6%) were shifted from ALL to AML protocols or vice versa. In conclusion the incidence of ALL is relatively low in Indonesia (Supriyadi et al., 2011), while the incidence of AML is similar to incidences reported from western countries. Therefore, we found that the relatively high percentage of AML patients (23%). Immunophenotyping in childhood leukemia in Indonesia played a role to improve the diagnosis, which was formerly based on the morphology only. Hence, improvement of prognosis is anticipated due to the right risk classification of children and subsequent selection of the most optimal treatment protocols
Improvement in immunophenotyping allows detecting mixed antigen expression on the ALL cells, including myeloid antigen, CD10 and CD34 expression in ALL. In
Chapter 4 we discuss the role of myeloid expression in childhood ALL & Chapter 5,
discussing of expression CD10, CD34 alone or in combination in 211 patients, and an expanded data on clinical significance of myeloid antigen co expression of 239 Indonesian children with ALL was analyzed. In Indonesian ALL population there was 25% myeloid antigen co expression. We found no association of myeloid expression with clinical and biological features, in overall patients. Kaplan Meier analysis based on leukemia free survival showed that myeloid-negative patients had a higher prognosis. In B-lineage ALL the proportion of myeloid was 27%, while in T-lineage was 18%. No difference in prognosis between positive and Myeloid-negative cases was found in B-lineage ALL. In T-ALL, myeloid antigen expression was found to be a significant adverse prognostic factor (p=0.04).
The expression of CD10 and CD34 also varies in different studies. In our study CD10 antigen was found in 148 patients (70%), CD34 expression was found in 112 patients (53%) of the whole patient cohort. Expression of CD10, CD34 and CD10 CD34 co-expression were associated with standard risk (SR) classification. Patients with CD10+ had a higher survival compared to CD10- (p<0.001). Expression of CD34 had no significant impact on prognosis, and co-expression of CD10 and CD34 had better prognosis. OS of the combination CD10+ and/or CD34+ was 61% vs. 31% in the double negative group, (p<0.001). This double negativity is also become an independent prognostic factor in our population besides age at diagnosis and risk classification.
In the B-lineage ALL group, the majority of our samples were CD10+ (78%.) Clinical significance of CD10 expression was varying. Survival was similar between CD10+ and CD10- patients. Expression of CD34 was 59% in B-lineage ALL. CD34 expression was associated with SR p=0.02. CD34+ alone was not found to be a predictive factor, and has no prognostic impact. Survival rate between CD34+ and CD34- patients were similar. The minority of CD10+CD34+ patients was high risk, whereas the majority of CD10-CD34- patients were classified as high risk. Expression of CD10 and/or CD34 was also associated with SR group. Beside age category, CD10-CD34- was a worse prognostic factor. EFS for CD10-CD34- significantly lower compared those who have CD10+ and/or CD34+.
In T-lineage ALL, CD10 expression was associated with lower WBC. In addition, CD34- was correlated with age over 10 years, and higher WBC. CD10 expression had a better survival. Survival for CD10 expression was 88% vs. 26% in CD10- (p=0.005). The expression of CD34 did not have a significant impact on outcome. In this T-lineage ALL, combination of CD10 and CD34 were significantly associated with OS. In multivariate analysis combination of CD10- and CD34- is the only significant poor prognostic factor, Hazard Ratio: 5.9, 95%CI: 1.6 – 21.3, p=0.007. The combination of CD10- and CD34- in T-lineage ALL had a significantly worse prognosis 13% vs. 77% in the combination of CD10+ and/or CD34+ (p=0.002).
In T-lineage patients, even though they are HR in particular the CD10+ patients and the combined CD10 and CD34 expression have a relatively good prognosis. A special care should be taken for those who are in T-lineage group with no expression of CD10 and/or CD34
The role of Prednisone-Dexamethasone randomization in our ALL patients was described in chapter 6. Clinical trials and in vitro studies have shown that dexamethasone is superior over the standard corticosteroid prednisone in killing
lymphoblasts. Dexamethasone is also benefit in lowering the incidence of CNS involvement and improving the cure rate of children with ALL.
Dexamethasone is an inexpensive drug, and always available in Indonesia. The Indonesia 2006 ALL protocol was a dexamethasone-based instead of prednisone-based since 1992 until present, but the toxicity was quite disturbing. In the Western studies, usage of dexamethasone resulted a higher event free survival, but also toxic deaths were more frequent. Although the difference did not reach statistical significance, the clinical importance was potentially high. Moreover, these findings are in line with the toxic death rate in a UK study, which was almost twice as high in the dexamethasone arm as in the prednisone arm.
In our study, dexamethasone and prednisone randomization showed that in overall patients, abandoned in the dexamethasone group and in the prednisone group were similar, also similar result found in death, leukemic events and continuous complete remission achievement. In SR patients showed trend for higher induction death in dexamethasone arm. In HR patients the randomization did not show statistical significance in death during induction and leukemic events. Treatment failures in our setting were mostly caused by abandonment and death during induction or in remission. The high abandonment rate during induction may be due to the higher incidence of behavioral changes of the child during corticosteroid treatment. On the other hand, abandonment is also related to social and economical aspects. Toxic death in our setting could be explained by the protocol that we used. Studies in Western countries concluded the addition of anthracycline showed more pronounced life-threatening conditions in a setting of a low-income country like Indonesia with a high incidence of infections and limited access to supportive care. Many international experts also gave comments to our ALL protocol, which has 4 doses of anthracycline during induction for both the standard risk and high-risk, that the protocol is too toxic! A reduction the 4-drug induction treatment for SR patients to become a 3-drug induction treatment is urgent and highly recommended. Methotrexate (MTX) is an important drug in the treatment of ALL, beside corticosteroid. The MTX inhibits several enzymes involved in folate homeostasis. Methylenetetrahydrofolate reductase (MTHFR) gene encodes for an enzyme, which plays a role in processing folic acid. This is very important for the synthesis of nucleic acids. Polymorphism of MTHFR C677T is associated with increased risk of MTX-induced toxicities.
Thymidylate synthase (TS) is a key-enzyme in de novo DNA synthesis. TS
metabolism of MTX, and consequently decreased sensitivity to MTX. TSER 3R was also reported to be associated with poorer outcome. TSER polymorphisms may affect the efficacy of MTX in ALL treatment. The prevalence of the TSER and MTHFR C677T polymorphisms may be different among populations. In chapter 7 and
8 we discussed the role of MTHFR C677T and TSER polymorphisms in Indonesian childhood leukemia in association with MTX treatment.
A frequent homozygous of TSER 3R/3R polymorphism was found in Indonesia. This finding might represent an under-treatment for Indonesian children with leukemia. In addition MTHFR, the frequency of TT genotype was very low in Indonesia both for healthy and leukemia children, compared to the Netherlands, resulting less toxicity to MTX in Indonesian children with respect to Caucasian children.
In Indonesian ALL protocol, the MTX dose is adopted from Western protocol. It might be under dosing. Since the use of the required higher dose of MTX in Indonesia might be limited by lack of financial resources and lack of supportive care, an alternative of less expensive treatments might be considered. An individualized treatment based on pharmacogenetic differences is recommended. This may increase effectivity of the protocol.
Recommendations:
1. Diagnosis based on immunophenotyping should be a part of routine procedure diagnosis and become a standard operating procedure in management of children with leukemia in Indonesia. This laboratory test should therefore be covered by government insurance anywhere in Indonesia (ASKES, JAMKESMAS, JAMKESDA, JAMKESOS)
2. A cell bank with samples from every patient with cryopreserved leukemic cell isolates and separate DNA-RNA isolation is an important tool for future studies and could be the responsibility of the Dr Sardjito Hospital/Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta.
3. A new Indonesian ALL protocol is urgently needed, by stopping Anthracycline in the standard risk ALL arm, together with other refinements.
4. Continuing Prednisone – Dexamethasone randomization in the new three-drug induction SR protocol is advisable.
5. Further development of the Indonesian Childhood Cancer Registry is needed. The importance of a correct and precise registry is very significant.
6. Implementing the supportive care in all aspects (infection control, isolation room, palliative care, psychologist mentoring) to increase the treatment results and the quality of life of children with cancers.
7. Developing the twinning program with developed countries remains one of the effective tools for improvement of the prognosis of children with cancer.
Future Research Possibilities.
1. Research on monitoring minimal residual disease of childhood leukemia with immunophenotyping.
2. Research on comparative genetics of childhood leukemia, which are related to drug sensitivity.
3. Studies on the impact of nutritional status on the prognosis of childhood cancer, including interventional approaches.
4. Study on cost-effectiveness of cancer treatment.
5. Prednisone - dexamethasone randomization should be continued after having a new Indonesian ALL protocol (3-drug based protocol on induction for SR patients)
6. Studies investigating ethnic differences in polymorphisms in enzymes that are related to sensitivity (antileukemic and toxic side effects) to MTX are highly relevant, especially since there is much debate about the best treatment schedule with either high or low dose MTX
7. Studies on molecular genetics to support individualized treatment-based on pharmacogenetic differences.
Samenvatting
Problemen bij het managen van kinderkanker, in het bijzonder van leukemie bij kinderen, werden in Yogyakarta, Indonesië, gecompliceerder sinds in 1998 de wereldwijde economische crisis begon. Het samenwerkingsverband tussen de Gadjah Mada Universiteit en het VU medisch centrum was daarom uiterst belangrijk. In 1998 werd in Yogyakarta een nationale bijeenkomst over leukemie bij kinderen gehouden. Deze bijeenkomst werd gesponsord door het Koningin Wilhelmina Fonds (KWF). Afgevaardigden van alle kinderoncologische centra namen deel aan deze bijeenkomst: kinderoncologen, pathologen, laboratoriummedewerkers en verpleegkundigen. Er werden aparte workshops en seminars gehouden voor zowel oncologen, laboratoriummedewerkers, verpleegkundigen en huisartsen. De deelnemers hebben tijdens de workshops hun kennis verrijkt. De workshop resulteerde in een kosten-effectief behandelingsprotocol voor kinderen met acute lymfatische leukemie (ALL), opgezet door drie internationale experts, Prof. AJP. Veerman uit Nederland, Prof. Günther Henze uit Duitsland en Prof. James Nachman uit de Verenigde Staten. Het protocol werd Wijaya Kusuma 2000-ALL genoemd, naar de bloem waarvan wordt gezegd alle ziektes te kunnen genezen. Ook werd in Yogyakarta een registratiesysteem voor kinderkanker opgezet. Eén van de toegewijde medewerkers, Purwanto, werd aangesteld als datamanager. De studie begon in maart 2006 en werd financieel ondersteund door het KWF.
Epidemiologie van leukemie
Het Dr. Sardjito Hospital is een academisch ziekenhuis dat een gebied bestrijkt van de Yogyakarta Special Provincie (YSP) en het zuidelijk deel van centraal Java. De bevolking in dit gebied wordt geschat op 5 miljoen. Alle patiënten met leukemie worden verwezen naar het Dr. Sardjito Hospital.
Data-analyse van alle kinderen (0-14 jaar) met leukemie, opgenomen tussen 1998 en 2009, liet zien dat het aantal kinderen met acute leukemie uit het verwijzing gebied van het Dr. Sardjito Hospital steeg van 45 per jaar in 1999, het tweede jaar van de studie, tot 91 per jaar in 2009. Dit resulteerde in een significante toename van de jaarlijkse incidentie ratio (AIR) van 34.6 in 1999 tot 70.0 in 2009. De AIR in 2009 was vele malen hoger dan de incidentie in de wereld, die 30-50 per miljoen mensen per jaar bedraagt. Om dit nader te onderzoeken werd een aparte analyse uitgevoerd van de patiënten die werden verwezen vanuit de YSP waarvan het inwonersaantal meer betrouwbaar bekend is. De gemiddelde jaarlijkse incidentie ratio (AAIR) in de YSP was 20.8 voor ALL en 8.0 voor acute myeloїde leukemie (AML) (Hoofdstuk 2). De incidentie van ALL was laag vergeleken met westerse landen. Ten gevolge van
deze relatief lage incidentie van ALL is het aandeel van AML relatief hoog (25%) vergeleken met westerse landen. Het aantal gevallen van kinderen met leukemie in de YSP en het zuidelijk deel van centraal Java steeg in de periode van 1998-2009. De stijging is mogelijk een gevolg van een toename in het aantal verwijzingen of een uitbreiding van het gebied waarvandaan kinderen met leukemie doorverwezen worden naar het Dr Sardjito Hospital.
erbeteren van de Diagnose
Voor deze studie begon werd de diagnose van kinderleukemie enkel gedaan met behulp van morfologie en cytochemische kleuringen. Deze methode is niet in overeenstemming met de standaard WHO-criteria, waarin ook de immunofenotypering is opgenomen. In 2006 werd de immunofenotypering opgezet in Yogyakarta (Hoofdstuk 3). Van maart 2006 tot november 2007 zijn 136 monsters geїmmunofenotypeerd. In deze eerste periode werd een enkel-kleurs methode gebruik met een set van 9 monoclonale antistoffen. De resultaten hiervan lieten een te grote discrepantie zien tussen morfologie en immunofenotypering bij AML, resulterend in een kappa score van 0.43 (matige overeenkomst). Een nieuwe methode werd toen opgezet. Vanaf december 2007 werd met deze nieuwe methode een 3-kleuren FACS-scan en een set van 15 antistoffen geïntroduceerd. Het panel antistoffen werd uitgebreid door toevoeging van antistoffen gericht tegen intracellulaire antigenen. Geïntroduceerd werden cytoplasmatisch CD3, een hoogst specifieke marker voor T-ALL, cytoplasmatisch CD79a voor B-ALL en cytoplasmatisch MPO en CD117 voor de myeloide cellijn (AML). Dit panel werd toegepast op 318 monsters en resulteerde in minder monsters met “zwakke marker expressie” (1%) vergeleken met de oude methode (14.7%). De overeenstemming tussen morfologie en immunofenotypering was toegenomen. De kappa score was 0.82 (bijna perfecte overeenkomst). De kwaliteitscontrole werd gedaan in het Cancer Center Amsterdam (CCA), alwaar onze resultaten werden gevalideerd. In ons patiënten cohort was een relatief groot gedeelte geclassificeerd als AML (23%). In de ALL-monsters was 83% B-ALL en 17% T-ALL. Negen van de, morfologisch, als ALL geclassificeerde monsters werden met de immunofenotypering als AML geclassificeerd en 12 van de 79, morfologisch als AML geclassificeerde monsters bleken met de immunofenotypering ALL. Dit betekent dat bij een aantal patiënten (6.6%) de diagnose uiteindelijk werd omgezet van ALL naar AML of vice versa. Concluderend, de incidentie van ALL is relatief laag in Indonesië (Supriyadi et al., 2011) terwijl de incidentie van AML vergelijkbaar is met de incidentie in westerse landen. Derhalve vonden we een relatief hoog percentage AML patiënten (23%). Immunofenotypering speelde een rol bij de verbetering van de diagnostiek van kinderleukemie in Indonesië, welke voorheen enkel was gebaseerd op morfologie.
Hierdoor wordt een verbetering van de prognose verwacht, aangezien door de verbeterde risico classificatie van kinderen met ALL het meest optimale behandelprotocol kon worden geselecteerd.
Verbetering van de immunofenotypering maakt gelijktijdige detectie van myeloїde antigenen, CD34 en CD10 in kinder ALL mogelijk. In Hoofdstuk 4 behandelen we de rol van myeloїde antigenen op kinder ALL en in Hoofdstuk 5 de expressie van CD10 en CD34 alleen of in combinatie. In Indonesië wordt op 25% van de ALL gevallen co-expressie van myeloide antigenen gevonden. Wij vonden in de meerderheid van de ALL gevallen geen myeloide co-expressie (p=0.02). Kaplan-Meier analyse van de ziekte-vrije overleving liet voor de myeloid negatieve populatie een duidelijk betere prognose zien. In B-ALL werd in 27% van de gevallen myeloide co-expressie gevonden tegen 17% bij T-ALL. In B-ALL liet myeloide co-expressie geen verschil in prognose zien. In T-ALL daarentegen liet myeloide co-expressie een aanmerkelijk slechtere prognose zien. Onze resultaten lieten zien dat de afwezigheid van myeloide antigenen een betere prognose gaf bij T-ALL.
De expressie van CD10 en CD34 varieerde in verschillende studies. In onze studie vonden we CD10 expressie in 148 patienten (70%) en CD34 expressie in 112 patienten (53%) over het patiëntencohort. Expressie van CD10 en/of CD34 was geassocieerd met de standaard risico (SR) classificatie. CD10 positieve patienten hadden een betere overleving vergeleken met CD10 negatieve patiënten (p<0.001). Expressie van CD34 had geen significante invloed op de prognose, en co-expressie van CD34 en CD10 was geassocieerd met een betere prognose. Overleving in deze CD34+ en/ of CD10+ groep was 61% tegenover 31% in de dubbel-negatieve groep (p<0.001). Deze dubbele negativiteit (CD34-,CD10-) is tevens, binnen onze patientenpopulatie, een onafhankelijke prognostische factor geworden, naast leeftijd ten tijde van de diagnose en risico classificatie, bij multivariabele analyse.
In de B-ALL groep was de meerderheid CD10+ (78%). Klinische significantie van CD10 expressie was variabel. Overleving was vergelijkbaar tussen de CD10+ en de CD10- groep. CD34 expressie werd gevonden in 59% van de gevallen. CD34 expressie was geassocieerd met SR (standaard risico). CD34 expressie alleen bleek geen voorspellende factor en had geen prognostische gevolgen. De minderheid van de CD34+,CD10+ gevallen was hoog risico (HR) terwijl de meerderheid van de CD34-, CD10- patiënten in de HR-groep viel. Expressie van CD34 en/of CD10 was ook geassocieerd met SR. Naast leeftijd was CD10-,CD34- een negatieve prognostische factor. De incident-vrije overleving in de CD10-,CD34- groep was significant lager vergeleken met de CD34+ en/of CD10+ groep.
In T-ALL was CD10 expressie geassocieerd met een laag aantal witte bloed cellen (WBC). Tevens was hier CD34- gecorreleerd met een leeftijd van >10 jaar en een hoger aantal WBC. CD10 expressie was geassocieerd met een betere overleving. Overleving bij CD10+ patiënten was 88% tegen 26% bij CD10- patiënten (p=0.002). De expressie van CD34 alleen had geen significante invloed op de uitkomst. In T-ALL was de combinatie van CD34 en CD10 significante geassocieerd met de algehele overleving. In de multivariabele analyse was de combinatie CD10-,CD34- de enige significante prognostische factor, Hazard Ratio: 5.9, 95%Cl: 1.6-21.3, p=0.007. De combinatie CD10-, CD34- in T-ALL had een significant slechtere prognose, 13% tegen 77% in de combinatie CD10 en/of CD34 (p=0.002).
Verbeteren van de Behandeling
De betekenis van prednison-dexamethason randomisatie bij onze ALL patiënten wordt beschreven in Hoofdstuk 6. Dexamethason is een goedkoop en makkelijk verkrijgbaar geneesmiddel in Indonesië. Het Indonesië 2006 ALL protocol was gebaseerd op het gebruik van dexamethason, in plaats van prednison, en vertoonde verontrustende toxiciteit. Klinische trials en in vitro studies hadden aangetoond dat dexamethason superieur is ten opzichte van prednison in het doden van lymfoblasten. Dexamethason vermindert bovendien de kans op uitbreiding naar het centraal zenuwstelsel en verbetert de kans op genezing voor kinderen met kanker in westerse landen. In westerse studies resulteerde het gebruik van dexamethason in een verhoogde incident-vrije overleving, echter, ook het aantal kinderen dat stierf ten gevolge van toxiciteit nam toe. Hoewel dit verschil niet statistisch significant was, had het een duidelijk klinisch belang. Deze bevindingen stemden tevens overeen met die van een studie in het Verenigd Koninkrijk waar het aantal toxische sterfte gevallen bijna verdubbeld was bij de groep patiënten die dexamethason gebruikten, ten opzichte van de groep patiënten met prednison.
De dexamethason en prednison randomisatie in onze studie toonde aan dat het aantal kinderen dat voortijdig de behandeling verliet in beide groepen vergelijkbaar was. Tevens werden een vergelijkbaar aantal sterfte gevallen, leukemie gerelateerde gebeurtenissen, en complete remissies gevonden. Bij standaard risico patiënten werd een hogere sterfte tendens ten tijde van de inductie fase gevonden bij kinderen uit de dexamethason groep. Bij hoog risico patiënten werd geen statistisch significant verschil in sterfte ten tijde van de inductie fase en leukemische events gevonden tussen beide groepen. Het falen van de behandeling werd voornamelijk veroorzaakt doordat patiënten voortijdig de behandeling verlieten en door sterfte gedurende de inductie of in remissie. Het hoge aantal kinderen dat voortijdig de behandeling stopt gedurende de inductie kan mogelijk verklaard worden uit de
gedragsveranderingen die optreden tijdens de behandeling met corticosteroїden. Anderzijds is het voortijdig staken van de therapie ook gerelateerd aan sociaal en economische factoren. Toxische sterfte kan mogelijk verklaard worden uit het gehanteerde protocol. Studies uit westerse landen concludeerden dat het toevoegen van anthracyclines in ontwikkelingslanden zoals Indonesië, waar hogere infectie incidenties voorkomen en verminderde toegang tot supportive care bestaat, resulteert in meer uitgesproken levensbedreigende condities. Diverse internationale experts gaven aan dat ons ALL protocol, dat 4 doseringen anthracycline gedurende de inductie fase bevat voor zowel de standaard risico als hoog risico patiënten, te toxisch is. Het verminderen van het aantal geneesmiddelen ten tijde van de inductie behandeling van 4 naar 3 medicijnen wordt sterk aanbevolen.
Methotrexaat (MTX) is een belangrijk geneesmiddel bij de behandeling van ALL, naast corticosteroїden. MTX remt diverse enzymen die betrokken zijn bij de folaat homeostase. Het methyleentetrahydrofolaat reductase (MTRHFR) gen codeert voor een enzym dat een rol speelt bij het verwerken van foliumzuur. Dit is erg belangrijk voor de synthese van nucleїne zuren. Polymorfisme van MTHFR C677T is geassocieerd met een verhoogd risico op MTX-geїnduceerde toxiciteiten.
Thymidylate synthase (TS) is een key-enzym bij de novo DNA synthese. TS
polymorfisme, in het bijzonder TSER 3R/3R resulteert in een hogere TS activiteit, een sneller metabolisme van MTX, en als gevolg daarvan in een verminderde gevoeligheid voor MTX. TSER 3R is ook geassocieerd met een slechtere prognose. TSER polymorfisme zou de effectiviteit van MTX in de ALL behandeling kunnen beїnvloeden. De prevalentie van de TSER en MTHFR C677T polymorfismen zou kunnen variëren tussen populaties met een verschillende ethniciteit. In Hoofdstuk 7 en 8 bespreken we de rol van MTHFR C677T en TSER polymorfismen bij kinderleukemie in Indonesië in relatie tot MTX behandeling. Een frequente homozygotie van TSER 3R/3R polymorfismen werd gevonden in Indonesië. Deze bevinding zou kunnen wijzen op onder-behandeling van Indonesische kinderen met ALL. Voor het enzym MTHFR was de frequentie van het TT genotype in Indonesië erg laag, voor zowel gezonde kinderen als kinderen met leukemie, ten opzichte van Nederland. Dit resulteert in verminderde toxiciteit ten gevolge van MTX bij Indonesische kinderen ten opzichte van Nederlandse kinderen.
In het Indonesische ALL protocol is de MTX dosering overgenomen van een westers protocol. Dit zou onder-behandeling kunnen betekenen. Aangezien het gebruik van de benodigde hogere MTX dosering in Indonesië mogelijk niet haalbaar is door gebrek aan financiële middelen en supportive care, zou een goedkopere behandeling kunnen worden overwogen. Een geїndividualiseerde behandeling gebaseerd op
farmacogenetische verschillen wordt aanbevolen. Dit zou de effectiviteit van het protocol kunnen verhogen.
Aanbevelingen:
1) Diagnostiek gebaseerd op immunofenotypering zou standaard onderdeel moeten uitmaken van het beleid bij kinderen met leukemie in Indonesië. Deze laboratorium test zou derhalve vergoed moeten worden door de diverse soorten ziektekostenverzekering in Indonesië (askes, jamkesmas, jamkesda, jamkesos)
2) Voor toekomstige studies is een biobank met daarin monsters van ingevroren leukemische cellen en aparte DNA-RNA-isolaties van elke patiënt essentieel en dit zou de verantwoordelijkheid kunnen zijn van het Dr Sardjito Hospital/ Faculty of Medicine, Gadjah Mada Universiteit, Yogyakarta
3) Er is dringend behoefte aan een nieuw Indonesisch ALL protocol, waarbij niet alleen gestopt wordt met anthracyclines bij standaard risico ALL patiënten maar tevens andere verfijningen worden aangebracht.
4) Wij adviseren de prednison-dexamethason randomisatie voort te zetten bij het nieuwe inductie protocol, waarin 3 geneesmiddelen worden gebruikt bij standaard risico patiënten.
5) Verdere ontwikkeling van de Indonesische Registratie van Kinderkanker is nodig. Correcte en precieze registratie is uitermate belangrijk.
6) Implementatie van alle aspecten van supportive care (infectie controle, isolatie kamer, palliatieve zorg, psychologische begeleiding) om de behandelingsresultaten en de kwaliteit van leven van kinderen met kanker te verhogen.
7) Het ontwikkelen van twinning programma’s met meer ontwikkelde landen blijft een effectieve methode om de prognose van kinderen met kanker te verbeteren.
Mogelijkheden voor toekomstig onderzoek:
1) Onderzoek naar “minimale restziekte” van leukemie bij kinderen met behulp van immunofenotypering.
2) Onderzoek naar genetische veranderingen bij kinderleukemie die gerelateerd zijn aan cytostatica gevoeligheid.
3) Onderzoek naar de impact van de voedingstoestand op de prognose van kinderkanker, inclusief implementatie van interventies.
4) Onderzoek naar de kosten-effectiviteit van kanker behandeling.
5) Prednison-dexamethason randomisatie zou moeten worden gecontinueerd bij het gebruik van een nieuw Indonesisch ALL protocol (inductie protocol met 3 geneesmiddelen voor standaard risico patiënten).
6) Onderzoek naar ethnische verschillen van enzym polymorfismen gerelateerd aan de gevoeligheid (antileukemische en toxische bijwerkingen) voor MTX zijn zeer belangrijk. Dit onderzoek zou kunnen bijdragen aan de discussie over het gebruik van een hoge of lage dosering MTX als beste behandeling.
7) Onderzoek naar moleculaire genetica om geїndividualiseerde behandeling mogelijk te maken, gebaseerd op farmacogenetische verschillen
Intisari
Krisis ekonomi yang terjadi pada tahun 1998 berdampak luas di berbagai sendi kehidupan di Indonesia. Termasuk diantaranya dalam diagnosis dan tatalaksana kanker anak, termasuk didalamnya leukemia. Namun dengan adanya kerjasama yang baik antara Universitas Gajah Mada dan VUUniversity Medical Centre yg dibina sejak awal tahun 1990an, kami mendapatkan manfaat yang sangat besar dan kekuatan dalam menghadapi krisis global tersebut. Dalam hal ini adalah manfaat dalam penanatalaksanaan kasus leukemia anak di Yogyakarta.
Pada awal tahun 1998 di Yogyakarta diadakan pertemuan nasional yang membahas permasalahan leukemia anak. Pertemuan ini disponsori oleh Koningin Wilhelmina Fonds (KWF) dari negara Belanda dan diikuti oleh seluruh pusat pendidikan kedokteran di Indonesia yang menangani kanker anak. Para peserta pada pertemuan tersebut adalah spesialis kanker anak (pediatric oncologyst), ahli patologi (klinik), laboran dan perawat. Berbagai seminar dan workshop terpisah masing masing untuk onkologis anak untuk aspek pengobatan, patologis dan laboran untuk aspek diagnostik serta perawat mengetengahkan aspek perawatan diselenggarakan pada pertemuan nasional tersebut. Pada akhir pertemuan nasional tersebut, disusunlah sebuah protokol nasional pengobatan leukemia anak. Protokol tersebut dibidani oleh 3 orang ahli dalam bidangnya yaitu Prof. AJP. Veerman dari Belanda, Prof. Gunter Henze dari Jerman dan Prof. James Nachman dari Amerika Serikat. Protokol pengobatan tersebut diberi nama ‘Wijaya Kusuma ALL 2000’ . Wijaya Kusuma adalah nama bunga dalam legenda pewayangan yang dipercaya dapat menyembuhkan semua jenis penyakit. Selain itu, secara khusus disusun juga sebuah sistem registrasi kanker anak di Yogyakarta, yang dikelola oleh seorang data manager (Purwanto) yang khusus membidangi masalah registrasi kanker anak.
Epidemiologi leukemia
Rumah Sakit DR Sardjito (RSS) merupakan sebuah rumah sakit pendidikan dan sekaligus merupakan rumah sakit rujukan tersier. RSS mempunyai jangkauan wilayah meliputi Propinsi Daerah Istimewa Yogyakarta (DIY) dan propinsi Jawa Tengah bagian selatan. Perkiraan populasi cakupan dari RS ini meliputi sekitar 5 juta jiwa, dan seluruh kasus leukemia pada wilayah jangkauan ini dirujuk ke RSS sebagai rumah sakit rujukan. Pada analisis yang dilakukan pada populasi anak (0-14 tahun) penderita leukemia yang dirawat antara tahun 1998 dan 2009 diperoleh hasil yaitu adanya peningkatan kasus baru leukemia akut dari 45 per tahun di tahun 1999 menjadi 91 per tahun di tahun 2009. Hasil ini menunjukkan peningkatan yang signifikan pada angka insidensi (Annual Incidence Rate /AIR) yakni dari 34.6
