General Discussion Summary Recommendation Samenvatting Intisari

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1 9 General Discussion Summary Recommendation Samenvatting Intisari General Discussion Summary Recommendation 169

2 9 General Discussion Summary Recommendation Samenvatting Intisari 170 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

3 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 Summary Recommendation 171

4 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 The situation is comparable to that in Western countries before 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 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 172 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

5 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 The Pediatric Leukemia Seminar and Workshop on improving leukemia diagnostics and patient care was then held in Yogyakarta in 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 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 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]. General Discussion Summary Recommendation 173

6 The annual incidence of ALL is about 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 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 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 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 174 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

7 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 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 General Discussion Summary Recommendation 175

8 [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 threecolor method was much better compared to one 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 and Kurec et al 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 176 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

9 analysis showed that age category was the only factor significant, HR 2.00 (95%CI: , 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). General Discussion Summary Recommendation 177

10 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< 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%. 178 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

11 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= 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 coexpression 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= CD10+ and/or CD34+ was found in 160/178 (90%). Expression of CD10 and/or CD34 was associated with risk classification, p= 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: , p = 0.013) for CD10-CD34- and 1.65 (95% CI: , 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: , p < 0.001) for CD10-CD34- and 1.74 (95% CI: , 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 In our study, only in T-ALL patients myeloid antigen expression was found to be a General Discussion Summary Recommendation 179

12 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: , 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 CD Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

13 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 General Discussion Summary Recommendation 181

14 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 182 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

15 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, ]. 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 [ ]. 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 General Discussion Summary Recommendation 183

16 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 C677T polymorphism 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]. 184 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

17 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 This resulted in a significant increase in AIR of 34.6 in 1999 to 70.0 in The Annual Incidence Rate (AIR) in 2009 was far above the world s incidence rate, which is 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 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 General Discussion Summary Recommendation 185

18 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 Myeloid-positive and Myeloidnegative 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). 186 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

19 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 coexpression 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: , p= 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 General Discussion Summary Recommendation 187

20 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 prednisonebased 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 polymorphism, in particular TSER 3R/3R results in a higher TS activity, faster 188 Diagnostic Advances for Improved Treatment of Childhood ALL in Indonesia

21 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. General Discussion Summary Recommendation 189