Health-care providers' perspectives on childhood cancer treatment in Manado, Indonesia.

BACKGROUND: Childhood cancer survival in low-income countries is low. OBJECTIVE: Our study investigated health-care providers' perspectives on childhood cancer treatment in Indonesia. Their health beliefs and attitudes toward parental financial difficulties, protocol adherence, parental education, and communication were explored. METHODS: A self-administered questionnaire was filled in by 222 health-care providers (156 doctors, 51 nurses, 6 social workers, 9 administrators) RESULTS: Health of children with cancer is beyond doctor's control and determined by luck, fate or God according to 35% of health-care providers, 30% were uncertain about this statement, and 35% disagreed. Combination of chemotherapy and alternative treatment is best to achieve cure according to 15% of health-care providers, 50% were uncertain, and 35% disagreed. Prosperous parents adhere better with treatment (67%). Doctors adhere better with cancer treatment for prosperous patients (55%). When dealing with poor families, less elaborate explanation is given (62%), more difficult vocabulary is used (49%), and less cooperation is offered (46%). Reasons for non-adherence with treatment protocol were as follows: financial difficulties parents (82%), side-effects (77%), lack of motivation parents (75%), and inadequate drugs supply at pharmacy (70%). Information about cancer and treatment makes parents more afraid or depressed about future, and parents prefer not to know according to 27% of health-care providers, 20% were uncertain, and 53% disagreed. Communication with parents is hindered by differences in status and social hierarchical structures (83%). CONCLUSIONS: Health-care providers' beliefs about childhood cancer treatment are characterized by much uncertainty and contradiction. This likely affects adherence of health-care providers, parents, and childhood cancer treatment outcome.

Varicella outbreak in a pediatric oncology ward: the Manado experience.

BACKGROUND: Varicella is highly contagious and dangerous disease, especially in immunocompromised patients. Children with cancer are at increased risk of severe illness and fatal cases occur. OBJECTIVE: To describe an outbreak of varicella among in-patient cancer children, family members and staff. Estella Children Cancer Center in Manado, Indonesia with 14 beds and a 15 bed capacity guest house for family members. METHODS: A retrospective study of patients, family members and staff who were diagnosed with varicella based on clinical appearance was performed. Follow up was until 28 days from the last patient diagnosis' date. RESULTS: From late February to early May 2009, varicella was affecting 4 among 8 children with leukemia, 1 family member and 1 housekeeping staff. Measurers taken after the index case were oral acyclovir both for patients and contacts, patient isolation, ward disinfection and some chemotherapy interruption. Nevertheless, a second and third wave of varicella occurred. The index case died due to encephalitis. Other patients were non-severe and cured, but one child was lost to follow up. CONCLUSIONS: This outbreak highlights the importance of proper prevention and prompt management of varicella in immunocompromised patients. Simple and locally applicable guidelines are needed.

NOTCH1 and/or FBXW7 mutations predict for initial good prednisone response but not for improved outcome in pediatric T-cell acute lymphoblastic leukemia patients treated on DCOG or COALL protocols.

Aberrant activation of the NOTCH1 pathway by inactivating and activating mutations in NOTCH1 or FBXW7 is a frequent phenomenon in T-cell acute lymphoblastic leukemia (T-ALL). We retrospectively investigated the relevance of NOTCH1/FBXW7 mutations for pediatric T-ALL patients enrolled on Dutch Childhood Oncology Group (DCOG) ALL7/8 or ALL9 or the German Co-Operative Study Group for Childhood Acute Lymphoblastic Leukemia study (COALL-97) protocols. NOTCH1-activating mutations were identified in 63% of patients. NOTCH1 mutations affected the heterodimerization, the juxtamembrane and/or the PEST domains, but not the RBP-J-κ-associated module, the ankyrin repeats or the transactivation domain. Reverse-phase protein microarray data confirmed that NOTCH1 and FBXW7 mutations resulted in increased intracellular NOTCH1 levels in primary T-ALL biopsies. Based on microarray expression analysis, NOTCH1/FBXW7 mutations were associated with activation of NOTCH1 direct target genes including HES1, DTX1, NOTCH3, PTCRA but not cMYC. NOTCH1/FBXW7 mutations were associated with TLX3 rearrangements, but were less frequently identified in TAL1- or LMO2-rearranged cases. NOTCH1-activating mutations were less frequently associated with mature T-cell developmental stage. Mutations were associated with a good initial in vivo prednisone response, but were not associated with a superior outcome in the DCOG and COALL cohorts. Comparing our data with other studies, we conclude that the prognostic significance for NOTCH1/FBXW7 mutations is not consistent and may depend on the treatment protocol given.

IKZF1 deletions predict relapse in uniformly treated pediatric precursor B-ALL.

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL) and is often difficult to predict. To explore the prognostic impact of recurrent DNA copy number abnormalities on relapse, we performed high-resolution genomic profiling of 34 paired diagnosis and relapse ALL samples. Recurrent lesions detected at diagnosis, including PAX5, CDKN2A and EBF1, were frequently absent at relapse, indicating that they represent secondary events that may be absent in the relapse-prone therapy-resistant progenitor cell. In contrast, deletions and nonsense mutations in IKZF1 (IKAROS) were highly enriched and consistently preserved at the time of relapse. A targeted copy number screen in an unselected cohort of 131 precursor B-ALL cases, enrolled in the dexamethasone-based Dutch Childhood Oncology Group treatment protocol ALL9, revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Separate analysis of ALL9-treatment subgroups revealed that non-high-risk (NHR) patients with IKZF1 deletions exhibited a approximately 12-fold higher relative relapse rate than those without IKZF1 deletions. Consequently, IKZF1 deletion status allowed the prospective identification of 53% of the relapse-prone NHR-classified patients within this subgroup and, therefore, serves as one of the strongest predictors of relapse at the time of diagnosis with high potential for future risk stratification.

Comparing childhood leukaemia treatment before and after the introduction of a parental education programme in Indonesia.

SETTING: Previously, treatment and the results of treatment for childhood acute lymphoblastic leukaemia (ALL) in Indonesia differed significantly between poor and prosperous patients. Poor patients received less individual attention from oncologists and access to parental education and donated chemotherapy was lacking. INTERVENTION: A structured parental education programme for both poor and prosperous parents was introduced in January 2004 to improve access to parental education and donated chemotherapy. The programme consisted of a video presentation, an information booklet, DVD, audiocassette, a statement-of-understanding for donated chemotherapy, and a complaints procedure. Informed consent was also sought. OBJECTIVE: Our study compared childhood ALL treatment outcome before and after the introduction of the parental education programme. DESIGN: The medical records of 283 children with ALL diagnosed before (1997-2002; n = 164) and after (2004-2006; n = 119) the introduction of the education programme were reviewed. Data on treatment results and parental socioeconomic status were collected. RESULTS: After the introduction of the education programme, treatment refusal decreased (from 14% to 2%) and event-free survival increased (from 13% to 29%) significantly among poor patients. Treatment dropout increased (from 0% to 13%) significantly among prosperous patients. Overall, toxic death (from 23% to 36%) increased significantly, but there was no significant difference in event-free survival. CONCLUSIONS: After introduction of the programme, treatment refusal decreased and event-free survival increased significantly among poor families. However, improved knowledge, skills and communication are still required to combat the high rates of toxic death and treatment dropout. Treatment intensity should be accompanied by improved supportive care.

Long-term results of Dutch Childhood Oncology Group studies for children with acute lymphoblastic leukemia from 1984 to 2004.

The Dutch Childhood Oncology Group (DCOG) has used two treatment strategies for children with acute lymphoblastic leukemia (ALL) based on Pinkel's St Jude Total Therapy or the Berlin-Frankfurt-Münster (BFM) backbone. In four successive protocols, 1734 children were treated. Studies ALL-6 and ALL-9 followed the Total Therapy approach; cranial irradiation was replaced by medium-dose methotrexate infusions and prolonged triple intrathecal therapy; dexamethasone was used instead of prednisone. Studies ALL-7 and ALL-8 had a BFM backbone, including more intensive remission induction, early reinduction and maintenance therapy without vincristine and prednisone pulses. The 5-year event-free survival and overall survival increased from 65.4 to 80.6% (P<0.001) and from 78.7 to 86.4% (P=0.07) in ALL-7 and ALL-9, respectively. In ALL-7 and ALL-8 National Cancer Institute (NCI) high-risk criteria, male gender, T-lineage ALL and high white blood cells (WBCs) predict poor outcome. In ALL-9 NCI criteria, gender, WBC >100 x 109/l, and T-lineage ALL have prognostic impact. We conclude that the chemotherapy-only approach in children with ALL in Total Therapy-based strategies and BFM-backbone treatment does not jeopardize survival and preserves cognitive functioning. This experience is implemented in the current DCOG-ALL-10 study using a BFM backbone and minimal residual disease-based stratification.

Pharmacokinetic, pharmacodynamic and intracellular effects of PEG-asparaginase in newly diagnosed childhood acute lymphoblastic leukemia: results from a single agent window study.

L-asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is thought to result from depletion of asparagine in serum and cells. We investigated the clinical response in vivo of 1000 IU/m(2) pegylated (PEG)-asparaginase and its pharmacokinetic, pharmacodynamic and intracellular effects in children with newly diagnosed ALL before start of combination chemotherapy. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common/pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (P=0.01) and BCRABL-positive ALL (P=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-asparaginase (P=0.02) and both were prognostic factors for long-term event-free survival (hazard ratio 6.4, P=0.004; hazard ratio 3.7, P=0.01). After administration of one in vivo dose of PEG-asparaginase no changes in apoptotic parameters or in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. One additional dose of PEG-asparaginase upfront ALL treatment did not lead to other severe toxicities.

Circumvention of glucocorticoid resistance in childhood leukemia.

In this study, we determined if in vitro resistance to prednisolone and dexamethasone could be circumvented by cortivazol or methylprednisolone, or reversed by meta-iodobenzylguanidine in pediatric lymphoblastic and myeloid leukemia. As there were strong correlations between the LC50 values (drug concentration inducing 50% leukemic cell kill, LCK) of the different glucocorticoids and median prednisolone/methylprednisolone, prednisolone/dexamethasone and prednisolone/cortivazol LC50 ratios did not differ between the leukemia subtypes, we conclude that none of the glucocorticoids had preferential anti-leukemic activity. Meta-iodobenzylguanidine however, partially reversed glucocorticoid resistance in 19% of the lymphoblastic leukemia samples.

Prognostic significance of molecular-cytogenetic abnormalities in pediatric T-ALL is not explained by immunophenotypic differences.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by chromosomal rearrangements possibly enforcing arrest at specific development stages. We studied the relationship between molecular-cytogenetic abnormalities and T-cell development stage to investigate whether arrest at specific stages can explain the prognostic significance of specific abnormalities. We extensively studied 72 pediatric T-ALL cases for genetic abnormalities and expression of transcription factors, NOTCH1 mutations and expression of specific CD markers. HOX11 cases were CD1 positive consistent with a cortical stage, but as 4/5 cases lacked cytoplasmatic-beta expression, developmental arrest may precede beta-selection. HOX11L2 was especially confined to immature and pre-AB developmental stages, but 3/17 HOX11L2 mature cases were restricted to the gammadelta-lineage. TAL1 rearrangements were restricted to the alphabeta-lineage with most cases being TCR-alphabeta positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. CALM-AF10 was associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcomes, respectively. Also cases with high vs low TAL1 expression levels demonstrated a trend toward good outcome. Most cases with lower TAL1 levels were HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for outcome. Classification into T-cell developmental subgroups was not predictive for outcome.

Causes of death--other than progressive leukemia--in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience.

We analyzed causes of death, other than resistant disease or relapse, in 875 children with acute lymphoblastic leukemia (ALL) and 229 with acute myeloid leukemia (AML), treated on three different Dutch Childhood Oncology Group (DCOG) ALL and three AML protocols. Overall, 23 (2.6%) ALL and 44 (19.2%) AML patients died. Early death (ED, before remission was reached) occurred in nine ALL (1%) and thirty AML (13.1%) patients, including three and ten deaths before treatment was initiated. Chemotherapy-related mortality in remission (CRM) occurred in nine ALL (1.1%) and eight AML (4.4%) patients. For ALL, both ED and CRM declined over time, although this was not statistically significant. For AML a decrease in ED was observed (from 26% to approximately 10%), but counter-balanced by an increase in CRM (from 3 to 8%), maybe related to the scheduling of intensification blocks in AML-92/94. Including transplant-related mortality, death in CR rates in AML increased from 3 to 15% in the last study. The main cause of ED was hemorrhage, often associated with hyperleucocytosis, and infection for CRM. We conclude that mortality dropped favorably in ALL, but not in AML. Especially for AML, effective but less toxic therapy and better supportive care guidelines need to be developed.

Online fluorescent method to assess BCRP/ABCG2 activity in suspension cells.

An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.

Glucocorticoid receptor alpha, beta and gamma expression vs in vitro glucocorticod resistance in childhood leukemia.

Alternative splicing of the primary glucocorticoid receptor (GR) transcript, resulting in glucocorticoid receptor alpha GRalpha, glucocorticoid receptor beta GRbeta and glucocorticoid receptor gamma GRgamma, may influence glucocorticoid (GC) resistance in childhood leukemia. To test this hypothesis, we determined GRalpha/beta protein and GRalpha/beta/gamma mRNA expression levels in 43 initial acute lymphoblastic leukemia (iALL), 10 initial myeloid leukemia (iAML), 11 relapsed ALL (rALL) samples and one rAML sample. The results were correlated with in vitro GC resistance. GRalpha mRNA correlated with protein expression (rho=0.39-0.56, P<0.05), but the protein to mRNA ratio was median 2.2-fold lower in rALL than in iALL (P<0.05). GRbeta mRNA was median 137-fold lower than GRalpha mRNA and correlated with GRalpha mRNA expression (rho=0.71, P<0.0001). GRbeta could not be detected at the protein level. GRgamma accounted for a median of 2.8% (range 0.95-7.4%) of all GR transcripts. GRalpha (protein and mRNA) and GRbeta (mRNA) expressions or GRalpha/GRbeta ratios did not correlate with in vitro GC resistance in iALL, but GRgamma (mRNA) did (rho=0.52, P=0.007). These results suggest that GRbeta is not involved in GC resistance in childhood leukemia. The association between GRgamma expression and in vitro GC resistance in iALL and the decreased protein/mRNA ratio in rALL, a subgroup resistant to GCs, warrants further exploration.

In vitro drug-resistance profile in infant acute lymphoblastic leukemia in relation to age, MLL rearrangements and immunophenotype.

Acute lymphoblastic leukemia (ALL) in infants under 1 year is strongly associated with translocations involving 11q23 (MLL gene), CD10-negative B-lineage (proB) immunophenotype, and poor outcome. The present study analyses the relationship between age, MLL rearrangements, proB-lineage, and in vitro drug resistance determined using the MTT assay. Compared to 425 children aged over 1 year with common/preB (c/preB) ALL, the 44 infants were highly resistant to steroids (for prednisolone (PRED) more than 580-fold, P=0.001) and L-asparaginase (L-ASP) (12-fold, P=0.001), but more sensitive to cytarabine (AraC) (1.9-fold, P=0.001) and 2-chlorodeoxyadenosine (2-CdA) (1.7-fold, P<0.001). No differences were found for vincristine, anthracyclines, thiopurines, epipodophyllotoxines, or 4-hydroperoxy (HOO)-ifosfamide. ProB ALL of all ages had a profile similar to infant ALL when compared with the group of c/preB ALL: relatively more resistant to L-ASP and PRED (and in addition thiopurines), and more sensitive to AraC and 2-CdA. Age was not related to cellular drug resistance within the proB ALL group (<1 year, n=32, vs >/=1 year, n=19), nor within the MLL-rearranged ALL (<1 year, n=34, vs >/=1 year, n=8). The translocation t(4;11)(q21;q23)-positive ALL cases were more resistant to PRED (>7.4-fold, P=0.033) and 4-HOO-ifosfamide (4.4-fold, P=0.006) than those with other 11q23 abnormalities. The expression of P-glycoprotein, multidrug-resistance protein, and lung-resistance protein (LRP) was not higher in infants compared to older c/preB ALL patients, but LRP was higher in proB ALL and MLL-rearranged ALL of all ages. In conclusion, infants with ALL appear to have a distinct in vitro resistance profile with the proB immunophenotype being of importance. The role of MLL cannot be excluded, with the t(4;11) being of special significance, while age appears to play a smaller role.

Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia.

PURPOSE: To confirm the prognostic value of a drug resistance profile combining prednisolone, vincristine, and l-asparaginase (PVA) cytotoxicity in an independent group of children with acute lymphoblastic leukemia (ALL) treated with a different protocol and analyzed at longer follow-up compared with our previous study of patients treated according to the Dutch Childhood Leukemia Study Group (DCLSG) ALL VII/VIII protocol. PATIENTS AND METHODS: Drug resistance profiles were determined in 202 children (aged 1 to 18 years) with newly diagnosed ALL who were treated according to the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92 protocol. RESULTS: At a median follow-up of 6.2 years (range, 4.1 to 9.3 years), the 5-year disease-free survival probability (pDFS) rate +/- SE was 69% +/- 7.0%, 83% +/- 4.4%, and 84% +/- 6.8% for patients with resistant (PVA score 7 to 9), intermediate-sensitive (PVA score 5 to 6), and sensitive (SPVA score 3 to 4) profiles, respectively (sensitive and intermediate-sensitive v resistant, P

Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia.

Bone marrow and peripheral blood samples from 362 patients with acute lymphoblastic leukaemia (ALL) proliferating cell and 90 patients with acute myeloid leukaemia (AML) were analysed for S-phase fractions, Ki67 antigen, and proliferating cell nuclear antigen expression. The S-phase fractions were correlated with in vitro drug resistance to 15 different anticancer agents. Leukaemia cells isolated from bone marrow had higher S-phase fractions than leukaemia cells isolated from peripheral blood (in initial ALL, median values resp. 6.9 and 2.7%, in initial AML resp. 5.3 and 1.3%; both P<0.01). Relapse ALL samples derived from bone marrow showed increased S-phase fractions (median 9.9%) compared with initial ALL samples (median 6.9%; P<0.01). ALL samples obtained at initial diagnosis showed higher S-phase fractions (median 6.9%) and higher Ki67 expression (median 30%) than initial AML samples (median resp. 5.3 and 14%; both P<0.05). The S-phase fractions were not related to white blood cell count, age, or gender. Within initial ALL, the S-phase fraction correlated significantly but modestly strong (rho=0.3-0.5; P<0.05) with sensitivity to antimetabolites (cytarabine, mercaptopurine, thioguanine), L-asparaginase, teniposide, and vincristine. Similar results were found within subgroups of initial ALL (nonhyperdiploid and common/precursor-B-lineage ALL). In relapsed ALL and AML such correlations were not found. In conclusion, cell proliferation differs between leukaemia subgroups and increased proliferation is associated with increased in vitro sensitivity to several anticancer agents in initial ALL.

Clinical significance of cellular drug resistance in childhood leukemia.

Cellular drug resistance is an important determinant of the response to chemotherapy, and its precise measurement may have clinical relevance. Potential applications are: prognostic factor for risk-group stratification, tailored chemotherapy for subgroups or individual patients with a specific cellular drug resistance profile, determination of cross-resistance patterns, study of drug interactions, study of resistance modulation or circumvention, selection of patients for phase II studies and screening for the cytotoxicity of novel compounds. The colorimetric 4-day MTT assay is a frequently used method. However, a distinction between malignant and non-malignant cells cannot be made, which should be taken into account. In the case of a relatively high percentage of contaminating non-malignant cells, the differential staining cytotoxicity (DiSC) assay can be used. The MTT assay's technical success percentage is about 80% for fresh ALL and AML samples. For methotrexate (MTX) a different assay must be used, such as the thymidylate synthase inhibition assay (TSIA). The MTT assay measures the number of living cells that survived drug exposure. Therefore, the effect of many if not most drugs to induce leukemia cell death by apoptosis is also included. This review mainly summarizes the data on cellular drug resistance in childhood leukemia, as obtained by the MTT assay and TSIA, in our laboratory in Amsterdam. These data clearly demonstrate the significant relation between in vitro cellular drug resistance and clinical and cell biological features and short- and long-term clinical outcome in childhood leukemia. In conclusion, cellular drug resistance testing provides clinically relevant information that can be available within 1 week and can be performed successfully in the vast majority of leukemia samples. The data are more and more being used and being considered for use in clinical trials in leukemia.