Immunobiological diversity in infant acute lymphoblastic leukemia is related to the occurrence and type of MLL gene rearrangement.

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period.

RAS pathway mutations as a predictive biomarker for treatment adaptation in pediatric B-cell precursor acute lymphoblastic leukemia.

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

Silencing of the tumor suppressor gene FHIT is highly characteristic for MLL gene rearranged infant acute lymphoblastic leukemia.

MLL rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL), diagnosed predominantly in infants (<1 years of age). Since current chemotherapy fails in >50% of patients with MLL, new therapeutic strategies are desperately needed. For this, understanding the biological features characterizing MLL is necessary. Analysis of gene expression profiles revealed that the expression of the tumor suppressor gene FHIT is reduced in children with MLL rearranged ALL as compared to ALL patients carrying germ line MLL. This finding was confirmed by quantitative real-time PCR. In 100% of the infant MLL cases tested, methylation of the FHIT 5'CpG region was observed, resulting in strongly reduced mRNA and protein expression. In contrast, FHIT methylation in infant and non-infant ALL patients carrying germ line MLL was found in only approximately 60% (P< or =0.004). FHIT expression was restored upon exposing leukemic cells to the demethylating agent decitabine, which induced apoptosis. Likewise and more specifically, leukemic cell death was induced by transfecting MLL rearranged leukemic cells with expression vectors encoding wild-type FHIT, confirming tumor suppressor activity of this gene. These observations imply that suppression of FHIT may be required for the development of MLL, and provide new insights into leukemogenesis and therapeutic possibilities for MLL.

Differential expression of p73 isoforms in relation to drug resistance in childhood T-lineage acute lymphoblastic leukaemia.

The T-lineage phenotype of childhood acute lymphoblastic leukaemia (ALL) is associated with an increased relapse-risk and in vitro resistance to drugs as compared to a precursor B phenotype. Antiapoptotic isoforms of p73 that lack part of the transactivation (TA) domain (DeltaTA-p73, i.e. p73Deltaex2, p73Deltaex3, p73Deltaex2/3 and DeltaN-p73) may cause resistance to anticancer agents through inhibition of p53 and/or proapoptotic p73 family members (TA-p73). We demonstrate in our study that the expression of total p73 mRNA was higher in childhood T-ALL compared to controls (P=0.004). In T-ALL, the relative contribution of antiapoptotic DeltaTA-p73 (88%) was larger than of proapoptotic TA-p73 (12%). Leukaemic cells of T-ALL patients expressing higher levels of antiapoptotic p73 were more resistant to the DNA-damaging drug daunorubicin compared to cells of patients with low or negative expression or these isoforms (P(trend)=0.045). Interestingly, p73Deltaex2 was the most abundantly expressed antiapoptotic isoform in daunorubicin-resistant patient cells (44% of total p73). No association was found between high expression of proapoptotic TA-p73 or antiapoptotic DeltaTA-p73 and relapse-risk. Our results suggest that childhood T-ALL is associated with a high expression of DeltaTA-p73. These isoforms may play a role in cellular resistance to DNA-damaging drugs in children at initial diagnosis of T-ALL.

Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)-positive pediatric ALL, and their relation with drug sensitivity and clinical outcome.

Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS +/- s.e. 53 +/- 17%) and of those with an extra der(21)t(12;21) (60 +/- 22%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (79 +/- 6%; P-trend = 0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to prednisolone (hazard ratio 5.78, 95% CI 1.45-23.0; P=0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

mRNA expression levels of (co)chaperone molecules of the glucocorticoid receptor are not involved in glucocorticoid resistance in pediatric ALL.

Resistance to glucocorticoids (GC) is an important adverse risk factor in the treatment of acute lymphoblastic leukemia (ALL). To induce apoptosis, GC bind to the GC receptor (GR), which is regulated by various (co)chaperone proteins such as heat-shock protein 70 (HSP-70), HSP-40, HIP (HSP-70-interacting protein), BAG-1 (BCL-2-associated gene product-1), HOP (HSP-70/HSP-90-Organizing protein), HSP-90, P-23, FKBP-51, FKBP-52 and CYP-40. In this study, we tested the hypothesis that mRNA expression levels of these molecules are determinants of GC resistance in childhood ALL. In all, 20 children with ALL cells in vitro sensitive to prednisolone (LC(50) < 0.1 microg/ml) were compared each with a resistant patient (LC(50) >150 mug/ml), matched for immunophenotype, age and white blood cell count. mRNA expression levels of the (co)chaperone molecules were measured by quantitative real-time RT-PCR and normalized to GAPDH and RNaseP levels. In vitro resistance to prednisolone was measured by MTT assay. HSP-90 mRNA expression levels were 2000-fold higher as compared to HSP-70. Using matched pair analysis, mRNA expression levels of the various (co)chaperone molecules were not significantly different between in vitro-sensitive and -resistant patients. GC resistance in childhood ALL cannot be attributed to different mRNA expression levels of the investigated (co)chaperone molecules involved in GC binding and transport to the nucleus.

Relation between genetic variants of the ataxia telangiectasia-mutated (ATM) gene, drug resistance, clinical outcome and predisposition to childhood T-lineage acute lymphoblastic leukaemia.

The T-lineage phenotype in children with acute lymphoblastic leukaemia (ALL) is associated with in vitro drug resistance and a higher relapse-risk compared to a precursor B phenotype. Our study was aimed to investigate whether mutations in the ATM gene occur in childhood T-lineage acute lymphoblastic leukaemia (T-ALL) that are linked to drug resistance and clinical outcome. In all, 20 different single nucleotide substitutions were found in 16 exons of ATM in 62/103 (60%) T-ALL children and 51/99 (52%, P = 0.21) controls. Besides the well-known polymorphism D1853N, five other alterations (S707P, F858L, P1054R, L1472W, Y1475C) in the coding part of ATM were found. These five coding alterations seem to occur more frequently in T-ALL (13%) than controls (5%, P = 0.06), but did not associate with altered expression levels of ATM or in vitro resistance to daunorubicin. However, T-ALL patients carrying these five coding alterations presented with a higher white blood cell count at diagnosis (P = 0.05) and show an increased relapse-risk (5-year probability of disease-free survival (pDFS) = 48%) compared to patients with other alterations or wild-type ATM (5-year pDFS = 76%, P = 0.05). The association between five coding ATM alterations in T-ALL, their germline presence, white blood cell count and unfavourable outcome may point to a role for ATM in the development of T-ALL in these children.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

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

Mechanisms of cellular anthracycline resistance in childhood acute leukemia.

Cellular resistance to anthracyclines is often associated with an unfavorable clinical outcome in a variety of cancers, including leukemia. Several mechanisms of anthracycline resistance may be involved such as an impaired transport of drugs across the plasma membrane, an increased drug efflux, an increased intracellular detoxification of drugs, a reduced availability of intracellular drug targets, or a reduced ability of cells to induce apoptosis. Here, we review the studies published about mechanisms which may explain resistance to anthracyclines in leukemic cells of pediatric patients.

Molecular determinants of glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia.

Glucocorticoids (GC) are probably the most important drugs in the treatment of ALL. Despite the extensive use of GC for many years, little is known about the molecular mechanisms of sensitivity and resistance. This review summarizes the knowledge on GC cytotoxicity in leukemia. The relevance of polymorphisms, splice variants and the number and regulation of the GC receptor are discussed. The role of multidrug resistance proteins, glutathione and glutathione S-transferase is evaluated, as well as the influence of the different heat-shock chaperone (hsp 90 and 70) and co-chaperone proteins (BAG-1 and others) which form a complex together with the GC receptor. Finally, the transactivation and transrepression (via NF-kappa B and AP-1 binding) of a wide range of genes (like c-myc) which initiates the final apoptosis pathway are discussed and suggestions for future directions of research in ALL patients are given.

Optimal immunocytochemical and flow cytometric detection of P-gp, MRP and LRP in childhood acute lymphoblastic leukemia.

The clinical relevance of multidrug resistance (MDR)-related proteins in childhood acute lymphoblastic leukemia (ALL) is largely unknown. The diversity of techniques, fixation methods, storage of cells (fresh or cryopreserved) etc, may contribute to discrepancies observed between several studies. We therefore optimized the detection of P-glycoprotein (P-gp), MDR-associated protein (MRP) and lung resistance-related protein (LRP) by immunocytochemistry and flow cytometry in childhood ALL cells. Thirteen fixation methods were compared using six antibodies in both immunocytochemistry and flow cytometry. The optimal fixation for P-gp (C219, MRK16), MRP (MRPr1) and LRP (LRP56) was a mixture of 2% (v/v) formaldehyde solution and acetone incubated for only 10 s at room temperature (FAc). For MRP recognized by MRPm6, the optimal fixation condition was acetone for 5 min at room temperature in immunocytochemistry, and methanol for 15 min at -20 degrees C in flow cytometry. P-gp staining by 4E3 was strongly antibody batch-dependent; on cytospins FAc fixation was optimal, but inconclusive data were obtained by flow cytometry. The optimized fixation conditions on fresh samples revealed a day-to-day variation in staining (both increasing and decreasing) in one third of the immunocytochemical tests. In flow cytometry the day-to-day variation in the fluorescence index was -1 +/- 22%. In both techniques, staining was comparable between fresh and cryopreserved cells. We recommend the use of the above mentioned fixation methods in order to study the clinical relevance of P-gp, MRP and LRP in childhood ALL.

The modulating effect of PSC 833, cyclosporin A, verapamil and genistein on in vitro cytotoxicity and intracellular content of daunorubicin in childhood acute lymphoblastic leukemia.

Resistance to anthracyclines is related to a poor prognosis in childhood acute lymphoblastic leukemia (ALL). Resistance to this class of drugs may (partly) be reversed by modulating agents, as has been demonstrated in a variety of cell lines. However, it is unknown which modulators may be of clinical benefit in childhood ALL. Therefore, we studied the modulating effect of PSC 833, cyclosporin A (CsA), verapamil (Vp) and genistein on daunorubicin (DNR) cytotoxicity, accumulation and retention in childhood ALL cells. DNR cytotoxicity was determined using the MTT assay; DNR accumulation, DNR retention and the expression of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and major vault protein/lung resistance protein (LRP) were determined by flow cytometry. In the majority of samples PSC 833 (19/26), CsA (22/26) and Vp (15/18) sensitized the cells to DNR whereas genistein made 25 out of 26 samples more resistant to DNR. The sensitizing effect on the cytotoxicity of DNR was median 1.2-fold using 2 microM PSC 833 (P = 0.025), 1.5-fold using 4 microM CsA (P = 0.003) and 1.6-fold using 6 microM Vp (P = 0.012) whereas the adverse effect of 25 microM genistein was median 1.8-fold (P < 0.0001). No relationship was found between the sensitizing effect of PSC 833, CsA or Vp and the degree of DNR resistance. In contrast, the adverse effect of genistein was largest in DNR sensitive samples (P = 0.003). The effect of each modulator on the cytotoxicity of DNR did not differ between initial and relapse ALL samples although the latter were median 1.4-fold more resistant to DNR (P = 0.005). Modulation of DNR cytotoxicity was not correlated with changes in the accumulated and retained intracellular DNR content or with the expression of P-gp, MRP and LRP. Besides genistein, PSC 833, CsA and Vp incidentally made ALL cells more resistant to DNR. CsA stimulated the leukemic cell survival in seven out of 26 samples, a phenomenon that was not related to the degree of DNR resistance. In conclusion, PSC 833, CsA and Vp but not genistein may be used to sensitize cells to DNR in childhood ALL. The data also indicate that not all patients may have a therapeutic benefit from these modulators. Therefore, an in vitro culture assay may be necessary to screen for patients who may benefit by a modulator in their therapy.

Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia--implications for treatment of infants.

The prognosis of infant ALL, characterized by a high incidence of the immature CD10 negative B-lineage ALL (proB ALL) is poor. This study aimed to determine the resistance profile of infant ALL cells. In vitro drug resistance was determined by the MTT assay of 395 children with ALL at initial diagnosis: there were 21 infants <1.5 years of which nine <1 year, 284 children aged 1.5-10 years (intermediate age group) and 90 children >10 years. Immunophenotyping resulted in 310 cALL/preB ALL, 69 T-ALL, 15 proB ALL and one unknown cases. The following drugs were tested: daunorubicin, doxorubicin, mitoxantrone, idarubicin (Ida), prednisolone (Pred), dexamethasone (DXM), vincristine (VCR), Asparaginase (Asp), 6-MP, 6-TG, AraC, VM26 and 4-HOO-ifosfamide (Ifos). Infants <1.5 years were significantly more resistant to Pred (>500-fold), Asp (11-fold) and VM26 (2.7-fold) but significantly more sensitive to Ara-C (2.3-fold) compared to the intermediate age group. When analyzing infants <1 year of age similar results were found. ProB ALL cells (seven infants <1.5 years; eight children >1.5 years) were significantly more resistant to glucocorticoids, Asp, thiopurines, anthracyclines and Ifos compared to cALL/preB ALL but more sensitive to Ara-C. Cells from children >10 years were significantly more resistant to Pred, DXM, Asp, Ida and 6-MP. T-ALL cells showed a strong resistance to Pred, Asp and VCR and a mild but significant resistance to all other drugs except thiopurines and VM26. We conclude that the poor prognosis of infant ALL is associated with a resistance to glucocorticoids and Asp. However, ALL cells from infants show a relatively high sensitivity to Ara-C which suggests that infants with ALL might benefit from treatment schedules that incorporate more Ara-C than the current treatment protocols.

Relationship between the intracellular daunorubicin concentration, expression of major vault protein/lung resistance protein and resistance to anthracyclines in childhood acute lymphoblastic leukemia.

In vitro resistance to anthracyclines is related to a poor prognosis in childhood acute lymphoblastic leukemia (ALL), but the underlying mechanisms are poorly understood. Using flow cytometry, we studied the contribution of daunorubicin (DNR) accumulation and retention, cell size, expression of the major vault protein/lung resistance protein (LRP), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) to the cytotoxicity of DNR (by MTT assay) in childhood ALL. The accumulated and retained DNR content was not related to the degree of DNR resistance, nor did the content differ between 53 initial and 20 relapse ALL samples (P >0. 05), although the latter were median two-fold more resistant to DNR (P = 0.004). Leukemic cell volume correlated with resistance to the anthracyclines DNR (Rs 0.32, P = 0.012) and idarubicin (Rs 0.46, P = 0.011) but not to other classes of drugs such as prednisolone, vincristine, L-asparaginase and etoposide. Relapsed patients had 1. 5-fold larger cells than patients at initial diagnosis of ALL (P = 0. 001). After cell volume correction, the intracellular DNR concentration was lower in relapsed compared with initial ALL cells (eg 60 min accumulation, P = 0.003). Moreover, the intracellular DNR concentration inversely correlated with DNR resistance, both in the accumulation (Rs -0.44, P < 0.001) and retention (Rs -0.33, P = 0. 016) test condition. The accumulated DNR concentration inversely correlated with expression of LRP (Rs -0.36, P = 0.012) but not with P-gp and MRP. Expression of LRP, but not of P-gp and MRP, significantly correlated with DNR resistance in childhood ALL (Rs 0. 33, P = 0.03). In conclusion, the intracellular DNR concentration and the expression level of LRP may contribute to DNR resistance in childhood ALL. The strength of the correlations also indicates that resistance to anthracyclines can not be explained by one single mechanism.

Multidrug resistance genes in infant acute lymphoblastic leukemia: Ara-C is not a substrate for the breast cancer resistance protein.

Infants with acute lymphoblastic leukemia (ALL) are more resistant to chemotherapeutic drugs than older children with ALL, except for Ara-C. Drug resistance mechanisms in infant ALL, however, remain unknown. Possibly, multidrug resistance (MDR) proteins like P-glycoprotein, MDR-associated protein (MRP1), lung resistance-related protein (LRP/MVP) and the breast cancer resistance protein (BCRP) play a role. Accordingly, we measured the mRNA levels of these proteins in infants (n=13) and non-infants (n=13) with ALL, using quantitative RT-PCR. Infants expressed 2.4-fold less BCRP mRNA (P=0.009) than non-infants with ALL. MDR1, MRP1 and LRP/MVP expression did not differ between both groups. MDR gene expression levels did not correlate to prednisolone, vincristine, daunorubicin or Ara-C cytotoxicity, except for BCRP expression, which correlated with resistance to Ara-C (Rs=0.53, P=0.012), suggesting that Ara-C might be a BCRP substrate. However, culturing patients ALL cells in the presence of the BCRP inhibitor Ko143 had no effect on Ara-C sensitivity. Inhibiting Bcrp1 in the Mdr1a-, Mdr1b- and Mrp1-deficient and Bcrp1-overexpressing mouse cell line Mef3.8/T6400, also did not modulate Ara-C cytotoxicity. Therefore, we conclude that Ara-C is not a substrate for BCRP and that MDR proteins do not play a significant role in drug resistance in infant ALL.

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.

PKCζ and PKMζ are overexpressed in TCF3-rearranged paediatric acute lymphoblastic leukaemia and are associated with increased thiopurine sensitivity.

Both tumour suppressor and oncogenic functions have been ascribed to the atypical zeta isoform of protein kinase C (PKCζ), whereas its constitutively active form PKMζ is almost exclusively expressed in the brain where it has a role in long-term memory. Using primers unique for either isoform, we found that both PKCζ and PKMζ were expressed in a subset of paediatric acute lymphoblastic leukaemia (ALL) cases carrying a TCF3 (E2A) chromosomal rearrangement. Combined PKCζ and PKMζ (PKC/Mζ) protein as well as phosphorylation levels were elevated in ALL cases, especially TCF3-rearranged precursor B-ALL cases, compared with normal bone marrow (P<0.01). Furthermore, high PKC/Mζ expression in primary ALL cells was associated with increased sensitivity to 6-thioguanine and 6-mercaptopurine (P<0.01), thiopurines used in ALL treatment. PKCζ is believed to stabilize mismatch-repair protein MSH2, facilitating thiopurine responsiveness in T-ALL. However, PKC/Mζ knockdown in a TCF3-rearranged cell line model decreased MSH2 expression but did not induce thiopurine resistance, indicative that the link between high PKC/Mζ levels and thiopurine sensitivity in paediatric precursor B-ALL is not directly causal. Collectively, our data indicate that thiopurine treatment may be effective, especially in paediatric TCF3-rearranged ALL and other patients with a high expression of PKC/Mζ.