ALBERT

Description: In children with acute lymphoblastic leukemia (ALL), response to treatment is assessed by bone marrow aspiration. We investigated whether minimal residual disease (MRD) can be effectively monitored in peripheral blood. We used flow cytometric techniques capable of detecting 1 leukemic cell among 10 000 or more normal cells to compare MRD measurements in 718 pairs of bone marrow and peripheral blood samples collected from 226 children during treatment for newly diagnosed ALL. MRD was detected in marrow and blood in 72 pairs and in marrow but not in blood in 67 pairs; it was undetectable in the remaining 579 pairs. Remarkably, findings in marrow and blood were completely concordant in the 150 paired samples from patients with T-lineage ALL: for each of the 35 positive marrow samples, the corresponding blood sample was positive. In B-lineage ALL, however, only 37 of 104 positive marrow samples had a corresponding positive blood sample. Notably, peripheral blood MRD in these patients was associated with a very high risk for disease recurrence. The 4-year cumulative incidence of relapse in patients with B-lineage ALL was 80.0% ± 24.9% for those who had peripheral blood MRD at the end of remission induction therapy but only 13.3% ± 9.1% for those with MRD confined to the marrow (P = .007). These results indicate that peripheral blood may be used to monitor MRD in patients with T-lineage ALL and that peripheral blood MRD may provide strong prognostic information in patients with B-lineage ALL.

Description: Background: Genetic polymorphisms in the TPMT gene cause inter-individual variability in 6MP metabolism and tolerability. In the context of ALL maintenance therapy (SJCRH Total XII) that was heavily reliant upon 6MP and methotrexate (MTX) and without individualizing 6MP based on TPMT genetics, dose-limiting toxicity due to TPMT defects resulted in interruptions of 6MP and MTX administration, which have been associated with worse ALL outcomes in some studies (McLeod, Br J Haematol 1999; Welch, Cancer Chemother Pharmacol 1996). Overall 6MP dose intensity (i.e. prescribed dose /protocol dose) was 83% (Relling, Blood 1999). Here we characterized the dose intensity (DI) and tolerance of 6MP among patients with different TPMT genotypes in a more contemporary ALL regimen that included multiple agents in addition to 6MP and MTX. Our goal was to determine the tolerability of 6MP in patients with heterozygous vs wild-type TPMT in the context of multi-agent ALL therapy. Method: 388 children with ALL on the SJCRH Total XV protocol were evaluable for this study. TPMT genotype was analyzed at day 5 of remission induction using PCR and exome sequencing. Of the 388 patients, 347 (89%) were classified as wild-type and 41 (11%) carried a low activity allele, including *2, *3A, *3C and 677G〉A (Udaka, Genet Test 2005). No homozygous deficient patients were identified. It was generally recommended that patients with TPMT heterozygosity receive a starting 6MP dose of 50-60 mg/m2/d regardless of risk arm. Wild-type patients on the low-risk (LR) arm (n = 202) received daily 6MP at 75 mg/m2, while those on the standard/high-risk (SHR) arm (n = 186) started with 50 mg/m2/d in weeks 1-16, then increased to 75 mg/m2/d until week 120 (girls) or week 146 (boys). No 6MP was given during reinductions I (weeks 7-9) and II (weeks 17-19). The daily dosage, days of treatment, and DI of 6MP were analyzed for each phase of maintenance therapy. 6MP dosages were re-evaluated every 8-16 weeks to maintain a desired degree of leukopenia. Dosage was decreased if patients had myelosuppression (WBC 〈 1000/mm3, ANC 〈 300/mm3 and platelet 〈 5 × 104/mm3). Dosage increases were considered if patients missed less than 25% of therapy but had persistently high WBC (〉 4000/mm3) and ANC (〉 1000/mm3). In addition to the two reinductions, SHR patients received weekly asparaginase during weeks 1-19 and monthly cyclophosphamide/cytarabine until week 68. After week 20, patients received daily 6MP and weekly MTX with addition of monthly vincristine/dexamethasone (VCR/DEX) pulses until week 96, after which only 6MP and MTX were given. Result: The median cumulative DI was 83% (range 14-135%) in wild-type and 68% (range 5-102%) in heterozygotes, similar to that reported for SJCRH Total XII (Relling, Blood 1999). The DI of each phase differed by TPMT genotype (Fig 1). During week 10-16, wild-type patients on the SHR arm had lower median DI (77%, range 12-173%) than those on the LR arm (85%, range 0-110%; P = 6.4 × 10-5) despite the lower protocol dose in the SHR arm, supporting the practice of lowering the protocol 6MP dose during the early intensive weeks of therapy for the SHR arm, and suggesting the higher dose of 75 mg/m2/d is well tolerated even during early intensive therapy for the LR arm. The DI was similar in weeks 20-95 (6MP/MTX plus monthly VCR/DEX pulses) and in weeks 96-146 (6MP/MTX only) after stratifying by genotype and risk arm (Fig 1), suggesting that VCR/DEX pulses do not alter 6MP tolerance. TPMT heterozygotes received lower median daily 6MP doses (61 mg/m2) than wild-type (73 mg/m2; P = 3.2 × 10-7) during maintenance therapy. By using the lowered daily dose in heterozygotes, we prevented dose interruptions: the median percentage of days with no 6MP therapy was similar in heterozygotes and wild-type (12% vs 11% missed, P = 0.5). Conclusion: Using the approach of lowering the 6MP dose from 75 to 50 mg/m2/d during early intensive therapy for SHR patients results in reasonable DI in both groups. The lower tolerated daily dose of 6MP 60 mg/m2 in heterozygotes, compared to 75 mg/m2 in TPMT wild-type patients, supports the recommendation for a lower starting dose of 6MP for TPMT heterozygotes. 6MP DI was similar during phases that did vs did not include VCR/DEX, and similar in this contemporary regimen that includes VCR/DEX pulses compared to older studies without VCR/DEX pulses. Fig 1 Fig 1. *P-value of comparison between TPMT genotypes, and #P-value of comparison between weeks 20-95 and weeks 96-146. Disclosures Evans: St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties. Relling:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties.

Description: The acquired genetic characteristics of acute lymphoblastic leukemia (ALL) blasts are often used to guide the intensity of therapy, whereas the germline host genetic characteristics of the patient generally have not been considered. Multiple common, functionally important polymorphisms affect genes whose products determine the pharmacokinetics and pharmacodynamics of antileukemic agents. It is not yet known how genetic polymorphisms may interact to affect the outcome of antileukemic therapy. Combining classification and regression tree with failure time analysis, we assessed whether 16 genetic polymorphisms, alone or in combination, predicted relapses in 246 children with ALL, 116 of whom were treated on the lower-risk (LR) and130 on the higher-risk (HR) arms of the St Jude protocol Total XIIIB. Genotyping was performed for the following polymorphic loci: CYP3A4*1B and CYP3A5*3; GSTP1 313A〉G, GSTM1 and GSTT1 deletions; MDR1 exon 21 (2677G〉T/A) and MDR1 exon 26 (3435C〉T); MTHFR 677C〉T and MTHFR 1298A〉C; NR3C1 1088A〉G; SLC19A1 80G〉A; TPMT 238G〉C, 460G〉A and 719A〉G; TYMS enhancer repeat; UGT1A1 promoter repeat polymorphism; VDR intron 8 G〉A and VDR FokI (start-site) T〉C. In all children with available RNA in their diagnostic ALL blasts, gene expression levels of prognostic genotypes were analyzed using the Affymetrix genechip array HG_U95Av2. Among the HR group, the glutathione S-transferase M1 (GSTM1) non-null genotype was associated with the risk of hematological relapse (5-year cumulative incidence, 17.1%±4.5% compared to 5.1%±2.9% for GSTM1 null genotype, p = 0.03), and among the non-null genotypes, the thymidylate synthetase (TYMS) 3/3 genotype was associated with a further increase in hematologic relapse risk (5-year cumulative incidence, 29.2%±9.5% compared to 10.9%±4.7% for TYMS 2/3 or 2/2 genotypes, p = 0.02). Increased expression levels of these two target genes (p 〈 0.0001 and p = 0.09, respectively) were consistent with resistance to the drugs interacting with these gene products. For central nervous system relapse, among the HR group, the vitamin D receptor (VDR) start site (p = 0.02) and intron 8 genotypes (p = 0.04) predisposed, whereas for LR patients the TYMS 3/3 genotype predisposed (p = 0.04). The genotypes associated with outcome have pharmacologic plausibility: e.g., high GST activity (GSTM1 non-null) could cause anticancer drug resistance; high TYMS activity (TYMS 3/3) would be less inhibited by antifolates. In conclusion, germline polymorphisms influence the outcome of antileukemic therapy, and therefore represent determinants of response that can be used to optimize therapy.

Description: There is conflicting information about the influence of body mass index (BMI) on the pharmacokinetics, toxicity, and outcome of chemotherapy. We compared pharmacokinetics, outcome, and toxicity data across 4 BMI groups (underweight, BMI ≤ 10th percentile; normal; at risk of overweight, BMI ≥ 85th and 〈 95th percentile; overweight, BMI ≥ 95th percentile) in 621 children with acute lymphoblastic leukemia (ALL) treated on 4 consecutive St Jude Total Therapy studies. Chemotherapy doses were not adjusted to ideal BMI. Estimates of overall survival (86.1% ± 3.4%, 86.0% ± 1.7%, 85.9% ± 4.3%, and 78.2% ± 5.5%, respectively; P = .533), event-free survival (76.2% ± 4.2%, 78.7% ± 2.1%, 73.4% ± 5.5%, and 72.7% ± 5.9%, respectively; P = .722), and cumulative incidence of relapse (16.0% ± 3.7%, 14.4% ± 1.8%, 20.6% ± 5.1%, and 16.7% ± 5.1%, respectively; P = .862) did not differ across the 4 groups. In addition, the intracellular levels of thioguanine nucleotides and methotrexate polyglutamates did not differ between the 4 BMI groups (P = .73 and P = .74, respectively). The 4 groups also did not differ in the overall incidence of grade 3 or 4 toxicity during the induction or postinduction periods. Further, the systemic clearance of methotrexate, teniposide, etoposide, and cytarabine did not differ with BMI (P 〉 .3). We conclude that BMI does not affect the outcome or toxicity of chemotherapy in this patient population with ALL.

Description: Acquired genetic characteristics of acute lymphoblastic leukemia (ALL) cells are used to individualize therapy, whereas germ line genetic characteristics generally are not. We determined whether ALL outcome was related to 16 genetic polymorphisms affecting the pharmacodynamics of antileukemic agents. Of 246 children, 116 were treated on the lower-risk (LR) and 130 on the higher-risk (HR) arms of a St Jude protocol. Patients in the HR group with the glutathione S-transferase (GSTM1) nonnull genotype had greater risk of hematologic relapse (P = .03), which was further increased by the thymidylate synthetase (TYMS) 3/3 genotype (P = .03). These genotypes remained predictive in multivariate analyses (P 〈 .001 and .003, respectively). No genotypes were predictive in the LR arm. Expression of these 2 genes in ALL blasts was lower in those with low-activity genotypes. For central nervous system relapse, among the HR group, the vitamin D receptor start site (P = .02) and intron 8 genotypes (P = .04) predisposed, whereas for LR patients the TYMS 3/3 genotype predisposed (P = .04). The GSTM1 non-null and TYMS 3/3 genotypes are plausibly linked to drug resistance. Polymorphisms interact to influence antileukemic outcome and represent determinants of response that can be used to optimize therapy. (Blood. 2005;105:4752-4758)

Description: Methotrexate (MTX) and mercaptopurine (MP) are widely used antileukemic agents that inhibit de novo purine synthesis (DNPS) as a mechanism of their antileukemic effects. To elucidate pharmacodynamic differences among children with acute lymphoblastic leukemia (ALL), DNPS was measured in leukemic blasts from newly diagnosed patients before and after therapy with these agents. Patients were randomized to receive low-dose MTX (LDMTX: 6 oral doses of 30 mg/m2) or high-dose MTX (HDMTX: intravenous 1 g/m2) followed by intravenous MP; or intravenous MP alone (1 g/m2), as initial therapy. At diagnosis, the rate of DNPS in bone marrow leukemia cells was 3-fold higher in patients with T-lineage ALL compared with those with B-lineage ALL (769 ± 189 vs 250 ± 38 fmol/nmol/h;P = .001). DNPS was not consistently inhibited following MP alone but was markedly inhibited following MTX plus MP (median decrease 3% vs 94%; P 

Description: By using rapid flow cytometric techniques capable of detecting one leukemic cell in 104 normal cells, we prospectively studied minimal residual disease (MRD) in 195 children with newly diagnosed acute lymphoblastic leukemia (ALL) in clinical remission. Bone marrow aspirates (n = 629) were collected at the end of remission induction therapy and at 3 intervals thereafter. Detectable MRD (ie, ≥0.01% leukemic mononuclear cells) at each time point was associated with a higher relapse rate (P 

Description: Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.

Description: The effect of traumatic lumbar puncture at the time of initial diagnostic workup on treatment outcome in children with newly diagnosed acute lymphoblastic leukemia (ALL) was investigated. The findings of the first 2 lumbar punctures performed on 546 patients with newly diagnosed ALL treated on 2 consecutive front-line studies (1984-1991) at St Jude Children's Research Hospital were retrospectively reviewed. Lumbar punctures were performed at the time of diagnosis and again for the instillation of first intrathecal chemotherapy. The event-free survival (EFS) experience for patients with 1 cerebrospinal fluid (CSF) sample contaminated with blast cells was worse than that for patients with no contaminated CSF samples (P = .026); that of patients with 2 consecutive contaminated CSF samples was particularly poor (5-year EFS = 46 ± 9%). In a Cox multiple regression analysis, the strongest prognostic indicator was 2 consecutive contaminated CSF samples, with a hazard ratio of 2.39 (95% confidence interval, 1.36-4.20). These data indicate that contamination of CSF with circulating leukemic blast cells during diagnostic lumbar puncture can adversely affect the treatment outcome of children with ALL and is an indication to intensify intrathecal therapy.