ALBERT

Description: Acute lymphoblastic leukemia cells from 19 children, including 7 who remain in first complete remission (CR1), were engrafted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-level infiltration of bone marrow, spleen, and liver was observed, with variable infiltration of other organs. The immunophenotypes of xenografts were essentially unaltered compared with the original patient sample. In addition, sequencing of the entire p53 coding region revealed no mutations in 14 of 14 xenografts (10 from patients at diagnosis and 4 at relapse). Cells harvested from the spleens of engrafted mice readily transferred the leukemia to secondary and tertiary recipients. To correlate biologic characteristics of xenografts with clinical and prognostic features of the patients, the rates at which individual leukemia samples engrafted in NOD/SCID mice were analyzed. Differences in biologic correlates were encountered depending on stage of disease: a direct correlation was observed between the rate of engraftment and length of CR1 for samples harvested at relapse (r = 0.96; P = .002), but not diagnosis (r = 0.38; P = .40). In contrast, the in vivo responses of 6 xenografts to vincristine showed a direct correlation (r = 0.96; P = .002) between the length of CR1 and the rate at which the leukemia cell population recovered following vincristine treatment, regardless of whether the xenografts were derived from patients at diagnosis or relapse. This study supports previous findings that the NOD/SCID model of childhood ALL provides an accurate representation of the human disease and indicates that it may be of value to predict relapse and design alternative treatment strategies in a patient-specific manner.

Description: Relapse following remission induction chemotherapy remains a barrier to survival in approximately 20% of children suffering from acute lymphoblastic leukemia (ALL). To investigate the mechanism of relapse, 27 matched diagnosis and relapse ALL samples were analyzed for clonal populations using polymerase chain reaction (PCR)–based detection of multiple antigen receptor gene rearrangements. These clonal markers revealed the emergence of apparently new populations at relapse in 13 patients. More sensitive clone-specific PCR revealed that, in 8 cases, these “relapse clones” were present at diagnosis and a significant relationship existed between presence of the relapse clone at diagnosis and time to first relapse (P 〈 .007). Furthermore, in cases where the relapse clone could be quantified, time to first relapse was dependent on the amount of the relapse clone at diagnosis (r = −0.84; P = .018). This observation, together with demonstrated differential chemosensitivity between subclones at diagnosis, argues against therapy-induced acquired resistance as the mechanism of relapse in the informative patients. Instead these data indicate that relapse in ALL patients may commonly involve selection of a minor intrinsically resistant subclone that is undetectable by routine PCR-based methods. Relapse prediction may be improved with strategies to detect minor potentially resistant subclones early during treatment, hence allowing intensification of therapy.

Description: Introduction Acute lymphoblastic leukaemia (ALL) in infants has poor overall survival despite being characterized by very few genetic aberrations per case. The most common genetic change, present in over 75% of cases, is the rearrangement of the mixed lineage leukaemia (MLL/KMT2A) gene (MLL-R) that also occurs in AML and mixed phenotype acute leukaemia (MPAL). Because infant ALL is rare and distinctive, most Australian and New Zealand patients are enrolled on specific clinical trials. In earlier infant ALL trials, MRD was not used for risk stratification firstly because of the difficulty of finding sensitive markers for specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements and the secondly because, in infant ALL, Ig/TCR markers are often present in sub-clones that can be lost at relapse due to clonal selection. MRD testing is performed in the current Interfant 06 trial preferentially using markers based on the genomic breakpoint sequences of MLL gene rearrangements. The treatment of infant ALL remains very challenging with relatively poor survival rates attributable to both toxicity and relapse. The objectives of this study were therefore to analyse MRD data for Interfant 06 patients enrolled at ANZCHOG centres and to perform a pilot experiment evaluating gene expression for key genes in infant ALL samples on a microfluidics platform, as a basis for identifying potential targeted therapies. Methods MRD was measured in bone marrow DNA from Interfant 06 patients enrolled since 2006, using sensitive Real-time Quantitative PCR (PCR-MRD) patient-specific assays to detect either MLL gene rearrangements (in 17) and/or conventional immunoglobulin and T-cell receptor (Ig/TCR) markers (21). Gene expression levels for 90 genes important in childhood cancers were measured by Taqman-based microfluidic assays in duplicate using cDNA from 2 micrograms of total RNA from 10 infant ALL samples (5 MLL, 5 non-MLL) at diagnosis (6) or relapse (4). Results Patient-specific PCR-MRD tests were developed for 27 out of 28 Australian and New Zealand infant ALL patients. 17/18 MLL-R ALL patients had MLL-R assays and 21/28 patients had Ig/TCR MRD tests, with only 1 (non-MLL) patient having no MRD markers. There was a wide range of MRD responses to induction therapy (Figure 1). Bone marrow MRD at the end of induction was high (〉1x10-3) in 44% of MLL-R ALL infants compared to 25% of non-MLL ALL infants and 15% of older children enrolled on ANZCHOG ALL8. In 8/13 MLL-R patients who had both types of marker, MRD levels were higher when measured by their MLL-R marker than by their Ig/TCR marker. In a set of 90 genes selected for expression analysis, higher levels were found for 17 genes in 2 or more of the 10 infant ALL samples evaluated. These more highly expressed genes included potential or known drug targets BCL2, ERBB2, ERBB4, ILRA2, CSF1R and PARP1. Conclusions The quantitation of MRD based on MLL rearrangements in ALL is effective and can also be used to monitor response to therapy in infant ALL as well as MLL-R cases of AML and MPAL. The combined application of MLL-R and Ig/TCR markers allowed 97% of infant ALL patients to be MRD monitored with a sensitive marker. In most patients with both type of MRD marker, higher levels of MRD were detected in end of induction samples using the MLL versus Ig/TCR tests. One interpretation is that Ig/TCR genes are rearranged after the MLL rearrangement in ALL sub-clones that are both more mature and more chemo-sensitive. This finding also confirms the current consensus that disease-related MLL-R markers provide better risk assessment than Ig/TCR markers. Our Fluidigm analysis has shown that quantitative measurement of multiple gene expressions is feasible on small RNA samples and can be used to rapidly screen for specific expression of genes coding for drug targets in ALL patients. Support: NHMRC Australia APP1057746, Sporting Chance Cancer Foundation. Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Disclosures No relevant conflicts of interest to declare.

Description: Introduction: While remission rates for childhood acute lymphoblastic leukemia (ALL) now exceed 80%, relapsed ALL remains the leading cause of non-traumatic death in children. Recently, a high-risk group of B-progenitor ALL patients has been identified. Such cases exhibit a gene expression profile similar to that of BCR-ABL1 positive (Ph+) ALL but are BCR-ABL1 negative, and also experience poor treatment outcomes. This subset, termed Ph-like ALL, is characterised by a range of genetic alterations that activate cytokine receptor and kinase signalling, allowing potential targeting by available tyrosine kinase inhibitors (TKI). The frequency of Ph-like ALL in the Australian community and the prognosis in the setting of the first MRD (minimal residual disease) intervention trial by the Australian and New Zealand Children's Haematology/Oncology Group (ANZCHOG ALL8) is unknown. Method: We retrospectively screened 250 unselected samples that were available from children diagnosed with B-ALL, for Ph-like ALL. The children, aged between 1 and 18 years, were enrolled on the ANZCHOG ALL8 trial and recruited from 2002-2011. The criteria for stratification to the high-risk group, based upon Berlin-Frankfurt-Munster (BFM) protocols, were BCR-ABL1 or MLL t(4;11) translocation; poor prednisolone response at day 8; failure to achieve remission by day 33 or high MRD (〉5 x10-4) at day 79. MRD was measured by RQ-PCR for patient-specific immunoglobulin and T-cell receptor rearrangements. All patients received a standard BFM four drug induction chemotherapy regimen including a prednisolone pre-phase and intrathecal methotrexate. High-risk patients received a further three novel intensive blocks of chemotherapy followed by transplant in most cases. Patients were screened for Ph-like ALL using a custom Taqman Low Density Array (TLDA) based upon previous reports. Fusions were then confirmed by RT-PCR for 30 known fusions, Sanger sequencing, mRNA sequencing and/or FISH. Results: Ten percent (25/250) of children in this cohort were identified as having Ph-like ALL, with most fusions converging on kinase activating pathways (Table 1). Three Ph-like ALL patients were considered high-risk, the remaining 22 (88%) were medium risk. Five children with Ph-like ALL, that did not have a fusion identified by RT-PCR, are currently under further investigation. Furthermore, 15 of the 20 (75%) of rearrangements involved CRLF2 with 10 (66%) of these children relapsing. Strikingly, 56% (14/25) of children in the ALL8 cohort who were identified as Ph-like subsequently relapsed compared to 16% (36/225) who were not, with significantly worse event free survival (p

Description: Despite significant advances in the treatment of childhood acute lymphoblastic (ALL) and acute myeloid (AML) leukemias, the prognosis of those with primary refractory, secondary and early relapsed disease remains extremely poor with survival reported in most studies at less than 20%. Salvage rates are particularly poor in children with ALL who relapse on therapy and in children with AML who relapse post autologous bone marrow transplant. Encouraging results using the re-induction combination regimen of Fludarabine, high dose Cytarabine and Idarubicin (FLAG-IDA) have been reported, however the majority of data is in adult patients. Here we report the largest series of pediatric patients with high risk acute leukemias receiving this regimen in combination with allogeneic hematopoietic cell transplantation as salvage therapy. Between 1999–2005 a toal of 32 patients at 2 Australian Pediatric Cancer Centers were given Fludarabine (30mg/m2 IV over 1 hour for 5 days); Idarubicin (8mg/m2 IV over 1 hour for 3 days); Cytarabine (2000mg/m2 IV over 4 hours for 5 days) and GCSF (5mcg/kg/day). There were 21 males and 11 females, with a median age of 10.4 years (range 1.7 to 15.5 years). All patients had high risk leukemias; relapsed ALL (n=13), primary refractory ALL (n=3), relapsed AML (n=13), primary refractory AML (n=1) and secondary AML (n=2). For the overall cohort median duration of first complete remission (CR1) was 16 months (range 2–34 months). Relapsed ALL patients were all considered extremely high risk with a CR1 duration of

Description: Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) 〈 0.05 and P value 〈 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

Description: Key Points PR-104 represents a potential novel treatment for relapsed/refractory T-ALL. AKR1C3 expression could be used as a biomarker to select patients who may respond to PR-104 in prospective clinical trials.

Description: The dihydropyranoindole scaffold was identified as a promising target for improving the anti-cancer activity of HDAC inhibitors from the preliminary screening of a library of compounds. A suitable methodology has been developed for the preparation of novel dihydropyranoindoles via the Hemetsberger indole synthesis using azido-phenylacrylates, derived from the reaction of corresponding alkynyl-benzaldehydes with methyl azidoacetate, followed by thermal cyclization in high boiling solvents. Anti-cancer activity of all the newly synthesized compounds was evaluated against the SH-SY5Y and Kelly neuroblastoma cells as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Biological studies showed that the tetracyclic systems had significant cytotoxic activity at higher concentration against the neuroblastoma cancer cells. More importantly, these systems, at the lower concentration, considerably enhanced the SAHA toxicity. In addition to that, the toxicity of designated systems on the healthy human cells was found to be significantly less than the cancer cells.

Description: Improvements in Outcome for Paediatric de novo Acute Myeloid Leukaemia Aditi Vedi1,2, Richard Mitchell1, Cecelia Oswald1, Glenn Marshall1,2, Toby Trahair1, David S Ziegler1,2 1Kids Cancer Centre, Sydney ChildrenÕs Hospital, Randwick, NSW, Australia, 2 School of Women and Children's Health, University of New South Wales, Randwick, NSW, Australia ABSTRACT The treatment for paediatric acute myeloid leukaemia (AML) has not changed significantly over the past 3 decades, yet outcomes have improved with cure rates increasing from 30% to over 50% of all newly diagnosed children over this period. This improvement in survival has been attributed to both treatment intensification and improved supportive care over the decades, although the precise impact of each remains unknown. Our group has retrospectively analysed a unique cohort of patients with de novo AML diagnosed in childhood (n=276), all treated with the same chemotherapy protocol over a 25-year period from 1986-2012. The contemporary cohort (2000-12), compared to historical cohorts (1986-99) had significantly improved overall survival (OS, 75% vs. 50%, p = 0.01), lower disease related mortality (38% vs. 19%, p = 0.02) and were significantly more likely to receive allogeneic transplant after relapse (SCT, 73% vs. 12%, p

Description: Abstract 939 Introduction: The glucocorticoids dexamethasone and prednisolone are critical components of combination chemotherapy regimens used to treat pediatric acute lymphoblastic leukemia (ALL). While approximately 80% of patients are cured, poor response to upfront prednisolone monotherapy in 5-10% of patients is a strong predictor of adverse treatment outcome. A greater understanding of the mechanisms responsible for glucocorticoid resistance in pediatric ALL is likely to result in the design of novel strategies to overcome resistance and improve outcome for patients with refractory disease. The pro-apoptotic BH3-only BCL-2 family member BIM (BCL-2L11) has previously been identified as a critical component of glucocorticoid-induced apoptosis in normal and malignant lymphocytes. The purpose of this study was to elucidate clinically relevant mechanisms of glucocorticoid resistance in pediatric ALL, and design and test resistance reversal strategies. Patients and Methods: The study included biopsy specimens obtained at diagnosis from a cohort of patients who received 7 days of prednisolone monotherapy (60 mg/m⋀2/day) and a single age-related dose of intrathecal methotrexate, and whose prednisolone response was determined on Day 8 to be Good (PGR, peripheral blast count 〈 1 × 10⋀9/L, n=11) or Poor (PPR, peripheral blast count 〉= 1 × 10⋀9/L, n=11). The study also included biopsies from a cohort of patients who experienced early relapse (within 2 years of diagnosis, n=12). Experimental models of the disease included xenografts (n=12) established in immune-deficient (NOD/SCID) mice using direct explants of patient biopsies. Methods of analysis included: real-time quantitative RT-PCR and immunoblotting of glucocorticoid-induced mRNA and proteins following dexamethasone treatment of ex vivo cultured xenograft cells; DNA methylation analysis of the BIM 5' Untranslated Region (5'UTR) by methylated DNA immunoprecipitation (MeDIP), bisulfite sequencing, and SEQUENOM MassArray Epityper analysis; real time and array based chromatin immunoprecipitation (ChIP) analysis of histone-H3K9 acetylation, H3K4 and H3K27 tri-methylation across the entire BIM locus; as well as assessment of the histone deacetylase inhibitor SAHA (Vorinostat) to reverse dexamethasone resistance both in vitro and in vivo. Results: Dexamethasone resistance in pediatric ALL xenografts was consistently associated with failure to up-regulate BIM mRNA and protein in response to dexamethasone treatment, despite verification that other known glucocorticoid-responsive genes (GILZ, FKBP5) were highly induced in all xenografts. These results indicate specific silencing of BIM in dexamethasone-resistant xenografts rather than a dysfunctional glucocorticoid receptor, leading us to focus on epigenetic regulation of BIM transcription. DNA methylation of the BIM 5'UTR was variable between xenografts, and showed no clear association with dexamethasone resistance. In contrast, the extent of H3K9 acetylation at the BIM locus significantly correlated with the ability of dexamethasone to up-regulate BIM expression in ALL xenografts (R=0.90; P