MLL Rearrangements Research Articles

Fluorescence in Situ Hybridisation (FISH) Detection of Prognostically Relevant Chromosomal Abnormalities in Childhood Acute Myeloid Leukaemia (AML)

Cytogenetics is important in diagnosis of acute myeloid leukaemia (AML) and a number of chromosomal abnormalities have prognostic significance. AML clinical trials use three prognostic cytogenetic groups: good, intermediate and poor risk, for risk stratification of newly diagnosed patients to the most appropriate treatment arm. Many AML studies have combined data from children and adults, although the biology of their disease and treatment responses differ. For example, chromosomal abnormalities are observed in 80% of childhood AML, compared to only 50% of adults. The recent opening of the International Randomised Phase III Clinical Trial in Children with Acute Myeloid Leukaemia, MyeChild01, required extensive review of existing and novel genetic risk factors for stratification of patients in this study. The final consensus was to include the following abnormalities: Good risk; t(8;21)(q22;q22)/RUNX1-RUNX1T1 (approximate expected incidence in childhood AML, 12%), inv(16)(p13q22)/CBFB-MYH11 (6%); Poor Risk: inv(3)(q21q26)/t(3;3)(q21;q26)/abnormalities of 3q/MECOM rearrangements (~1%), monosomy 5/deletion of the long arm of chromosome 5 (5q) (~1%), monosomy 7 (4%), t(6;9)(p23;q34)/DEK-NUP214 (~1%), t(9;22)(q34;q11)/BCR-ABL1 (~1%), t(6;11)(q27;q23)/MLL-MLLT4/t(4;11)(q21;q23)/MLL-AFF1/t(10;11)(p11-p14;q23)/MLL-MLLT10 (5%), t(5;11)(q35;p15.5)/NUP98-NSD1(<5%),abnormalities of the short arm of chromosome 12 (12p) (~4%), inv(16)(p13.3q24.3)/CBFA2T3-GLIS2 (<2%); Intermediate risk; t(9;11)(p21;q23)/MLL-MLLT3/t(11;19)(q23;p13.3)/MLL-MLLT1/other MLL rearrangements (11%) and all other cases (25%).A pilot study was performed to evaluate the role of FISH in accurate detection of these abnormalities using a retrospective cohort of 158 paediatric AML patients. Patients were initially classified according to available karyotype, as t(8;21)(q22;q22) (n=15, 9%), inv(16)(p13q22) (n=12, 8%), t(15;17)(q24;q21) (n=8, 5%, although these patients are excluded from MyeChild01), monosomy 7/abnormalities of long arm of chromosome 7 (7q) (n=13, 8%), chromosome 5 abnormalities (n=3, 2%), t(9;22)(q34:q11) (n=1, 0.6%) and MLL rearrangements (n=49, 31%). Those patients with normal karyotype and other abnormalities were grouped together as other (n=57, 36%). They were analysed using a range of specific FISH probes, either commercially available from CytoCell or Kreatech or home grown, for the abnormalities listed above. No additional patients were identified with RUNX1-RUNX1T1, PML-RARα, CBFβ-MYH11 or BCR-ABL1, although copy number changes involving the chromosomal regions covered by these probes were indicated. No patients were found with the poor risk abnormality, inv(16)(p13.3q24.3)/CBFA2T3-GLIS2, likely due to its rarity.However, a number of previously undetected abnormalities were identified: MLL rearrangement, with poor risk translocations excluded (n=2), MLL-MLLT3 (n=1), 12p abnormalities (n=6), NUP98-NSD1 (n=3), DEK-NUP214 (n=1) and MECOM rearrangement (n=1). These latter 11 patients, accounting for 19 % of the other group, originally classified as intermediate risk became re-classified as poor risk following FISH screening.Cytogenetics is important to identify those significant chromosomal abnormalities involved in paediatric AML, which are used to stratify patients for treatment. FISH enables accurate detection of rare cryptic abnormalities associated with poor risk, meaning that more patients can benefit from appropriate risk stratification. Thus we are confident that FISH provides a reliable detection method to be implemented in MyeChild01, alongside screening for those mutations of prognostic relevance: NPM1, CEBPA and FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Relapse Prediction By Flow Cytometric Minimal Residual Disease Assessment in Infants with Acute Lymphoblastic Leukemia

Abstract Acute lymphoblastic leukemia (ALL) in infants (less than 1 year old) is a unique tumor with distinct biological features and poor outcome even when modern treatment schemes are used. Rearrangementsof MLL-gene, located in 11q23 region, are detected in approximately 75-80% of infants with ALL and lead to treatment resistance and high relapse rate. Nevertheless patients with germline MLL also demonstrate inferior outcome compared to ALL in older children. Thus additional risk factors implementation is one of the crucial points in infant ALL management. Minimal residual disease (MRD) measurement by multicolor flow cytometry (FCM) or various PCR technics is a well-standardized method of treatment response evaluation in childhood ALL, although in infants MRD data is not so widely applied. The aim of the study was to evaluate the relapse prediction feasibility in infant ALL by FCM MRD assessment during remission induction of MLL-Baby protocol. Methods. Totally 89 infants aged from 5 days to 11 months were enrolled in the present study. In 23 cases (25.8%) MLL gene was germline (MLL-g group), 33 patients (37.1%) had MLL-AF4 fusion while in remaining 33 cases (37.1%) other types of MLL-rearrangements were found. All patients were diagnosed as B-cell precursor ALL and all were treated by well established in Russia and Belarus MLL-Baby protocol, which is specially designed for infant ALL management. MRD was measured by 6-10-color FCM in bone marrow (BM) samples obtained at day 15 and at the end of remission induction (day 36). The availability of samples at at least one of these time-points was the only criteria for study group completion. In 43 patients with known types of MLL-rearrangements MRD was also assessed by fusion gene transcript (FGT) detection in RQ-PCR after first consolidation or first high risk block (for intermediate risk and high risk groups respectively). Relapse risk was investigated by cumulative incidence of relapse (CIR) estimation. Median of follow-up was 3 years 10 months. Results. Finally at day 15 MRD was studied in 71 cases. 17 patients (23.9%) were tested MRD-negative while remaining ones displayed various levels of MRD-positivity: 8 cases (11.3%) - from 0.01% to 0.1%; 14 (19.7%) - from 0.1% to 1%; 22 (31.0%) - from 1% to 10%; 10 (14.1%) - more than 10%. Proportion of MRD-positivity was lower in MLL-g group compared to MLL-rearranged (MLL-r) patients (58.8% and 81.5% respectively, p=0.06). Prognostic impact of day 15 MRD differed due to MLL-status. In MLL-r group significant differences between MRD(+) and MRD(-) patients were observed (n=10, CIR 0.28(0.18) and n=37, CIR 0.67(0.08) respectively, p=0.025). At the same time in MLL-g group these outcome differences were not significant (n=7, CIR 0 and n=9, CIR 0.22(0.14) correspondingly, p=0.197). Interestingly, in patients carrying MLL-AF4 fusion, known to be one of the most adverse types of MLL-rearrangements, day 15 MRD-negativity predicted low relapse incidence (n=5, CIR 0 and n=16, CIR 0.68(0.12) respectively, p=0.045). Thus day 15 MRD-negativity allows to detect low-risk MLL-r infants but it is not applicable in MLL-g group. It was previously shown that any detectable level of FGT after first consolidation or first high risk block predicts very poor outcome in MLL-r cases (G. Tsaur et al, ASH-2011). In current series RQ-PCR data in patients, FCM MRD(+) at day 15, distinguished groups of intermediate and very high relapse risk (n=17, CIR 0.51(0.13) and n=18, CIR 0.84(0.09) respectively, p=0.017). At day 36 FCM MRD was assessed in 82 infants. Among them 35 (42.7%) were tested negative while remaining 47 (57.3%) were MRD(+) at various levels. Prognostic value of day 36 FCM MRD data in MLL-r group was not significant: MRD(+) patients (n=23) had CIR of 0.71(0.08) while in MRD(-) cases (n=33) CIR was 0.49(0.12), p=0.153. Conversely, in MLL-g group low end-induction MRD (less than 0.1%) lead to excellent outcome compared to patients with higher MRD (n=14, CIR 0 and n=7, CIR 0.22(0.20) respectively, p=0.010). Conclusions. Thus FCM MRD data could distinguish infants with low risk of ALL relapse, but in MLL-r and MLL-g groups different time-points are prognosticaly significant. In MLL-g patients tandem application of FCM at early time-point and RQ-PCR later could help to define groups with low, intermediate and high relapse risk. MRD data could be added to MLL-Baby protocol risk group stratification, which is currently based on type of MLL-rearrangement. Disclosures No relevant conflicts of interest to declare.

Oncogenic Feedback Activation Between BCL6 and MLL Promotes Malignant Transformation in MLL-RearrangedAcute Lymphoblastic Leukemia

Background:BCL6 is known as a protooncogene and transcriptional repressor in diffuse large B cell lymphoma, where it is frequently involved in chromosomal rearrangements. We recently identified BCL6 as a novel mediator of drug resistance to tyrosine kinase inhibitors (TKI) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). In addition,BCL6 directly competes with the tumor suppressor BACH2 for p53 promotor binding to protecting cells from p53-mediated apoptosis in multiple ALL subgroups (Swaminathan et al., Nature Medicine 2013). Based on this, our current study is focusing on the function of BCL6 in different subtypes of human ALL.Results: Analysis of gene expression data from 207 children with high-risk B cell precursor ALL (COG P9906) showed that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (p=0.007). Furthermore, 49 matched sample pairs from patients at diagnosis and relapse showed increased BCL6 levels at relapse compared to diagnosis (p=0.003). To test whether or not there are specific subtypes of leukemia with high BCL6 expression levels, we studied BCL6 expression via western blot and immunohistochemistry staining in Non-Ph+ cell lines and ALL patient samples (n=76). Interestingly, BCL6 levels are particularly elevated in MLL-rearranged (MLLr) ALL cases. In addition, patients from the clinical trial that had high BCL6 levels and had MLL rearrangements had the worst clinical outcome (p=0.0009). We next tested if MLLr oncogenes drive aberrant BCL6 expression. First, we performed a ChIP-analysis using antibodies against MLL, AF4, and ENL, which provided evidence for direct binding to the BCL6 promoter. We then performed a BCL6 Western blot analysis of inducible MLL-AF4-transgenic and retrovirally transduced MLL-ENL pre-B cells, demonstrating that both oncogenes are sufficient to induce ~10-fold upregulation of BCL6 protein levels. Additionally, we used a newly developed conditional BCL6 knock out/reporter mouse model to decipher the function of BCL6. We transduced B cell progenitor cells from BCL6fl/fl mice with MLL-ENL and either with a control or Cre-expression vector. Using the BCL6 reporter capability of the mouse we found that BCL6 is significantly higher expressed in MLL-ENL transduced cells. To test if MLL is required for BCL6 upregulation, we used a conditional MLL knock out mouse and found that after Cre-mediated deletion of MLL, pro-B, mature-B, and MLL-AF4 transduced ALL cells almost lost the ability to upregulate BCL6. Interestingly, using inducible BCL6 transgenic and knockout as well as retrovirally transduced pre-B and ALL cells showed that overexpression of BCL6 leads to higher expression levels of MLL and deletion of BCL6 results in lower expression levels of MLL. Therefore, we conclude that MLL and BCL6 both cooperate and activate each other's expression in an activating feedback loop. Strikingly, Cre-mediated BCL6- deficiency results in apoptosis of MLL-ENL transduced cells.Clinical relevance: To verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human ALL xenografts with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if pharmacological inhibition of BCL6 is of potential use for patients with MLLr leukemia, we treated multiple MLL-AF4 rearranged human xenograft cases with a RI-BPI a BCL6 peptide inhibitor. Strikingly, treatment with RI-BPI not only compromised colony formation in methylcellulose it also prevents leukemia-initiation in transplant recipient mice. RI-BPI also had a strong synergistic effect when combined with the chemotherapy drug Vincristine, which represents the backbone for most high risk regimen in pediatric ALL.Conclusions: These findings identify BCL6 as a central factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat MLL-rearranged ALL. Aberrant expression of BCL6 in MLLr ALL is the direct consequence of the MLLr oncogenic activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome. DisclosuresArmstrong:Epizyme, Inc: Consultancy. Ernst:Amgen: Other: stocks. Melnick:Janssen: Research Funding.

Genomic Landscape of Pediatric Mixed Phenotype Acute Leukemia

▪Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor.Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases.Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations.In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases.Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. [Display omitted] [Display omitted] DisclosuresLoh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

Comparison of MRD Levels and Gene Expression Patterns in MLL-R Versus Non-MLL Infant ALL

Abstract 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 (&gt;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.

Clusters of Childhood Mixed Phenotype Acute Leukemia Diagnosed By Nation-Wide Immunophenotyping in Japan

[Background] Acute leukemia of ambiguous lineage is a very rare subtype defined by WHO 2008 classification. It is classified as mixed phenotype acute leukemia (MPAL), acute undifferentiated leukemia (AUL) and natural killer lymphoblastic leukemia. Basically, these category of leukemia consist of heterogenous pathogenesis. Determination of the lineage were usually decided by major lineage markers such as myeloperoxidase (MPO), CD19, and cytoplasmic CD3. However, more detailed immunophenotyping is necessary to confirm the cell lineages, especially in case of MPAL. Japanese pediatric leukemia/lymphoma study group (JPLSG) started nation-wide immunophenotyping with unified panel including more than 50 antigens for childhood hematological malignancy. Throughout this study, we found 32 acute leukemia of ambiguous lineage, including 3 AUL and 29 MPAL cases. MPAL cases were divided into several peculiar groups by our detailed phenotyping. We report here the characteristics of pediatric AUL/MPAL cases, including clinical manifestations and peculiar immunophenotype.[Result] In 3,229 case from January 2012 to December 2015, 32 cases were diagnosed as acute leukemia of ambiguous lineage. Gender ratio (M:F) was 18:14 and median age were 8.4 years (0.3 to 18.1) respectively. These cases were consisted of 3 AUL and 29 MPAL. According to detailed immunophenotyping, MPAL cases could be divided into 4 distinct clusters, (1) 7 T cell (T-) acute lymphoblastic leukemia (T-ALL)/acute myelogenous leukemia (AML)-M1 biphenotypic, (2) 4 early T-cell precursor (ETP)-like, (3) 8 B cell precursor (BCP-) ALL/Myelomonocytic bilineal, and (4) 10 BCP-ALL with MPO expression subtypes. T-ALL associated cases indicated relatively older age; (1) 12.3 and (2) 10.2 vs (3) 5.9 and (4) 7.5 respectively. 3 of (4) cases indicated characteristic genotypes; 1 minor bcr-abl, 1 major bcr-abl, and 1 AML1-ETO/Flt3-ITD. 4 out of 8 bilineal cases (3) were infantile ALL with MLL rearrangement, but other 4 older cases did not indicate MLL rearrangement.[Discussion] Acute leukemia of ambiguous lineage is a rare subtype of leukemia. According to WHO 2008 classification, it is further classified as MPAL, AUL and natural killer lymphoblastic leukemia. In this study, we evaluate 32 patients of acute leukemia of ambiguous lineage. It is of note that we could not identify no natural killer lymphoblastic leukemia in this childhood leukemia cohort. We also evaluate 29 MPAL cases, which showed 4 distinct subgroups. T-ALL/AML-M1 biphenotypic cases indicated relatively older age, usually diagnosed as AML-M1 with morphologic FAB classification, and received mostly myeloid oriented therapy. ETP-ALL like cases were positive for T cell antigens such as CD2 and/or CD7 and also some myeloid antigens but lacked cytoplasmic CD3 expression. BCP-ALL related cases were also divided into two groups, BCP-ALL/Myelomonocytic bilineal and BCP-ALL with MPO expression. Bilineal leukemia often found in infantile ALL with MLL gene rearrangement. In this study, we also found 4 non-infantile bilineal leukemia without MLL gene rearrangement. These cases occasionally cause lineage-switch relapse, so diagnosis of bilineal leukemia seems to be very significant. Recent studies showed EP300-ZNF384 fusion indicate CD10 negative or low BCP-ALL with MPO expression. Our cases also tended to indicate negative or low CD10 expression. In conclusion, we identified a spectrum of immunophenotype in MPAL patients. In childhood cases, most of MPAL cases were divided into 4 distinct categories. It must be useful information before examination of genetic abnormality. DisclosuresNo relevant conflicts of interest to declare.

Identification of Two Distinct Poor Prognostic Subgroups Related to High Expression of BMP2 or PRDM16 in Pediatric AML

[Background]Pediatric acute myeloid leukemia (AML) remains to be a disease with a poor prognosis, with a long-term survival rate of approximately 60%. We have recently reported that high PRDM16 expression (high-PRDM16) was common in t(5;11)(q35;p15.5)/NUP98-NSD1-positive patients, and that high-PRDM16 was found to be a novel poor prognostic factor in this disease. However, this finding was not observed in FAB-M7. The presence of inv(16)(p13.3q24.3)/CBFA2T3-GLIS2 was postulated to be a reason. This fusion was reported as the most frequent fusion gene associated with a poor prognosis in FAB-M7, and we found that all CBFA2T3-GLIS2-positive patients had a low PRDM16 expression (low-PRDM16). Furthermore, some studies reported that bone morphogenetic protein 2 (BMP2) was frequently and highly expressed in CBFA2T3-GLIS2-positive patients, indicating the importance of BMP2 in pathogenesis in a subset of AML. In this study, we analyzed the expression status of BMP2 and PRDM16 and their relationship to prognosis in pediatric AML.[Patients and Methods]A total of 369 AML patient samples, who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial, were analyzed. Expression level of BMP2 and PRDM16 is analyzed by real-time quantitative PCR (Taqman Gene Expression Assay), using ABL1 as a control gene. The cutoff value for high-BMP2 was defined as the lowest expression level among 11 CBFA2T3-GLIS2-positive patients, and that for high-PRDM16 was defined as the lowest expression level among 10 NUP98-NSD1-positive patients. We also screened fusion genes and gene mutations of CBFA2T3-GLIS2, NUP98-JARID1A, RBM15-MKL1, MLL rearrangements, NUP98-NSD1, FUS-ERG, DEK-NUP214, RUNX1-RUNX1T1, CBFB-MYH11, and gene mutations of KIT, NRAS, KRAS, WT1, NPM1, MLL-PTD, and FLT3-ITD.[Results]Out of 369 patients, high-BMP2 and high-PRDM16 were identified in 47 (12.7%) and 44 (11.9%) patients, respectively. Only three (0.8%) of 369 patients had both high-BMP2 and high-PRDM16 (high-BMP2/PRDM16). In 44 high-BMP2 patients and 41 high-PRDM16 patients (3 high-BMP2/PRDM16 patients were excluded), MLL rearrangements were only found in high-BMP2 patients (25.5% vs 0%, p < 0.001), whereas FLT3-ITD was significantly much more frequent in high-PRDM16 patients (4.5% vs 56.1%, p < 0.001). Core-Binding-Factor (CBF)-AML was rare in both groups (4.5% and 0%, p = 0.495). Kaplan|Meier analyses revealed that high-BMP2 patients and high-PRDM16 patients had a low 5-year overall survival (OS) (46.3% and 29.3%, respectively). In molecular subgroup analyses, the prognosis of MLL-rearranged patients, including 12 high-BMP2 patients (21.8%), was not influenced by BMP2 expression (5-year OS: high-BMP2 66.7% vs low-BMP2 64.9%, p = 0.488), suggesting that the prognosis of MLL-rearranged patients might primarily be defined by their fusion partner genes and other gene aberrations as previously reported (e.g., EVI1 expression). Considering this result, further survival analyses of the patients lacking CBF-AML or MLL-rearrangements were performed (n = 174, 5-year OS: 50.5%). In this subgroup, 5-year OS of high-BMP2 patients (n = 30) and high-PRDM16 patients (n = 41) were significantly lower than that of low-BMP2/low-PRDM16 patients (36.7% vs 67.2%, p < 0.001, and 29.3% vs 67.2%, p < 0.001, respectively).[Conclusions]High-BMP2 and high-PRDM16 could identify distinct subgroups with poor prognosis in pediatric AML. Further investigation of molecular-targeted therapy against these genes may contribute to the improvement of clinical outcome in these subgroups. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Differential Expression of Adhesion Molecule Receptors May Influence Bone Marrow Microenvironment-Mediated Protection of Leukemia-Initiating Cells (LICs) in Infant MLL-rearranged (MLL-R) Acute Lymphoblastic Leukemia (ALL)

Background: Compared to older children with ALL who achieve a minimal residual disease (MRD)-negative remission, infants with ALL who achieve an MRD-negative remission have a significantly higher rate of relapse. This suggests that, despite the achievement of MRD negativity, a very small amount of leukemia cells persist and contribute to disease relapse in infant ALL; LICs are likely to represent this persistent population of cells. Previously, we demonstrated that infant MLL-R ALL derive significantly more protection from co-culture with bone marrow (BM) stromal cells than non-MLL-R ALL. Therefore, protection by the BM microenvironment may contribute to LIC persistence in infant MLL-R ALL. We hypothesized that infant MLL-R ALL LICs have increased interactions with the BM microenvironment, through increased surface expression of adhesion molecule receptors. We also hypothesized that co-culture with normal BM stroma would protect LICs from spontaneous and chemotherapy-induced apoptosis, and AMD3100 (plerixafor) would decrease stromal protection through inhibition of CXCR4-CXCL12 signaling.Methods/Results: We analyzed 9 viably cryopreserved diagnostic samples collected from infants with MLL-R ALL (n=4 MLL-AF4, n=5 MLL-ENL). Using flow cytometry, we identified 2 phenotypically-defined LIC subpopulations, CD34+CD38+CD19+ and CD34-CD19+ (Aoki et al 2015), within the bulk leukemic blast population (CD45+). Consistent with previous findings, 34+38+19+ LICs were predominant in MLL-AF4 samples, while 34-19+ LICs were predominant in MLL-ENLsamples. Next, we measured surface expression of the adhesion molecule receptors CXCR4, CD49d (VLA-4), CXCR7, and CXCR3 (quantified as mean fluorescence intensity, MFI). Overall, surface expression of CXCR4, CD49d, CXCR7, and CXCR3 was higher in the 34+38+19+ LICs, compared to 34-19+ LICs (e.g., CXCR4 MFI 142 in 34+38+19+ vs. 76 in 34-19+, p=0.02).Next, samples were treated with dose ranges (0-30 µM) of AraC or etoposide (Etop) and cultured for 48 hours in 3 conditions: 1) off stroma, 2) on stroma, or 3) on stroma with the CXCR4 inhibitor AMD3100 (10 μM). Stromal cells were cultured from a healthy BM donor. In vehicle control-treated 34+38+19+ LICs, stromal co-culture led to increased surface expression of CD49d (avg 64% increase, p=0.03) and CXCR7 (avg 67% increase, p=0.02). Conversely, in vehicle control-treated 34-19+ LICs, stromal co-culture did not affect surface expression of the measured adhesion molecule receptors. In all 3 culture conditions, surface expression of CXCR4, CD49d, and CXCR7 was significantly higher in 34+38+19+ LICs compared to 34-19+ LICs (p≤0.01).We then measured apoptosis by flow cytometry and Annexin V binding. In the absence of chemotherapy, stromal co-culture significantly protected 34+38+19+ LICs from spontaneous apoptosis (p<0.001), but not 34-19+ LICs (p=0.16). Treatment with AMD3100 decreased stromal protection from spontaneous apoptosis in 34+38+19+ LICs (p<0.05), but not 34-19+ LICs (p=0.13). Notably, 34-19+ LICs were more resistant to spontaneous apoptosis even when cultured without stromal support (avg Annexin V-negative fraction 76.5% in 34-19+ vs. 62.8% in 34+38+19+, p=0.005). In spite of differences in stromal-mediated protection from spontaneous apoptosis, stromal co-culture protected both LIC populations from AraC- or Etop-induced apoptosis (p<0.001 for 34+38+19+, p<0.01 for 34-19+). Also, 34-19+ LICs were more resistant to Etop than 34+38+19+ LICs in the presence (p<0.001) or absence (p<0.01) of stroma. Finally, AMD3100 decreased stromal protection from AraC- or Etop-induced apoptosis (p<0.001 for 34+38+19+, p<0.05 for 34-19+).Conclusions: Our results suggest that the two immunophenotypically distinct LIC populations in infant MLL-R ALL may rely differently on the BM microenvironment. Higher surface expression of adhesion molecule receptors may explain why 34+38+19+ LICs derive more benefit from stromal co-culture, while lower surface expression of adhesion molecule receptors may explain decreased reliance on stroma by 34-19+ LICs. There may also be differences in chemotherapy resistance between the LIC populations. Despite these differences, CXCR4 inhibition enhanced chemosensitivity in both LIC populations. These results support the development of new therapeutic strategies that target leukemia-stroma interactions to improve outcomes for infants with MLL-R ALL. DisclosuresNo relevant conflicts of interest to declare.

Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate.The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML.In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs.The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells.To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells.Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo.Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation.Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. DisclosuresNo relevant conflicts of interest to declare.

The Role of MLL1 and MLL2 in MLL Fusion Oncoprotein-Initiated Leukemia

The MLL1 histone methyltransferase gene at 11q23 undergoes many distinct chromosomal translocations to yield poor prognosis leukemia. In these MLL-rearranged (MLL-r) leukemias, the remaining wild-type allele is typically expressed, and the fusion oncoprotein lacks the C terminal histone methyltransferase domain. The role of the wild-type, endogenous MLL1 allele in MLL-fusion protein mediated leukemogenesis is not fully understood, with some evidence suggesting an important functional role. On the other hand, the wild-type allele is lost in some patients and in the MLL-r ML2 cell line, suggesting that the wild-type allele is not absolutely required. Here we rigorously address the role of endogenous MLL1 in MLL-r leukemia. Using precise and independent animal models, we show that MLL1 is dispensable for MLL-AF9- or MLL-AF6-mediated acute myelogenous leukemia (AML). In parallel experiments, deletion of Menin, encoding a chromatin targeting cofactor for MLL-fusion proteins, has severe effects on the proliferation and survival of MLL-r leukemia, as previously published. Potential redundancy between MLL family methyltransferases was addressed by co-deleting the closest paralog, Mll2. Surprisingly, deletion of Mll2 by itself had a significant impact on survival of MLL-AF9 leukemia cells, and co-deletion of Mll1 and Mll2 more effectively killed these cells, suggesting a redundant or synthetic lethal relationship between the two genes. A comparison of gene expression and histone modification changes in Mll1, Mll2 and Mll1;Mll2 double knockout leukemia cells showed that 1) global defects in histone H3, lysine 4 methyation can be exclusively attributed to MLL2, 2) MLL1- versus MLL2-dependent genes are largely distinct, and 3) Mll1;Mll2 double knockout leukemia cells exhibit deficiencies in NFκB and integrin ß3 pathways, but exhibit little evidence of deregulation of either MLL1- or MLL-fusion protein target genes. These findings reshape our thinking about the role of endogenous MLL family proteins in MLL-fusion protein leukemogenesis. The data presented here, in conjunction with the minimal effect of Mll2 deletion on hematopoietic stem and progenitor cells make MLL2 an attractive candidate for targeted therapy in MLL-r leukemia. DisclosuresErnst:Amgen: Other: stocks. Yokoyama:Sumitomo Dainippon Pharma: Research Funding. Neff:Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel; Epizyme: Patents & Royalties: patent filed.

Predictive Value of Minimal Residual Disease in WHO2016-Defined Mixed Phenotype Acute Leukemia (MPAL)

▪BACKGROUND: Mixed phenotype acute leukemia (MPAL) as defined according to the World Health Organization 2016 guidelines (WHO2016) comprises approximately 2-3% of acute leukemia. In other acute leukemias, minimal residual disease (MRD) has emerged as the predominant predictor of relapse. MRD at end of Induction (EOI) is therefore a key risk-stratifying feature for lymphoid and myeloid disease regimens. To our knowledge, no studies to date have reported on the predictive value of MRD for treatment of MPAL. Although the absence of randomized trials for MPAL precludes a clear determination of optimal regimen, acute lymphoblastic leukemia (ALL) directed-therapy is increasingly considered frontline treatment internationally. The primary objective for this study was to evaluate the association of EOI MRD with event free survival (EFS) from ALL regimens used for treatment of MPAL.METHODS: Flow cytometry data for all acute leukemias diagnosed 2008-2015 and treated with Children’s Oncology Group regimens for ALL were examined by two flow cytometrists blinded to clinical outcomes (MO, SL). Data was analyzed using CellQuestPro and FACSDiva software (BD Biosciences, San Jose, CA). A comprehensive panel of 21 B-, T-, and myeloid markers were evaluated in all cases. A diagnosis of MPAL was strictly established according to the WHO2016 definition. EOI MRD was performed routinely by flow cytometry using a "difference from normal" approach. Cases with insufficient flow cytometry to either (1) establish the diagnosis of MPAL or (2) evaluate the presence and quantity of MRD were excluded. Due to the rarity of the T/myeloid phenotype in the dataset (n=1), the analysis was restricted to B/Myeloid. The primary endpoint was EFS; secondary outcomes explored predictors of EOI MRD positivity and the prevalence of recurrent cytogenetic abnormalities. MRD was categorized as a binomial (negative <0.01%) or ordinal (<0.01%, <0.1%, ≥0.1%) variable in separate models. EFS was calculated by Kaplan-Meier analysis. Step-wise multivariable Cox proportional hazard models included MRD and demographic/disease candidate predictors (age [years], sex, initial white blood cell count [WBC], central nervous system disease, cytogenetic abnormality, isolated myeloperoxidase expression). As some patients were classified as MPAL retroactively, category of treatment regimen (ALL high-risk [36/53] versus standard-risk [17/53]) was "forced" into the final multivariable model. Due to the non-normal distribution of MRD, predictors of MRD positivity were assessed by rank-sum test.Results: Of 59 cases identified as MPAL, 53 met inclusion criteria for analysis (1 Induction death, 1 insufficient MRD for analysis, 2 received AML therapy, 1 T/myeloid, 1 MPAL lymphoma). At a mean follow-up time of 3.5±1.7 years, EFS for the overall cohort was 88.0±4.6%. However, EFS was significantly greater for those MRD- versus those MRD+ (94.4±3.9% vs 67.5±12.1%, p=0.0005; Figure 1). Multivariable analysis confirmed MRD as a significant predictor of EFS even after accounting for treatment regimen (hazard ratio 5.0, 95%CI 1.7-15.3, p=0.0036). No other candidate predictors remained significant in the final multivariable models. Although different levels of MRD positivity were significantly associated with poorer EFS, visual examination of the dataset showed the 0.01% threshold to be most representative. Recurrent cytogenetic abnormalities were found in 15/53 children (Trisomy 4+10 n=11/53, TELAML n=1/53, iAMP21 n=2/53, BCRABL n=1/53). No MLL rearrangements were identified. The single patient with BCRABL received continuous imatinib during therapy (no event). No differences at diagnosis were present in those EOI MRD+ versus MRD-, although a WBC count ≥50K/uL was borderline significant (p=0.051).Conclusion: Using a "difference from normal" approach, EOI MRD at a threshold of 0.01% significantly predicts risk for strikingly poorer EFS in children with B/Myeloid MPAL. Children who achieve an EOI MRD-negative remission demonstrate excellent survival with ALL chemotherapy alone. Future investigation is necessary to prospectively validate these findings, examine MRD in T/myeloid and cytogenetic subgroups, and to explore the success of myeloid salvage therapy ±SCT to "rescue" those with a poor response to ALL therapy. [Display omitted] DisclosuresO'Gorman:Becton, Dickinson and Company: Consultancy, Research Funding.

Comprehensive Genomic Profiling for Improved Diagnosis and Therapy of Pediatric Acute Leukemias

Subsets of pediatric acute leukemias are refractory to frontline therapy or relapse with standard treatment. Pediatric leukemias are associated with distinct molecular and genomic alterations. Clinical genomic profiling has been used effectively to improve the diagnosis of adult leukemias. However, its use and utility for pediatric leukemias are not well defined. Here, we used FoundationOne Heme, a clinical grade, high-throughput, hybridization capture-based next-generation sequencing (NGS) assay for targeted sequencing of all exons of 405 genes as well as RNA sequencing of 265 genes. We analyzed the results of samples from 71 patients sequenced on the FoundationOne® Heme assay from children and young adults with acute lymphoblastic leukemia (ALL) (n= 34); early T cell precursor ALL (ETP) (n=3); acute myelogenous leukemia (AML) (n= 19); ambiguous lineage leukemia (n=3); and myelodysplastic syndrome (MDS) (n=12) who were treated at Memorial Sloan Kettering Cancer Center in the Department of Pediatrics over a 26 month period (January 2014-March2016). Age of patients ranged from 1-24 years of age (median =10 years). 42 samples were from patients who had yet to receive cytotoxic therapy (newly diagnosed) and 29 samples were from patients who had relapsed (n=24) or had refractory disease (n=5).The current average turnaround time for FoundationOne® Heme is 12-14 days. Overall 99% (70/71) of specimens were successfully profiled. We identified 230 genomic alterations, including 59.1% (136/230) missense or nonsense mutations of genes, 15.6% (36/230) copy number alterations, and 25.6% (59/230) gene fusions or rearrangements. Molecular aberrations were detected in 81% (9/11) of patients with normal karyotypes; 55% (5/9) of these were cytogenetically cryptic genomic rearrangements including NUP98-NSD1 (n=2) and 1 MLL-PTD (n=1). We identified one novel translocation involving SATB1-PDGFRB in a sample from a patient with pre B ALL. Another patient with pre B ALL was found to have a unique combination of genomic alterations including an MLL-PTD and SSBP2-JAK2. Two patients with MDS were identified as harboring germline GATA2 mutations, indicating the likely benefit of an allogeneic hematopoietic stem cell transplantation. Three patients had therapy modifications based on findings with the addition of dasatinib (ZMIZ1-ABL1), ruxolitinib (PCM1-JAK2) and trametinib (PTPN11mutation), and two patients had risk stratification altered based on MLL rearrangements which were not identifiable by conventional cytogenetics.Overall, the use of comprehensive genomic profiling led to improved accuracy of diagnosis or alteration of therapy in 10% (7/71) cases, including molecular based therapy selection (n=3), escalation of conventional chemotherapy due to high-risk features (n=2), and inclusion of stem cell transplantation (n=2). Thus, clinical genomic profiling of pediatric acute leukemia led to discovery of new pathobiology, improved accuracy of clinical diagnosis, and inclusion of targeted molecular therapies. [Display omitted] DisclosuresKobos:Janssen: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Roshal:BD Biosciences: Consultancy. Zhong:foundation medicine: Employment. Balasubramanian:Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation medicine: Employment. Vergilio:Foundation Medicine: Employment. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Retrospective Evaluation of Correlations Between Genetic Backgrounds and Stem Cell Transplantation for De Novo Pediatric Acute Myeloid Leukemia: A Study from the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Clinical Trial

IntroductionPediatric acute myeloid leukemia (AML) comprises less than 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Approximately 40% of patients still have a relapse after first-line therapies, and the expected long-term survival rate decreases following relapse. Stem cell transplantation (SCT) was a conclusive strategy for de novo AML patients with a high risk and relapsed or refractory patients with standard and intermediate risk in a recent clinical trial. AML is a molecularly and clinically heterogeneous disease caused by various genetic alterations. Thus, it is difficult to accurately evaluate risk stratification even if known representative molecular markers, including KIT, FLT3-ITD, t(8;21)/RUNX1-RUNX1T1, and KMT2A (also known as MLL)-rearrangements, are used.MethodsWe investigated differences in the genetic background between SCT and non-SCT groups in participants of the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Among 369 patients with de novo AML, 175 patients received SCT. A standardized form was used to record clinical variables, including patient demographic information. The clinical data of patients in each risk group were followed for 3 years after the date of final registration. JPLSG performed a central review of morphologic classification and karyotyping based on the World Health Organization Classification, French-American-British classification, and cytogenetic analysis using conventional G-banding. Molecular characterization included mutational analyses of KIT (exons 8 and 17), N- and K-RAS (exons 1 and 2), NPM1 (exon 12), CEBPA (exon 12), FLT3-ITD, NUP98-NSD1,and CBFA2T3-GLIS2 gene rearrangement, as well as KMT2A- partial tandem duplication (MLPA methods). We also evaluated the gene expression of MECOM (also known as EVI1) and PRDM16 (also known as MEL1) because their high expressions are known poor prognostic markers. Overall survival (OS) was defined as the time from AML diagnosis to death or censorship at the last follow-up. Event-free survival (EFS) was defined as the time from AML diagnosis to treatment failure, relapse, death, or last follow-up.ResultsThe 3-year OS among SCT patients (n = 175) was approximately 50%. It was significantly worse than that of non-SCT patients (n = 194, 90%; P < 0.001). Among 137 CBF-AML patients, 44 patients (32%) received SCT and their 3-year OS was 80%. On the other hand, among 232 non-CBF-AML patients, 131 patients (57%) received SCT, and their 3-year OS was only 35%. This result indicated that SCT is beneficial for relapsed CBF-AML, whereas most non-CBF patients who received SCT did not obtain the clinical benefit. In terms of the molecular characteristics, among 58 patients with high EVI1 expression, 39 patients (67%) received SCT, and their 3-year OS was approximately 35%. EVI1 expression was especially useful when using with MLL rearrangement because prognosis of patients with both MLL rearrangement andhigh EVI1 expression were extremely poor. On the other hand, among 84 patients with high PRDM16 expression, 63 patients (75%) received SCT, and their 3-year OS was approximately 20%. Although all AML patients with FLT3-ITD were assigned to receive SCT in the AML-05 trial, FLT3-ITD(+) patients withlow PRDM16 expression had a better outcome than FLT3-ITD(+) patients with high PRDM16 expression (3-year OS: 70% vs. 21%; P < 0.001). This result indicated that FLT3-ITD itself might not necessarily be associated with poor prognosis. PRDM16 gene expression is a useful marker to select patients needing SCT, particularly for patients with FLT3-ITD.ConclusionSCT was beneficial for patients with CBF-AML, whereas SCT was insufficient to rescue patients with non-CBF AML who relapsed, particularly patients with both FLT3-ITD(+) and high PRDM16 expression. New strategies, such as gemtuzumab ozogamicin or haploidentical SCT, are urged to rescue high risk/refractory patients. DisclosuresNo relevant conflicts of interest to declare.

TP53 Mutations in AML Predict Adverse Outcome in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant

Despite significant progress in elucidating the predictive value of somatic mutations in AML following conventional chemotherapy, the implications of extended genomic profiling in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT) are poorly understood. Moreover, little work has been performed correlating somatic mutations identified on an extended gene panel with AML relapse and survival following allo-HSCT, and comparison of the prognostic significance of somatic mutations, transplant conditioning intensity, stem cell source, AML disease types and cytogenetics in an unselected cohort of AML patients undergoing allo-HSCT has not been described.We performed mutation profiling on diagnostic bone marrow samples from 153 patients with AML who underwent allo-HSCT. Median age was 58 years (19-78), and median follow-up after allo-HSCT was 8 months (1-33). Three sequencing platforms were used (Foundation Medicine Hematology NGS panel, N=32; Institutional 28 gene myeloid panel, N=51; 6 gene PCR panel, N=70). Hazard ratios (HR) for relapse and OS were calculated for genes mutated in 5 or more patients (IDH 1/2, JAK2, DNMT3A, FLT3 ITD & TKD, NPM1, TET2, RUNX1, ASXL1 and TP53). Secondary AML was defined as AML arising from a prior myeloid disorder or therapy related AML. Categorical variables were compared with Fischers exact test and continuous variables using Kruskal-Wallis test. Cox regression estimated hazard ratios (HR) for factors associated with relapse and OS post-allo-HSCT. Significant factors were included in multivariate analysis.Factors associated with significantly reduced OS following allo-HSCT included blasts over 5% prior to allo-HSCT, secondary AML, therapy-related AML, monosomal karyotype, mutant RAS and mutant TP53. Blasts over 5% before allo-HSCT, complex or monosomal karyotype and TP53 mutation were associated with risk of relapse (Table 1). No other somatic mutations predicted relapse. Median OS from diagnosis for TP53-mutated AML patients undergoing allo-HSCT was 6 months (Fig 1). Allo-HSCT characteristics were not associated with relapse or OS in this cohort (Table 1).Given its association with OS and relapse, we focused subsequent analyses on patients with mutant TP53. No significant difference in conditioning or donor source was noted between TP53-mutated and wild-type patients. Mutations in TP53 were associated with NCCN poor risk AML, complex karyotype and monosomal karyotype (Table 2). Three patients (33%) with TP53 mutations did not have a complex or monosomal karyotype but did have either MLL rearrangement (N=1) or adverse cytogenetics with del(5q) (N=2). While the number of patients with TP53 mutations was small in this unselected cohort (9/83, 11%), multivariate analysis found that mutations in TP53 and presence of over 5% blasts before allo-HSCT were both predictive of relapse after transplant (table 3).In the TP53-mutant group, 4/9 patients relapsed and 5/9 patients died. 3 died due to non-relapse mortality, and 2 died due to AML. 2 relapsed patients remained alive at last follow-up; one received azacitidine/DLI and attained a flow-cytometry-based MRD-positive CR before progressing, and the other had progressive disease following Decitabine. 3/4 relapsed patients had sequencing at the time of relapse. TP53 mutations were identified at relapse in all 3 patients, indicating that the TP53 mutated clone was involved in relapse.In summary, we found that TP53 mutations and pre-transplant bone marrow blasts were associated with relapse after allo-HSCT in this unselected cohort of AML patients. TP53 mutations were associated with NCCN high-risk AML, complex and monosomal karyotype and therapy-related AML. We did not find other somatic mutations to be associated with relapse. Further study in a larger cohort is necessary to confirm the prognostic implications of TP53 mutations and to assess the value of other mutations in predicting outcomes after allo-HSCT. The poor outcomes observed for patients with TP53-mutated AML highlight the need to assess TP53 mutation status to identify patients who may benefit from maintenance strategies or other pharmacologic or cellular approaches after transplant to reduce the risk of relapse in these patients. [Display omitted] [Display omitted] [Display omitted] DisclosuresLevine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Complementary Molecular Biology Techniques for the Detection of Unbalanced Chromosomal Abnormalities in Acute Myeloid Leukemias and Myelodysplastic Syndromes: Comparison of Fluorescence In Situ Hybridization (FISH) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background: The accurate detection of cytogenetic abnormalities in Acute Myeloid Leukemias (AML) and Myelodysplastic Syndromes (MDS) play an important role in prognostic value and often assist in therapeutic choice. The most commonly used genetic test for the diagnostics of these conditions is karyotype (KT), which is able to detect frequent non-random chromosomal abnormalities. However, this technique requires metaphases and offers low resolution (5Mb), which may lead to the loss of clinically important smaller genomic rearrangements. Therefore, a complementary technique is sometimes needed. FISH, for example, is a highly reliable technique, it can be performed in interphases, has higher resolution, and can detect balanced translocations (important for prognosis mainly in AML), although it is very expensive. As an alternative method, MLPA is usually less costly, is able to evaluate more genomic regions at once and is performed with genomic DNA, which is convenient for biological material availability. On the other hand, it is not able to detect balanced translocations due to its semi-quantitative PCR-based method. The aim of this study was to evaluate MLPA as a complementary technique to KT, and to compare the outcomes of MLPA and FISH panels for the high resolution detection of common unbalanced genetic alterations in MDS and AML.Methods: A total of 23 samples from patients diagnosed with MDS (n=16)/AML(n=6) were tested for KT and MLPA. 20 metaphases were analyzed in G-banding KT. MLPA (Kits P144 and P145, MRC-Holland) was performed with genomic DNA extracted from bone marrows. A subset of 10 samples was tested for FISH MDS/AML panel (PML/RARA, CBFB/MYH11, RUNX1/RUNX1T1, MLL rearrangement, del5q, del7q, de17p and del20q, Cytocell), and the outcomes were compared to MLPA. Reagent costs were also compared between MLPA and FISH techniques.Results: Outcomes from KT and MLPA were mostly concordant: 91.3% of samples had either concordant or partially concordant results (considering "partially concordant" when MLPA could detect smaller genetic alterations not achieved by KT). Discordance between KT and MLPA occurred in two cases: (i) MLPA detected a deletion of RUNX1 gene (chr 21) in a previously normal KT, and (ii) MLPA showed different abnormalities than those in a complex KT case, with low mitotic index. When comparing regions that are notoriously related to MDS/AML (-5/del5q, -7/del7q, +8, del11q, del13q, del17p, del20q and del21q), all results were concordant between MLPA and KT. Comparison of MLPA and FISH was performed with 10 samples, of which 4 were negative for genetic alterations and 6 were positive. 90% of samples had concordant results, and 10% (one sample) showed extra genetic alterations in MLPA (del7q; del11q and del20q). This is expected since MLPA analyzes more genetic regions then FISH MDS/AML panel at once. Genomic regions accessed both by MLPA and FISH included -7, +8, del17p, del21q.Regarding financial expenses (cost of commercial MDS/AML panels and additional reagents), MLPA costs approximately U$280.00 (considering test sample plus internal controls), while FISH costs U$555.00 (values were converted from quotes provided by regional distributors and converted to dollars).Conclusions: MLPA results are equivalent to FISH for the detection of MDS/AML frequent unbalanced genetic alterations. Both techniques are reliable as complementary molecular biology tests for KT. Moreover, MLPA is a friendly and less expensive technique for the detection of high resolution genomic unbalanced abnormalities, frequently adding extra information to KT results. Nevertheless, it is important to notice that only FISH can detect balanced translocations with significant prognostic value in AML. DisclosuresNo relevant conflicts of interest to declare.

Landscape of Driver Mutations and Their Clinical Impacts in Pediatric Acute Lymphoblastic Leukemia

IntroductionB-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner.MethodsWe enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection.ResultsThe median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]).Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation.Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified.Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q < 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL.ConclusionsWe revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Novel Targets Identified By in Vitro Screening of Patient Samples with Adult Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL) is considered a success story in pediatrics, with cure rates greater than 85%. In contrast, the estimated cure rate for adolescent and young adult (AYA) patients is estimated at 60% and is just 20-40% for adults. This is thought to be secondary to chemotherapy associated toxicities for older patients as well as different disease biology. In the AYA population, more patients carry the chemo-resistant Ph-like phenotype, with an estimated incidence of up to 27%. However, the genetic profile of adult ALL has not been well described. In addition, although the ABL specific kinase inhibitors and JAK inhibitors are predicted to be active in Ph-like ALL, there are no reports on functional drug sensitivity data in AYA or adult ALL. Our group has been collecting functional data on both newly diagnosed and relapsed AYA and adult patients since 2010; in this study we summarize the functional data obtained during this time. Materials and Methods: All clinical samples were obtained with informed consent and approval of the institutional review board at OHSU. Data obtained on both newly diagnosed and relapsed patients obtained from Jan 2010 to present was reviewed. At the time of diagnosis or at relapse, mononuclear cells were isolated from bone marrow aspirates of patients and exposed to a library of 130 small molecule inhibitors at varying concentrations. After 72 hours, cell viability was assessed via an MTS based assay and drug efficacy was assessed based on the IC50 of of each panel drug against each patient sample. The calculated IC50 for each drug was compared to the median IC50 of over 1000 patient samples and was deemed hypersensitive if the IC50 value was below the 20th percentile. A pathway was considered to be actively targeted if patients had more than one inhibitor targeting that pathway identified as hypersensitive, or a single drug was considered valid if it was hypersensitive in replicate testing.Results: We identified a total of 42 adults with either B-cell or T-cell ALL who had analyzable data; those with Ph+ ALL were excluded. Of these, 28 had B cell ALL including the MLL rearrangement and 14 had T cell ALL. For the B cell ALL group, 17 had data obtained at diagnosis and 11 at relapse. 75% of patients had at least one active inhibitor identified, with the range being from 2-25 inhibitors per patient targeting from 1-5 different pathways per patient. We found that 35% of patient samples were sensitive to a JAK inhibitor, an ABL inhibitor or both. In addition, we found overlapping sensitivities with 43% of patient samples sensitive to PI3K/AKT/mTOR inhibitors, 43% of samples were sensitive to IGF1R inhibitors, 49% were sensitive to PDGFR/VEGFR inhibitors, 18% were sensitive to p38MAPK inhibitors and 11% each were sensitive to CSF1R or FLT3 inhibitors. Of note, all of the FLT3 inhibitor sensitivities were within the MLL-rearranged subgroup. Among the T-ALL group, 10 had data obtained at diagnosis and 4 at relapse. 52% of the samples had at least one inhibitor identified, with the range being from 1-20 inhibitors targeting from 1-4 pathways per patient. Drugs targeting the following pathways were identified: JAK family (57.1%), ERBBB family (57.1%), SYK (28.5%), and HDAC, MDM2 and aurora kinase (14.3% each).Discussion: This study provides the evidence that there are multiple novel potentially targetable pathways present in AYA and adult patients with ALL. Given the poor outcomes in this group of patients, further exploration of these potentially targetable pathways is warranted. DisclosuresTyner:Agios Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Inctye: Research Funding; Janssen Research & Development: Research Funding; Seattle Genetics: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Druker:Curis: Patents & Royalties; Pfizer: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; AstraZeneca: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute.

Comparison of Efficacy and Safety of Two Types of E.coli Asparaginase (Native or Pegylated) for Treatment of Adult Patients with High-Risk (HR), Philadelphia (Ph) Chromosome-Negative ALL Included in the Prospective MRD-Oriented Protocol ALL-HR-11 from the Spanish Pethema Group

Background and objective. Asparaginase (ASP) is an essential drug for ALL treatment. Two types of E.coli ASP (native and pegylated) are used in clinical trials for treatment of children and adults with ALL, but to our knowledge direct comparison between these two types of ASP in the same protocol has not been performed. This study aimed to compare the efficacy and safety of native vs. PEG-ASP in adult patients with HR, Ph-negative ALL.Patients and methods. HR ALL included one or more of the following parameters at baseline: age 30-60 yr., WBC count >30x109/L for B-cell precursor ALL or >100x109/L for thymic T-ALL, pro-B, early or mature T-ALL, 11q23 or MLL rearrangements or complex karyotype. Induction therapy consisted of vincristine, prednisone, daunorubicin and ASP (native 10,000 IU/m2, i.v., days 16-20 and 23-27 or PEG 2,000 IU/m2, iv, day 15 depending on center decision) for 4 weeks (Induction-1). FLAG-Ida was administered as intensified induction (Induction-2) in patients not achieving CR or in those in CR with MRD≥0.1% at the end of induction, and those patients proceeded to allogeneic HSCT if CR was attained. Patients in CR and MRD<0.1% proceeded to early consolidation therapy including 3 cycles with rotating cytotoxic drugs with high-dose methotrexate, high-dose ARA-C and ASP (native 20,000 IU/m2 on day 3 of each cycle or PEG 2,000 IU/m2 on day 3 of each cycle). Patients continued with delayed consolidation (identical to that of early consolidation) followed by maintenance therapy up to 2 yr. in continuous CR if MRD levels after consolidation were <0.01%, otherwise they were assigned to allogeneic HSCT. The cumulated doses of native ASP and PEG-ASP for patients who completed the induction and early+delayed consolidation were 220,000 IU/m2 and 14,000 IU/m2, respectively. No ASP levels were assessed.Results. Ninety-onepatients received native ASP and 35 PEG-ASP in Induction-1. The two groups of patients were comparable for the main clinical and hematologic ALL parameters. No differences were observed in the CR rate (86% vs. 86%), in the frequency of MRD level <0.1% after Induction-1 (63% vs. 70%), and that of MRD level <0.01% after early consolidation (74% vs. 92%, p=0.19), as well as in the proportion of patients submitted to allogeneic HSCT (20% vs. 14%). No differences were found in overall survival or disease-free survival probabilities at 3-years according to the type of ASP (OS: 60% [95%CI: 47% ; 73%] vs. 57%[95%CI: 36% ; 78%], p=0.872 and DFS: 40% [95%CI: 25% ; 55%] vs. 58% [95%CI: 36% ; 80%], p=0.302). Abnormalities in the coagulation parameters were significantly more frequent in patients receiving PEG-ASP in Induction-1 (18% vs. 35%, p=0.045). These abnormalities, together with hepatic toxicity were significantly more frequent in patients receiving PEG-ASP in early consolidation (1% vs. 13%, p=0.003, and 5% vs. 34, p<0.001, respectively). A trend for more allergic reactions was seen in patients receiving native ASP (18% vs. 5%, p=0.1) No differences in the frequency of ASP discontinuation rate were observed (6 % vs. 3%).Conclusions. In HR, Ph-negative adult ALL patients included in the PETHEMA ALL-HR 11 protocol the type of E.coli ASP (native vs. PEG) did not have impact on response and outcome. Allergic reactions were more frequently seen with native ASP and coagulation abnormalities and hepatic toxicity were most frequent with PEG-ASP. Most of the differences in toxicity can be explained by the schedule of ASP given in consolidation (single dose of native ASP vs. single dose of PEG-ASP in each cycle).Supported in part by grants RD12/0036/0029 (RTICC, FEDER), PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain DisclosuresNo relevant conflicts of interest to declare.

Outcomes of Childhood Acute Lymphoblastic Leukemia with CNS-2, CNS-3 and Traumatic Lumbar Puncture with Blasts in Two Eras of Taiwan Pediatric Oncology Group (TPOG)-ALL-2002 Study with or without Cranial Radiation

IntroductionThe nationwide TPOG-ALL-2002 protocol for treating children with acute lymphoblastic leukemia (ALL) was activated in January 2002 in Taiwan. To eliminate cranial radiation (CrRT)-related sequelae and minimize the adverse prognostic impact of traumatic lumbar puncture with blasts (TLP), we conducted a prospective study beginning in 1999 to modify CNS-directed therapy with delayed first triple intrathecal therapy (TIT) and omission of CrRT for all risk groups of newly diagnosed ALL. This approach improved CNS control at the single-institution study (J Clin Oncol, 2014) and has been used in whole TPOG since January 2009.Patients with non-CNS-1 status (i.e., CNS-2, CNS-3 and TLP) have higher risk of CNS relapse and need intensive CNS therapy. On behalf of TPOG members, we reported the outcomes of these non-CNS-1 patients in the two eras (2002-2008 and 2009-2012) with or without the modified CNS therapy.MethodsFrom 2002 to 2012, all newly diagnosed ALL children in Taiwan were enrolled in TPOG-ALL-2002 protocol (Leukemia, 2010). B-precursor ALLs were stratified into standard risk (SR), high risk (HR) and very high risk (VHR) groups, according to age, WBC count, cytogenetics and molecular analysis at diagnosis. T-cell ALL was designated as VHR. Since 2009, patients had been treated with TIT alone without CrRT. The first TIT was performed until the disappearance of blasts from peripheral blood (PB), no later than 10 days. SR patient with detectable PB blasts on day 8 was upgraded to HR group.SR with CNS-1 received 14 doses of TIT, and those with CNS-2, or TLP got 2 additional TITs during induction and 3 additional TITs during maintenance therapy. Totally, SR with CNS-2 or TLP received 19 doses of TIT. And SR patients with CNS-3 or cranial nerve palsy at diagnosis were classified as HR. For HR with CNS-1, 17 doses of TIT were given. If HR groups with CNS-2, CNS-3, or TLP, they will receive total 25 doses of TIT including 2 additional ones during induction and 6 additional ones during maintenance therapy. Before 2009, only SR and HR with refractory CNS leukemia (failure to clear CSF blasts after 3 consecutive TITs) and those with CNS relapse will receive CNS radiation.For VHR group before 2009, CrRT of 18 Gy was given to all patients at the end of remission induction. Instead, TIT was given for age < 2 years. After 2009, CrRT was totally omitted. VHR with CNS-1 received 19 doses of TIT. For VHR patients with CNS-2, CNS-3 or TLP at diagnosis, TIT was administered once/week until achieving CSF remission and then once 4 weeks during maintenance therapy.ResultsSince January 2009, all patients received TIT alone, irrespective of risk groups. The two cohorts, totally 1,366 patients, 909 treated with CrRT (2002-2008) and 457 without CrRT (2009-2012) were balanced in presenting features and distribution of CNS status. There were no significant differences between two eras in the event-free survival (EFS), overall survival (OS), incidences of isolated CNS relapse and any CNS relapse (Pediatr Blood Cancer, accepted June 2016).The number of patients with CNS-2, CNS-3 and TLP were 35, 26 and 19 before 2009; and 18, 10 and 9 after 2009, respectively. Patients with younger age (< 1 year), higher WBC counts (> 100 X 109/L), HR/VHR or MLL rearrangement had significantly more non-CNS-1 status in the era of 2002-2008. And the significant factors prone to be non-CNS-1 were male, T-lineage and HR/VHR with the first TIT delaying. There were no significant differences between the two eras in the 5-year EFS and OS. Though the incidence of 5-year any CNS relapse was not significantly different, fewer relapses were observed in the era of 2002-2008 (Table 1). Before 2009, eleven CNS relapses experienced between 1 month and 3.1 years (median, 10 months) after remission. Two relapses after 2009 occurred on 1 month and 7 months, respectively.ConclusionsDelayed first TIT until the clearance of circulating blasts and total omission of CrRT did not compromise the survivals and CNS control of childhood ALL patients with CNS-2, CNS-3 and traumatic lumbar puncture with blasts. Moreover, this approach prevents the adverse sequelae caused by CrRT. Furthermore, a skillful TIT is mandatory to improve results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunophenotypic Profiling of Childhood B-Cell Precursor Acute Lymphoblastic Helps Identifying Genetic Subtypes, Including Recently Identified Ones

Background B-cell precursor acute lymphoblastic leukemia (B-ALL) comprises distinct entities characterized by recurring genetic alterations. These classifying alterations are associated with gene expression profiles, reflecting the specific biology of each genetic subgroup. In patients with none of these classifying alterations, referred as "B Other", RNAseq recently led to the identification of a huge number of in frame fusions which are likely to define novel genetic subgroups. Leukemic blasts often show aberrant antigen expression, giving rise to leukemia-associated immunophenotypic profiles (LAIP) distinct from their normal immature B-lineage counterparts. These variations in protein expression are likely to reflect the specific biology of B-ALL. We evaluated how LAIP were associated with the various classifying genetic lesions, including recently identified ones.Materials and Methods A cohort of 299 childhood B-ALL was studied prospectively. Genetic typing by cytogenetics, MLPA and RT-PCR identified high hyperdiploidy (Heh, n=77), ETV6-RUNX1 (n=81), TCF3-PBX1 (n=21), TCF3-HLF (n=3), MLL rearrangement (n=25), BCR-ABL/Phie-like fusions (n=10), ERGdel (n=8), TSLPR (n=8), iAMP21 (n=6), Hypodiploidy (n=6). The remaining patients were considered "B-Other" (n=54). Immunophenotyping was performed by 6-8-colors flow cytometry with a panel of 29 antibodies targeting surface proteins routinely used for ALL diagnosis and MRD assessment. The log of Ratio Fluorescence Intensity (RFI) of B-ALL/normal B precursor cells was calculated for each marker. RNAseq was performed in 18 B-Other and 8 BCR-ABL/Phie-like. Fusions were detected using TopHat2. Samples were classified using hierarchical clustering and/or principal component analyses (R software).Results Unsupervised clustering identified 6 groups with shared pattern of LAIP. Four groups were associated with known classifying lesions: ETV6-RUNX1 (of which 87% co-clustered), Heh (82%), TCF3-PBX1 (100%) and MLL (96%). ALL that did not cluster according to their genetic group mostly clustered (23/26; 89%) in two groups, hereafter named "B Other-1" and "B Other-2", which mainly contained ALL lacking any previously known cytogenetic lesion. Interestingly, 3 of 5 ETV6-RUNX1 with IKZF1del clustered in the "B Other 2", suggesting that misclassifications may not be due to poor LAIP specificity but rather reveal distinct biological features in some ALL.Although comprising too few cases to define specific groups, 8/10 B-ALL with BCR-ABL/Phie-like, 3/3 TCF3-HLF, and 8/8 TSLPR co-clustered in "B Other 2", and 7/8 ERGdel in "B Other 1". No clear clustering was observed for Hypodiploid and iAMP21. B-Other ALL were allocated either to oncogenic groups, e.g. TCF3-PBX1 (n=7) or MLL (n=7), or to the "B Other" groups (n=35). Only one B Other, harboring an IGH-IL3 fusion, co-clustered with BCR-ABL/Phie-like ALL. RNAseq of 18 B-Other identified no additional Phie-like fusion and none of these B-Other co-clustered with a group of 8 controls BCR-ABL/Phie-like. This makes unlikely that some Phie-like cases remained unidentified. However, other fusions involving MEF2D (n=3), PAX5 (n=5) or ZNF384 (n=2) were evidenced in B Other ALL. Cases with MEF2D or ZNF384 clustered both at the transcriptome and protein level. Using LAIP clustering, MEF2D ALL layed close to those with TCF3 fusions. ZNF384 cases clustered in the MLL group, consistent with a lack of CD10 expression combined to myeloid antigen expression. PAX5 ALL were allocated to the "B Other 2" group with no clear clustering and, more surprisingly, no consistent effect on CD19 expression. Secondary aberrations such as deletions in IKFZ1 or CDKN2A/B genes did not significantly affect clustering.Conclusion Unsupervised clustering based on surface protein expression allowed to classify most childhood ALL genetic groups, including Phie-like, MEF2D or ZNF384, which are challenging to identify due to the diversity of the underlying gene fusions. LAIP can thus rapidly orientate diagnosis, in particular for Phie-like which are candidates to targeted therapies. Interestingly, B-Other that clustered in known genetic groups may present biologically equivalent alterations undetected in our routine workup. Further studies will allow to assess whether cases co-clustering with ETV6-RUNX1 (n=2) or ERGdel (n=6) correspond to the recently described ETV6-RUNX1-like or DUX4 group respectively. DisclosuresBaruchel:Servier: Consultancy; Novartis: Consultancy; Jazz: Consultancy; Baxalta: Research Funding; Celgene: Consultancy.