Japanese Pediatric Leukemia/Lymphoma Study Group Research Articles

Negative CD19 Expression Is Associated with Inferior Relapse-Free Survival in RUNX1-RUNX1T1-Positive Acute Myeloid Leukemia; The Japanese Pediatric Leukemia/Lymphoma Study Group Experience from the AML-05 Study

Background: Acute myeloid leukemia (AML) with RUNX1-RUNX1T1 and CBFB-MYH11 have been recognized as core-binding factor (CBF) AML accounting for 25% of the pediatric AML patients. CBF-AML patients have been considered to have a good prognosis, but 30 - 50 % of the RUNX1-RUNX1T1-positive AML patients experience relapse. This finding suggests that some population of them have risk factors associated with poor outcome. Previous studies revealed that KIT activating mutation as a predictor of poor outcome. In addition, previous studies of relatively small number of patients also revealed that CD56 positivity or CD19 negativity were also poor prognostic factors. However, the relationship between KIT activating mutation and specific pattern of cell surface antigens has not been fully investigated.Aim and methods: We performed a retrospective analysis of RUNX1-RUNX1T1-positive AML patients treated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 protocol to determine risk factors of relapse using the integration of data including pattern of cell surface markers on leukemic cell at diagnosis, genetic abnormalities, and clinical characteristics. Flow cytometric analysis of immunophenotyping was performed in the central laboratories using same panel of antibodies. Conventional cytogenetic analysis using G-banding was performed as part of the routine work-up. Molecular study using quantitative RT-PCR for the detection of RUNX1-RUNX1T1 and PCR for the detection of FLT3-ITD was also performed as part of the routine work-up. Screening of mutation of 8 genes, such as NRAS, KRAS, KIT, WT1, C/EBPA, ASXL1, ASXL2, and CSF3R, was performed by genomic PCR and Sanger sequencing. A total of 106 AML patients enrolled in the JPLSG AML-05 study were RUNX1-RUNX1T1-positive AML, but we could not obtain the data of cell surface marker in 6 of them. Thus, we analyzed 100/106 (94.3%) patients with RUNX1-RUNX1T1-positive AML. Statistical analysis was performed by Kaplan-Meier method with log-rank test. A Cox proportional hazards model was used to determine risk factors for survival and relapse. Results are reported as adjusted odds ratios with 95% confidence intervals. Statistical significance was set at p < 0.05.Results: In entire study population, 8 of the 100 patients died and 24 of the 100 patients experienced relapse, respectively. The 3-year overall survival (OS) and relapse-free survival (RFS) rates were 91.7 % (95 % CI; 83.2 - 96.0) and 69.5 % (95 % CI; 59.0 - 77.9), respectively. In terms of genetic analysis, 21 / 100 (21.0%) patients had KIT exon 17 mutation, 11/100 (11.0%) had KIT exon 8 mutation, 6/100 (6%) had KRAS mutation, 16/100 (16%) had NRAS mutation, 2/100 (2%) had C/EBPA mutation, 2/100 (2%) had WT1 mutation, 6/100 (6%) had ASXL1 mutation, 9/100 (9%) had ASXL2 mutation, and 6/100 (6%) had CSF3R mutation. FLT3-ITD was also identified in 3 of the 100 (3%) patients. In terms of cell surface marker expression pattern, CD19 expression was negative in 59 / 100 (59.0%) patients, and CD56 expression was positive in 43 / 100 (43.0%) patients. Patients with KIT exon 17 mutation were significantly accumulated in the CD19 negative (CD19 (-)) population (18 / 59 vs. 3 / 41, p < 0.001). Survival analysis revealed that KIT exon 17 mutation and CD19 (-) were associated with inferior 3y-RFS (KIT exon 17 mutation: negative vs positive, 74.6 vs 50%, p<0.01, CD19 expression: positive vs negative, 83.1 vs 59.8 %, p<0.01). On the contrary, CD56 (+) was not associated with poor RFS (positive vs negative, 66.1 vs 74.7%, p=0.15). In addition, CD19 (-) was associated with poor RFS even in the patients without KIT exon 17 mutation (positive vs negative, 85.4 vs 65.2%, p=0.04). Finally, CD19 (-) was the sole significant risk factor of relapse (hazard ratio; 3.09, 95% CI; 1.26 - 7.59, p < 0.01) by multivariate analysis.Discussions: This study revealed that CD19 negativity might be a distinct character of poor prognostic subgroup of RUNX1-RUNX1T1-positive AML. On the contrary, CD56 positive was not poor prognostic factor of RUNX1-RUNX1T1-positive AML. Although majority of the patients with KIT exon 17 activating mutation belonged to CD19(-) patients, CD19 (-) patients without KIT exon 17 mutation still showed inferior RFS, suggesting biological difference between CD19 (+) and CD19 (-) RUNX1-RUNX1T1 AML should be investigated. DisclosuresNo relevant conflicts of interest to declare.

Recurrent Genomic Aberrations of D-Type Cyclins Are Therapeutic Targets of CDK4/6 Inhibitors in t(8;21) and MLL-Rearranged Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease, characterized by expansion of undifferentiated myeloid precursor cells. The outcome for AML has improved through optimal treatment protocols, new drugs, and better supportive care; however, relapse remains common and patients with relapsed AML have poor prognosis. Recent genome-wide analyses revealed several recurrently mutated genes in AML, however, few of these driver mutations have been developed as therapeutic targets to date. In AML, t(8;21) and MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape is limited.Patients and Methods:The AML-05 study is a Japanese nationwide multi-institutional study of children (age < 18 years old) with de novo AML, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). The trial was registered with the UMIN Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm; number UMIN000000511) and conducted in accordance with the principles set down in the Declaration of Helsinki, and approved by the Ethics Committees of all participating institutions. All patients, or their parents / guardians, provided written informed consent. For whole-exome sequencing (WES), whole-exome capture was accomplished by liquid phase hybridization of sonicated genomic DNA using a bait cRNA library (SureSelect Human ALL Exon V5 or V5 Inc RNA 5 kit), following the manufacturer's protocol. Massively parallel sequencing of the captured targets was performed using a HiSeq 2000/2500 (Illumina) with the paired-end 126-133 bp read option. For targeted sequencing, target enrichment was performed using a SureSelect custom kit (Agilent) designed to capture all coding exons of the 338 genes. Similarly, CCND1, CCND2, and CCND3 were also captured and sequenced in t(8;21) AML samples. For cell cycle analysis, cell lines were treated with DMSO, palbociclib (500 nM), or abemaciclib (500 nM) for 24 h. Then, cells were stained with propidium iodide and analyzed using a FACS Canto II flow cytometer (BD Biosciences).Results and Discussion:First, we analyzed paired AML tumor and germline samples from nine pediatric MLL-rearranged AML patients by WES. In total, 52 mutations (mean, 5.8 mutations/patient) were identified, including known mutational targets in AML, such as FLT3, BRAF, SETD2, BCORL1, and WT1. Moreover, novel CCND3 mutation was detected in one patient. Next, we analyzed 56 samples from patients with pediatric MLL-rearranged AML enrolled in the JPLSG AML-05 study, using targeted sequencing. We selected 338 genes, among which were previously reported and putative driver genes, including CCND3, for targeted sequencing. We identified eight mutations in CCND3 in five pediatric MLL-rearranged AML patients (8.9%). All mutations were clustered in the PEST domain. Four of the eight mutations were R271fs, which is a known hot-spot mutation in lymphoid malignancies. Mutations of the other D-type cyclins (CCND1, CCND2) have been reported in t(8;21) AML (Leukemia, 2017); therefore, we also searched for mutations in CCND1, CCND2, and CCND3 by targeted sequencing of samples from pediatric t(8;21) AML patients (n=105). CCND1 (n=3, 2.9%) and CCND2 (n=8, 7.6%) mutations were detected; however, no mutations of CCND3 were detected. By contrast, there were no mutations of CCND1 or CCND2 in MLL-rearranged AML (n=56), suggesting that mutations of D-type cyclins exhibit a subtype-specific pattern in AML. A recent study demonstrated that genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4/6 inhibitor (Cancer Cell, 2017), therefore, we also examined the effects of CDK4/6 inhibitors (abemaciclib and palbociclib) on two t(8;21) AML cell lines (Kasumi-1 and SKNO-1) and five MLL-rearrangement AML cell lines (ML-2, MV4-11, MOLM-13, THP-1, and NOMO-1) were analyzed. All cell lines described above exhibited impaired proliferation after treatment with CDK4/6 inhibitors. Furthermore, treatment of these cell lines with CDK4/6 inhibitors resulted in detection of lower frequencies of S/G2/M phase cells by flow cytometry, suggesting that cells were arrested in G1 phase via CDK4/6 inhibition. These data provide further insights into the genetic basis of, and potential therapeutic strategies in t(8;21) and MLL-rearranged acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Transcriptome Analysis Offers a Comprehensive Illustration of the Genetic Background of Pediatric Acute Myeloid Leukemia: Japan Pediatric Leukemia/Lymphoma Study Group AML-05

Background Recent advances in the genetic understanding of acute myeloid leukemia (AML) have improved clinical outcomes in pediatric patients. However, approximately 40% of patients with pediatric AML relapse, contributing to a relatively low overall survival (OS) rate of approximately 70%. The objective of this study was to reveal the comprehensive genetic background of pediatric AML. Methods We performed transcriptome analysis (RNA-seq) in 139 of the 369 patients with de novo pediatric AML who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial and investigated correlations between genetic aberrations and clinical information. Findings Using RNA-seq, we identified 54 in-frame gene fusions and one RUNX1 out-of-frame fusion in 53 out of 139 patients. Moreover, we found at least 258 gene fusions in 369 patients (70%) through RT-PCR and RNA-seq. Six gene rearrangements were newly identified, namely, NPM1-CCDC28A, TRIP12-NPM1, NPM1-DNAJC1, TBL1XR1-RARB, ETV6-CTNNB1, and RUNX1-FNBP1. In addition, we found rare gene rearrangements, namely, MYB-GATA1, NPM1-MLF1, ETV6-NCOA2, ETV6-MECOM, RUNX1-PRDM16, RUNX1-CBFA2T2, and RUNX1-CBFA2T3. Among the remaining 111 patients, KMT2A-PTD, biallelic CEBPA, and NPM1 gene mutations were found in 11, 23, and 17 patients, respectively. These mutations were completely mutually exclusive with any gene fusions. Interpretation RNA-seq unmasked the complexity of gene rearrangements and mutations in pediatric AML. We identified potentially driver alterations in nearly all patients with AML, including novel gene fusions. Based on the results of the RNA-seq, risk stratification should be reconsidered. Funding Statement: Ministry of Education, Culture, Sports, Science, and Technology of Japan and Japan Agency for Medical Research and Development. Declaration of Interests: I have no financial relationships to disclose. Ethics Approval Statement: The present study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review boards of Gunma Children’s Medical Center and the participating institutes and the ethical review board of the JPLSG AML-05 trial.

Monitoring of fusion gene transcripts to predict relapse in pediatric acute myeloid leukemia.

In acute myeloid leukemia (AML), accurate detection of minimal residual disease (MRD) enables better risk-stratified therapy. There are few studies, however, on the monitoring of multiple fusion transcripts and evaluation of their accuracy as indicators of MRD at multiple time points. We retrospectively examined RNA obtained from 82 pediatric AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study. The expression of six important fusion transcripts (AML1(RUNX1)-ETO, CBFB-MYH11, MLL(KMT2A)-AF9, MLL-ELL, MLL-AF6, and FUS-ERG) was analyzed at five time points 30-40days apart following diagnosis. In patients with AML1-ETO (n=36 at time point 5), all six patients with >3,000 copies and four of 30 patients with ≤3,000 copies relapsed. AML1-ETO transcripts persisted during treatment even in patients without relapse, as well as CBFB-MYH11 transcripts. In contrast, in patients with MLL-AF9 (n=9 at time point 5), two patients were positive for MLL-AF9 expression (>50 copies) and both relapsed. Only one of seven MLL-AF9-negative patients relapsed. In the AML1-ETO group, MRD-positive patients (>3,000 copies at time point 5) had significantly lower relapse-free survival (RFS; P<0.0001) and overall survival (OS; P=0.009) than MRD-negative patients. Similarly, in the MLL-AF9 group, MRD-positive patients (>50 copies at time point 5) had significantly lower RFS (P=0.002) and OS (P=0.002) than MRD-negative patients. Detection of MLL-AF9 transcripts on real-time quantitative polymerase chain reaction is a promising marker of relapse in pediatric AML. In contrast, the clinical utility of detecting AML1-ETO and CBFB-MYH11 expression is limited, although higher AML1-ETO expression can be a potential predictor of relapse when assessed according to an optimal threshold.

Multiplex fusion gene testing in pediatric acute myeloid leukemia.

Gene abnormalities, particularly chromosome rearrangements generating gene fusion, are associated with clinical characteristics and prognosis in pediatric acute myeloid leukemia (AML). Karyotyping is generally performed to enable risk stratification, but the results are not always consistent with those of reverse transcription-polymerase chain reaction (RT-PCR), and more accurate and rapid methods are required. A total of 487 samples from de novo AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study (n = 448), and from acute promyelocytic leukemia (APL) patients enrolled in the JPLSG AML-P05 study (n = 39) were available for this investigation. Multiplex quantitative RT-PCR was performed to detect eight important fusion genes: AML1(RUNX1)-ETO(RUNX1T1), CBFB-MYH11, MLL(KMT2A)-AF9(MLLT3), MLL-ELL, MLL-AF6(MLLT4), FUS(TLS)-ERG, NUP98-HOXA9, and PML-RARA. Fusion genes were detected in 207 (46.2%) of the 448 AML-05 patient samples. After exclusion of two samples with PML-RARA, no chromosomal abnormalities were identified on karyotyping in 19 of 205 patients (9.3%) positive for fusion genes on RT-PCR. Fusion genes were confirmed on fluorescence insitu hybridization (FISH) in 11 of these 19 patients. In contrast, fusion genes were detected in 37 of 39 patients (94.9%) from the AML-P05 study, and 33 of these results were consistent with the karyotyping. There were discrepancies in four patients (10.8%), three with normal karyotypes and one in whom karyotyping was not possible. All four of these patients were PML-RARA positive on FISH. Multiplex quantitative RT-PCR-based fusion gene screening may be effective for diagnosis of pediatric AML.

Clinical and Biological Features of Pediatric Acute Myeloid Leukemia with Primary Induction Failure in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Study

INTRODUCTIONTreatment outcome of pediatric acute myeloid leukemia (AML) patients has improved over the past few decades due to intensive chemotherapy. Primary induction failure (PIF), defined as failure to achieve complete remission after the second induction chemotherapy, occurs in around 10% of children with newly diagnosed AML. Effective treatment strategy for these patients is not established and their poor prognoses have been reported elsewhere. Here we investigated the clinical and biological features of pediatric AML patients with PIF who were registered in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Study (AML-05). METHODSWe conducted a retrospective survey of patients who were registered in the AML-05. We analyzed probabilities of overall survival (OS) according to gender, age at diagnosis, white blood cell count (WBC) at diagnosis, FAB classification, etc. Genetic analysis of primary leukemic blasts was performed. We screened fusion genes and gene mutations as follows: RUNX1-RUNX1T1, CBFB-MYH11, NUP98-NSD1, DEK-NUP214, MLL-PTD, WT1, NPM1, and MLL-rearrangements, gene mutations of KIT and FLT3-ITD. Furthermore, we evaluated the clinical and biological background of these PIF patients to define their prognostic factors statistically. RESULTS: Among the 443 patients registered in the AML-05 between November 2006 and December 2010, 43 patients (9.7%) had PIF. The observation period from the date of PIF was 5-78 months (median, 20 months). The age at diagnosis was 0.1-17 years (median, 6.1 years). There was no significant difference in gender (28 males vs15 females). The 3-year OS rate was 19.0% (95% confidence interval 1.0%-38.0%). This was extremely poor compared to the patients who achieved complete remission (CR): 66.7% for high-, 73.2% for intermediate-, and 92.6% for low-risk groups, respectively. Disease recurrence is the major cause of death (n=19, 46.3%). The mean WBC at diagnosis of PIF patients was 96 x 109/L: this is significantly higher than that of non-PIF patients (51 x 109/L, P<0.01, Cox regression analysis). Morphologically, FAB classification M7 was more frequently detected in the PIF group compared with non- PIF group, although not statistically significant: 11 patients (25.6%) vs 43 patients (9.1%) (P=0.07). Unfavorable prognostic cytogenetics such as FLT3-ITD (n=16, 37.2%), NUP98-NSD1 (n=6, 15.4%), and complex karyotype (n=9, 20.9%) were detected more frequently in PIF patients, as well as MLL-PTD (n=7, 16.2%), c-KIT mutations (n=6, 14.0%), DEK-NUP214 (n=2, 4.7%), and monosomy 7 (n=6, 14%). There were no cases with WT1 or NPM1 mutations. Only 3 patients had core binding factor leukemia with RUNX1-RUNX1T1 or CBFB-MYH11 in the PIF patients. Thirty-seven PIF patients received stem cell transplantation (SCT). The patients without SCT did not achieve CR, and died of primary disease. The patients who had achieved CR before SCT showed significantly better OS (P=0.03). There was no significant difference in donor source and conditioning regimen. Younger age at diagnosis (<10 years of age, P=0.02) was associated with better outcome. High-dose cytarabine-containing re-induction chemotherapy seemed effective on univariate analysis, which indicates the type of re-induction chemotherapy to be prognostic. Finally, multivariate analysis showed only age at diagnosis (<10 years of age) to be a significant prognostic factor. CONCLUSIONSThe prognosis of pediatric AML patients with PIF was extremely poor. In our survey, unfavorable molecular biological background of leukemia was associated with PIF. It is necessary to explore more effective treatment strategies based on leukemia biology for these patients in the future. DisclosuresNo 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.

Relapsed/refractory pediatric B-cell non-Hodgkin lymphoma treated with rituximab combination therapy: A report from the Japanese Pediatric Leukemia/Lymphoma Study Group.

Pediatric relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL) has been reported to be extremely difficult to cure, but rituximab is expected to be effective in improving the prognosis of pediatric R/R B-NHL patients. Here, we assessed the treatment and prognosis of pediatric R/R B-NHL in the rituximab era in Japan. We collected information on patients with R/R B-NHL who were registered with the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) B-NHL03 protocol, a nationwide multicenter trial for newly diagnosed patients. We assessed the treatment and outcome of 33 pediatric R/R B-NHL cases. Twenty-eight patients received rituximab combination therapy as salvage treatment. R-ICE (rituximab, ifosfamide, carboplatin, and etoposide), the most common regimen, was used in 22 patients as first-line salvage therapy. Twenty-three patients received hematopoietic stem cell transplantation (HSCT). Among all 33 patients, 23 (70.0%) achieved partial remission or complete remission. Their 5-year overall survival rate was 48.5%, which was far superior to that in both our previous study and in another study on pediatric R/R B-NHL. Four toxic deaths associated with viral infection occurred after allogeneic HSCT following on rituximab combined salvage therapy. Risk factor multivariate analysis for survival in patients receiving rituximab combination therapy showed central nervous system combined relapse had inferior outcome. The prognosis of pediatric R/R B-NHL in a Japanese cohort remained poor but is showing improvement in the rituximab era. Rituximab combination therapy is effective for R/R B-NHL patients who did not receive rituximab as primary treatment. We need to consider possible viral infections in allogeneic HSCT after rituximab treatment.

Prognostic Significance of CXCR4 Overexpression in Pediatric Acute Myeloid Leukemia with Low-Risk: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Background:The outcome of acute myeloid leukemia (AML) has been improved by advanced risk-stratified chemotherapy based on prognostic factors. However, the long-term survival rate remains 60-70% and further investigation is needed. Core binding factor (CBF)-AML, characterized by t(8;21) or inv(16)/t(16;16), is the most frequent subtype in pediatric AML. Although CBF-AML is generally classified as a low-risk (LR) group, approximately 30% of the patients relapsed within 3 years in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. This finding suggests that the LR patients had other risk factors that could account for their adverse outcome. CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor that helps leukemia cells adhere to bone marrow stromal cells that affect the cell survival and proliferation. There are several reports that CXCR4 overexpression is an adverse prognostic factor in adult AML; however, few studies have examined their significance in pediatric AML. Here, we retrospectively examined whether CXCR4 overexpression can be an adverse prognostic factor in pediatric AML with LR.Patients and Methods:From 2006 to 2010, 485 de novo pediatric AML patients aged <18 years were registered in the AML-05 trial. Overall, 42 patients were excluded, mainly because of misdiagnosis. The risk groups were defined as follows: all of the CBF-AML patients with complete remission (CR) after the initial induction course without FLT3 -ITD were included in the LR group, while patients with -7, 5q-, t(16;21)(p11;q22), Ph1, or non-CR after the initial induction course or with FLT3-ITD were included in the high-risk (HR) group. Patients with no LR or HR features were in the intermediate-risk. CXCR4 mRNA expression was analyzed by quantitative RT-PCR using diagnostic bone marrow samples. CXCR4 expression levels were dichotomized based on the average expression level.Results and Discussion:A total of 248 samples were available for this study. No significant differences in the clinical outcomes were observed between these 248 patients and the 195 patients who did not have CXCR4 data (EFS, P =0.55; OS, P =0.87). CXCR4 overexpression (CXCR4 +) was present in 81 patients (32.7%). CXCR4 + was not correlated with age, sex, and WBC count. CBF-AML was similarly distributed between CXCR4 + and CXCR4 - patients. The frequency of FLT3 -ITD in CXCR4 + patients was significantly higher in CXCR4 - patients (P =0.049), while there were no significant differences in that of KIT and CEBPA mutation (P =0.08 and 0.71, respectively). In total (n=248), no significant differences were observed between CXCR4 + and CXCR4 - patients in 3-year OS (68.6% vs. 75.1%, P =0.42) and 3-year EFS (65.6% vs. 58.9%, P =0.27). However, in the LR group (n=93), a significant difference was observed between CXCR4 + and CXCR4 - patients in 3-year OS (79.2% vs. 98.3%, P =0.008), whereas no significant difference was observed in 3-year EFS (83.3% vs. 69.6%, P =0.33). We have revealed that KIT mutations are adverse prognostic factors in t(8;21) pediatric AML in our recent study (Leukemia, 2015). In t(8;21) AML without KIT mutations (n=44), a significant difference was observed between CXCR4 + and CXCR4 - patients in 3-year OS (76.1% vs. 100.0%, P =0.01), whereas no significant difference was observed in 3-year EFS (77.8% vs. 74.2%, P =0.85). Multivariate Cox regression analysis, including CXCR4 +, KIT mutation, and age identified CXCR4 + as the only prognostic factor predicting poor OS in the cohort of AML with LR (hazard ratio, 11.01; P =0.01), but not EFS (hazard ratio, 0.54; P =0.21). The results suggest that CXCR4 + is associated with an adverse prognosis in pediatric AML with LR. The adverse outcome was restricted in OS, not in EFS, suggesting that CXCR4 + may be associated with the poor prognosis after recurrence, although the underlying mechanism remains unknown. Furthermore, it is noteworthy that the OS of LR patients with CXCR4 - was quite high, which should be confirmed in larger studies.Conclusions:We identified the adverse prognostic significance of CXCR4 overexpression in pediatric AML with LR. The adverse prognosis may be restricted after recurrence. Further studies of the underlying biology are required. There are several promising CXCR4 antagonists including AMD3100, therefore, our findings will help to develop better therapeutic approaches in pediatric AML with LR. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Characteristics of Blastic Plasmacytoid Dendritic Cell Neoplasms Presenting Leukemia Dissemination, Diagnosed By Nation-Wide Immunophenotyping for Pediatric Hematological Malignancy in Japan

[Background] According to the nation-wide retrospective study for pediatric leukemia phenotyping, immunological diagnostic committee of Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) established diagnostic criteria and panel to pediatric hematological malignancies in 2008. Then Nation-wide standardized central immnophenotyping in Japan was started from 2011, and done in more than 2,500 cases by the end of 2014. Most cases were determined immunological diagnosis, but it was of note that we found 11 acute unclassified leukemia cases, including typical and atypical blastic plasmacytoid dendritic cell neoplasms (BPDCN). BPDCN is a rare subtype of leukemia/lymphoma, which has been categorized in the 2008 World Health Organization (WHO) classification of hematological diseases under acute myeloid leukemia and related precursor neoplasms. It was well known for high incidence of extra medullary regions. Most cases of BPDCN were found in elder people, so pediatric cases were extremely rare. We report here the characteristics of pediatric BPDCN cases, including clinical manifestations and peculiar immunophenotype.[Materials and Methods] Immunophenotyping was done on bone marrow (BM) aspiration samples according to diagnostic panel and criteria established by JPLSG immunological diagnostic committee. Immunophenotyping data from July 2011 to June 2015 were collected from JPLSG immunophenotyping centers and analyzed. BPDCN cases were screened by typically CD4+ 56+ HLA-DRhi profile. Other lineage markers such as CD7, CD34, CD13, CD33, CD36, CD64, CD123, and NG2 were also analyzed. As it is well known that existence of atypical phenotype in a substantial proportion, including absence of CD56 and/or CD4, immunophenotype data were prudently investigated and compared with 21 acute myelogenous leukemia (AML) presenting M5 characteristics in FAB criteria.[Result] Out of more than 2,500 cases, 4 cases were diagnosed as typical BPDCN (CD4+ 56+ HLA-DRhi), and 2 atypical BPDCN (1 CD56-, 1 CD4-) by immunophenotyping. All 6 cases presented leukemic dissemination, but only 4 typical cases indicated extra medullary regions. Gender ratio (M: F) was 3:1 and 0:2, and median age were 6.9 years (3.3 to 11.3) and 13.8 (12.8, 14.7) respectively. All 6 cases were negative for CD34 and CD13, and positive for NG2. CD123 was also positive in all (5/5) cases. CD33 and CD36 were positive in 5 cases respectively. It was of note that CD64, typical monocytic marker, was negative in all 6 cases although it was positive in all AML-M5 cases. All BPDCN cases could be distinguished with expression patterns of NG2, CD7, CD33, CD36 (positive) or CD13, CD64 (negative). Additionally, CD2 was positive in 3/6 cases, TdT was positive in 2/6 cases. CD117 was positive in 2 cases (1 typical, 1 atypical).[Discussion] BPDCN is a rare, clinically aggressive hematologic malignancy that most commonly manifests as cutaneous lesions with or without bone marrow involvement and leukemic dissemination. Diagnosis of BPDCN has been usually made by histopathologic examination with cutaneous lesions, which is typically positive for interleukin-3 receptor (CD123), blood dendritic cell antigen 2 (BDCA2)/CD303, and TCL-1. Morphologic and immunophenotypic studies were usually performed on BM and/or peripheral blood prior to histopathologic examination, however diagnosis with these examinations as BPDCN is not so easy because of the lack of morphological peculiarity or traditional lineage-specific markers. Treatments for BPDCN patients have been also inconsistent, because there are no prospective clinical trial data to define the optimal frontline treatment. AML-like or acute lymphoblastic leukemia (ALL)-like regimens have been used for induction therapy, as well as lymphoma-like regimens. The difficulty of BPDCN diagnosis might affect to choose inconsistent frontline therapy. Recently some reports documented ALL oriented therapy indicated more effectiveness to BPDCN, so the diagnosis should be made precisely to evaluate the treatment effectiveness. Here we indicated new diagnostic strategy by immunophenotyping with NG2, CD7, CD33, CD36, CD13, and CD64 as well as CD4, CD56 and HLA-DR. This approach contributes not only to select treatment regimen but also to investigate molecular pathogenesis such as whole exome sequence. DisclosuresNo relevant conflicts of interest to declare.

The Outcome of Relapsed Childhood Core Binding Factor Acute Myeloid Leukemia: A Report from the JPLSG AML-05R Study

BACKGROUND: Core binding factor acute myeloid leukemia (CBF-AML), which is characterized by the chromosomal abnormalities of t(8;21) and inv(16), is the most frequent subtype of pediatric AML. Patients with CBF-AML have a better prognosis than patients with a normal karyotype when they receive chemotherapy of appropriate intensity; however, in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study, the excessive treatment reduction was observed to induce a higher incidence of relapse in children with CBF-AML. We investigated the outcomes of patients with relapsed CBF-AML who were registered in the JPLSG AML-05.PATIENTS & METHODS: We conducted a retrospective analysis of the JPLSG AML-05R study using a questionnaire. Furthermore, a mutation analysis was performed to identify NRAS, KRAS, KIT, WT1, NPM1, NUP98-NSD1, FLT3-ITD and MLL-PTD mutations using the available samples. We investigated the probability of overall survival (pOS) after relapse according to the patient's age, time from diagnosis to relapse, site of relapse, FAB classification, t(8;21) versus inv (16), reinduction chemotherapy regimen, obtaining a second complete remission before stem cell transplantation (SCT), the method of SCT and the presence of additional mutations.RESULTS: Forty-one of the 154 children with CBF-AML who were registered in the JPLSG AML-05 study experienced a relapse. The information from 32 of these 41 patients was collected using a questionnaire. Twenty-seven had t(8;21), and 5 had inv (16). It was possible to perform molecular analyses for 28 of the patients. KIT mutations were identified in 16 of 28 patients (57.1%). The follwing mutations were also detected: NRAS (n=4), KRAS (n=1), and WT1 (n=1). There were no cases with FLT3 -ITD, NUP98-NSD1, MLL-PTD or NPM1 mutations. Twenty-five cases achieved a complete remission after the initial reinduction (78.1%). The pOS was 65.6% (21/32) after a mean observation period of 2.7 years. The significant prognostic factors influencing pOS included a failure to obtain complete remission prior to SCT (p=0.03), reinduction chemotherapy regimen [p=0.02, a FLAG-based or ECM (etoposide, cytarabine and mitoxantrone)-based regimen was better than other modalities], donor source (p<0.01, cord blood was better than bone marrow and peripheral blood) and HLA matching between the donor and recipient (p=0.03, a mismatch was better than a full-match). Additional mutations, including the presence of the KIT gene, did not have any effect on pOS (p=0.64).CONCLUSIONS: Relapsed childhood CBF-AML in the JPLSG AML-05 study was still associated with a better prognosis, because almost two thirds of the patients were successfully treated with SCT. Obtaining complete remission prior to SCT was associated with a good prognosis. A FLAG- or ECM-based regimen is therefore recommended for reinduction chemotherapy. Unexpectedly, cord blood transplantation and HLA-mismatched SCT were associated with a better prognosis. Further investigations are necessary to clarify the genes that are associated with a poor prognosis in patients with CBF-AML. DisclosuresNo relevant conflicts of interest to declare.

Distinct Clinical and Cytogenetic Characteristics and Poor Prognosis in Children with Acute Erythroid Leukemia: A Report from the JPLSG AML-05 Study

BACKGROUND: Acute erythroid leukemia (AEL) is a rare subtype of acute myeloid leukemia (AML) that represents less than 5% of AML in children. However, its clinical picture is not fully understood.PATIENTS & METHODS: We conducted a retrospective analysis on AEL using data from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study (registered at http://www.umin.ac.jp/ctr/index.htm as UMIN000000511) in which patients were enrolled between 2006 and 2010. Morphological, immunophenotypic, cytogenetic, and molecular diagnoses were confirmed by the integrated prospective central diagnostic system.RESULTS: Among the 443 children (age 0 to 18 years old at diagnosis) with de novo AML (acute promyelocytic leukemia and Down syndrome patients are excluded), ten AEL patients (2.2%) were identified. All the cases were classified as M6 in French-American-British classification (eight as M6a, one as M6b, and one as M6). Notably, 8 were classified as AML with myelodysplasia-related changes (5 with morphological dysplasia and 3 with myelodysplasia-related cytogenetic abnormalities) and 2 as AEL in World Health Organization classification (ver.4). There were 6 males and 4 females. Median age and leukocyte count at diagnosis was 3.5 years old (range, 1-15 years old) and 4.7 x 109/L (1.1-16.1 x 109/L), respectively. Cytogenetically, 3 had complex karyotype, 2 had monosomy 7, and 5 had normal karyotype. Molecular analysis showed one case with NRAS mutation, one with both mutated KIT and WT1, and one with MLL-PTD. No case was FLT3 -ITD positive. Eight cases achieved complete remission after initial induction but 3-year event-free survival and overall survival rates were 30.0% (SE 14.4%) and 41.1% (SE 17.5%), respectively.CONCLUSIONS: AEL has distinct features such as high frequency of myelodysplasia- related features and poor risk cytogenetics. Because the outcome seems poor, further genetic analysis is required for development of effective novel therapy. DisclosuresNo relevant conflicts of interest to declare.

A Combination of EVI1 and PRDM16 Expression Clarified the Clinical Features of Intermediate/High Risk Patients in Pediatric Acute Myeloid Leukemia

BackgroundSeveral molecular markers, such as FLT3-internal tandem duplication (ITD), NPM1, CEBPA are well known to correlate with mortality in patients with acute myeloid leukemia (AML). Recently, a number of gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT and FLT3. However, DNMT3A, IDH1/2, and TET2 are rare in pediatric patients with AML, thus accurate risk evaluation remained challenging even after incorporating these molecular markers.On the other hand, overexpression of the EVI1 gene is reported to be associated with adverse outcome in pediatric AML. Moreover, we have previously reported that measuring of PRDM16 gene expression was a powerful tool to predict the prognosis of pediatric AML. PRDM16 gene is highly homologous to the MDS1/EVI1 gene, which is an alternatively spliced transcript of the EVI1 gene. In this study, we investigated EVI1 gene expression to verify the prognosis of EVI1 gene expression and the relationship between EVI1 and PRDM16 gene expression.MethodsBetween 2006 and 2010, 485 de novo pediatric AML patients participated in the Japanese AML-05 study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Among them, 116 patients were excluded from the study because of misdiagnosis and unavailability of their RNA samples. Therefore, 369 patients were analyzed. Quantitative RT-PCR analysis was performed in these patients using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to EVI1 and PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between these gene expressions and other genetic alterations, and clarified the prognostic impact of EVI1 and association between EVI1 and PRDM16 genes.ResultsA total of 58 of 369 patients (15.7%) showed high expression of EVI1 gene. Overexpression of EVI1 gene was strongly associated with dismal prognosis; low risk (LR; 1 of 123 patients, or 0.8%); intermediate risk (IR; 38 of 147 patients, or 25.9%); high risk (HR; 6 of 50 patients, or 12%); and non-complete remission (Non-CR; 13 of 49 patients, or 26.5%), (P < 0.001). Overexpression of EVI1 correlated with the following characteristics: younger age at diagnosis; M4, M5, and M7 subtype; higher coincidence of MLL-rearrangement; and lower coincidence of t(8;21), and inv(16). EVI1 overexpression was very frequent among patients with de novo pediatric AML and IR/non-CR groups. Furthermore, more than half of patients in M6 (5 of 8 patients, or 62.5%) were EVI1 high expression. Interestingly, no patients with EVI1 high expression in M7 had a fusion of CBFA2T3-GLIS2. Patients with EVI1 overexpression also more frequently harbored a complex karyotype and monosomy 7. The frequencies of patients with high or low EVI1 expression differed widely with respect to each genetic alteration, as follows: t(8;21), 1% vs 99%, P < 0.001; inv(16), 0% vs 100%, P < 0.001; NUP98-JARID1A, 83% vs 17%, P < 0.001; OTT-MAL, 100% vs 0%, P = 0.02; and KIT, 5% vs 95%, P = 0.003. The overall survival (OS) and event-free survival (EFS) among patients with EVI1 high expressions were significantly lower than that among patients without such gene aberrant expression (3-year OS 54% vs. 77%, P=0.008G3-year EFS: 34% vs 58%, P<0.001), respectively.On the other hand, a total of 84 of 369 patients (22.8%) showed high expression of PRDM16 gene. The OS and EFS among PRDM16 overexpressing patients were significantly worse than those among low expression group (3-year OS: 51% vs 81%, P<0.001; 3-year EFS: 32% vs 64%, P<0.001), respectively. Remarkably, concerning 125 patients with high EVI1 and/or PRDM16 expression, their prognosis was much worse than that of patients without these high expression (3-year OS: 54% vs 84%, P<0.001; 3-year EFS: 32% vs 68%, P<0.001) , respectively.ConclusionsWe investigated EVI1 and PRDM16 gene expression in de novo pediatric AML patients, and their high expression was associated with inferior survival, respectively. We suggest that high EVI1 and/or PRDM16 expression is useful marker for adverse outcome. On the other hand, low EVI1 and PRDM16 expressions are useful to elucidate low risk patients. DisclosuresNo relevant conflicts of interest to declare.

Individual Pharmacokinetic Targeting of Busulfan in Allogeneic Hematopoietic Stem Cell Transplantation Is Essential for Infants with MLL Gene-Rearranged Acute Lymphoblastic Leukemia: A Report from the JPLSG MLL03 Study

Dose adjustment of busulfan (BU) based on individual pharmacokinetics (PK) has been established as an important clinical tool in conditioning of hematopoietic stem cell transplantation (HSCT). Wide interindividual variety of achievable BU concentration in infants by recommended formula is a problematic issue. Nevertheless, there is no sizable BU PK data in infants. We report here the characteristics of BU PK data in infants with MLL gene-rearranged acute lymphoblastic leukemia (MLL-r ALL) and its relevance to the outcome of HSCT.MLL03, a nationwide study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG), enrolled 62 infants with MLL-r ALL from 2004 to 2009 and tested whether an early intervention of allogeneic HSCT in first complete remission (1CR) could prevent early relapse and produce improved event-free survival (EFS) (Koh K, et al. Leukemia 2015). Protocol determined conditioning consisted of BU combined with etoposide and cyclophosphamide. Plasma BU concentration was measured by high performance liquid chromatography assay based on one-compartment model. The initial BU dose was determined according to an individual BU PK test measured one week prior to the start of HSCT conditioning, and further adjusted on the PK results.Among the 43 infants who received HSCT in 1CR, complete BU PK data were available in 30 cases (21 cases were transplanted from unrelated cord blood donor and 9 from family donor). Oral (PO) BU was given in 18 cases and intravenous (IV) BU in 12 cases. These 30 cases were analyzed for correlation with the clinical outcome. We set the target range of steady state concentration (Css) of BU calculated from PK within 600-900 ng/ml. As a result of BU PK test, the level of estimated Css in the group receiving PO-BU and IV-BU reached the range of 405-949 ng/ml and 484-1247 ng/ml, respectively; 61% of PO-BU group and 46% of IV-BU group were within the desired range. There was a trend of IV-BU group to attain higher targeted Css level with relatively lower dose of recommended formula, while higher dose of PO BU was necessary to achieve proper Css level. Thus, higher level of Css could be easily reached in the IV-BU group compared to the PO-BU group. After adjustment of BU dose based on the PK test results, no case showed abnormally high Css and one case showed extremely low Css than the targeted range despite twice as higher recommended PO BU dose were given. Despite wide diversity of PK data observed in each individual, permissible Css levels could be attained in most of the cases.All the cases achieved full engraftment. There was no statistical significance in the outcome including overall survival, EFS, relapse rate, and treatment-related mortality according to the level of Css nor BU formula. Sinusoidal obstruction syndrome (SOS) occurred in 6 cases (20%; two grade 3, three grade 2, and one grade 1), although Css levels of all the 6 cases did not exceed the upper limit (median 638 ng/ml, range 540-790 ng/ml). However, SOS occurred in another 5 cases out of 13 patients who did not undergo BU PK adjustment, with trend of more severe disease (38%; one grade 4, three grade 3, and one grade 2). We therefore consider that the BU dose adjustment based on PK test is useful to reduce both severity and incidence of SOS. Besides SOS, one case died of transfusion related lung injury.In conclusion, wide interindividual variety of BU PK was observed in infants with MLL-r ALL and dose adjustment of BU based on PK test would be essential for BU containing conditioning of allogeneic HSCT for infants in terms of both safety and efficacy. DisclosuresOff Label Use: Off-label use of CliniMACS purified CD34+ cells.

Respiratory syncytial virus infection in infants with acute leukemia: a retrospective survey of the Japanese Pediatric Leukemia/Lymphoma Study Group.

Respiratory syncytial virus (RSV) can cause life-threatening complications of lower respiratory tract infection (LRTI) in young children with malignancies, but reports remain limited. We performed a retrospective nationwide survey to clarify the current status of RSV disease among infants with hematological malignancies. Clinical course, treatment, and outcome of patients with hematological malignancies who suffered from RSV infections at the age of <24 months during anti-tumor therapy from April 2006 to March 2009 were investigated by sending a questionnaire to all member institutions of the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Twelve patients with acute leukemia were identified as having experienced RSV disease. The primary diseases were acute myeloid leukemia (n = 8) and acute lymphoblastic leukemia (n = 4). RSV infection occurred pre- or during induction therapy (n = 8) and during consolidation therapy (n = 4). Eight patients developed LRTI, four of whom had severe pneumonia or acute respiratory distress syndrome; these four patients died despite receiving intensive care. In our survey, the prognosis of RSV disease in pediatric hematological malignancies was poor, and progression of LRTI in particular was associated with high mortality. In the absence of RSV-specific therapy, effective prevention and treatment strategies for severe RSV disease must be investigated.

Prognostic impact of cytogenetic abnormalities in children and adolescents with mature B-cell non-Hodgkin lymphoma: A report from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG).

Little information is available on cytogenetic abnormalities and their prognostic importance in childhood mature B-cell non-Hodgkin lymphoma (B-NHL). We performed a review of 79 abnormal karyotypes in childhood B-NHL treated by a uniform protocol. Del(17p) was independently associated with significantly inferior event-free survival in Burkitt or Burkitt-like lymphoma. The adverse prognosis of MYC/8q24 rearrangement, +7q or del(13q), was not observed, which had been suggested as risk factors in FAB/LMB96. Our results imply the possible existence of a biological difference among ethnicities and should be useful to narrow down the gene causing poor prognosis in childhood B-NHL.

Differentiating Burkitt Lymphoma from Burkitt-like Lymphoma in Pediatrics: Report from the Japanese Pediatric Leukemia/Lymphoma Study Group B-NHL03 Study

Introduction: Pediatric mature B-cell non-Hodgkin lymphoma (B-NHL) is mostly classified as Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). It has a predominantly germinal center origin. In pediatric BL, some cases are difficult to distinguish from DLBCL, which has been referred to as atypical BL or Burkitt-like lymphoma (BLL). In the recent World Health Organization classification of 2008, B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (IDB) was defined as a new but heterogeneous category. This group has morphological and genetic features of both DLBCL and BL. Therefore, IDB includes atypical BL or BLL in children. To the best of our knowledge, little is known about the exact difference between pediatric BL and IDB, and a distinction needs to be made between the clinical aspects of these two groups. In this study, we performed a clinicopathological study using the cohort from Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) B-NHL03 study, which was the nation-wide prospective study for pediatric B-NHL in Japan, to understand the meaning of the classification of BL in children.Methods: From November 2004 to January 2011, 346 cases of newly diagnosed pediatric B-NHL were enrolled in the B-NHL03 study. Of these, 208 cases were diagnosed in the central pathology review system with reference laboratory results. Seventy-eight cases were subclassified as BL (37.5%), 42 as IDB (20.2%), 57 as DLBCL (27.4%), and 31 as not subclassified (14.9%). Finally, 120 cases of BL or IDB were included in our study cohort. The pathological features and diagnostic and prognostic factors of those cases were analyzed.Results: The median age was 9.0 years (range, 1.9–15.5) in BL patients and 9.1 years (3.0–15.8) in IDB patients. Male (M) predominance was observed in both groups and the M/female (F) sex ratio was 5.5 (M/F 66/12) in BL and 9.5 (38/4) in IDB cases. According to the Murphy staging system, 7.7%/24.4%/39.7%/28.2% of BL patients and 14.3%/40.5%/26.2%/19.0% IDB patients were at stage I/II/III/IV, respectively. The frequency of the cases with central nervous system diseases and cerebrospinal fluid (CSF) blasts were 10.3% and 5.1% in BL and 7.1% and 2.4% in IDB, respectively. In addition, the number of the cases with elevated lactate dehydrogenase (LDH) levels (more than twice the institutional upper limit) was 43.6% for BL and 33.3% for IBD. 4-year overall survival and event-free survival (EFS) were 93.6% and 91.0% for BL and 95.2% and 85.7% for IBD, respectively. In general, there is little statistical difference in clinical presentation between both groups. Pathological examination showed that BL has a significantly high frequency of C-myc translocation (57.1% vs. 33.3%, p = 0.03). Immunohistochemistry demonstrated that most cases in both groups were CD10 positive (BL 97.4%, IBD 95.1%), Bcl-6 positive (both groups 100%), and overexpressed Ki67 (≥90%; BL 93.6%, IBD 92.9%). Moreover, 11.1% of BL and 5.7% of IBD patients were Bcl-2 positive. Because a large difference was not observed in the clinicopathological presentation of the two patient groups, we combined both to analyze prognostic factors. To determine the effects of various risk factors of EFS, log-rank testing with stratification for age (≥10 or <10), sex (M or F), stage (I & II or III & IV), CSF blasts level (±), LDH level (more than or less than twice the institutional upper limit), C-myc translocation(±), immunohistochemical staining of CD10 (±), Bcl-2 (±), Bcl-6 (±), and Ki67 (≥ or <90%) were examined. High staging (83.3% vs. 97.9%, p = 0.012), CSF blast+ levels (40.0% vs. 91.3%, p = 0.001), and high LDH levels (77.1% vs. 97.2%, p = 0.001) significantly correlated with inferior outcome.Conclusions: We conclude that in our cohort, the distinction between BL and IDB does not have much meaning in B-NHL in children. Furthermore, our study shows that the prognostic factors in childhood B-NHL identified in clinical trials in Western countries are identified in patients with the broad BL in Japan. DisclosuresNo relevant conflicts of interest to declare.

Risk-Oriented Therapy for Myeloid Leukemia of Down Syndrome: A Nationwide Prospective Study By the Japanese Pediatric Leukemia / Lymphoma Study Group (JPLSG)

Introduction: Asmyeloid leukemia in Down syndrome patients (ML-DS) is known to have higher sensitivity against cytotoxic agents, children with ML-DS are treated with less intensive ML-DS-oriented protocol in recent clinical studies. On the basis of results of previous Japanese trials for ML-DS, we have evaluated an efficacy of the risk-oriented therapy in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-D05 study.Patients and Methods: Between January 2008 and December 2010, seventy-four patients with newly diagnosed ML-DS from 122 hospitals in Japan were enrolled in this study. All patients received induction chemotherapy consisted of pirarubicin (25 mg/m2/d for 2 days), cytarabine (100 mg/m2/d for 7 days), and etoposide (150 mg/m2/d for 3 days). Patients who achieved complete remission (CR) after initial induction therapy were stratified to the Standard Risk (SR) and received four courses of reduced dose intensification therapy. Patients who did not achieve CR were stratified to the High Risk (HR) and received intensfied therapy consisted of continuous and high-dose cytarabine. Prophylaxis for CNS leukemia was not included throughout the therapy.Results: Out of 74 patients registered in this study, two patients were excluded (one because of uncontrolled cardiac failure, another of non-Down syndrome), and 72 were eligible and were evaluated. Male/Female ratio was 40/32. The median age at diagnosis was 19 months (range, 10 months and 17 years old). Median follow-up period was 3.64 years (range, 0.05 -5.96 years. One patient died of sepsis during initial induction therapy. Sixty-nine patients were stratified to SR and 2 patients to HR. Both of the two HR patients achieved CR but one relapsed. No therapy-related death was observed during intensification therapy. The 3-year event free and overall survival rate was 83.3% ± 4.4% and 87.5% ± 3.9 %, respectively. Age at diagnosis less than 2 years old was significant favorable prognostic factor for relapse (p=0.009). Sex, history of TAM, and chromosomal abnormalities (Normal karyotype or monosomy 7) did not influence the risk of relapse.Conclusion: This study succeeded the previous Japanese strategy with very low-intensive chemotherapy regimen for ML-DS, and despite the dose reduction of chemotherapeutic agents compared to the previous studies, the overall outcome was good and further dose reduction might be possible for specific subgroups. However, considering that most relapse occurred in the SR group defined by morphological treatment response and that relapsed cases are rarely salvageable, more accurate method for identification of the poor prognostic subgroup is needed.This trial is registered with UMIN Clinical Trials Registry (UMIN-CTR, URL: http://www.umin.ac.jp/ctr/index.htm), number UMIN000000989. DisclosuresNo relevant conflicts of interest to declare.

High Event-Free Survival Rate with Minimum-Dose-Anthracycline Treatment in Childhood Acute Promyelocytic Leukemia: A Nationwide Prospective Study By the Japanese Pediatric Leukemia / Lymphoma Study Group (JPLSG)

Introduction: Anthracyclines (Atc) are the key drugs in the treatment of acute promyelocytic leukemia (APL) combined with all-trans retinoic acid (ATRA), however, dose-dependent risk of long-term cardiac toxicity is sometimes problematic especially in pediatric patients. To evaluate efficacy of the treatment with reduced cumulative doses of Atc in childhood APL, we conducted a nationwide non-randomized prospective study, AML-P05, in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG).Patients & Methods: Between April 2006 and March 2011, forty-six children with newly diagnosed APL were enrolled in this study. All patients received at least 3-day precedence of ATRA monotherapy (45mg/m2/d for 35 days in total), followed by chemotherapeutic agents consisted of cytarabine (200mg/m2/d for 7 days) and daunorubicin (DNR, 45mg/m2/d for 3 days) as the first induction therapy. The second induction therapy and subsequent 3 courses of consolidation therapy consisted of ATRA, either high- or intermediate-dose of cytarabine, triple intrathecal injection, and single dose of Atc; 10mg/m2 of mitoxantrone (MIT) in the first 2 courses and 45mg/m2 of pirarubicin (THP) in the last 2 courses. Finally, patients received one-year maintenance therapy with ATRA for 15 days every 3 months.Results: Among the 46 patients registered in this study, 3 were excluded because of PML/RARA negativity, and the remaining 43 patients including 2 with molecular variants were evaluated. The median age at diagnosis was 9 years (range, 11 months to 15 years old). The median follow up period was 4.47 years (0.00-7.45 years). FLT3-ITD was positive in 6 out of 40 patients examined. Two patients died during induction therapies; one of coagulopathy in first course and the other of infection in second course. Two patients developed differentiation syndrome but resolved with temporary cessation of ATRA and supportive therapies. Three patients did not achieve complete remission (CR) after 2 courses of induction therapy, and overall CR rate was 85.7% (36/42, 95%CI: 71.5-94.6%). Neither cardiac adverse events nor treatment-related death were observed during consolidation and maintenance therapies. Three patients exhibited relapse during or after maintenance therapy, and another one developed secondary acute myeloid leukemia. Consequently, the 3-year event-free (EFS) and overall survival rate were 83.6% (95%CI: 68.6-91.8%) and 90.7% (95%CI: 77.1-96.4%), respectively. Age less than 4 years old at diagnosis and non-M1 marrow after first induction therapy were associated with lower EFS. High WBC count (>10,000/μL), low platelet count (<40,000/μL), FLT3-ITD positivity, and additional chromosomal abnormality did not influence the risk of events.Conclusions: According to the JPLSG criteria of Atc equivalents, cumulative Atc dose in this study was converted to 246mg/m2 of doxorubicin (for 1:0.83 for DNR, 1:4 for MIT, and 1:0.6 for THP). The dose was much lower than that in recent US or European protocols, especially in those designed primarily for adult APL patients where more than 600mg/m2 of Atc were used. Comparing with these studies, we have achieved equivalent good results with minimum does of Atc. Therefore, single administration of Atc in each consolidation phases seems sufficient in the treatment of childhood APL.This trial is registered with UMIN Clinical Trials Registry (UMIN-CTR, URL: http://www.umin.ac.jp/ctr/index.htm), number UMIN000000645 DisclosuresNo relevant conflicts of interest to declare.