Diagnostic Bone Marrow Research Articles

Signaling Responses to Stroma-Derived Soluble Factors Are Associated with Outcome and with Expression of Microenvironment-Related Genes in Pediatric AML

Pediatric AML has a relapse rate approaching 40%, indicating that resistance to chemotherapy remains a critical problem. We are focused on identifying and overcoming environment-mediated mechanisms of resistance. We previously reported that the sensitivity of G-CSF- and IL-6-induced STAT3 signaling was significantly associated with outcome in pediatric AML patients. In this follow-up study, we used conditioned medium (CM) from HS5 stromal cells as a more physiological stimulus to evaluate the capacity of primary AML cells to activate signaling pathways.We studied 111 diagnostic bone marrow samples from patients who enrolled on Children's Oncology Group (COG) AML treatment studies, AAML03P1 or AAML0531, and provided informed consent to bank bone marrow. The same chemotherapy backbone was used in both trials. All samples had at least 70% viability after thawing. Thawed cells were divided into aliquots for stimulation with 50% CM, 10 ng/ml G-CSF, or 5 ng/ml IL-6. Unstimulated cells were used for isotype controls and for determination of basal signaling levels. Following 15 min stimulation, cells were fixed and processed for FACS analysis of CD45, pY-STAT3, pY-STAT5, pERK1/2, and pAKT. Responses were expressed as the fold change in mean fluorescence intensity for stimulated cells over unstimulated cells (ΔMFI).The ΔMFIs for pY-STAT3 and pY-STAT5 varied between 0.1 and 31.8. The ΔMFIs for pERK1/2 and pAKT rarely exceeded 2. Samples with a robust response to one stimulus generally responded robustly to the others. The Pearson correlation coefficient (r) for CM-induced pY-STAT3 v. G-CSF-induced pY-STAT3 was 0.8511 (p<0.00001). The CM-induced pY-STAT5 and G-CSF-induced pY-STAT5 responses were significantly but less strongly correlated (r=0.2765; p=0.0043). Cut point analyses identified response thresholds that distinguished patients with higher EFS from those with lower EFS. We found that higher CM-induced pY-STAT5 was significantly associated with an inferior EFS (Figure 1), with HR 2.06 for those with ΔMFI > 1.96 (p=0.034). Previously we found that higher inducible pY-STAT3 was associated with significantly better EFS (Redell, et al, Blood, 2013; Long, et al, Oncotarget, 2017). In this study, no cut point for CM-induced pY-STAT3 ΔMFI distinguished patients with good or poor EFS. Cytogenetics and FLT3/ITD were not significantly different for groups above and below the ΔMFI cut points. The finding that robust STAT5 signaling is associated with inferior EFS suggests that a factor that signals primarily through STAT5 and not STAT3 (e.g. GM-CSF, IL-3, FL) could contribute to treatment resistance and relapse.Interestingly, most of the samples that failed to activate STAT3/5 pathways in response to G-CSF also failed to respond to the cocktail of factors in CM, suggesting a generalized signaling dysfunction. To investigate intrinsic gene expression differences, we leveraged existing RNA-seq data for the samples for which we generated signaling responses. We obtained RNA-seq data for 27 samples from the NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. RNA-seq data for an additional 45 samples were provided through collaboration with the University of Washington. Of these 72 samples, 29 samples were resistant to CM-induced pY-STAT3 (ΔMFI <2, CM-R) and 43 samples were sensitive (ΔMFI > 2, CM-S). Raw counts were normalized and analyzed by EdgeR with GLM algorithm and blocking to control for batch effects from the two datasets. We found that 219 features were significantly differentially expressed (DE; FDR <0.05), with 192 being expressed more highly in the CM-R group. Gene set enrichment analysis identified the “matrisome” gene set, including genes encoding growth factors and matrix proteins, as being significantly enriched in the CM-R group. For example, genes encoding G-CSF, GM-CSF, WNT7B, and integrin B3 were upregulated in CM-R samples. Additionally, a number of non-HOX homeobox genes, including DLX2, DLX3 and MSX2, were increased in CM-R samples. There was no difference in the mean %blasts for the samples in the CM-R and CM-S groups, arguing against the increased expression of matrix-related genes being due to a higher proportion of non-blast cells in the CM-R samples. Our integration of gene expression with inducible STAT3/5 responses will yield novel insights into extrinsic survival signaling and mechanisms of dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Clinical Review of the Co-Occurrence of Myeloproliferative and Lymphoproliferative Neoplasms

The co-occurrence of myeloproliferative (MPN) and lymphoproliferative neoplasms (LPN) is rare and many publications have been limited to small case reports. Others have involved a considerable number of patients, but the coexistence remains underreported and inadequately studied. A recent retrospective review reported that a MPN patients have a 2.8-fold higher relative risk of developing LPN.A database developed at Weill Cornell Medicine (WCM) was queried for patients with ≥3 visits between 1998-2018 with a diagnostic code for a MPN and lymphoma or myeloma subtype. Patients identified were verified to ensure that study inclusion criteria were satisfied. Observed co-occurrence was compared to nation-wide reported prevalence.Demographic and clinical details of 24 patients with a MPN and LPN were recorded (Table 1). The ratio of males to females was 1.7. Essential thrombocythemia and polycythemia vera, and chronic lymphocytic leukemia (CLL) were the leading MPN and LPN subtypes, respectively. Patients were assigned to risk or staging categories at diagnosis based on subtype-specific criteria (Table 2).Median values for diagnostic bone marrow biopsy findings in 14 patients were 2% [0-5] for myeloblasts and 80% [15-100] for cellularity. Additionally, 10 patients had evaluated reticulin fibrosis: 5 (50.0%) presented as MF-0, 4 (40.0%) as MF-1, and 1 (10.0%) as MF-2.Progression to myelofibrosis was confirmed by morphology in 1 (4.2%) patient 10.1 years after a polycythemia vera diagnosis and 5.4 years after a diffuse large B-cell lymphoma (DLBCL) diagnosis. Progression to acute myeloid leukemia was confirmed by morphology in 1 (4.2%) patient 2.4 years after a chronic myelomonocytic leukemia diagnosis and 1.2 years after a smoldering myeloma (SM) diagnosis.Interphase fluorescence in situ hybridization (iFISH) detected cytogenetic abnormalities in 5/8 (62.5%) CLL patients: 2/5 (40.0%) and 5/5 (100.0%) patients harbored deletions in trisomy 12 and 13q14, respectively. Immunoglobulin heavy chain variable region gene (IGVH) status was unmutated in 2 (25.0%) patients. One (12.5%) patient was CD38+ and 2/6 (33.0%) patients were ZAP-70+. At diagnosis, all 8 patients presented with early stage disease (Rai stage 0-II). Based on the CLL-specific international prognostic index (IPI), 3/8 (37.5%) and 5/8 (62.5%) presented as low-risk and intermediate-risk, respectively.Of the 6 lymphoma patients: 5 (83.0%) patients presented with Ann Arbor stage-IV disease at diagnosis. Four (66.7%) patients presented as low/intermediate-risk, and 2 (33.3%) presented as high-risk based on disease-specific IPIs. One patient presented with -17p by iFISH. All 4 patients that were evaluated for Ki-67 had moderate/high expression.Of the 7 multiple myeloma (MM) patients, 6 (85.7%) presented as stage 1 and 1 (14.3%) as stage 3. Of the 3 SM patients, all 3 presented as low risk [12]. In addition, these patients were categorized as IgG-K (4; 40.0%), IgG-L (2; 20.0%), IgA-K (1; 10.0%), IgA-L (1; 10.0%), IgM (1; 10.0%), and biclonal IgG-L/IgA-L (1; 10.0%) [13].Mutation statuses were identified by commercially tested myeloid or lymphoid molecular panels. As expected in this MPN subtype distribution, 11 (45.8%) are JAK2+, 2 (8.3%) are MPL+, 1 (4.2%) is BCR-ABL+, and 1 (4.2%) is CALR+ (Table 3).The risk of a co-occurrent MPN and LPN is higher than expected if they are mutually exclusive (Table 4A-4B, 5). Of interest, 13 (54.2%) patients were diagnosed with a MPN 11.8±18.8 years prior to a LPN; conversely, 11 (45.9%) were diagnosed with a LPN 6.5±6.2 years prior to a MPN. In addition, MPN therapy was started 2.0±2.3 years after a MPN diagnosis, and LPN therapy was started 2.6±4.0 years after a LPN diagnosis. A review of survival analysis requires larger subtype populations since the degree of survival can vary greatly, but it has been reported that patients with a MPN or LPN have significantly reduced life expectancy when compared to the general population. Median follow-up for our patient is 8.2 years (1.5-28.0) with 17/24 (70.8%) patients still being actively followed at our institution, 6 (25.0%) are been lost to follow-up, and 1 (4.2%) is deceased.The significant prevalence of these hematologic malignancies in combination emphasizes the importance of performing a bone marrow biopsy, which we espouse at our institution, cytogenetic analysis, and myeloid and lymphoid molecular testing to identify mutations. [Display omitted] DisclosuresFurman:Loxo Oncology: Consultancy; Gilead: Consultancy; Verastem: Consultancy; Acerta: Consultancy, Research Funding; TG Therapeutics: Consultancy; Incyte: Consultancy, Other: DSMB; Pharmacyclics LLC, an AbbVie Company: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Niesvizky:Amgen Inc.: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Ritchie:Bristol-Myers Squibb: Research Funding; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Speakers Bureau; ARIAD Pharmaceuticals: Speakers Bureau.

Developmental Ontogeny of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Revealed By Recurrent High Burden Clonal Hematopoiesis, Including in “Skin-Only” Disease

INTRODUCTION:Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic neoplasm with poor outcomes and limited therapeutic options. BPDCN pathogenesis is not clear, given that half of patients present with skin tumors without overt bone marrow involvement (“skin-only” disease). Most patients with “skin-only” BPDCN will eventually develop disease progression with bone marrow infiltration. Accordingly, a model of BPDCN pathogenesis has been proposed wherein malignant transformation of a plasmacytoid dendritic cell first occurs in the skin and is followed by dissemination to the bone marrow. Formal evidence to support this model is lacking.METHODS:To study the developmental ontogeny of BPDCN, we performed DNA sequencing of diagnostic bone marrow and skin biopsies from 10 patients. Four patients had “skin-only” disease with negative marrow assessments by histology and flow cytometry (Fig 1A-B). Two patients had skin tumors with limited marrow involvement (≤10% of the cellularity) and 4 patients had skin tumors with extensive concurrent marrow involvement. Six of ten patients had marrow sequencing performed serially before and after therapy. Exon capture and amplicon-based targeted sequencing assays covering recurrently mutated genes in blood cancers were used. Whole exome sequencing (WES) was also performed on germline DNA, “uninvolved” marrow, and a skin tumor from one patient with “skin-only” BPDCN.RESULTS:All patients with “skin-only” disease and negative bone marrow assessment at diagnosis harbored high variant allele frequency (VAF) mutations in the bone marrow, consistent with clonal hematopoiesis (Fig 1C). Two patients with cutaneous disease and only limited marrow involvement also demonstrated multiple high VAF mutations in the marrow that exceeded the degree of BPDCN tumor involvement. Mutations observed in these patients' bone marrows included known pathogenic variants in genes recurrently mutated in BPDCN and other blood cancers, including ASXL1 (4/6 cases; 45-71% VAF), TET2 (4/6; 31-45% VAF), SF3B1 (1/6; 22% VAF), ZRSR2 (1/6; 86% VAF), CUX1 (1/6; 88% VAF), and EZH2 (1/6; 79% VAF).Post-therapy analysis also suggested underlying clonal hematopoiesis in BPDCN. In a patient with 10% bone marrow involvement by tumor at diagnosis, persistent high VAF TET2 and ZRSR2 mutations were noted post-therapy despite a reduction in tumor burden to <1% of the cellularity (Fig 1D, patient 5). Similarly, 2 patients with more extensive marrow involvement at diagnosis (>30% of the cellularity) showed persistence of pathogenic mutations in JAK2, SRSF2, and GNB1 post-therapy despite a reduction in the marrow tumor burden to <1% of the cellularity (Fig 1D, patients 4 and 8).Most marrow mutations were also detected in paired biopsies of skin tumors, suggesting clonal progression from a pre-malignant marrow precursor. To further validate the clonal relationship between these anatomic compartments in BPDCN, we performed WES in one patient with “skin-only” disease who showed identical ASXL1 and TET2 mutations in the marrow and skin at diagnosis. Interestingly, by WES, 87% of all somatic single nucleotide variants (SNVs) were unique to the skin tumor, 11% of SNVs were unique to the bone marrow, and only 2% were shared between both anatomic sites (Fig 1E). This supports a model of branching pre-malignant evolution in the marrow, with one sub-clone seeding the skin and acquiring additional mutations during malignant transformation.CONCLUSION:Clonal hematopoiesis is a defining feature of BPDCN. High VAF mutations in the bone marrow of BPDCN patients without overt tumor involvement likely reflects extensive pre-malignant clonality rather than infiltration by tumor cells arising in the periphery. In contrast to clonal hematopoiesis of indeterminant potential (CHIP), which usually has a single gene mutated at low VAF, uninvolved marrow in BPDCN often harbors multiple mutations at high clonal burden. These data suggest that the earliest events in BPDCN pathogenesis occur in hematopoietic progenitor cells, which then seed peripheral sites during complete malignant transformation. These findings also raise concern about the use of morphologically “normal” stem cells for autologous transplantation in patients with BPDCN. [Display omitted] DisclosuresGriffin:Moderna Therapeutics: Consultancy. Lane:N-of-one: Consultancy; Stemline Therapeutics: Research Funding.

Higher Number of Cycles of Cytarabine Consolidation Improves Outcomes in Elderly AML Patients

Introduction: Determining the optimal post-remission management for elderly patients with acute myeloid leukemia (AML) remains a challenge. AML is primarily a disease of the elderly with >60% of newly diagnosed patients aged 60 years and older. For younger AML patients, post-remission therapy in the form of high-dose cytarabine (HIDAC) consolidation or allogeneic hematopoietic stem cell transplant (HSCT) is critical in maintaining durable disease control and improving survival. However, post-remission HIDAC in elderly AML patients is associated with higher rates of neurotoxicity and decreased survival benefit compared to younger patients. The optimal post-remission therapy for elderly AML patients remains undefined. The objective of this study is to determine the outcomes of using varying doses (0.25-3 g/m2) of cytarabine as post-remission consolidation therapy in an elderly AML population.Methods: We conducted a single-center retrospective analysis of AML patients aged 60 years and older treated with cytarabine as consolidation therapy at UPMC Hillman Cancer Center between January 1, 2005 and December 31, 2014. Patients were excluded if they were not in complete morphologic remission prior to proceeding with cytarabine consolidation or if they proceeded directly to HSCT after consolidation therapy. Relapse-free survival (RFS) was measured from the time of post-induction bone marrow biopsy confirming remission to the date of relapse or death. Overall survival (OS) was defined as the duration from diagnostic bone marrow biopsy confirming AML diagnosis to the date of death. Kaplan-Meier analysis was used to estimate the survival distributions and log-rank test was used to compare survival functions from groups. Multivariate analysis was performed using Cox proportional hazard regression to determine the relationship of RFS and OS to patient's age, cytogenetic risk group, AML type (de novo vs secondary AML), transplant status, cytarabine dosage and number of cycles. All data was analyzed using SAS v9.4 (SAS Institute, Cary, NC, USA).Results: Out of 156 identified patients, 121 were eligible for analysis. Median age at diagnosis was 66 years (range 59-78) with 77 (64%) male patients. Thirty-six patients (30%) had secondary AML. Cytogenetic abnormalities were favorable in 7 (6%), intermediate in 86 (71%), unfavorable in 24 (20%), and unknown in 4 (3%) patients. Cytarabine was administered at a mean dose of 1.44 g/m2 (range 0.25-3.0, SD 0.84) and a median number of cycles of 4 (range 1-4). One patient (0.8%) developed reversible grade 3 neurotoxicity during cycle 2 at a dose of 2 g/m2 with ataxia, dysmetria, and tremors. For all patients, the median RFS was 12.5 months (95% CI 9.7-15.6). Median RFS was significantly different among the cytogenetic risk groups (p=0.0008 via log-rank test): favorable-risk RFS had not been reached because most subjects were in remission and alive at time of analysis, intermediate-risk RFS 13.6 months (95% CI 11.0-17.3), unfavorable-risk RFS 8.3 months (95% CI 3.5-8.9). In this study population, the median OS was 20.5 months (95% CI 16.4-27.1). Median OS was significantly different among the cytogenetic risk groups (p=0.003 via log-rank test): favorable-risk OS 131.3 months (95% CI 12.9-upper limit not reached), intermediate-risk OS 25.1 months (95% CI 18.9-39.4), and unfavorable-risk OS 10.8 months (95% CI 10.4-15.8). Average cytarabine dose did not significantly correlate with RFS or OS in the model. Multivariate analysis revealed a 27% decreased rate of relapse (HR 0.73, 95% CI 0.58-0.92, p=0.008), and 24% decreased mortality rate (HR 0.76, 95% CI 0.59-0.96, p=0.024) with each additional cytarabine cycle given.Conclusions: Our study supports the use of cytarabine as a tolerable and effective option for post-remission therapy in elderly AML patients, particularly those with favorable-risk cytogenetics. Each additional cycle administered was associated with improved RFS and OS rates in this study population. DisclosuresNo relevant conflicts of interest to declare.

WHO-2016 Classification in ALL By Cytogenetics, FISH and Molecular Biology - Experience of Two Reference Centers in Brazil

IntroductionAlmost all neoplasms have their prognosis determined by cytogenetic and molecular factors, which determines a tailored approach for each case. According to the World Health Organization, every case of Acute Lymphoblastic Leukemia (ALL) must be classified not only immunophenotypically but also from a genetic point of view. Over the last few years, it has been recognized the differences in incidence and genetic profile of ALL from Latin America (LA). In Brazil, a large country with a distinctive ethnical background when compared to the rest of LA, few reports on ALL have been published and there is a limited availability of genetic tests for classification, especially in public health service.ObjectiveTo describe cytogenetic (karyotype and fluorescence in situ hybridization, FISH), molecular markers and World Health Organization (WHO) -2016 classification of diagnostic samples sent to two reference centers in Brazil.Material and MethodsThis is a cross-sectional study involving patients diagnosed with ALL from January 2014 to May 2018. Diagnostic bone marrow specimens were analyzed by G band conventional karyotyping (Nomenclature according International System for Human Cytogenomic 2016) (n: 157), molecular markers [(genic fusions by RT-PCR - BCR-ABL1 (n: 96), TCF3-PBX1 (n: 77), ETV6-RUNX1 (n: 83), KMT2A-AFF (n:74)] and B-ALL multi-probe FISH (n:54) were employed according to manufacturer's recommendations and following international guidelines. It was attempted to classify all cases by WHO-2016, as well as by age. Samples with no metaphases, non-conclusive or without molecular tests were considered non-classifiableResultsSamples from 157 ALL patients at diagnosis were analyzed. Median age was 8 years old (1 month - 88 years) [6 (3.8%) cases had less than 12 months; 99 (63%) between 1 to 15 years; 22 (14%) between 16 and 39 years and 30 (19.1%) older than 40 years]. The majority were B cell lineage ALL (93%). Thirty cases were excluded due to missing immunophenotyping, karyotype and/or molecular studies. Alterations were detected in 94 (74%) of 127 cases [88 (76%)/116 in B-ALL and 6 (54%)/11 in T-ALL. It was possible to classify by WHO 2016 B-ALL subtypes 116 cases, all of them stratified by age groups (Graphic 1).ConclusionOur results are consistent with the literature, showing that KMT2A rearrangement predominates in infant ALL, while a pediatric age range from 1 to 15 years old, with a predominance of good prognostic subtypes: hyperdiploidy and ETV6-RUNX1. In adults, there is predominance of the BCR-ABL1 subtype, which seems to be more frequent than non-Latin cohorts. B-ALL NOS was detected in 27.8% of the cases, predominating in the age group of 16 to 39 years, which may correspond to Ph-like ALL, a recently described subtype linked to a worse prognosis and seemingly more common in latinos. DisclosuresNo relevant conflicts of interest to declare.

The Mutational Profile of Pediatric Therapy-Related Myeloid Neoplasms

We and others recently showed that the mutational spectrum of de novo pediatric myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is different than those in adults. MDS and AML also occur in children as a consequence of cytotoxic therapies used to treat childhood malignancies and are collectively referred to as therapy-related myeloid neoplasms (tMN). The incidence of pediatric tMN is ~1% in the pediatric cancer population. These secondary malignancies are usually resistant to conventional chemotherapy and managed with hematopoietic cell transplantation (HCT). These patients have a dismal prognosis. TP53 mutations and somatic alterations in chromatin modifiers predominate in adults with tMN, yet whether children with tMN have a similar constellation of genetic alterations remains unclear since comprehensive genomic profiling has not been completed in a large pediatric tMN cohort. We hypothesize that the mutational profile of pediatric tMN will be different than adult tMN given the patients' younger age and the different spectrum of primary tumor types and chemotherapies.Here we describe the somatic mutational profile of pediatric tMN (including tMDS & tAML) using whole exome (WES) and RNA-sequencing. We evaluated 65 diagnostic bone marrow samples from 61 unique patients, obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 & 2018. The cohort contains 26 tMDS and 39 tAML cases; in 4 patients both tMDS and tAML samples were included. Primary tumors included hematological malignancies (n=45), bone and soft tissue solid tumors (n=14), and brain tumors (n=2); acute lymphoblastic leukemia (ALL) was the most common primary tumor (n = 38, 62%). WES was completed for 61 tumor/normal pairs using Nextera Rapid Capture Expanded Exome (Illumina), while WGS was completed on 4 pairs. Normal comparator genomic DNA was obtained from flow-sorted lymphocytes. Median sequencing coverage for the tumor and normal samples were 107x and 95x, respectively. An average of 49 variants/patient (range: 6-217) was observed in the tMN cohort, including coding, non-coding, silent, and splice site variants, which is significantly different than our previously reported 5 variants/patient in pediatric primary MDS (p = 1x10-6). There was not a significant difference in the number of mutations/patient when tMDS was compared to tAML. Mutational signature analysis (https://cancer.sanger.ac.uk/cosmic/signatures) identified 3 major signatures, the most predominant was characterized by a strong bias for C>A mutations (Signature 24), followed by a signature with strong transcriptional strand bias for T>A mutations (Signature 27) and then a smaller subset resembling MDS and AML (Signature 1). Interestingly, patients with Signature 1 had an inferior 2-year overall survival than the other mutational signatures, with a median survival of 0.3 years (p = 0.0005). WES data and conventional karyotyping showed that chromosome 7 deletions (del(7)) were frequent (n=21, 32%), followed by deletions involving chromosome 5 (del(5)) (n=10, 15%). All of the cases with del(5) had complex cytogenetics and 6 of the 10 cases also had del(7). Ras/MAPK pathway mutations were present in 37% of the cases (40 total mutations in 27 cases). Canonical KRAS (n = 14), NF1 (n = 8), and NRAS (n = 7) mutations were the most frequent coding mutations present overall. Only 5 patients had somatic TP53 mutations, all of which had complex karyotypes. RNA sequencing was performed on 55 samples with suitable RNA. KMT2A rearrangements (KMT2Ar) were common (n = 29, 53%), 4 of which were cytogenetically cryptic. KMT2A rearrangements were more common in tAML (n = 25) but were present in tMDS (n = 4). Nearly half of these KMT2Ar cases also harbored an additional Ras/MAPK mutation. Fusions involving NUP98, RUNX1, MECOM, and ETV6 were also detected.In conclusion, we show that the mutational profile of pediatric tMN has fewer TP53 mutations and more KMT2Ar than adults, as well as a unique set of mutational signatures. These differences are likely a reflection of age-specific chemotherapeutic strategies and fewer pre-existing TP53 mutant hematopoietic clones in children. Future studies understanding the clonal evolution of pediatric tMN development will be helpful in describing pediatric tMN further. DisclosuresNo relevant conflicts of interest to declare.

Immunophenotypic and Genetic Overlap between JMML and CMML

Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution.Methods:The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel.CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis.Results:We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance.We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining.Conclusions:Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. DisclosuresTasian:Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Characterization of Genomic Landscape and Risk Stratification of De Novo Cytogenetically Normal Acute Myeloid Leukaemia

Background. Acute myeloid leukaemia (AML) is a group of heterogeneous diseases with distinct clinicopathologic, cytogenetic and genetic features, sharing in common an abnormal increase in myeloblasts in blood or bone marrow (BM). Induction and consolidation chemotherapy as well as allogeneic haematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However, treatment outcome is unsatisfactory and only 30-40% patients achieve durable remission. Disease heterogeneity in AML may account for different treatment responses. In this study, we examined the mutation spectrum of cytogenetically normal AML (CN-AML) patients in Hong Kong, where AML treatment and indications for allogeneic HSCT are uniform, and evaluated their clinical outcome with respect to the specific gene mutations.Patients. Young adult patients (18-60 years old) with AML diagnosed between August 2003 to September 2017 in 8 regional hospitals in Hong Kong were included. Their clinicopathologic and cytogenetic features as well as treatment outcome were examined.Treatment and endpoints. Treatment comprised induction (daunorubicin 60-90 mg/m2 for 3 days; cytarabine 100 mg/m2 for 7 days) and consolidation (cytarabine 3 gram/m2 for 4-6 doses) for 3-4 courses. BM examination was performed on day 28 or until white cell and platelet counts recovered. Non-remission or relapsed cases were treated with salvage chemotherapy comprising combinations of cytarabine with idarubicin/etoposide, fludarabine, mitoxantrone or clofarabine. Complete remission (CR) was defined as circulating and BM blasts of ≤5% with neutrophil (≥1x109/L) and platelet count (≥100x109/L) recovery. CR with incomplete haematological recovery (CRi) was defined as circulating and BM blasts of ≤5% without complete neutrophil or platelet recovery.Mutation profiling. Next generation sequencing (NGS) for 36 recurrently mutated genes in AML was performed in diagnostic BM samples. Sequencing data were analyzed by in-house bioinformatics pipeline.Statistical evaluation. Patient survivals were calculated by Kaplan-Meier analysis and compared by log-rank test. P-values of <0.05 were considered statistically significant.Results. One hundred and seventy five patients were studied, with CR achieved in 150 cases after 1 (N=105, 70%), 2 (N=27, 18%) or ≥ 3 (N=18, 12%) courses of induction chemotherapy; and not achieved in 25 cases (primary refractory). Allogeneic HSCT was performed in 64 patients (at CR1,N=35; CR2, N=21; ≥ CR3, N=2; relapsed / persistent leukaemia, N=6). Relapse-free-survivals (RFS) at 1, 2 and 5 years were 41.3%, 27.3% and 9.3%; with median at 0.84 year. Overall survival (OS) at 1, 2 and 5 years were 69.1%, 38.3% and 15.4%, with median at 1.51 years. Mutations of ≥ 1 genes occurred in 169 cases (96%); involving with highest frequencies NPM1 (44%), FLT3-ITD (35.3%), DNMT3A (31%), IDH2 R140Q + R172K (14.3%) and CEBPA (bi-allelic, 13.7%). Individually these gene mutations had no impact on CR. However, FLT3-ITD + wildtype NPM1 and FLT3-ITD + mutated DNMT3A portended extremely poor RFS and OS. Bi-allelic CEBPA mutations portended significantly better OS but not RFS, suggesting that these cases were susceptible to relapse but were salvageable with re-induction and allogeneic HSCT. Co-existing mutations of NPM1, DNMT3A and FLT3 were common. Other mutations were mostly solitary, including bi-allelic CEBPA and IDH2 R172K mutations. NRAS and FLT3 mutations were mutually exclusive.Conclusion. CN-AMLs were intrinsically heterogeneous. Mutational profile might be useful for personalized treatment. The cooperativity and mutual exclusivity of gene mutations may be of pathogenetic significance and can inform patient-specific therapeutic strategies. DisclosuresKwong:Bayer: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Leung:Daiichi: Research Funding; Novartis: Speakers Bureau.

Human Acute Myeloid Leukemia Inhibits Normal Erythroid Differentiation through the Paracrine Effects of IL-6

Acute Myeloid Leukemia (AML) is an aggressive cancer resulting in severe cytopenias related to bone marrow (BM) failure. The common assumption for AML-induced BM failure is overcrowding due to clonal expansion of immature myeloid blasts, leading to failure of normal hematopoiesis. However, in a cohort of 293 AML patients, we found that disease burden (% of blasts determined on diagnostic BM aspirate) did not predict severity of cytopenia (hemoglobin rs=-0.053; p=0.49; WBC rs=-0.030, p=0.70; platelet rs=0.091, p=0.026), strongly arguing against simple crowding as the main mechanism underlying AML-induced BM failure. Thus, the goal of our study is to identify novel mechanism(s) associated with AML-induced BM failure, potentially enabling development of new therapies to improve AML management and reverse morbidity.Conventional xenograft models of human AML do not typically exhibit cytopenias, making them unsuitable to study AML-induced BM failure. We speculated that in the mouse, increased splenic extramedullary hematopoiesis compensated for failed intramedullary hematopoiesis due to AML. To test this hypothesis, we performed surgical splenectomy on NSG mice prior to their transplantation with human AML. Strikingly, splenectomized NSG mice engrafted with primary human AML at a 30-70% disease burden (n=8 for primary AML samples, n=5-10 for each group of splenectomized NSG mice) developed leukopenia and severe anemia compared to sham-operated AML-engrafted controls. AML-engrafted splenectomized NSG mice showed early mortality compared to AML-engrafted NSG mice with intact spleens (10.1 weeks vs. 34.2 weeks p<0.0001). Thus, our splenectomized murine xenograft model uniquely recapitulates human disease and allows for novel characterization of AML-induced BM failure.Utilizing our model, splenectomized NSG mice engrafted with human AML demonstrated depletion of normal hematopoietic progenitors (HSPCs) including HSCs (11.1 fold p<0.0001), MPPs (8.90 fold, p<0.001)), CMPs (11.4 fold p<0.0001), MEPs (1.76 fold p=0.0005) and GMPs (5.07 fold p<0.0001). Focusing on anemia, AML also depleted erythroid progenitors including proerythroblasts (1.39 fold p=0.04), normoblasts (4.96 fold p<0.0001), late normoblasts (10.0 fold p<0.0001), and reticulocytes (4.34 fold p=0.007). The relative preservation of proerythroblasts and depletion of normoblasts indicated that AML blasts impart a specific in vivo differentiation blockade in erythroid differentiation.To explore mechanisms by which AML blasts inhibit normal HSPCs, we generated conditioned media (CM) from patient-derived AML blasts, and found that AML-CM suppressed BFU-E colony formation from normal HSPCs (3.1-5.1 fold). Furthermore, in a direct co-culture system with AML blasts and CD34+ HSPCs, AML blasts inhibited erythroid differentiation from the CFU-E to normoblast stage by 81%. Removing CD34+ cells from the AML co-culture allowed cells to resume differentiation to the proerythroblast stage. These experiments demonstrate that AML imparts a differentiation blockade along the MEP-proerythroblast axis in a cell non-autonomous, reversible fashion.Using cytokine array analysis, we identified elevated IL-6 levels in AML sample-derived CM (n=10, 2841±766.4 pg/ml, 7.80 fold increase, p=0.03) compared to CD34+-derived CM (n=5, 364±36.0 pg/ml). Increased IL-6 was also found in BM aspirates from human AML engrafted splenectomized NSG mice (715±125pg/ml, p=0.001) compared to mice engrafted with normal CD34+ HSPCs (undetectable). Thus, we hypothesized that IL-6 produced by AML blasts acts as a paracrine factor to suppress erythropoiesis. Consistent with this hypothesis, an IL-6 neutralizing antibody reversed the inhibition of BFU-E formation imparted by AML-CM. Furthermore, the addition of recombinant IL-6 to liquid cultures of erythroid differentiation resulted in a 23% reduction in proerythroblasts.Together, our data suggests that (1) overcrowding is not the primary mechanism resulting in BM failure in AML; (2) AML blasts play a previously unrecognized role in imparting a differentiation blockade along the MEP-proerythroblast axis, resulting in progressive anemia; and (3) this differentiation blockade is at least partially attributable to IL-6 secreted from AML blasts as a paracrine factor. DisclosuresNo relevant conflicts of interest to declare.

The Common Causes Leading to Pancytopenia at Tertiary Care Hospital Hossain

Pancytopenia is a triad of simultaneous presence of anemia, leucopenia and thrombocytopenia. It is result from a number of disease processes. Both hematopoietic and nonhematopoietic conditions manifest with features of pancytopenia.Methods: This was a cross-sectional study carried out in the Department of Medicine, Dhaka Medical College Hospital between December 2016 to July 2017. We included adult patients of both sexes having age 16 years and above. Criteria for inclusion were persistent pancytopenia on peripheral blood film of more than one week duration. All patients underwent a detailed medical history and full physical examination followed by blood sampling for the investigations. After taking all the aseptic measures and with standard technique the diagnostic bone marrow aspiration and trephine biopsy were done.Results: There were 36 patients with pancytopenia which were included in this study. There were 20 (56%) males and 16 (44%) females with a 1.25:1 male to female ratio and a mean age 47.30 years ± 15.01 SD. The most common complaints were bleeding manifestation (29/36), followed by generalized weakness (27/36) and fever (25/36). Anaemia was the most common (100%) clinical feature followed by jaundice and splenomegaly (6/36). Aplastic anemia was the commonest cause that was observed in 27.78% (10/36) cases followed by Megaloblastic anemia 16.67% (6/36) and erythroid hyperplasia 13.88% (5/36).Conclusion: Aplastic anaemia is leading cause of pancytopenia in this study followed by megaloblastic anaemia and hypersplenism being second and third common causes respectively. Pancytopenia should be suspected on clinical grounds when a patient presents with unexplained anemia, prolonged fever and tendency to bleed.J Dhaka Medical College, Vol. 26, No.2, October, 2017, Page 157-161

High-Throughput Copy Number Profiling by Digital Multiplex Ligation-Dependent Probe Amplification in Multiple Myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease with a diverse clinical outcome. Copy number alterations (CNAs), including whole chromosome and subchromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. We tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA), a novel technique combining MLPA and next-generation sequencing, to detect disease-related CNAs. Copy number status at 371 genomic loci was simultaneously analyzed in 56 diagnostic bone marrow samples, which were also examined by conventional MLPA and interphase fluorescence in situ hybridization (iFISH). On average, digitalMLPA identified 4.4 subchromosomal CNAs per patient. The increased number of probes compared with conventional MLPA allowed a detailed mapping of CNAs, especially on chromosome 1, where 24 different patterns were observed in 38 patients harboring loss(1p) and/or gain(1q). iFISH, MLPA, and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). In addition, we provide proof of principle that digitalMLPA can detect known point mutations, in this case the BRAFV600E. Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen point mutations in MM, which could efficiently be used in myeloma diagnostics.

Two novel fusion genes, AIF1L-ETV6 and ABL1-AIF1L, result together with ETV6-ABL1 from a single chromosomal rearrangement in acute lymphoblastic leukemia with prenatal origin.

Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and 2 novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood. We cloned coding sequences of AIF1L-ETV6 and ABL1-AIF1L in-frame fusion transcripts from the patient's leukemic blasts and we show that the chimeric protein containing the DNA binding domain of ETV6 is expressed from the AIF1L-ETV6 transcript and localized in both the cytoplasm and nucleus of transfected HEK293T cells. Transcriptomic and genomic profiling of the diagnostic bone marrow sample revealed Ph-like gene expression signature and loss of the IKZF1 and CDKN2A/B genes, the typical genetic lesions accompanying ETV6-ABL1-positive ALL. The prenatal origin of the rearrangement confirms that ETV6-ABL1 is not sufficient to cause overt leukemia, even when combined with the 2 novel fusions. We did not find the AIF1L-ETV6 and ABL1-AIF1L fusions in other ETV6-ABL1-positive ALL. Nevertheless, functional studies would be needed to establish the biological role of AIF1L-ETV6 and ABL1-AIF1L and to determine whether they contribute to leukemogenesis and/or to the final leukemia phenotype.

Wilms Tumor 1 Expression at Diagnosis Correlates With Genetic Abnormalities and Polymorphism But Is Not Independently Prognostic in Acute Myelogenous Leukemia: A Hokkaido Leukemia Net Study

BackgroundThe prognostic effect of Wilms tumor 1 (WT1) expression at the diagnosis of acute myelogenous leukemia (AML) has been controversial. The aim of the present study was to determine the correlations of WT1 expression at the diagnosis of AML with established prognostic alterations. Patients and MethodsWe analyzed diagnostic bone marrow samples from 252 patients. WT1 expression, single nucleotide polymorphism (SNP) in the WT1 gene (rs16754), and Fms-like tyrosine kinase receptor-3 internal tandem duplication (FLT3-ITD) mutation were analyzed for all patients. The nucleophosmin 1 (NPM1) mutation and CCAAT/enhancer-binding protein-α (CEBPA) double mutation were analyzed for cytogenetically normal (CN)-AML. The KIT mutation was analyzed for core-binding factor AML. ResultsWithin the cytogenetically favorable prognosis group, WT1 expression in AML with inv(16) or t(15;17) was significantly greater than that in AML with t(8;21). In cases with CN-AML, FLT3-ITD and NPM1 mutations both correlated with greater expression of WT1, and the CEBPA double mutation was related to lower WT1 expression. The existence of both FLT3-ITD and NPM1 mutations showed synergistically greater expression of WT1 in CN-AML. SNP in the WT1 gene (rs16754) was significantly associated with lower expression of WT1. The WT1 levels were not prognostic factors in the total cohort or any cytogenetic group or stratified by SNP status. ConclusionBecause WT1 expression has correlated with known prognostic factors, the prognostic effect of WT1 levels could be misunderstood depending on the distribution of the collaborative mutations in each cohort. We have concluded that the prognostic significance of WT1 at the diagnosis of AML is weak compared with the other established prognostic factors.

Prospective investigation of applicability and the prognostic significance of bone marrow involvement in patients with neuroblastoma detected by quantitative reverse transcription PCR.

Detection of bone marrow (BM) involvement in patients with neuroblastoma is crucial for staging and defining prognosis. Furthermore, the persistence of residual tumor cells in the BM is associated with an unfavorable outcome. Expression of PHOX2B, TH, ELAVL4, and B4GALNT1 (GD2-synthase) was analyzed by quantitative polymerase chain reaction in neuroblastoma cell lines, control BM samples, and in BM samples from patients. The threshold level of expression for each gene was established through receiver operator characteristic analysis and used to determine the diagnostic test performance. The prognostic significance of BM involvement was assessed by survival rates calculations. The median of follow-up time was 36.1 months. Neither PHOX2B nor TH expression was detected in control BM, while expression of ELAVL4 was found in 20 (76.9%) and GD2-synthase in 15 (57.7%) of 26 samples. The overall correct predictive value for TH, ELAVL4, and GD2-synthase, based on thresholds levels, was 0.952, 0.828, and 0.767, respectively, whereas the overall correct predictive value for PHOX2B was 0.994. The PHOX2B/TH expression in diagnostic BM of patients with neuroblastoma corresponded with a decreased survival rate (P<0.001) in the total cohort and in different risk groups. Predominance of normalized expression of PHOX2B over TH>1.68 in the diagnostic BM samples demonstrated an adverse prognostic effect (P=0.006). Persistence of PHOX2B/TH expression in the BM during and after induction chemotherapy resulted in dismal outcome (P=0.022 and P=0.012). PHOX2B and TH are the most optimal markers for detection of BM involvement, allowing identification of high-risk patients. Predominance of PHOX2B expression over TH has a strong adverse prognostic impact.

Impact of Bone Marrow Aspirate Tregs on the Response Rate of Younger Newly Diagnosed Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is widely considered a distinct clinical entity with a well-defined molecular and genetics-based prognosis. Particularly in a younger patient, the therapeutic approach depends largely on diagnostic risk stratification, which has an impact on the outcome after therapy. We added Treg evaluation to the usual molecular and cytogenetics profile in the AML younger patients' diagnostic bone marrow aspirate (dBMA) in order to search for any correlation between Tregs and overall response (OR) as well as survival (OS) rates. We studied 23 AML young patients, all treated with standard induction chemotherapy: OR (complete remission (CR) + CR incomplete (CRi)) was documented in 10 of 23 patients (44%); there were two partial responder patients. The optimal dBMA Treg cut-off value for predicting response to treatment (≥21/μL) was obtained by ROC curve analysis. However, in multivariate analysis, apart from the expected impact of the molecular/cytogenetic risk (p = 0.049) and NPM mutation (p = 0.001), dBMA Tregs ≥ 21/μL was not correlated with OR. Actually, higher dBMA Tregs were associated with the good intermediate molecular/cytogenetic risk group (p = 0.02), whose median OS was confirmed to be better as compared with that of the poor risk group (18 versus 5 months, p = 0.05) and equal to the dBMA Tregs ≥ 21/μL group (5 versus 5 months, p = 0.902), respectively. The possible prognostic value of such an immunological player as BMA Tregs in the diagnostic and successive phases of AML needs to be confirmed in larger patient numbers.

An evaluation of c-myc aberrations in patients with newly diagnosed multiple myeloma (NDMM).

e20006 Background: Multiple myeloma (MM) is a plasma cell dyscrasia marked by clonal evolution and preceded by a premalignant stage, which progresses via pathway deregulation, including MYC activation. Activation of MYC, an oncogenic protein that is part of the MAP kinase growth and proliferation pathway, has been implicated in the progression to active multiple myeloma. Methods: We performed a retrospective review of clinical features of patients with NDMM and myc aberrations between Oct 2015 and Nov 2017. c-myc abnormalities were found on diagnostic bone marrow evaluations in 19 patients by conventional karyotype and/or fluorescent in situ hybridization assays. Results: 10 (53%) of these patients had a myc 8;14 translocation, 5 (26%) had an overexpression of myc, and 4 (21%) had an underexpression of myc, with a laboratory cutoff for expression at 4% for each of these mutations. 14 (76%) of patients were under 65 years, whereas 5 (24%) of patients were 65 years or older. Evaluation of CRAB features in these myc-mutated newly diagnosed MM patients were as follows: 5 (26%) presented with renal disease (Cr > 2mg/dL); 5 (26%) with hypercalcemia (Ca > 11mg/dL); 16 (84%) with osteolytic lesions found on radiography; 11 (58%) with anemia (Hgb < 10g/dL). This is consistent with previous data of presenting symptoms in NDMM. R-ISS staging for c-myc mutated NDMM patients included: 3 (16%) Stage I; 9 (47%) Stage II, 4 (21%) Stage III, and 3 (16%) with missing staging data. Conclusions: These data suggest that the presence of a myc mutation is not explicitly associated with features thought to otherwise confer a poor prognosis including high-risk cytogenetics, high beta 2 microglobulin, high LDH, and decreased albumin. Further evaluation is necessary to confirm these findings in a larger group of patients and to evaluate the apparent age discrepancy noted in this small retrospective study. Characteristics No. of Patients (N = 19) Percent of Patients (%) Male 10 53% Female 9 47% Age < 65 14 74% Age ≥ 65 5 26% White 8 42% Black 3 16% Asian 1 5% Other 7 37% R-ISS Stage I 3 16% R-ISS Stage II 9 47% R-ISS Stage III 4 21% R-ISS Missing 3 16% Hypercalcemia (Ca > 11mg/dL) 5 26% Renal Disease (Cr > 2mg/dL) 5 26% Anemia (Hgb < 10g/dL) 11 58% Radiographic Osteolytic Lesions 16 84%

Prevalence and Clinicopathologic Significance of BRAF V600E Mutation in Chinese Multiple Myeloma Patients

BackgroundPrevious studies in Western countries demonstrated BRAF V600E mutation only in a small subset of multiple myeloma (MM) patients. However, the prevalence and clinicopathologic significances of this mutation remain unclear in Chinese MM patients. Patients and MethodsWe studied diagnostic bone marrow samples from 205 Chinese MM patients by allele-specific PCR to detect BRAF V600E mutation and by high-resolution melting assay to detect KRAS and NRAS mutations. The mutations were confirmed by independent assays. ResultsBRAF V600E mutation was found in 9.3% of the cases, the highest prevalence hitherto reported. In addition, the mutation was significantly associated with hypercalcemia and a male predominance but not with aggressive extramedullary diseases or a high serum creatinine level as reported in Western studies. Importantly, BRAF V600E mutation was an adverse prognostic factor for overall survival in younger MM patients by subgroup analysis. Concurrent analysis of RAS mutations highlighted differential alteration spectrum of RAS signaling between Chinese and Western MM, which may suggest a unique myeloma-related genetic profile in Chinese patients. ConclusionOur study revealed a higher prevalence of BRAF V600E mutation in Chinese MM patients. The associated prognostic impacts on younger patients could be beneficial to risk stratification and potential application of BRAF-targeted therapies in Chinese MM management. This is the first large-scale study revealing the prevalence and clinicopathologic significances of BRAF V600E mutation in Chinese myeloma.

Association of mutations with morphological dysplasia in de novo acute myeloid leukemia without 2016 WHO Classification-defined cytogenetic abnormalities.

Despite improvements in our understanding of the molecular basis of acute myeloid leukemia (AML), the association between genetic mutations with morphological dysplasia remains unclear. In this study, we evaluated and scored dysplasia in bone marrow (BM) specimens from 168 patients with de novo AML; none of these patients had cytogenetic abnormalities according to the 2016 World Health Organization Classification. We then performed targeted sequencing of diagnostic BM aspirates for recurrent mutations associated with myeloid malignancies. We found that cohesin pathway mutations [q (FDR-adjusted P)=0.046] were associated with a higher degree of megakaryocytic dysplasia and STAG2 mutations were marginally associated with greater myeloid lineage dysplasia (q=0.052). Frequent megakaryocytes with separated nuclear lobes were more commonly seen among cases with cohesin pathway mutations (q=0.010) and specifically in those with STAG2 mutations (q=0.010), as well as NPM1 mutations (q=0.022 when considering the presence of any vs. no megakaryocytes with separated nuclear lobes). RAS pathway mutations (q=0.006) and FLT3-ITD (q=0.006) were significantly more frequent in cases without evaluable erythroid cells. In univariate analysis of the 153 patients treated with induction chemotherapy, NPM1 mutations were associated with longer event-free survival (EFS) (P=0.042), while RUNX1 (P=0.042), NF1 (P=0.040), frequent micromegakaryocytes (P=0.018) and presence of a subclone (P=0.002) were associated with shorter EFS. In multivariable modeling, NPM1 was associated with longer EFS, while presence of a subclone and frequent micromegakaryocytes remained significantly associated with shorter EFS.

Abstract 28: The novel role of FOXM1 in AML

Abstract Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces the expression of genes involved in the execution of the mitotic program of normal cells, while it overexpressed in the majority of human cancer cells. In addition, FOXM1 regulatory network was a major predictor of adverse survival outcomes. We have previously shown that FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines cellular localization of FOXM1. Mutations in NPM1 result in cytoplasmic relocalization of NPM (NPM1mut) and favorable outcome for the patients. We have also shown by immunofluorescence in the AML cell line OCI-AML3 and in AML primary samples with NPM1mut, FOXM1 and mutant NPM co-localize in the cytoplasm. Here we show the evidence that improved outcomes in the subset of NPM1mut AML may be partially explained by the cytoplasmic relocalization and consequent functional inactivation of FOXM1. First, we confirmed the colocalization of FOXM1 and NPMmut in cytoplasm of AML primary patient samples using Vectra imaging of AML diagnostic bone marrow biopsies. Strong cytoplasmic expression of FOXM1 was seen only in NPM1mut AML cells. We proceeded to test the role of FOXM1 in mediating chemoresistance in leukemia cell lines with nuclear FOXM1. Stable knockdown of FOXM1 in AML cell lines KG-1 with nuclear FOXM1/wild-type NPM resulted in increased sensitivity to the chemotherapeutic agent cytarabine. We overexpressed FOXM1 in the OCI-AML3 cell line with cytoplasmic localization of FOXM1 and confirmed overexpression of exogenous FOXM1 in the nuclear compartment by fractionation. Furthermore, we showed that nuclear overexpression of FOXM1 switches the sensitivity of these leukemia cell line to resistant phenotype. These data imply that suppressing of FOXM1 in AML could increase sensitivity to standard chemotherapy, while overexpression of FOXM1 increases chemoresistance of AML cells. Our data suggest that FOXM1 inhibitors may be useful for AML patients with FLT3 wild-type/NPM1 wild type with nuclear FOXM1. We used two compounds to target FOXM1 in AML: honokiol and ixazomib. Honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression and honokiol's inhibitory effect on FOXM1 is a result of direct binding of honokiol to FOXM1. We found that that honokiol suppresses FOXM1 in AML cell lines and sensitizes AML cells with nuclear FOXM1 to cytorabine. We also tested novel proteasome inhibitor ixazomib (Takeda) as a potential FOXM1 inhibitor in AML. We found that similarly to several other proteasome inhibitors ixazomib inhibits transcriptional activity of FOXM1, FOXM1 mRNA, and FOXM1 protein expression in primary AML cells and in AML cell line. In addition, it shows potential synergy with AML's most common drug cytarabine. Combination of cytarabine and ixazomib in AML KG1 cells led to strong apoptosis correlated with FOXM1 suppression. Since ixazomib is already approved for patients with multiple myeloma and it works as FOXM1 inhibitor in AML, we propose to test it as FOXM1 inhibitor in vivo. These results substantiate FOXM1 targeting to improve chemosensitivity for AML treatment. Our work is based on an intriguing premise that nuclear export of FOXM1 by mutant NPM1 in a subset of favorable risk AML is responsible for outcome. Furthermore, the idea that FOXM1 inactivation leads to favorable outcome gives rise to several strategies to develop FOXM1 inhibitors to increase the efficacy of chemotherapy in AML patients. Citation Format: Irum Khan, Marianna Halasi, Andrei L. Gartel. The novel role of FOXM1 in AML [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 28.

Abstract PR09: Single-cell transcriptional profiling of acute myeloid leukemia identifies self-renewing stem cells

Abstract Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the leukemia cells with self-renewal potential and ability to recapitulate the disease. Most anticancer therapies are designed to inhibit proliferation. Yet, in hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to target LSCs and eradicate AML. Our goal is define the self-renewing LSCs in order to develop therapeutics that target them and eliminate AML relapse. We previously showed that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation of a transgenic mouse model of AML (Mll-AF9/NRASG12V) (Sachs et al. Blood 2014; Kim et al. Blood 2009). We hypothesize that self-renewal capacity and the NRAS-activated pathways required for self-renewal are limited to a subpopulation of LSCs. We used single-cell RNA sequencing to identify the self-renewing cells among the LSC-enriched subgroup in this model (Mac1LowKit+Sca1+, “MKS”). We identified three discrete transcriptional profiles among the LSC-enriched subpopulation and found that that two of these profiles (Profile 1 and Profile 2) are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted the MKS LSCs based on CD36 and CD69 expression. Sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that MKS-CD36-CD69+ cells (consistent with Profile 1) rapidly transferred leukemia with high penetrance in 20 of 22 mice. In contrast, MKS-CD36+CD69- cells (Profile 2) failed transfer leukemia in most mice; only 2 of 25 of these mice developed AML (p &amp;lt; 0.004). In our previous work, we demonstrated that the NRASG12V-activated self-renewal gene expression profile that we identified in our murine model was expressed in human AML, suggesting that the gene expression behavior of LSCs from this model may recapitulate the gene expression behavior of human LSCs (Sachs et al. Blood 2014). In order to determine if the single-cell transcriptional profiles of our murine AML can be found in primary human AML precursors, we performed single-cell RNA sequencing on CD34+ human AML cells obtained from a diagnostic bone marrow specimen. Analogous to our murine model, we found that these human AML cells express 2 distinct single-cell transcriptional profiles and they differentially express RAS-activated gene expression profiles and profiles of hematopoietic differentiation. Next, we used our murine single-cell self-renewal transcriptional profile to define a 96-gene panel consisting of 88 genes from this profile and 8 housekeeping genes. We sorted primary, diagnostic human AML cells for leukemia stem and progenitor cells (CD34+CD38-) and performed single-cell qPCR on these cells using our 96-gene panel. We found that a subset of these cells preferentially expresses Profile 1, the self-renewal gene expression profile that we identified in our murine model, and another subset preferentially expresses Profile 2 (the profile associated with no leukemia-reconstituting capacity). In these experiments, we use a murine model of AML to define the LSC self-renewal gene expression profile at the single-cell level and functionally validate this profile in vivo. Analogous to the murine model, a subset of human AML stem and progenitor cells expresses this LSC self-renewal gene expression profile at the single-cell level. These data suggest that single-cell gene expression profiling can delineate leukemia cells with true self-renewal capacity. This abstract is also being presented as Poster 43. Citation Format: Klara E. Noble-Orcutt, Karen Sachs, Connor Navis, Alexandria Hillesheim, Ian Nykaza, Rebecca S. LaRue, Conner Hansen, Ngoc Ha, Michael A. Linden, David A. Largaespada, Zohar Sachs. Single-cell transcriptional profiling of acute myeloid leukemia identifies self-renewing stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR09.