Cytogenetic Subtypes Research Articles

Cytogenetic Characteristics and Outcomes of Patients Receiving CTL019 CAR T Cell Therapy

Background: CTL019 is a therapy derived from autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) that was approved by the FDA in August 2017 (tisagenlecleucel). Complete and durable remissions have been seen in the setting of pediatric and young adult patients with relapsed and refractory B cell acute lymphoblastic leukemia (ALL) (Maude NEJM 2018). Initial case reports suggested that there may be differential outcomes mediated by cytogenetic characteristics of the leukemia at CAR T cell infusion. Here, we report results from a single institution experience of 112 patients. Methods: Patients with relapsed/refractory ALL were identified as having received CTL019 either in the context of a clinical trial (NCT02906371) or commercial product (tisagenlecleucel) at Children's Hospital of Philadelphia from October 2016 to April 2019. Patients who received prior CAR T therapy were excluded. Demographic, cytogenetic, and outcome data were manually abstracted from the medical record or clinical trial datasets. High risk lesions were defined as MLL(KMT2A) rearrangements, Philadelphia-chromosome (Ph+), Ph-like, hypodiploidy, and TCF3/HLF fusion. Favorable cytogenetics were defined as the presence of hyperdiploidy or ETV6/RUNX1fusion and intermediate were defined as iAMP21, IKZF1deletion, or TCF3/PBX1. Patients were classified according to their highest risk cytogenetic characteristic and stratified by cytogenetic risk category present at CAR T cell infusion. Relapse-free survival (RFS) and overall survival (OS) was described for cohorts with more than 10 patients. Results: One hundred and twelve patients were included in the analysis, with a median age of 11 years (range 1-29) at infusion, of which 32% had had a previous allogeneic hematopoietic stem cell transplant (alloHSCT). Disease burden at the time of CTL019 infusion was heterogenous, with 61% having detectable disease in the bone marrow and 21% having more than 25% blasts by flow cytometry. Thirty-six patients (32%) had leukemias with high-risk genetic lesions at infusion, including 12 with MLL rearrangements and 18 with Ph+ or Ph-like lesions (Table 2). Thirty-one patients (28%) had hyperdiploidy or ETV6/RUNX1; 3 additional were in conjunction with high-risk cytogenetics (t(17;19) and 2 with Ph+), and 3 in the setting of intermediate-risk cytogenetics (iAmp21, TCF3/PBX1, IKZF1deletion). Figure 1 demonstrates RFS for those patients in remission at day 28 following infusion, stratified by cytogenetic risk category. Complete remission (CR) rate in the high-risk cytogenetics group was 94%. RFS at 12 months was 69% (0.50-0.82), 69% (0.40-0.86), and 67% (0.48-0.80) for non-informative, favorable, and high-risk cytogenetic groups, respectively. Figure 2 shows OS of patients infused with CTL019, again stratified by cytogenetic categories of interest, with a maximum follow-up time of 30 months. OS at 12 months was 84% (0.68-0.93) and 76% (0.56-0.88) for the non-informative and high-risk cytogenetic groups, respectively. There were no deaths in that time period for the favorable risk category. There was no statistically significant difference in RFS or OS for patients with high-risk cytogenetics. The intermediate-risk cytogenetics group (n<10) was excluded from these analyses. Conclusion: Durable remissions can be achieved with CTL019 across several high-risk cytogenetic subtypes of B-ALL. Stratifying outcomes by cytogenetic risk category in this unadjusted analysis does not show a statistically significant difference in either RFS nor OS. Further investigation is needed to parse out the contribution of individual cytogenetic lesions as well as the effects of other relapse and survival risk factors at play. Figure Disclosures Rheingold: Novartis: Consultancy; Pfizer: Research Funding. Callahan:Novartis: Consultancy. Hunger:Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Equity Ownership; Jazz: Honoraria; Novartis: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Maude:Kite: Consultancy; Novartis: Consultancy.

Overexpression of chromatin remodeling and tyrosine kinase genes in iAMP21-positive acute lymphoblastic leukemia

Intrachromosomal amplification of chromosome 21 (iAMP21) is a cytogenetic subtype associated with relapse and poor prognosis in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). The biology behind the high relapse risk is unknown and the aim of this study was to further characterize the genomic and transcriptional landscape of iAMP21. Using DNA arrays and sequencing, we could identify rearrangements and aberrations characteristic for iAMP21. RNA sequencing revealed that only half of the genes in the minimal region of amplification (20/45) were differentially expressed in iAMP21. Among them were the top overexpressed genes (p < 0.001) in iAMP21 vs. BCP ALL without iAMP21 and three candidate genes could be identified, the tyrosine kinase gene DYRK1A and chromatin remodeling genes CHAF1B and SON. While overexpression of DYRK1A and CHAF1B is associated with poor prognosis in malignant diseases including myeloid leukemia, this is the first study to show significant correlation with iAMP21-positive ALL.

Circular RNA differential expression in blood cell populations and exploration of circRNA deregulation in pediatric acute lymphoblastic leukemia

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.

Predisposing germline mutations in high hyperdiploid acute lymphoblastic leukemia in children.

High hyperdiploidy (HD) is the most common cytogenetic subtype of childhood acute lymphoblastic leukemia (ALL), and a higher incidence of HD has been reported in ALL patients with congenital cancer syndromes. We assessed the frequency of predisposing germline mutations in 57 HD-ALL patients from the California Childhood Leukemia Study via targeted sequencing of cancer-relevant genes. Three out of 57 patients (5.3%) harbored confirmed germline mutations that were likely causal, in NBN, ETV6, and FLT3, with an additional six patients (10.5%) harboring putative predisposing mutations that were rare in unselected individuals (<0.01% allele frequency in the Exome Aggregation Consortium, ExAC) and predicted functional (scaled CADD score ≥ 20) in known or potential ALL predisposition genes (SH2B3, CREBBP, PMS2, MLL, ABL1, and MYH9). Three additional patients carried rare and predicted damaging germline mutations in GAB2, a known activator of the ERK/MAPK and PI3K/AKT pathways and binding partner of PTPN11-encoded SHP2. The frequency of rare and predicted functional germline GAB2 mutations was significantly higher in our patients (2.6%) than in ExAC (0.28%, P = 4.4 × 10-3 ), an observation that was replicated in ALL patients from the TARGET project (P = .034). We cloned patient GAB2 mutations and expressed mutant proteins in HEK293 cells and found that frameshift mutation P621fs led to reduced SHP2 binding and ERK1/2 phosphorylation but significantly increased AKT phosphorylation, suggesting possible RAS-independent leukemogenic effects. Our results support a significant contribution of rare, high penetrance germline mutations to HD-ALL etiology, and pinpoint GAB2 as a putative novel ALL predisposition gene.

RUNX1-ETO: Attacking the Epigenome for Genomic Instable Leukemia.

Oncogenic fusion protein RUNX1-ETO is the product of the t(8;21) translocation, responsible for the most common cytogenetic subtype of acute myeloid leukemia. RUNX1, a critical transcription factor in hematopoietic development, is fused with almost the entire ETO sequence with the ability to recruit a wide range of repressors. Past efforts in providing a comprehensive picture of the genome-wide localization and the target genes of RUNX1-ETO have been inconclusive in understanding the underlying mechanism by which it deregulates native RUNX1. In this review; we dissect the current data on the epigenetic impact of RUNX1 and RUNX1-ETO. Both share similarities however, in recent years, research focused on epigenetic factors to explain their differences. RUNX1-ETO impairs DNA repair mechanisms which compromises genomic stability and favors a mutator phenotype. Among an increasing pool of mutated factors, regulators of DNA methylation are frequently found in t(8;21) AML. Together with the alteration of both, histone markers and distal enhancer regulation, RUNX1-ETO might specifically disrupt normal chromatin structure. Epigenetic studies on the fusion protein uncovered new mechanisms contributing to leukemogenesis and hopefully will translate into clinical applications.

CD79a Is Associated with Central Nervous System Infiltration of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

▪Despite the advances in the treatment of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), infiltration of the central nervous system (CNS) remains a clinical challenge. Certain cytogenetic subtypes such as E2A-PBX1-and BCR-ABL-positive BCP-ALL confer a higher risk for CNS involvement initially and for CNS relapse. Novel strategies to predict CNS and to eradicate leukemic cells from the CNS are subjects of ongoing research.In order to identify targets with diagnostic and therapeutic relevance, comparative RNA-sequencing was performed with patient derived xenograft (PDX) blasts from 5 E2A-PBX1-positive patients, recovered from the bone marrow (BM) and from the CNS of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Differential gene expression analysis revealed the upregulation of various genes of the pre-B cell receptor complex, particularly the signaling component CD79a (Igα) in blasts recovered from the CNS as compared to blasts from the BM. We then investigated the impact of CD79a on CNS infiltration in vivo and in patients. CD79a was downregulated by short-hairpin RNA (shRNA) mediated knockdown in the E2A-PBX1 positive cell line 697. Proliferation rates of 697-shCD79a cells and control-transfected 697 cells (697-shCtrl) in vitro were similar. Furthermore, NSG mice injected with 697-shCD79a cells showed comparable survival times, as well as similar blast infiltration in spleen and BM as animals injected with 697-shCtrl cells. However, downregulation of CD79a led to a significantly lower number of CNS-positive mice (4/15, 26%) as compared to control animals (7/10, 70%) (p=0.0486, Figure A). This indicates that CD79a is not critically involved in proliferation and peripheral engraftment, but in CNS infiltration of E2A-PBX1 positive 697 cells in vivo.To test if CD79a also affects CNS involvement in BCR-ABL-positive leukemia, a murine/murine transplantation model was used. B-cells isolated from CD79a-knockout (CD79a-KO) or wildtype mice (CD79a-Ctrl) were stably transfected with a BCR-ABL fusion gene and cultured independent of cytokines, thereby inducing malignant transformation. Both cell lines were subsequently injected into recipient NSG mice (n=8/group) and leukemic development was followed. The experiment was terminated when all control mice had developed leukemic symptoms and mice were analyzed for leukemic engraftment. A further CD79a-KO group was included for survival analysis. Median spleen volume as a surrogate of leukemic infiltration was significantly lower in mice injected with CD79a-KO as compared to CD79a-Ctrl cells (0.35 cm³ vs. 0.06 cm³; p=0.0001). Median blast percentages in spleens and BM were also markedly reduced (75.3% vs. 5.8%; p=0.0001 and 61.0% vs. 4.5%; p=0.0001, respectively). Importantly, none of the animals in the CD79a-KO group showed blasts in the CNS as assessed by histology whereas blasts were present in all of the animals in the CD79a-Ctrl group. Finally and most importantly, NSG-mice injected with CD79a-KO cells showed a highly significant prolongation in median survival as compared to mice with CD79a-Ctrl cells (29 days vs. 95 days; p=0.0001, Figure B). Altogether, these data suggest that in a model of BCR-ABL-positive leukemia, absence of CD79a impacts the engraftment of blasts in vivo, in the CNS and other leukemic niches.To further validate our findings in patient material, we measured CD79a protein expression in PDX cells from an E2A-PBX1- and a BCR-ABL-positive patient serially transplanted into NSG mice for three passages. For both entities and in all passages, CNS blasts showed a higher CD79a expression than blasts isolated from the bone marrow. In order to assess if CD79a can be used as a marker to predict CNS involvement in patients, CD79a mRNA levels were measured in a selected cohort of 98 pediatric BCP-ALL patients, which contained 26 CNS-positive patients matched to 72 CNS-negative patients. CNS-positive patients showed significantly higher mRNA levels of CD79a than CNS-negative patients (p=0.0225, unpaired t-test, Figure C) suggesting that CD79a may be of value as a potential diagnostic marker for initial CNS involvement in BCP-ALL.Our results indicate a role of CD79a in proliferation and CNS infiltration of BCP-ALL blasts in experimental settings and patients. We intend to prospectively evaluate CD79a as a prognostic marker, which may also be a therapeutic target in CNS-positive BCP-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NUTM1 Is a Rare but Recurrent Fusion Gene Partner in B Cell Precursor Acute Lymphoblastic Leukemia Associated with Increased Expression of Genes on Cytoband 10p12.31

▪INTRODUCTION In 20-25% of the pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, the driving cytogenetic aberration is unknown. It is important to identify more primary lesions in this remaining B-other group to provide better risk stratification and identify possible treatment options. In this study, we aimed to identify novel recurrent fusion genes in BCP-ALL through RNA sequencing.METHODS We used paired-end total RNA Illumina sequencing to detect fusion genes with STAR-fusion and FusionCatcher in a population-based ALL cohort (n=71). We used Affymetrix U133 Plus2 expression arrays in a larger population-based ALL cohort (n=661) and an infant ALL cohort (n=70) to compare gene expression levels. Fluorescent in situ hybridization (FISH) was performed using Cytocell NUTM1 break-apart probe set MPH4800.RESULTS We identified an in-frame SLC12A6-NUTM1 fusion transcript composed of exons 1-2 of SLC12A6 fused to exons 3 to 8 of NUTM1 by RNA sequencing. Both genes are located on 15q14 within 5.3 Kb distance on opposite strands, and the fusion could result from an inversion. The fusion transcript is predicted to encode almost the total NUTM1 protein including the acidic binding domain for the histone acetyltransferase EP300. The SLC12A6-NUTM1 fusion case showed high NUTM1 expression, while NUTM1 expression was absent in the remaining cases.Using gene expression profiling, we identified four additional pediatric and two non-KMT2A-rearranged infant BCP-ALL cases with high NUTM1 expression. In the population-based cohort reflecting all different cytogenetic subtypes, these cases were restricted to the B-other group without known sentinel cytogenetic abnormalities. FISH showed a NUTM1 break apart pattern in all four tested NUTM1-positive cases indicative of a balanced translocation. RNA sequencing confirmed an ACIN1-NUTM1 fusion in one of the infant cases. We conclude that NUTM1 is normally not expressed in leukemic lymphoblasts, and that its expression can be induced by a gene fusion. The karyotypes of the predicted NUTM1 fusion cases combined with RNA sequencing data suggest that different chromosomal rearrangements are involved, likely resulting in different NUTM1 fusion partners. In literature, BRD9-NUTM1, IKZF1-NUTM1, and CUX1-NUTM1 fusions were reported in pediatric B-other cases, and BRD9-NUTM1 and ACIN1-NUTM1 fusions were reported in non-KMT2A-rearranged infants. Our combined aberrant gene expression and FISH results indicate that NUTM1 fusions occur in 2.4% (5/210) of pediatric and in 28% (2/7) of infant BCP-ALL cases without a sentinel cytogenetic aberration.The recurrence of NUTM1 aberrations in BCP-ALL cases without a known driver and the resulting expression of NUTM1 suggests that this fusion could be a new oncogenic driver in leukemia. All seven patients with a NUTM1 fusion achieved continuous complete remission with a median follow-up time of 8.3 years (range 4.8-13.8 years), suggesting that NUTM1 fusions in BCP-ALL have a favorable prognosis.To get an insight in the underlying biology, we compared gene expression between NUTM1-positive and NUTM1-negative pediatric B-other cases. We identified 130 differentially expressed probe sets (FDR ≤0.01) with a peculiar enrichment of those located on chromosome band 10p12.31 (Bonferroni adjusted p=4.05E-04). The genes in cytoband 10p12.31, including BMI1, were variably upregulated in 6/7 NUTM1-positive cases and positively correlated to NUTM1 expression levels. The NUTM1 protein is capable of binding and hereby stimulating the histone acetyltransferase activity of the EP300 protein. The EP300 protein preferentially binds a risk allele of BMI1 associated with increased risk for BCP-ALL. The BMI1 protein has been shown to convert BCR-ABL1-positive progenitor cells into BCR-ABL1-positive BCP-ALL cells. Hence, we postulate that NUTM1 fusion proteins contribute to leukemogenesis by stimulating EP300, leading to upregulation of BMI1 and other 10p12.31 genes in BCP-ALL.CONCLUSIONNUTM1 fusions are a rare but recurrent event in BCP-ALL that seems to have a good prognosis. The NUTM1 fusions result in expression of the normally silent NUTM1 gene and are associated with upregulation of a cluster of genes on 10p12.31 including the leukemogenic BMI1 gene. DisclosuresNo relevant conflicts of interest to declare.

Genomic Abnormalities Among African Individuals with Monoclonal Gammopathies Using Calculated Ancestry

Monoclonal gammopathies, including multiple myeloma (MM), represent a group of plasma cell (PC) disorders that comprise of mostly incurable hematopoietic malignancies with an increasing incidence in the US. Previous epidemiological studies demonstrated a 2-3 fold higher incidence of monoclonal gammopathy of undetermined significance (MGUS) and a similarly higher incidence of MM along with a ~4-year younger age of onset among African Americans (AA) compared to European Americans (EAs) (Fonseca, Leukemia, 2017). This suggests a possible ancestral-associated genetic predisposition of AAs to the development of monoclonal gammopathies (Landgren, Blood, 2006). When access to care is equal, AAs have a better overall survival compared to EAs suggesting that AAs may have a genetic predisposition that renders them better responders to treatment or have more favorable cytogenetic subtypes of MM (Waxman, Blood, 2010). Previous efforts to understand this disparity have relied on self-reported race rather than genetic ancestry. We hypothesize that quantifying genetic ancestry is necessary to fully understand the genetic mechanisms of racial disparities of monoclonal gammopathies. We describe our study of 881 patients with monoclonal gammopathies who had undergone uniform testing to identify MM-specific cytogenetic abnormalities. DNA from the bone marrow samples was genotyped on the Precision Medicine Research Array. Biogeographical ancestry was assessed using the Geographic Population Structure Origins tool, which provides the ancestral breakdown of 36 admixture components for each individual representing different geographic regions and outputs admixture proportions corresponding to those ancestries (Elhaik, Nat Commun, 2014). We evaluated whether an increase in the percentage of African Ancestry altered the odds of any primary cytogenetic abnormality. We demonstrated that a 10% increase in the percentage of African ancestry was associated with a 6% increase in the odds of detecting either an t(11;14), t(14;16) or t(14;20) (odds ratio = 1.06, 95% CI: 1.02 - 1.11; p-value = 0.05). Although we observed an increase in the odds of each of the individual translocations with 10% increase in African ancestry, l t(11;14) (OR=1.03) t(14;16) (OR=1.11) and t(14;20) (OR=1.10), these three abnormalities were combined to achieve greater statistical power. The differences in cytogenetic abnormalities were most striking among individuals with ≥80.0% African (n=120) compared to individuals with We complement our past studies that relied on self-reported race and characterized patients9 demographic and uniformly collected cytogenetic data in relation to genetically defined African ancestry. We demonstrate the prevalence of having one of three specific subtypes (i.e., t(11;14), t(14;16), or t(14;20)) was significantly higher in those with highest (≥80%) compared to lowest ( Disclosures Elhaik:DNA Diagnostics Center: Consultancy. Baird:DNA Diagnostics Center: Employment. Cerhan:Jannsen: Other: Scientific Advisory Board; Nanostring: Research Funding; Celgene: Research Funding. Stewart:Amgen Inc., BMS, Celgene, Takeda, Roche, Seattle Genetics, Janssen, Ono: Consultancy; Amgen Inc., Celgene, Roche, Seattle Genetics: Research Funding. Dispenzieri:Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, GSK: Research Funding. Kumar:Novartis: Research Funding; KITE: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity9s Board of Directors or advisory committees; AbbVie: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity9s Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity9s Board of Directors or advisory committees; AbbVie: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Roche: Research Funding.

Germline GAB2 Mutations in Childhood Acute Lymphoblastic Leukemia

Recent studies using next-generation sequencing of selected individuals, such as those with familial leukemia or congenital syndromes, have identified rare and highly penetrant germline mutations that predispose to childhood acute lymphoblastic leukemia (ALL). High hyperdiploidy (HD), the most common cytogenetic subtype of childhood ALL, is enriched in children with RASopathies who develop ALL and, similarly, a high proportion of ALL patients with germline ETV6 or IKZF1 mutations presented with the HD subtype. Here, we aimed to identify novel predisposition genes in a selected group of HD-ALL patients.Targeted sequencing of 538 cancer-relevant genes was carried out using the UCSF500 Cancer Gene Panel in diagnostic bone marrow (i.e. tumor) DNA from 57 HD-ALL patients from the California Childhood Leukemia Study (CCLS). HD-ALL patients were selected based on absence of somatic KRAS or NRAS hotspot mutations detectable by Sanger sequencing, and absence of somatic copy number deletions from multiplex ligation-dependent probe amplification (MLPA) assays. After filtering out likely somatic mutations (mutant allele fraction <0.44), and restricting to variants with low frequency in unselected individuals (allele frequency <0.01% in the Exome Aggregation Consortium, ExAC) and with predicted functional effects (Combined Annotation Dependent Depletion, CADD score ≥20), we identified 151 putative predisposing mutations. Of 41 mutations of interest selected for validation, 37 (90.2%) were confirmed as germline in origin via Sanger sequencing of remission or newborn bloodspot DNA.Rare and predicted functional germline mutations in known (NBN, SH2B3, ETV6, CREBBP, MSH6) or suspected (MLL, ABL1, FLT3, MYH9) ALL predisposition genes were identified in nine out of 57 patients (15.8%). Three additional patients harbored germline mutations in the GRB2-associated binding protein 2 (GAB2), a known binding partner of PTPN11-encoded SHP2 and activator of the ERK/MAPK and PI3K/AKT pathways. Two GAB2 mutations, a missense mutation S592F and frameshift mutation P621fs, were predicted to be highly functional (CADD scores = 34 and 36 respectively) and absent in ExAC. Frequency of rare and damaging GAB2 mutations was significantly higher in our patient set (2.6%) than in ExAC (0.28%, P = 2.70 x 10-6). We replicated this finding in sequencing data from 309 ALL patients in the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project (0.81% vs. 0.28%, P = 0.015).Patient GAB2 mutations were cloned into HEK293 cells and, following EGF stimulation, we found that the P621fs mutation reduced SHP2 binding and ERK1/2 phosphorylation but increased AKT phosphorylation. This suggested possible Ras-independent leukemogenic effects, supported by a lack of somatic Ras pathway mutations in the three GAB2 mutant patients. Additional functional analyses and sequencing of larger patient cohorts will be required to elucidate the role of germline GAB2 mutations in childhood ALL. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Targeting of the Ubiquitin Conjugating Enzyme UBE2N in Myeloid Malignancies

Inflammatory and innate immune signaling pathways are activated in leukemic stem and progenitor cells and contribute to the pathogenesis of hematologic malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). UBE2N is a ubiquitin (Ub) conjugating enzyme that catalyzes lysine 63 (K63)-linked Ub chains on substrates that are critical for signal transduction of broad innate immune signaling pathways. Here we report that UBE2N is required for leukemic cell function by mediating oncogenic innate immune signaling, and identified a novel chemical class of small molecule inhibitors that target UBE2N enzymatic activity.Upon UBE2N downregulation with two lentivirally expressed shRNAs, MOLM-13 and THP-1 cells lose their clonogenic potential and undergo cell death. Unlike for AML cells, UBE2N was dispensable for colony formation and viability of healthy cord blood CD34+ cells. The active site of UBE2N contains a cysteine (Cys) at position 87, which is essential for binding and transfer of Ub to its substrates. We performed in silico structure- and in vitro cell-based screens to identify small molecules that dock to the active site of UBE2N and covalently bind to Cys-87, as an approach to inhibit Ub transfer to substrates. Two structurally-related candidates, UC-764864 and UC-764865, emerged as inhibitors of UBE2N, as they specifically blocked the E1-UBE2N thioester transfer in vitro. Treatment of MDS/AML cell lines and patient-derived primary cells with UC-764864 and UC-764865 suppressed innate immune signaling and induced cytotoxic effects in MDS/AML cell lines and primary cells while sparing healthy hematopoietic cells in vitro and in vivo.To identify the molecular basis of UBE2N inhibition, we performed a global Ub screen for changes in ubiquitinated substrates by mass spectrometry and evaluated changes in gene expression by RNA-seq in MOLM-13 cells treated with vehicle control or the newly derived UBE2N inhibitors. RNA-seq of MOLM-13 cells treated with UC-764864 revealed that inhibition of UBE2N in leukemic cells targets oncogenic innate immune pathways, including NF-kB and Type I interferon signaling networks. UC-764864 and UC-764865 reduced the ubiquitination status of UBE2N, and altered the ubiquitination of proteins involved in innate immune signaling and the DNA damage response by primarily reducing K63-linked Ub modifications. Two substrates identified by the Ub screen, DDB1 and UBE2M, are components of the CUL4-CRBN E3 ligase complex and a target of the anti-leukemic therapy, Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and UBE2N inhibitors on MDS/AML cells. As compared to individual treatments, the combination of LEN and UC-764864, UC-764865 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and UBE2N inhibitors impaired MDS/AML cells that are refractory to treatment of LEN or UBE2N inhibitors alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS or AML. In summary, we implicate the Ub conjugating enzyme UBE2N as a target in MDS/AML, and identified novel small molecule inhibitors that target UBE2N and modify the function of Ub E3 ligases that are important for UBE2N-associated diseases, including autoinflammatory and autoimmune disorders, and hematologic malignancies. DisclosuresNo relevant conflicts of interest to declare.

Differences in genomic abnormalities among African individuals with monoclonal gammopathies using calculated ancestry

Multiple myeloma (MM) is two- to three-fold more common in African Americans (AAs) compared to European Americans (EAs). This striking disparity, one of the highest of any cancer, may be due to underlying genetic predisposition between these groups. There are multiple unique cytogenetic subtypes of MM, and it is likely that the disparity is associated with only certain subtypes. Previous efforts to understand this disparity have relied on self-reported race rather than genetic ancestry, which may result in bias. To mitigate these difficulties, we studied 881 patients with monoclonal gammopathies who had undergone uniform testing to identify primary cytogenetic abnormalities. DNA from bone marrow samples was genotyped on the Precision Medicine Research Array and biogeographical ancestry was quantitatively assessed using the Geographic Population Structure Origins tool. The probability of having one of three specific subtypes, namely t(11;14), t(14;16), or t(14;20) was significantly higher in the 120 individuals with highest African ancestry (≥80%) compared with the 235 individuals with lowest African ancestry (<0.1%) (51% vs. 33%, respectively, p value = 0.008). Using quantitatively measured African ancestry, we demonstrate a major proportion of the racial disparity in MM is driven by disparity in the occurrence of the t(11;14), t(14;16), and t(14;20) types of MM.

Inotuzumab ozogamicin in pediatric patients with relapsed/refractory acute lymphoblastic leukemia

Although inotuzumab ozogamicin (InO) is recognized as an effective agent in relapsed acute lymphoblastic leukemia (ALL) in adults, data on safety and efficacy in pediatric patients are scarce. We report the use of InO in 51 children with relapsed/refractory ALL treated in the compassionate use program. In this heavily pretreated cohort, complete remission was achieved in 67% of patients with overt marrow disease. The majority (71%) of responders were negative for minimal residual disease. Responses were observed irrespective of cytogenetic subtype or number or type of prior treatment regimens. InO was well-tolerated; grade 3 hepatic transaminitis or hyperbilirubinemia were noted in 6 (12%) and grade 3/4 infections in 11 (22%) patients. No patient developed sinusoidal obstruction syndrome (SOS) during InO therapy; however, 11 of 21 (52%) patients who underwent hematopoietic stem cell transplantation (HSCT) following InO developed SOS. Downregulation of surface CD22 was detected as a possible escape mechanism in three patients who developed a subsequent relapse after InO. We conclude that InO is a well-tolerated, effective therapy for children with relapsed ALL and prospective studies are warranted. Identification of risk factors for developing post-HSCT SOS and strategies to mitigate this risk are ongoing.

Expression Analysis of PVT1, CCDC26, and CCAT1 Long Noncoding RNAs in Acute Myeloid Leukemia Patients.

Recent evidence indicates that the PVT1, CCDC26, and CCAT1 long noncoding RNAs (lncRNAs) are involved in the leukemogenic process. This study quantified the expression levels of the PVT1, CCDC26, and CCAT1 lncRNAs in patients with acute myeloid leukemia (AML) and also correlated their expression levels with the clinicopathological features of the patients. The expression levels of the PVT1, CCDC26, and CCAT1 lncRNAs were analyzed using quantitative reverse transcription-polymerase chain reaction of bone marrow specimens obtained from 86 AML patients, 48 AML-M3 patients, and 40 normal controls. No differences were found between the combined AML patient populations and the healthy controls with respect to the expression levels of PVT1, CCDC26, and CCAT1 (p = 0.35, p = 0.09, and p = 0.77, respectively). However, compared with the controls, the AML-M3 patients had higher PVT1 expression (p = 0.017). Furthermore, high-risk AML-M3 patients manifested higher expression levels of PVT1 than low- and intermediate-risk groups. In addition, distinctive CCDC26 and CCAT1 expression levels were observed among patients with different French-American-British subtypes (p = 0.001 for CCDC26 and p = 0.013 for CCAT1). Compared with the healthy controls, AML-M4 and M5 had higher CCAT1 expression (p = 0.04) and AML-M2 and AML-M4/M5 patients had higher CCDC26 expression (p < 0.001 and p = 0.02, respectively). In addition, different patterns of CCDC26 expression were found among the different cytogenetic risk subtypes (p = 0.005). Finally, patients with intermediate cytogenetic risk showed higher CCDC26 expression levels. The differential expression of the PVT1, CCDC26, and CCAT1 lncRNAs in different AML subtypes suggests that the deregulation of these transcripts may function in the multistep leukemogenic process and that they may serve as new therapeutic targets for this malignancy.

Improved cytogenetic characterization and risk stratification of pediatric acute lymphoblastic leukemia using single nucleotide polymorphism array analysis: A single center experience of 296 cases.

Single nucleotide polymorphism array (SNP-A) analyses are increasingly being introduced in routine genetic diagnostics of acute lymphoblastic leukemia (ALL). Despite this, only few studies that have compared the diagnostic value of SNP-A with conventional chromosome banding have been published. We here report such a comparison of 296 ALL cases, the largest series to date. Only genomic imbalances >5 Mb and microdeletions targeting the BTG1, CDKN2A/B, EBF1, ERG, ETV6, IKZF1, PAX5, and RB1 genes and the pseudoautosomal region 1 (PAR1) were ascertained, in agreement with recent guidelines. Of 36 T-cell ALL cases, the karyotypes of 24 cases (67%) were revised by SNP-A analyses that either revealed additional imbalances >5 Mb or better characterized the changes found by G-banding. Of 260 B-cell precursor (BCP) ALL cases, SNP-A analyses identified additional copy number alterations, including the above-mentioned microdeletions, or better characterized the imbalances found by G-banding in 236 (91%) cases. Furthermore, the cytogenetic subtype classification of 41/260 (16%) BCP ALL cases was revised based on the SNP-A findings. Of the subtype revisions, 12/41 (29%) had clinical implications as regards risk stratifying cytogenetic groups or genotype-specific minimal residual disease stratification. We conclude that SNP-A analyses dramatically improve the cytogenetic characterization of both T-cell and BCP ALL and also provide important information pertinent to risk stratification of BCP ALL.

Risk of MGUS in relatives of multiple myeloma cases by clinical and tumor characteristics.

We and others have shown increased risk of monoclonal gammopathy of undetermined significance (MGUS) in first-degree relatives of patients with multiple myeloma (MM). Whether familial risk of MGUS differs by the MM proband's age at onset, tumor or clinical characteristics is unknown. MM and smoldering MM (SMM) cases (N = 430) were recruited from the Mayo Clinic in Rochester, Minnesota between 2005-2015. First-degree relatives over age 40 provided serum samples for evaluation of MGUS (N = 1179). Age and sex specific rates of MGUS among first-degree relatives were compared to a population-based sample. Cytogenetic subtypes were classified by Fluorescence in situ hybridization. MGUS was detected in 75 first-degree relatives for an age- and sex- adjusted prevalence of 5.8% (95% CI: 4.5-7.2). Prevalence of MGUS in first-degree relatives was 2.4 fold (95% CI: 1.9-2.9) greater than expected rates. Familial risk did not differ by proband's age at diagnosis, gender, isotype, IgH translocation, or trisomy. This study confirms first-degree relatives of MM cases have a significantly higher risk of MGUS compared to the general population, regardless of age, gender, or tumor characteristics. In selected situations, such as multiple affected first-degree relatives, screening of first-degree relatives of MM cases could be considered for follow-up and prevention strategies.

Trisomy 12 chronic lymphocytic leukemia expresses a unique set of activated and targetable pathways.

Although trisomy 12 (+12) chronic lymphocytic leukemia (CLL) comprises about 20% of cases, relatively little is known about its pathophysiology. These cases often demonstrate atypical morphological and immunophenotypic features, high proliferative rates, unmutated immunoglobulin heavy chain variable region genes, and a high frequency of NOTCH1 mutation. Patients with +12 CLL have an intermediate prognosis, and show higher incidences of thrombocytopenia, Richter transformation, and other secondary cancers. Despite these important differences, relatively few transcriptional profiling studies have focused on identifying dysregulated pathways that characterize +12 CLL, and most have used a hierarchical cytogenetic classification in which cases with more than one recurrent abnormality are categorized according to the abnormality with the poorest prognosis. In this study, we sought to identify protein-coding genes whose expression contributes to the unique pathophysiology of +12 CLL. To exclude the likely confounding effects of multiple cytogenetic abnormalities on gene expression, our +12 patient cohort had +12 as the sole abnormality. We profiled samples obtained from 147 treatment-naïve patients. We compared cases with +12 as the only cytogenetic abnormality to cases with only del(13q), del(11q), or diploid cytogenetics using independent discovery (n=97) and validation (n=50) sets. We demonstrate that CLL cases with +12 as the sole abnormality express a unique set of activated pathways compared to other cytogenetic subtypes. Among these pathways, we identify the NFAT signaling pathway and the immune checkpoint molecule, NT5E (CD73), which may represent new therapeutic targets.

MicroRNA, an Antisense RNA, in Sensing Myeloid Malignancies.

Myeloid malignancies, including myelodysplastic syndromes and acute myeloid leukemia, are clonal diseases arising in hematopoietic stem or progenitor cells. In recent years, microRNA (miRNA) expression profiling studies have revealed close associations of miRNAs with cytogenetic and molecular subtypes of myeloid malignancies, as well as outcome and prognosis of patients. However, the roles of miRNA deregulation in the pathogenesis of myeloid malignancies and how they cooperate with protein-coding gene variants in pathological mechanisms leading to the diseases have not yet been fully understood. In this review, we focus on recent insights into the role of miRNAs in the development and progression of myeloid malignant diseases and discuss the prospect that miRNAs may serve as a potential therapeutic target for leukemia.

Gene and protein analysis reveals that p53 pathway is functionally inactivated in cytogenetically normal Acute Myeloid Leukemia and Acute Promyelocytic Leukemia

BackgroundMechanisms that inactivate the p53 pathway in Acute Myeloid Leukemia (AML), other than rare mutations, are still not well understood.MethodsWe performed a bioinformatics study of the p53 pathway function at the gene expression level on our collection of 1153 p53-pathway related genes. Publically available Affymetrix data of 607 de-novo AML patients at diagnosis were analyzed according to the patients cytogenetic, FAB and molecular mutations subtypes. We further investigated the functional status of the p53 pathway in cytogenetically normal AML (CN-AML) and Acute Promyelocytic Leukemia (APL) patients using bioinformatics, Real-Time PCR and immunohistochemistry.ResultsWe revealed significant and differential alterations of p53 pathway-related gene expression in most of the AML subtypes. We found that p53 pathway-related gene expression was not correlated with the accepted grouping of AML subtypes such as by cytogenetically-based prognosis, morphological stage or by the type of molecular mutation. Our bioinformatic analysis revealed that p53 is not functional in CN-AML and APL blasts at inducing its most important functional outcomes: cell cycle arrest, apoptosis, DNA repair and oxidative stress defense. We revealed transcriptional downregulation of important p53 acetyltransferases in both CN-AML and APL, accompanied by increased Mdmx protein expression and inadequate Chk2 protein activation.ConclusionsOur bioinformatic analysis demonstrated that p53 pathway is differentially inactivated in different AML subtypes. Focused gene and protein analysis of p53 pathway in CN-AML and APL patients imply that functional inactivation of p53 protein can be attributed to its impaired acetylation. Our analysis indicates the need in further accurate evaluation of p53 pathway functioning and regulation in distinct subtypes of AML.

Classification of pediatric acute myeloid leukemia based on miRNA expression profiles.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease with respect to biology as well as outcome. In this study, we investigated whether known biological subgroups of pediatric AML are reflected by a common microRNA (miRNA) expression pattern. We assayed 665 miRNAs on 165 pediatric AML samples. First, unsupervised clustering was performed to identify patient clusters with common miRNA expression profiles. Our analysis unraveled 14 clusters, seven of which had a known (cyto-)genetic denominator. Finally, a robust classifier was constructed to discriminate six molecular aberration groups: 11q23-rearrangements, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17) (q21;q22), NPM1 and CEBPA mutations. The classifier achieved accuracies of 89%, 95%, 95%, 98%, 91% and 96%, respectively. Although lower sensitivities were obtained for the NPM1 and CEBPA (32% and 66%), relatively high sensitivities (84%−94%) were attained for the rest. Specificity was high in all groups (87%−100%). Due to a robust double-loop cross validation procedure employed, the classifier only employed 47 miRNAs to achieve the aforementioned accuracies. To validate the 47 miRNA signatures, we applied them to a publicly available adult AML dataset. Albeit partial overlap of the array platforms and molecular differences between pediatric and adult AML, the signatures performed reasonably well. This corroborates our claim that the identified miRNA signatures are not dominated by sample size bias in the pediatric AML dataset. In conclusion, cytogenetic subtypes of pediatric AML have distinct miRNA expression patterns. Reproducibility of the miRNA signatures in adult dataset suggests that the respective aberrations have a similar biology both in pediatric and adult AML.

Space-Time Clustering of Childhood Leukemia: Evidence of an Association with ETV6-RUNX1 (TEL-AML1) Fusion.

BackgroundMany studies have observed space-time clustering of childhood leukemia (CL) yet few have attempted to elicit etiological clues from such clustering. We recently reported space-time clustering of CL around birth, and now aim to generate etiological hypotheses by comparing clustered and nonclustered cases. We also investigated whether the clustering resulted from many small aggregations of cases or from a few larger clusters.MethodsWe identified cases of persons born and diagnosed between 1985 and 2014 at age 0–15 years from the Swiss Childhood Cancer Registry. We determined spatial and temporal lags that maximized evidence of clustering based on the Knox test and classified cases born within these lags from another case as clustered. Using logistic regression adjusted for child population density, we determined whether clustering status was associated with age at diagnosis, immunophenotype, cytogenetic subtype, perinatal and socioeconomic characteristics, and pollution sources.ResultsAnalyses included 1,282 cases of which 242 were clustered (born within 1 km and 2 years from another case). Of all investigated characteristics only the t(12;21)(p13;q22) translocation (resulting in ETV6-RUNX1 fusion) differed significantly in prevalence between clustered and nonclustered cases (40% and 25%, respectively; adjusted OR 2.54 [1.52–4.23]; p = 0.003). Spatio-temporal clustering was driven by an excess of aggregations of two or three children rather than by a few large clusters.ConclusionOur findings suggest ETV6-RUNX1 is associated with space-time clustering of CL and are consistent with an infection interacting with that oncogene in early life leading to clinical leukemia.