Diagnostic Bone Marrow Research Articles

TP53 mutations identify younger mantle cell lymphoma patients who do not benefit from intensive chemoimmunotherapy

AbstractDespite recent advances in lymphoma treatment, mantle cell lymphoma (MCL) remains incurable, and we are still unable to identify patients who will not benefit from the current standard of care. Here, we explore the prognostic value of recurrent genetic aberrations in diagnostic bone marrow (BM) specimens from 183 younger patients with MCL from the Nordic MCL2 and MCL3 trials, which represent current standard-of-care regimens. In the univariate model, mutations of TP53 (11%) and NOTCH1 (4%), and deletions of TP53 (16%) and CDKN2A (20%), were significantly associated with inferior outcomes (together with MIPI, MIPI-c, blastoid morphology, and Ki67 > 30%); however, in multivariate analyses, only TP53 mutations (HR, 6.2; P < .0001) retained prognostic impact for overall survival (OS), whereas TP53 mutations (HR, 6.9; P < .0001) and MIPI-c high-risk (HR, 2.6; P = .003) had independent prognostic impact on time to relapse. TP53-mutated cases had a dismal outcome, with a median OS of 1.8 years, and 50% relapsed at 1.0 years, compared to a median OS of 12.7 years for TP53-unmutated cases (P < .0001). TP53 mutations were significantly associated with Ki67 > 30%, blastoid morphology, MIPI high-risk, and inferior responses to both induction- and high-dose chemotherapy. In conclusion, we show that TP53 mutations identify a phenotypically distinct and highly aggressive form of MCL with poor or no response to regimens including cytarabine, rituximab, and autologous stem-cell transplant (ASCT). We suggest patients with MCL should be stratified according to TP53 status, and that patients with TP53 mutations should be considered for experimental frontline trials exploring novel agents.

A novel immunohistochemical score to predict early mortality in acute myeloid leukemia patients based on indoleamine 2,3 dioxygenase expression

Indoleamine 2,3 dioxygenase-1 (IDO-1) is an enzyme in the kynurenine pathway which augments tumor-induced immune tolerance. Previous studies in childhood acute myeloid leukemia (AML) have shown a negative correlation of IDO-1 mRNA expression with outcomes. The aim of our study was to develop a practical and objective immunohistochemical technique to quantify IDO-1 expression on diagnostic bone marrow biopsies of AML patients in order to facilitate its use in routine clinical practice. IDO-1 mRNA was extracted from diagnostic bone marrow specimens from 29 AML patients. IDO-1 protein expression was assessed in 40 cases via immunohistochemistry and quantified by a novel ‘composite IDO-1 score’. In a univariate analysis, higher age (p = 0.0018), male gender (p = 0.019), high risk cytogenetics (p = 0.002), higher IDO-1 mRNA (p = 0.005), higher composite IDO-1 score (p < 0.0001) and not undergoing allogeneic stem cell transplant (SCT, p = 0.0005) predicted poor overall survival. In a multivariate model that included the aforementioned variables, higher composite IDO-1 score (p = 0.007) and not undergoing allogeneic SCT (p = 0.007) was found to significantly predict poor outcomes. Further, patients who failed induction had higher composite IDO-1 score (p = 0.01). In conclusion, ‘composite IDO-1 score’ is a prognostic tool that can help identify a certain subset of AML patients with ‘early mortality’. This unique subset of patients can potentially benefit from specific IDO-1 inhibitor therapy, currently in clinical trials.

Clinical impact of KMT2C and SPRY4 expression levels in intensively treated younger adult acute myeloid leukemia patients.

To evaluate the prognostic impact of gene expression levels (ELs) of two tumor suppressor genes, sprouty 4 (SPRY4, located on 5q) and lysine methyltransferase 2C (KMT2C, located on 7q) in correlation with clinical characteristics and genetic abnormalities assessed at initial diagnosis in acute myeloid leukemia (AML). Gene expression levels were measured on cDNA by RT-qPCR from diagnostic bone marrow samples of 275 intensively treated adult AML patients (median age, 48years). KMT2C ELs were significantly lower in abn7q/-7 (P=.001), whereas SPRY4 ELs were not associated with abn5q/-5. Higher KMT2C and SPRY4 ELs were significantly associated with lower genetic risk groups as defined by the European LeukemiaNet classification. Additionally, KMT2C ELs were lower in cytogenetically normal patients with DNMT3A (P=.01) or FLT3-ITD mutations (P=.05). KMT2C ELs were not associated with prognosis, whereas higher SPRY4 ELs showed a favorable impact on event-free (EFS, P=.01), relapse-free (RFS, P=.01) and in-trend on overall survival (P=.06) for cytogenetically abnormal patients, which was confirmed in multivariable analysis for EFS (HR, 0.84; 95%-CI, 0.73-0.97; P=.02) and RFS (HR, 0.85; 95%-CI, 0.73-0.98; P=.02). Our data indicate that KMT2C ELs are associated with specific genetic features and that SPRY4 ELs may add prognostic information.

Abstract 2416: Transcriptome and cluster analysis of infant acute lymphoblastic leukemia cases with and without MLL (KMT2A) rearrangement

Abstract Background: Infant acute lymphoblastic leukemia (ALL) is a high-risk cancer with poor survival due to treatment failure and relapse. The majority of infant ALL cases possess an MLL (KMT2A) translocation (MLL-R), where MLL joins with AF4 (AFF1), ENL (MLLT1), or another partner gene to create a novel fusion gene. A subset of infant ALL cases do not involve an MLL rearrangement (non-MLL-R). Gene expression profiling delineates MLL-R from non-MLL-R cases. Here, we describe transcriptome profiling and cluster analysis of MLL-R and non-MLL-R cases and compare between partner genes. Methods: We performed RNA sequencing (RNA-seq) on diagnostic blood or bone marrow samples from 40 infants with ALL. Cohort A included 16 patients with MLL-R ALL known to have relapsed; cohort B, 10 patients with MLL-R ALL not known to have relapsed; and cohort C, 14 patients with non-MLL-R ALL whom to date have also not relapsed. RNA-seq was performed to an average of 8.8Gb and analyzed using Bowtie2, RSEM, and limma-voom. Multiple different groups of cases (e.g. cohort A versus B) were compared for differential gene expression; the resulting genes were ranked by adjusted P value with a significance cutoff of 0.01 and further evaluated for pathway analysis with Ingenuity software. Hierarchical clustering of samples was conducted using distance measures computed from normalized gene expression. Results: Transcriptome analysis of MLL-R versus non-MLL-R cases found 2,334 differentially expressed genes. Ingenuity pathway analysis of these genes showed enrichment in specific gene sets, such as those involved in PTEN, EGF, and NF-κB signaling. Ingenuity predicted increased PTEN signaling and diminished EGF and NF-κB signaling in MLL-R cases relative to non-MLL-R cases. Transcriptome analysis of relapsed versus non-relapsed MLL-R cases found no differentially expressed genes meeting significance criteria, while analysis of MLL fusion partners identified 46 differentially expressed genes including BMI1 and MYC. Cluster analysis demonstrated a distinct grouping of non-MLL-R cases relative to MLL-R cases. Additionally, MLL-R samples sub-clustered according to their MLL fusion gene partners. However, no difference in clustering was appreciated between MLL-R cases that later went on to relapse versus those that did not. Conclusions: Our findings show that MLL-R infant ALL has a distinct gene expression signature at diagnosis that correlates with the type of MLL translocation present. This is important because it suggests treatment strategies could be tailored to the MLL partner gene. Risk of relapse in MLL-R cases could not be predicted by this signature due to a paucity of differentially expressed genes between relapsed and non-relapsed groups at diagnosis. Similar to prior profiling studies, we find that non-MLL-R cases independently cluster together and have a gene expression signature distinct from MLL-R cases.[B.Y. and M.F. contributed equally to this work.] Citation Format: Byunggil Yoo, Midhat S. Farooqi, Rumen Kostadinov, Neil Miller, Jeff Johnston, Emily Farrow, Shannon Kelley, Margaret Gibson, Patrick Brown, Erin Guest. Transcriptome and cluster analysis of infant acute lymphoblastic leukemia cases with and without MLL (KMT2A) rearrangement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2017-2416

Clonal selection and asymmetric distribution of human leukemia in murine xenografts revealed by cellular barcoding

AbstractGenetic and phenotypic heterogeneity of human leukemia is thought to drive leukemia progression through a Darwinian process of selection and evolution of increasingly malignant clones. However, the lack of markers that uniquely identify individual leukemia clones precludes high-resolution tracing of their clonal dynamics. Here, we use cellular barcoding to analyze the clonal behavior of patient-derived leukemia-propagating cells (LPCs) in murine xenografts. Using a leukemic cell line and diagnostic bone marrow cells from 6 patients with B-progenitor cell acute lymphoblastic leukemia, we demonstrate that patient-derived xenografts were highly polyclonal, consisting of tens to hundreds of LPC clones. The number of clones was stable within xenografts but strongly reduced upon serial transplantation. In contrast to primary recipients, in which clonal composition was highly diverse, clonal composition in serial xenografts was highly similar between recipients of the same donor and reflected donor clonality, supporting a deterministic, clone-size–based model for clonal selection. Quantitative analysis of clonal abundance in several anatomic sites identified 2 types of anatomic asymmetry. First, clones were asymmetrically distributed between different bones. Second, clonal composition in the skeleton significantly differed from extramedullary sites, showing similar numbers but different clone sizes. Altogether, this study shows that cellular barcoding and xenotransplantation providea useful model to study the behavior of patient-derived LPC clones, which provides insights relevant for experimental studies on cancer stem cells and for clinical protocols for the diagnosis and treatment of leukemia.

Diagnostic bone marrow sampling in cats.

This review, aimed at all veterinarians who treat cats, highlights the reasons a clinician may elect to sample bone marrow, and details the collection of bone marrow for both cytology and histopathology. Potential complications and limitations of each procedure are also reviewed. Evidence base: The collection of bone marrow for cytology and histopathology is not new to feline medicine and several research studies have investigated various sampling modalities in cats. This article offers a comprehensive review of the currently accepted best practices.

Morphologic and Molecular Characteristics of De Novo AML With JAK2 V617F Mutation.

Background:JAK2 V617F mutation (mut) in acute myeloid leukemia (AML) is rare. We describe the clinicopathologic findings of a single-institution series of 11 de novo AML cases with JAK2 V617. Methods: We identified cases of de novo AML with JAK2 V617F over a 10-year period. We reviewed diagnostic peripheral blood and bone marrow (BM) morphologic, cytogenetic, and molecular studies, including next-generation sequencing. The control group consisted of 12 patients with JAK2 wild-type (wt) AML matched for age, sex, and diagnosis. Results: We identified 11 patients (0.5%) with JAK2 V617F, with a median age at diagnosis of 72.5 years (range, 36-90 years). Ten neoplasms were classified as AML with myelodysplasia-related changes and 1 as AML with t(8;21)(q22;q22). All JAK2mut AML cases showed at least bilineage dysplasia, 7 of 11 showed fibrosis, 8 of 11 had an abnormal karyotype, and 5 had deletions or monosomy of chromosomes 5 and 7. Using the European LeukemiaNet (ELN) classification, 9 patients (82%) with JAK2mut AML were intermediate-2 and adverse risk. Cases of JAK2mut AML did not have mutations in other activating signaling pathways (P=.013); 7 (64%) showed additional mutations in at least one gene involving DNA methylation and/or epigenetic modification. Patients with JAK2mut AML had a significantly higher median BM granulocyte percentage (12% vs 3.5%; P=.006) and a higher frequency of ELN intermediate-2 and adverse risk cytogenetics (P=.04) compared with those with JAK2wt AML. JAK2mut AML showed higher circulating blasts, but this difference was not significant (17% vs 5.5%; P=not significant). No difference was seen in the median overall survival rate of patients with JAK2mut AML versus those with JAK2wt AML (14 vs 13.5 months, respectively). Conclusions: De novo JAK2mut AML is rare and frequently found in patients with dysplasia, BM fibrosis, and abnormal karyotype with intermediate- or high-risk features; gene mutations in DNA methylation and epigenetic-modifying pathways; and absence of gene mutations in activating signaling pathways.

Presumed primary immune‐mediated neutropenia in 35 dogs: a retrospective study

To describe, in a cohort of dogs with presumed primary immune-mediated neutropenia, the presenting clinical characteristics, haematology results, bone marrow characteristics, therapies used (drugs and doses), clinical response to treatment, relapse and outcome at six months and one year. Multi-institutional recruited retrospective descriptive case series with voluntary submissions. Presumed immune-mediated neutropenia was diagnosed based on a neutrophil concentration <1·5×109 cells/L on a minimum of two complete blood counts, exclusion of other causes of neutropenia based on a diagnostic bone marrow aspirate or biopsy, and exclusion of secondary immune-mediated neutropenia. Dogs meeting these diagnostic criteria between 2006 and 2013, and that had a haematocrit of ≥29% and minimum of two complete blood clounts performed after initiation of therapy, were included. Information on 35 dogs was included. Neutropenia was less than 0·5×109 cells/L in most cases (21 dogs), 0·5 to ·99×109 cells/L in 11, and 1.0 to 1·49×109 cells/L in three. Eight dogs had thrombocytopenia, which was severe (<49·9×109 cells/L) in three. [Correction added on 23 May 2017, after first online publication: the cell numbers were incorrect due to errors in the conversion of cell measurements to international units. The numbers have been corrected throughout the article and Table 2.] Twenty-three dogs had myeloid hyperplasia, 10 dogs had myeloid hypoplasia and two dogs had normal myelopoiesis. Neutropenia resolved in 32 of 33 dogs within two weeks of starting corticosteroid therapy and in all dogs within one month. Relapse of neutropenia occurred in 12 cases within one year. Initial response of presumed primary immune-mediated neutropenia cases to corticosteroid therapy can be excellent. Long-term monitoring for relapse is warranted because 34% of cases relapsed during or after taper of immunosuppressive medications.

Prognostic Value of Diagnostic Bone Marrow Plasma Cell Percentage in Multiple Myeloma

Prognostic Value of Diagnostic Bone Marrow Plasma Cell Percentage in Multiple Myeloma

Association of CNS involvement in childhood acute lymphoblastic leukaemia with cholesterol biosynthesis upregulation

BackgroundAcute lymphoblastic leukaemia is the most common childhood cancer. Despite advances in treatment, meningeal-infiltrating CNS disease (found in up to 90% of children if untreated) remains a challenge. Every child with acute lymphoblastic leukaemia receives intensive and toxic treatment targeting CNS disease, yet CNS involvement at relapse is common. We tested our hypothesis that leukaemic cells must adapt metabolically to survive in the nutritionally poor CNS microenvironment. MethodsHuman acute lymphoblastic leukaemia cell lines (SEM and REH) were xenografted into immunodeficient (NSG) mice. Leukaemic cells purified from the CNS and spleen underwent RNA-sequencing (NextSeq 500, Illumina, SanDiego, CA, USA). Clinical validity of findings was explored in publicly available microarray datasets (GSE11877/TARGET and GSE60926) with gene set enrichment and pathway analyses. The effect of simvastatin on SEM cells in vitro, and the development of CNS leukaemia in vivo, was investigated. FindingsRNA-sequencing analysis showed upregulation of the cholesterol biosynthesis pathway in CNS compared with that in systemic leukaemia. GSE60926 microarray data confirmed this finding in leukaemic cells from cerebrospinal fluid in children with acute lymphoblastic leukaemia at CNS relapse versus cells from bone marrow at diagnosis and relapse. Analysis of GSE11877/TARGET data showed that upregulation of two or more cholesterol synthesis genes (z-score ≥2) in the bone marrow blasts at diagnosis was associated with an increased rate of CNS relapse (10 [30·3%] of 33 patients with upregulation vs 15 [8·6%] of 174 patients without; log-rank [Mantel-Cox] p<0·0001). In-vitro study showed that disruption of cholesterol synthesis via hydroxymethylglutaryl-CoA reductase inhibition with simvastatin decreased leukaemic cell proliferation and induced apoptosis. Interestingly, oral simvastatin treatment increased CNS leukaemia in male though not female NSG mice (three per group, Student's t test p=0·0004). InterpretationOur findings suggest that cholesterol metabolism could be important in CNS acute lymphoblastic leukaemia. Cholesterol biosynthesis is upregulated in CNS leukaemia, and a cholesterol gene signature in diagnostic bone marrow predicts CNS relapse. Disruption of cholesterol synthesis is toxic in vitro but not effective in vivo. Therefore, reduced circulating cholesterol might induce a CNS-like phenotype in systemic acute lymphoblastic leukaemia cells, increasing cholesterol synthesis and adaptation for the CNS niche. FundingWilliam and Elizabeth Davies Foundation, Chief Scientist Office (ETM/374).

Coexisting and cooperating mutations in NPM1-mutated acute myeloid leukemia

NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n=120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0–11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.

Visceral Leishmaniasis in an Immunocompromised Patient: Diagnostic Bone Marrow Cytomorphology.

Visceral Leishmaniasis in an Immunocompromised Patient: Diagnostic Bone Marrow Cytomorphology.

Mass Cytometry As a Modality to Identify Candidates for Immune Checkpoint Inhibitor Therapy within Acute Myeloid Leukemia

Introduction: Immune checkpoint inhibitors (ICIs) have shown significant efficacy in solid malignancies and recently in Hodgkin's disease. However, there are presently no biomarkers that predict which cancer types and/or individual patients will benefit from ICIs. Despite intensification of chemotherapeutic regimens, survival rates in acute myeloid leukemia (AML) have plateaued. We are therefore, performing studies to characterize the immune phenotype and genetic profile of AML patients who may respond to ICI therapy. Specifically, we are identifying cell subsets present in the marrow of patients with AML, evaluating their differentiation and activation status, and quantifying the density of immune modifiers. We are also performing functional assays to define the status of T cells in the AML microenvironment by evaluating the responsiveness of these cells to stimulation. Additionally, these studies assess the impact of several ICIs on the proliferative potential and cytokine production capacity of T cells resident in the bone marrow of AML patients.Methods: Immunophenotyping was performed on the diagnostic bone marrow (Dx BM), diagnostic peripheral blood (Dx PB), and remission bone marrow (CR BM) for a single patient with AML. These studies used a time-of-flight mass cytometer (CyTOF) by staining samples with a panel containing 35 antibodies to surface markers and cytokines (Table 1). T cells were defined and then differentiated based on the presence of CD3 and CD4 or CD8. T cells were further classified into naive, central memory (CM), effector memory (EM), and effector cells. Functional assays measured proliferative capacity and cytokine production profile of bone marrow T cells in response to CD3 stimulation for the Dx BM and Dx PB. Changes to the baseline T cell functional profile in the presence of antibodies against PD1, CTLA4, TIM3, and TIGIT were measured in the Dx BM.Results: T cell percentages were 3.14%, 5.46%, and 17.1% for the Dx BM, Dx, PB, and CR BM, respectively. CD4:CD8 ratios were 3.5, 2.9, and 4.5, respectively. EM cells made up the largest percentage of CD8+ T cells in the Dx BM (40.99%), Dx PB (57.06%), and CR BM (53.55%). Naïve (52.49%), EM (37.63%) and CM (44.3%) cells made up the largest percentages of CD4+ T cells in the Dx BM, Dx PB, and CR BM, respectively (Figure 1). Checkpoint expression was absent (median intensity < 1) on all T cells except for some CD8+ subsets (Figure 2). Within the Dx BM, the effector (1.93) and EM (4.34) cells expressed TIGIT and the EM cells expressed PD1 (1.92). Within the Dx PB, the effector (1.81) and EM (4.0) cells expressed TIGIT. Within the CR BM, the effector cells (2.3) expressed TIGIT. In response to CD3 stimulation, 57.5% of the Dx PB T cells divided while there was no proliferation observed in the Dx BM. In response to CD3 stimulation and PD1 blockade, 28.2% of the Dx BM T cells divided and there was a 4-fold increase in IFNγ production. There was no division in the Dx BM T cells in response to TIGIT blockade but there was a 10-fold and 121-fold increase in IL-2 and IFNγ production, respectively. There was no significant proliferation or cytokine production in response to TIM3 or CTLA4 blockade.Conclusion: We observed differential T cell responses to stimulation in the presence of ICIs in this patient. The Dx PB T cells were capable of proliferating in the presence of CD3 stimulation alone whereas the Dx BM T cells were only able to divide in the presence of PD1 blocking antibody. PD1 blockade elicited proliferation and low levels of IFNγ production whereas TIGIT blockade elicited negligible proliferation but high levels of IFNγ production. There was low but differential checkpoint expression on T cells, with PD1 and TIGIT having the highest expression, specifically on CD8+ T cell subsets. The predominant CD8+ T cell subset, along with differential checkpoint expression, persisted into the CR BM but there was a shift in the predominant CD4+ T cell subset. These data suggest the presence of immune suppression in the bone marrow microenvironment that may be amenable to checkpoint inhibition. [Display omitted] [Display omitted] [Display omitted] DisclosuresHuang:Janssen Research & Development, LLC: Employment, Equity Ownership. Sasser:Janssen Research & Development, LLC: Employment. Adams:Janssen Research & Development, LLC: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Lind:Janssen: Research Funding; Fluidigm: Honoraria.

Monitoring Minimal Residual Disease in AML By Patient Specific Mutational Fingerprint Using Multiplex PCR and Deep Sequencing

Abstract Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of abnormal hematopoietic progenitor cells. With induction chemotherapy, patients attain a high rate of complete remission as measured by cytogenetic and flow cytometry markers; however, the majority eventually experience relapse. Accurate monitoring of minimal residual disease (MRD) can provide important information for relapse prediction, but current techniques rely on single somatic mutations or a small number flow cytometry markers. It was recently shown that subsets of leukemia-associated mutations can persist after treatment, even if standard clinical evaluation suggests complete remission. Such patients have an increased risk of relapse and reduced overall survival. It is however difficult to foresee which mutations at diagnosis that will persist and contribute to the leukemic relapse. To reliably monitor MRD status, a compelling strategy would be to ascertain as many mutations as possible. We here demonstrate how this can be achieved using an automated design of multiplex PCR primers followed by deep sequencing of the PCR products, enabling monitoring of MRD and mutation pattern based on each patients initial unique mutational fingerprint. Methods: We selected five patients with AML or high risk myelodysplastic syndrome (MDS) with whole exome sequencing (WES) data available from a diagnostic bone marrow or a peripheral blood sample together with a matched skin biopsy for identification of somatic variants. All five cases had material available from presentation, at least one follow up time point, and at relapse. All somatic coding mutations with a variant allele frequency (VAF) above 5% from the WES that passed the sequencing quality threshold were included, constituting the patients mutational fingerprint. The number of mutations ranged from 9 to 33 per patient. Individualized multiplex PCR assays (1-2 multiplex PCR assays/patient) were designed towards all fingerprint mutations using in-house software together with MPprimer. The multiplex PCRs were performed using Qiagen multiplex PCR kit (Qiagen). Each patient specific fingerprint analysis was performed on paired diagnosis, follow up and relapse samples. Sequencing libraries were generated using Nextera XT DNA library prep kit (Illumina) and sequencing was performed on a NextSeq500 (Illumina). Variant recalling was performed using freebayes and only variants with a VAF&gt;5% and coverage above 100X in the diagnostic sample were considered successful MRD markers. Results: Automatic primer design was possible for 84 out of the total 88 mutations (95%). 75 of the targets (89%) were regarded successfully amplified in the multiplex PCR (sequencing coverage above 100X) and had a median coverage of 6566X. The error rate was estimated to around 1%. This multiplex PCR and sequencing approach allowed us to track each patient's unique mutation pattern in the follow up samples and at relapse. We could identify three patients in which all mutations were cleared in the follow up samples prior to relapse (Fig. 1a, b, c) and two patients in which not all mutations were cleared in the follow up samples (Fig. 1d, e). We could also identify which of the mutations at diagnosis that were present also at relapse (Fig. 1a-e). Hence, this approach is a relatively cost effective, fast and reliable assay for monitoring the disease-causing AML clone during a follow up. Conclusions: Traditional MRD monitoring by detection of single mutations or aberrant expression of flow cytometry markers is a proven and powerful method for identifying patients with a higher risk of relapse. However, a known problem with this approach is the risk that some markers are lost at relapse. We here describe a straight forward method allowing the diagnostic patient specific mutational fingerprint to be followed, which should serve as a more stable disease marker of the aberrant clone. In the five patients investigated, we could track mutations that were cleared, persisted despite clinical signs of remission, and mutations that were retained or lost at relapse. In a clinical setting, following an initial screen for somatic variants by WES, the individual multiplex PCR MRD assay could easily and at a relatively low cost be performed in any small NGS lab, thus allowing improved risk stratification and follow up of patents diagnosed with AML and other malignant hematologic disorders. Disclosures Fioretos: Cantargia: Equity Ownership.

Minimal Residual Disease in Patients with Follicular Lymphoma Treated with Obinutuzumab or Rituximab As First-Line Induction Immunochemotherapy and Maintenance in the Phase 3 GALLIUM Study

Minimal Residual Disease in Patients with Follicular Lymphoma Treated with Obinutuzumab or Rituximab As First-Line Induction Immunochemotherapy and Maintenance in the Phase 3 GALLIUM Study

Clinical Heterogeneity in RUNX1-Associated Familial Myelodysplastic Syndrome - Report of Two Novel Pedigrees with Childhoodleukemia

RUNX1-associated familial leukemia is a rare hematologic malignancy. It is inherited in an autosomal dominant manner and mainly causes myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Since constitutional mutations in RUNX1 can lead to thrombocytopenia or dysfunctional platelets, the disease is termed familial platelet disorder with associated myeloid malignancies (FPDMM, MIM 601399). Here, we report two novel families with early onset MDS illustrating the variability of the disorder.The index patient (P1) of family 1 (Fig. 1A) was known to have asymptomatic thrombocytopenia (90 G/l) since his neonatal period. Platelet assays revealed impaired in vitro aggregation. Asymptomatic thrombocytopenia was also known in his younger sister, his mother and his maternal grandfather. A maternal uncle had died from leukemia at age 4. While P1 was treated with antibiotics for a middle ear infection, he developed petechiae and hematoma. He was found to have a platelet count of 11 G/l with a normal white blood count (WBC), hemoglobin concentration (Hb) and MCV. Bone marrow investigations led to the diagnosis of MDS with excess blasts (MDS-EB) with normal karyotype. Sequencing of RUNX1 identified a heterozygous truncating RUNX1 mutation in P1 that was also present in his mother: c.90_117dup; p.(Leu40AlafsX80). P1 underwent hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD) following a preparative regimen consisting of busulfan, cyclophosphamide and melphalan (BuCyMel). He is alive and well 4 years post HSCT. Two years after his diagnosis, a 17-year-old maternal uncle of P1 (P2) was also diagnosed with MDS-EB with normal karyotype. Successively, the disorder progressed to AML and karyotyping showed a clonal derivative chromosome 7 due to an unbalanced translocation der(7)t(2;7)(p12;q21), and later on, a clonal evolution with trisomy 12. RUNX1 sequencing indicated that P2 also carries the familial RUNX1 mutation. Finally, the causative RUNX1 mutation was also identified in the younger sister and two further maternal uncles, all showing mild, asymptomatic thrombocytopenia without evidence of malignant transformation.In the second family (Fig. 1B), the male index patient (P3) had congenital, asymptomatic thrombocytopenia with subsequent platelet counts of 60-80 G/l. At age 3.5, platelet count declined over several months reaching values of 3-20 G/l. WBC, Hb and MCV were normal. The patient had recurrent nose bleeds and prolonged bleeding after cuts and blood drawing. Diagnostic bone marrow examination showed MDS-EB with normal karyotype. Since, thrombocytopenia was also known in P3’s mother, RUNX1 was sequenced and showed a typical heterozygous truncating mutation in P3 and his mother: c.415C>T; p.(Arg139X). The mutation was absent in P3’s brother. At age 16, a maternal aunt of P3 had died due to a cervical cancer. Her DNA was not available for testing. P3 received a MUD HSCT following a BuCyMel preparative regimen and is alive and well 2 years post HSCT.These two new pedigrees with germline RUNX1 mutation emphasize a few interesting points. For both index patients the history of familial thrombocytopenia had been known since birth, but genetic work-up was only initiated after the platelet count had rapidly decreased and bleeding had become obvious. At the time of genetic confirmation, MDS-EB had to be diagnosed despite normal values and blood morphology for white cells and red cells. Thus, decreasing platelet counts in patients with known RUNX1 germline mutations or long-standing thrombocytopenias of unknown cause should result in prompt bone marrow examination, even in the absence of other signs or symptoms suspicious of hematopoietic neoplasia. Furthermore, the platelet count might be a suitable screening instrument in surveillance of patients known to carry germline RUNX1 mutations. Although both index patients had been successfully transplanted following a standard myeloablative regimen, earlier diagnosis of MDS might have saved toxicity and late effects. With our current knowledge of myeloid neoplasms with germline predisposition and pre-existing platelet disorders, children with thrombocytopenia and a family history should have complete genetic work-up for currently known alterations. Consensus guidelines need to be developed by specialists in hematology/oncology, coagulation, general pediatrics and human genetics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeted Sequencing of Diagnostic Samples Correlated to Clinical Outcome: Data from the Nordic Mantle Cell Lymphoma (MCL2 and MCL3) Studies with Long-Term Follow-up

INTRODUCTIONDuring the past decades, the outcome of Mantle Cell Lymphoma (MCL) treatment has improved substantially in younger patients. In a recent update of the Nordic MCL2 trial we show very long response durations after a median follow-up of 11.4 years, but we also observe a continuous pattern of relapses even after 10 years of remission.(Eskelund et al, 2016) The course of this disease remains very heterogeneous, and with the current prognostic indexes we are unable to identify patients who might be cured by the current standard-of-care and others who would possibly benefit from alternative frontline approaches. Recently, next-generation sequencing (NGS) studies have explored the mutational landscape of MCL, however, in inhomogeneous and diversely treated cohorts or with short follow-up. Still, TP53 and NOTCH1/2 mutations have been shown to be prognostic markers. In our current study we examine the prognostic impact of aberrations in the most frequently mutated genes in MCL in a homogenously and optimally treated patient cohort, with a long-term follow-up. MATERIAL AND METHODSFreshly frozen DNA from diagnostic bone marrow samples from patients included in two prospective Nordic trials, MCL2 and MCL3 were analysed. In both trials patients received intensified first line induction therapy with alternating courses of R-CHOP and R-HD-Cytarabine and consolidation with high-dose therapy and ASCT. All patients signed an informed consent.(Geisler et al, 2008; Kolstad et al, 2014). NGS was performed using the Ion Torrent Technology. A targeted panel of 8 genes frequently mutated in MCL was constructed on the basis of previous NGS studies.(Bea et al, 2013; Zhang et al, 2014) The panel included all coding regions of the following genes: ATM, CCND1, TP53, KMT2D, NOTCH1, NOTCH2, WHSC1 and BIRC3. Cut-off for calling a mutation was set to a variant allele frequency >3%. Mean depth was >1500X in all patients. RESULTSSo far, we have mutational data from 72 patients. Patients were previously untreated and <66 years (median 58, range 37-65). Fifty-three percent were either MIPI intermediate or high risk. Eighty-five percent of patients had bone marrow involvement at diagnosis. After a median follow-up of 11.6 years, median overall (OS) and progression-free survival (PFS) of all 72 patients were 12.4 and 9.8 years, respectively.Of the 72 patients, mutations were detected in 33 cases. Twenty-one patients carried 1 mutation and 12 patients had >1 mutation (2-4). Mutations were distributed as follows: ATM 15 (21%), KMT2D 11 (15%), WHSC1 7 (10%), TP53 6 (8%), CCND1 5 (7%), NOTCH2 4 (6%), NOTCH1 3 (4%), BIRC3 2 (3%). In univariate analyses, mutations in TP53 were highly predictive of an inferior outcome (median OS and PFS were 14 and 10 months, respectively; p<0.0001 for both outcomes) (Fig). Likewise, patients with either NOTCH1 or NOTCH2 mutations displayed worse OS (median OS 8.1 years, p=0.023), and there was a trend towards inferior PFS (median PFS 4.5years, p=0.087). No other mutations predicted better or worse outcomes, nor did carrying any one or >1 mutations. In multivariate analyses, the only prognostic significant mutations were TP53 (p<0.0001, HR=15.9) and NOTCH1 (p=0.012, HR=5.3).TP53 mutations were associated with increased Ki67 expression, median 30% (range 20-80%), higher risk MIPI/MIPI-B/MIPI-C, and 4 out of 6 patients had blastoid morphology at diagnosis. All 6 TP53 mutations were found in the DNA-binding domain, and 5 were missense mutations while the last was a frameshift mutation. Patients with missense mutations all died within 3 years (range 6-34 months) due to relapsing or progressive disease, while PFS and OS of the latter patient was 4.6 years and 8.4 years, respectively.We found no predictors for late relapses. CONCLUSIONHere we evaluate the prognostic impact of mutations in eight genes that are commonly involved in MCL in a cohort of 72 younger patients who received intensified induction therapy and ASCT in the Nordic MCL2 trial. In line with previous reports, we demonstrate a negative prognostic impact of TP53 and NOTCH1/2 mutations; however, in multivariate analyses only TP53 and NOTCH1 mutations held significance. Missense mutations in the DNA binding domain of TP53 seem to predict an exceedingly poor short term outcome. At the annual meeting, we will present mutational analyses and long term follow up of >150 patients from the combined MCL2 and MCL3 cohort. [Display omitted] DisclosuresJerkeman:Amgen: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Mundipharma: Research Funding. Geisler:Roche: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy.

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

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

Immunoproteasome Inhibition to Target AML with Activated RAS Pathways

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 only leukemia cells with self-renewal potential and ability to recapitulate the disease. Our goal is to develop therapeutics that target and eradicate AML LSCs. We showed that activated NRAS facilitates self-renewal in a murine model of AML; mTORC1 activation by NRAS is important for this effect (Sachs et al. Blood 2014). RAS activates mTORC1 through the PI3K/AKT/mTOR pathway which is highly upregulated in AML (Xu et al. Blood 2003, Altman et al. Oncogtarget 2011). Recently, we found that RAS-activated malignancies, including AML, induce immunoproteasome formation via mTORC1 (Yun et al. Mol Cell 2016). Pan-proteasome inhibitors, such as bortezomib and carfilzomib, are widely used in the treatment of lymphoid malignancies and have some activity in AML. We hypothesize that specific inhibition of the immunoproteasome may target NRAS-mediated self-renewal in LSCs. We use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). We investigated the possible role of the immunoproteasome in our murine model and found that abolishing NRASG12V transgene expression led to a loss of immunoproteasome component genes expression (p<0.001). We also found that the LSC-enriched subpopulation preferentially upregulated expression of immunoproteasome component genes (p<0.002) in comparison to non-stem cells from this model. Accordingly, we found that primary human AML CD34+ precursors upregulated immunoproteasomal components relative to CD34- AML cells by up to 3.7 fold. mTOR inhibition with everolimus diminished expression of immunoproteasome component genes (p=0.05) suggesting that immunoproteasome activity may be NRASG12V/mTOR-dependent in this model as well. Next, we sought to identify the self-renewing cells among immunophenotypically defined LSC-enriched subgroup in this model. We performed single-cell RNA sequencing on the LSC-enriched population from this model. We identified three discrete trasncriptional profiles among the LSCs. Comparing the single-cell transcriptional profiles of NRASG12V-expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of these profiles are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted LSCs based on CD36 and CD69 expression as well as established immunophenotypic markers, Mac1, Kit, Sca-1 (LSC-enriched supopulation in this model is Mac1LowKit+Sca1+). These 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 CD36-CD69+Mac1LowKit+Sca1+ cells (consistent with Profile 1 gene expression) rapidly transferred leukemia with high penetrance. In contrast, CD36+CD69- Mac1LowKit+Sca1+ cells (consistent with Profile 2 gene expresison) did not transfer leukemia to mice (p < 0.004). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature at the singel-cell level and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. We performed single-cell RNA sequencing on CD34+ human AML precursors 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 that differentially express RAS-activated gene expression profiles. Finally, we found that, PR957, a specific immunoproteasome inhibitor, inhibits in vitro colony formation more effectively than carfilzomib (a second generation pan-proteasome inhibitor) in our murine model of AML suggesting that immunoproteasome inhibition may be specifically target self-renewal in AML. In these experiments, we use single-cell RNA sequencing to identify the self-renewing subset of RAS-driven LSCs at the single-cell level and implicate immunoproteasome inhibition as an approach to target RAS-mediated self-renewal. DisclosuresNo relevant conflicts of interest to declare.

The Genomic Landscape of Pediatric Myelodysplastic Syndromes

Myelodysplastic syndromes are uncommon in children (incidence of ~2 cases/million) and have a poor prognosis. Despite the wealth of knowledge about the genomic landscape of adult MDS, much less is understood about pediatric MDS, and many recurrent mutations found in adults are not common in children (Hirabayashi, Blood 2012). Furthermore, the clinical presentation, bone marrow morphology, and cytogenetic abnormalities are also different when comparing adult and pediatric MDS, suggesting disparate underlying mechanisms.Here we describe the somatic and germline genomic landscape of pediatric MDS using whole exome sequencing (WES) and RNA-sequencing. We evaluated 88 diagnostic bone marrow samples obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1988 and 2015. This cohort contains 34 primary MDS, including Refractory Cytopenia of Childhood/RCC (n=19) and Refractory Anemia with Excess Blasts/RAEB (n=15). For comparison, we also included 32 treatment-related (tMDS), 14 MDS/MPN (including 10 Juvenile Myelomonocytic Leukemia/JMML), and 8 cases of AML with Myelodysplasia-Related Changes/AML-MRC (including 5 cases that previously would have been classified as RAEB in transformation/RAEB-T). WES was completed for 87 tumor/normal pairs (tumor only, n=1) using the Nextera Rapid Capture Expanded Exome (Illumina). Normal comparator gDNA was obtained from flow-sorted lymphocytes and variants were classified as germline if present at greater than 30% variant allele frequency (VAF) in the lymphocyte sample; thus, bone marrow mosaicism cannot be excluded. Mean sequencing coverage for the tumor and normal samples were 102x and 105x, respectively. An average of 7.9 variants were observed per patient in the primary MDS cohort (RCC=6.3, RAEB=10.2), compared to 25.5/patient in the tMDS cohort (p=0.001). Copy number information, obtained from WES data, determined that deletions involving chromosome 7 were frequent (n=28, 32%). Approximately 50% of RCC cases had deletions involving chromosome 7 (9 of 19), compared to only 20% of RAEB cases (3 of 15). Amplification of chromosomes 8 (n=7, 8%) and 21 (n=6, 7%), and deletions of 17 (n=5, 6%) were present at low frequency. In total, we detected 43 additional copy number abnormalities (including 9 cryptic chromosome 7 abnormalities) with WES compared to standard conventional karyotyping. RAS/MAPK pathway mutations were present in 42% of the patients (49 total mutations in 37 cases, including 4 germline variants). Fourteen of the 34 primary MDS cases (41%) had at least one RAS/MAPK mutation, including 13 somatic and 2 germline variants. Mutations in RNA splicing genes (germline, n=0; somatic, n=7; 8% of cohort) were less common, unlike what is observed in adult MDS. As expected, 2 patients with JMML harbored germline variants in PTPN11 and NF1. Surprisingly, presumed germline variants were detected in RRAS and NF1 in patients with primary MDS. Germline variants in transcription factors seen in familial MDS/AML (e.g., RUNX1, CEBPA, ETV6, GATA2) were uncommon, although a germline GATA2 variant was found in a single AML-MRC case. RNA-seq using the TruSeq (Illumina) Stranded RNA protocol was performed on 70 samples with suitable RNA. Fusion transcripts were uncommon in primary MDS, while fusions involving KMT2A, NUP98, RUNX1, MECOM, and ETV6were detected in the tMDS and AML-MRC cohorts.Although many of the mutations affecting the RAS/MAPK pathway were in common genes (NRAS, PTPN11, NF1, or CBL), many other mutations were in genes less frequently reported to be mutated in myeloid neoplasms, such as BRAF, SOS1, RIT1 and RRAS. We demonstrated that the mutations in BRAF (G469A, D594N, N581I) and SOS1 (E433K, G328R, S548R) found in our cohort activate the RAS/MAPK pathway to variable levels, as measured by ERK phosphorylation. In addition, expression of the BRAFvariants conferred IL3-independence in Ba/F3 cells.In conclusion, we show that the genomic landscapes of pediatric and adult MDS are different, namely in patterns of RAS/MAPK pathway and RNA splicing gene mutations, thus supporting the notion that MDS in adults and children comprise unique biological entities. The enrichment of RAS/MAPK mutations in pediatric MDS suggests biologic overlap with JMML and may provide direction for future therapeutic options. DisclosuresNo relevant conflicts of interest to declare.