BM Samples Research Articles

Leukemogenesis in Seven Donor Cell Derived Myeloid Neoplasms Patients. Whole Exome Sequencing Reveals Clonal Dynamics

IntroductionDonor cell derived myeloid neoplasm (DCMN), defined as the development of de novohematological malignancies from cells of donor origin,is a late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We report on seven cases of DCMN in which whole-exome sequencing (WES) at different time-points after allo-HSCT, as well as in a sample from each donor, was performed. The ultimate objective was to accurately described the clonal architecture, spatial heterogeneity and identify somatic mutations that are induced in the process of leukemogenesis and clonal evolution of myeloid neoplasm. Donors were also analyzed to detect underlying condition predisposing to the development of DCMN.Patient and MethodsSeven patients with a confirmed diagnosis of DCMN and their donors were recruited from different Spanish institutions. This cohort included a total of 32 BM samples at different time points after allo-HSCTand one PB sample from each donor (one case received dual allo-HSCT, CB and PB from both donors were obtained), which provided a total of 40 samples.Genomic DNA samples were prepared according to Agilent SureSelect-XT Human exon 50Mb enrichment kit (Agilent Technlogies, Santa Clara, CA) preparation guide and libraries were sequenced on Illumina HiSeq platform (Illumina, San Diego, CA). DNA sequencing data from recipient post-transplant BM samples, were matched against their donor PB sample and previous post-transplant BM samples to identify the acquisition of mutations along the post allo-HSCT period.Germline variants in donors were studied in order to detect mutations that predisposed to the development of a myeloid neoplasm.The research protocol was approved by the Ethic Committee of Gregorio Marañón General University Hospital. Patients´ and donors´ information was collected from their medical records.ResultsClinical and biological characteristics of the 7 patients with DCMN and their donors are shown in Table 1.Mutational profiles obtained from the follow-up samples at different time-points post-HSCT demonstrated high intra-tumor genetic heterogeneity and clonal dynamic for all cases. The number of variants are increased over time and at the moment of DCMN diagnosis, the median number of variants was 28, ranging from 18 to 92 variants (Figure 1).WES identified in DCMN patients gene mutations commonly seen in adult AML or MDS, such as in SETBP1, DNMT3A, TET2, RUNX1, CSF3R, EP300and IDH2.In addition, others non-silent variants were acquired in all cases. Among the additional novel alterated genes, we found 23 strong candidateswith oncogenic potential. LUC7L2, NOP14, LAMA5, SKOR2, EML1, SNX13, RHPN2, IRS1, MTG2, TENM2, MEFV, GSE1, NOTCH4, DTX1, CNOT4, PNKP, GRB7,SENP7,TAF1L, ZKSCAN2, ZBTB20, ZNF461 and MEGF10.Analysis of CNVs revealed numerical alterations across the post allo-HSCT samples in patients 1, 2, 3, 4, 5 and 7. The most common chromosomal alterations in DCMN were monosomy 7 and other chromosome 7 abnormalities, which detected in the 86% (6/7) of the patients.Although none of the donors developed a myeloid neoplasm at the moment of diagnosis of DCMN in recipient, donor 1 revealed an abnormal karyotype (45,XY,-7) at the moment of the allo-HSCT. All other donors harbored at least one pathogenic or probably-pathogenic variants, most probably of germline origin, in genes involved in hematological or solid tumor predisposition.ConclusionsThe development of DCMN involves dynamic genomic processes that begin months before the clinical onset. In this study, our integrated multi-step analysis revealed the intra-tumor heterogeneity and evolutionary history of seven DCMN.The present study reveals a process of sequential clonal expansions promoted by the acquisition of somatic mutations in donor hematopoietic cells.Detection of heritable or acquired gene mutations in donors associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-HSCT.Leukemic transformation ofdonor hematopoietic stem cells provides a useful in vivomodel to study the mechanisms involved in leukemogenesis.Novel approaches based on high-depth next generation sequencing to study consecutive samples from post-transplant period in these patients, appear promising to discover new genes involved in myeloid neoplasm and to decipher the mechanisms of leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-Transplant NPM1 Mutant Transcript Level Is Highly Predictive of Outcome after Allograft and Thresholds Are Dependent on FLT3 ITD Status

IntroductionRelapse remains the most common cause of treatment failure for patients with acute myeloid leukaemia (AML) who undergo allogeneic stem cell transplantation (SCT) and carries a grave prognosis. Multiple studies have identified the presence of minimal residual disease (MRD) prior to SCT assessed by flow cytometry (FCM) as a strong predictor of relapse: however, little is known about the impact of molecular MRD pre-SCT. Molecular techniques can provide 100-1000 fold greater sensitivity than FCM in NPM1 mutated AML allowing identification of patients with very low level MRD prior to SCT. Peri-transplant management decisions for patients with positive molecular MRD are highly challenging due to the absence of robust outcome data.MethodsBetween 2009-14 the NCRI AML17 study enrolled 3215 adult non-APML patients aged 16-77 eligible for intensive chemotherapy. Central screening for NPM1 mutations was positive in 861/2949 (29%); 530 patients provided serial samples for MRD monitoring. Paired blood (PB) and bone marrow aspirates (BM) were requested after each chemotherapy cycle and then quarterly; additional samples were requested pre- and post-SCT. Samples were analysed by RT-qPCR using a suite of 27 mutation-specific reverse primers. Results were only fed back to clinicians after June 2012 when patients could be treated for confirmed re-emergent or persistent molecular positivity. Post-remission treatment was determined according to the validated NCRI risk score, with poor-risk patients recommended for SCT during first complete remission (CR1). Survival analysis was performed using the logrank test.ResultsIn total 108/530 patients received SCT (CR1 57 (52%), after molecular relapse or progression (MR) 30 (28%), CR2 21 (19%)). Five-year survival post-SCT (5y-OS) was 65% in CR1, and 54% in MR/CR2 (p=0.3). After MR 26/30 patients received chemotherapy prior to SCT. Evaluable pre-SCT PB and BM samples taken within 60 days of SCT were available for 104 and 78 patients (both available n=74).Considering PB samples, 5y-OS was 73% (median OS (mOS) not reached (NR)) for MRD-ve patients (n=74) vs. 30% (mOS 8.1 m) for any PB positivity (n=30) (p<0.0001). Patients with a negative pre-SCT BM had 5y-OS of 79% (mOS NR) vs 47% (mOS 11.9 m) if the BM was positive (p=0.002).Of the 47 patients who received additional chemotherapy for morphologic or molecular relapse or progression, 26 (55%) converted to MRD negativity accounting for 44% of 59 patients who were MRD negative pre-SCT. Ninety-three per cent of 59 pre SCT-MRD negative patients had been MRD negative in the PB after the second cycle of induction (a previously identified marker of favourable outcome).A threshold of 200 mutant NPM1 transcripts/105 ABL copies in the pre-SCT PB sample split patients into 3 groups with 2y-OS of 81% (negative, n=74), 54% (low, n=13) and 9% (high, n=17; p<0.0001). In the BM a threshold of 1000 copies defined 3 groups with a 2y-OS of 86% (negative, n=36), 56% (low, n=32) and 12.5% (high, n=8; p<0.0001).Thirty four patients were positive for FLT3-ITD at diagnosis (5y-OS 55%) and 74 were negative (5y-OS 62%; p=0.3). For patients without FLT3 ITD, negative, low and high levels of MRD in the pre-SCT PB sample were associated with 2y-OS of 79% (n=53), 88% (n=8) and 0% (n=9) (p<0.0001). For BM samples, 2y-OS was 78% (n=28), 62% (n=24) and 0% (n=5; p=0.0001). For patients with FLT3 ITD, corresponding 2y-OS for PB samples were 89% (n=21), 0% (n=5) and 25% (n=8; p=0.0005); for BM samples 2y-OS was 83% (n=9), 38% (n=8) and 25% (n=4; p=0.02).We assigned 80 patients to high and low risk groups based on FLT3 ITD status and pre-SCT MRD level in PB and BM. High-risk patients (FLT3 ITD and any detectable level of MRD in the PB or BM, or FLT3 ITD negative with > 200 copies in the PB or > 1000 copies in the BM) had 3y-OS of 14% (n=27, mOS 6.5 months) compared with 80% for low-risk patients (n=53, p<0.0001) (fig 1).ConclusionsPatients who test negative for NPM1 mutant transcripts immediately before SCT have a favourable outcome which is also observed in FLT3 ITD negative patients who are MRD positive at levels which do not exceed 200 copies in the PB or 1000 copies in the BM. FLT3 ITD mutated patients with any level of MRD prior to SCT, and FLT3 ITD negative patients with transcript levels above these thresholds, have a very high risk of relapse and may benefit from further chemotherapy or FLT3 inhibition prior to or after SCT; studies to investigate this are urgently needed. [Display omitted] DisclosuresHills:Daiichi Sankyo: Consultancy, Honoraria. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Russell:Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau.

Mice Expressing MYC and NrasQ61R in Germinal Center B Cells Develop Highly Aggressive Multiple Myeloma

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression.Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients.To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients.We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results.Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. DisclosuresNo relevant conflicts of interest to declare.

Tryptophan Deprivation Promotes an Adaptive Response and Contributes to Bioenergetics in Multiple Myeloma

Background and Objective:There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy.In multiple myeloma (MM) disease progression depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth.Our previous work showed that i) MM cells have metabolic plasticity due to availability of external nutrients; ii) aminoacid shortage due to the tryptohan (trp) degrading enzyme IDO-1 was associated to clinical MM progression. Thus, we tested bio-energetic changes in MM upon trp deprivation.Experimental design:We evaluated the trascriptome changes in the adaptive response in four human MM cell lines (MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c-myc and primary BM samples of MGUS and MM to trp shortage.The pathway was confirmed looked at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293).ResultsTrp deprivation induced an adaptive response through increased expression, time and dose-dependent, of ATF4-ASNS-CHOP-GADD34, part of GCN2 signaling, associated to increased expression of p62 (the main autophagic receptor in MM) followed by autophagy flux induction, as shown by flow cytometry and immunofluorescence evaluations.In MM cell lines, trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation, inducing in vitro increase of lactate dehydrogenase (LDH) and intracellular lactate and NAD/NADH with down-regulation of surface expression of the NAD-ase CD38. Similarly, short-term trp deprivation in CD138+ plasma cells isolated by magnetic sorting from MM patients, downregulated surface expression of CD38 and intra-cellular amount of c-myc, as detected by flow cytometry.This phenomenon was associated to increased isocitrate dehydrogenase (IDH-1), enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production.In the GSE66293 MM progression was associated to increased expression of ENO-1, PGK-1, and DLD, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant.ConclusionOverall, our study reveals that trp deprivation promotes an adaptive response of MM offering new putative targets in the bio-energetic pathway for synthetic lethality. DisclosuresPalumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria.

Exome Sequencing Identifies Mecom Missense Variant As Prognostic Marker for Overall Survival of Elderly Acute Myeloid Patients Treated with Azacitidine

Hypomethylating agents (HMA) azacitidine (AZA) and decitabine are approved low-intensity frontline therapy for AML pts, but patient selection between intensive versus low-intensity therapy remains mater of debate. To date, description of biomarker of response to HMA has brought few convincing findings. Most studies have focused on somatic mutations in candidate epigenetic genes such as DNMT3A, TET2, and IDH1/2 or tumor suppressor TP53 and led to controversies with none of this gene currently used to guide therapy decision in clinical practice. We performed an hypothesis-free, innovative, exome sequencing study to identify predictive biomarkers of outcome of AML pts treated with AZA.From Jan 2007 to Dec 2016, 279 newly diagnosed non-M3 AML pts were consecutively treated with AZA in the regional cancer network ONCOMIP. The median age was 76 yrs (45-93 yrs). Median follow-up was 5.5 yrs. AML was secondary of MDS, MPN or therapy related in 141 pts (50.5%). Karyotype was adverse in 135 pts (49.1%). Median WBC count was 2.7 G/L. Overall, 54 pts obtained CR/CRi (19,4%) and median OS was 10.6 months. Among the 279 pts, we identified a discovery cohort of 49 pts with extreme phenotype consisting in 26 pts achieving CR/CRi versus 23 pts in failure despite at least 3 cycles of AZA, and a validation cohort of 175 pts with baseline DNA sample and evaluable for response. Adverse karyotypes were more frequent in the validation set (30% vs 53% respectively, p=.004), which was likely due to an enrichment of responders pts in the discovery cohort.For exome sequencing of the discovery cohort, libraries were captured from baseline DNA BM samples with Sureselect all-exome V4 kit (Agilent) followed by paired-end, 150bp reads sequencing on a NextSeq sequencer (Illumina). Sequences were aligned with Nextgene and non-synonymous SNV were filtered according to 3 different patterns defined on gene function (nucleotide analogs metabolism/COSMIC census genes/AML genes), read depth, variant allele frequency and recurrence. An elastic-net regularization combining LASSO and Ridge penalties simultaneously (Zou & Hastie, 2005) was used to identify association between variants and clinical response. Using a resampling approach, bootstrap stability selection (BSS) were computed for each variant and only those with a BSS≥75% were selected. The exon capture baits targeted 98% of genes in the Consensus CDS database. A mean coverage depth of 85× per sample was achieved, with 92.5% of targets covered at ≥ 20× depth. From the 3 gene/SNV filtering patterns, bootstrapping of the data obtained with elastic-net regularization identified 4 candidate SNV in C11orf80, DZIP, ZNF543 and MECOM genes. Inspection of the exome-seq data for MECOM revealed that 11 of the 26 individuals in the responders sample had an heterozygous missense variant in the coding sequence of MECOM at position 169098992 (rs7622799; encoding p.Pro120Ser; MAF = 0.13 in the Exome Aggregation Consortium[ExAC]).We screened the 4 candidates SNV by targeted sequencing in the validation set and identified MECOM rs7622799, in 34/175 pts (19.4%). Response rates did not differ between pts with rs7622799 (8.8% CR/CRi) and without rs7622799 (9.2% CR/CRi, p=1.00). We then assessed whether genotype rs7622799 predicted OS in time dependent analysis using a Piecewise Cox model and demonstrated that pts with rs7622799 had improved OS from 3 months after diagnosis compared with non-rs7622799 (HR 0.56 95%CI[0.34;0.93]; p=0.024). The 3 other SNVs were not associated with patient outcome.We used an extreme phenotype study design, to discover that the missense variant rs7622799 of MECOM is correlated in time dependent analysis with the overall survival of older AML pts treated with AZA. MECOM is a complex locus gene which encodes for several isoforms namely EVI1, a very aggressive oncogene when overexpressed in AML, and MDS1-EVI1 (or PRDM3), which has epigenetic function as histone methyltransferase activity. Both isoforms directly interact with epigenetic modifiers (HDAC, DNMT3A). Further analyses are needed to best understand the functional impact of rs7622799. Although our findings could indicate that rs7622799 is associated with outcome of older AML pts in the setting of AZA, it remains unknown if it could be used to stratify pts between intensive and low-intensity therapy. Targeted sequencing of this SNV in a control cohort treated with intensive chemotherapy is ongoing [Display omitted] DisclosuresFornecker:Takeda: Honoraria; Servier: Honoraria.

Identification of Individualized Minimal Residual Disease in Acute Myeloid Leukemia By Flow Cytometry and Its Application of Prognostic Significance

▪Acute myeloid leukemia (AML) is a group of hematological malignancies with high heterogeneity in clinical characteristics and prognosis. Based on the leukemia-associated immunophenotypes (LAIPs) of AML, minimal residual disease (MRD) which is related to the outcome of the patients could be detected by multiparameter flow cytometry. Although 0.1% was commonly used to discriminate outcome in most studies so far, measurable MRD or MRD level below 0.1% has also been associated with prognostic significance, the precise threshold of MRD for prognosis prediction in AML still remains controversial.In this study, a total of 292 adult patients diagnosed as AML (non-M3) were enrolled, and 36 kinds of LAIPs were detected by flow cytometry. Based on the expression level of these LAIPs in 47 normal or regenerating bone marrow samples, the individual baseline level of each kind of LAIP was established, which ranged from <2.00×10-5 to 5.71x10-4, much lower than 0.1%. MRD statement based on 0.1% or individual baseline expression level of each LAIP were termed as 0.1%-MRD and individual-MRD respectively.The survival analysis showed that, comparing with the generally used MRD cut off value of 0.1%, individual-MRD threshold distinguished the patients with different outcome more precisely. A total of 273,162 and 163 samples were detected at the time point of achieved complete remission (post CR), after the first (post Con1) and the second (post Con2) consolidation course, respectively. At all three time points, the patients of individual MRDneg showed significantly better survival compared with those of 0.1%-MRDneg/individual-MRDpos or 0.1%-MRDpos (3y-OS: P<0.001, P<0.001; 3y-EFS: P<0.001, P<0.001). Multivariate analysis also showed that individual-MRD status presented independent prognostic value at each of three time point.Notably, in patients of low or intermediate risk groups (LR or IR) according to the NCCN risk stratification, the Individual-MRD status at post CR could identify the patients with significantly different outcome. The higher 3-year estimated OS and EFS rate were observed in the patients with individual-MRDneg in both LR and IR groups (LR group: 3y-OS: 92.2% vs 65.6%, p=0.003; 3y-EFS: 72.9% vs 44.6%, p=0.001; IR group: 3y-OS: 60.6% vs 37.3%, p=0.023; 3y-EFS: 53.3% vs 23.3%, p=0.006), while 0.1%-MRD status could not distinguish these differences clearly. Furthermore, among the patients of LR or IR group which received chemotherapy only, those with individual-MRDneg status presented favorable survival which was even comparable with the patients accepted allogeneic hematopoietic stem cell transplantation (ASCT) (3y-OS: 77.8% vs 77.2%, p=0.941, 3y-EFS: 64.2% vs 66.5%, p=0.611).In conclusion, our study established the individual MRD threshold according to LAIP baseline levels in normal or regenerating BM samples. The individual MRD status could predict prognosis more precisely, especially in NCCN low or intermediated risk cohorts. In addition, with combination of individual MRD status and cytogenetic-molecular risk classification, we distinguished the patients with superior survival, which might help to guide the choose of ASCT strategy in clinical practice. DisclosuresNo relevant conflicts of interest to declare.

APT-studies of phase formation features in VVER-440 RPV weld and base metal in irradiation-annealing cycles

This paper presents a thorough APT study of VVER-440 RPVs WM and BM samples cut out from the inner RPV surface. The features of phase formation (number densities, sizes and composition) in VVER-440 RPV steels under primary irradiation, recovery annealing, re-irradiation, recovery re-annealing and subsequent accelerated re-irradiation are identified to justify its lifetime extension up to 60 years by recovery re-annealing.It is shown that primary and re-irradiation is accompanied by formation of radiation-induced Cu-P-based precipitates whereas recovery annealing leads to their partial dissolution and coarsening of the undissolved ones. At this, for both WM and BM under primary annealing, Cu dissolves in the matrix, while under re-annealing Cu almost doesn't return to the matrix. In WM P fully dissolves in the matrix under annealing while in BM there is gradual matrix P depletion under operation (including recovery annealing) due to formation of P grain boundary segregation. Under the third irradiation cycle, Cu almost doesn't contribute to precipitation: in WM radiation-induced precipitates are P-based, enriched with Si, Ni and Mn, while in BM there are Si-based precipitates, enriched with Ni and Mn. In BM number density of precipitates is lower than in WM during all operation stages that causes its lower radiation embrittlement.

Mixed-phenotype acute leukemia characteristics: first report from Iran.

Mixed-phenotype acute leukemia (MPAL) is the infrequent type of acute leukemia characterized by immunophenotypic and/or cytochemical features of both lineages, but the diagnosis of this disease still is a challenge. In this study, we analyzed immunophenotyping, cytochemistry and frequency of MPAL patients to better diagnosis of MPAL characteristics according to WHO 2016 criteria for the first time in Iran. In this retrospective study, 27 patients were diagnosed as MPAL based on WHO 2016 criteria during 2014-2017. Flow cytometric immunophenotyping was performed on PB and BM samples evaluation of different CD marker expressions in MPAL subsets. RT-PCR was performed for the analyses of BCR/ABL1 fusion in MPAL subsets. Among 27 cases, (70.4%) 19 cases were B + My, (22.22%) 6 cases were T + My, and 2 cases (7.40%) were B + T + My. CD34, CD19, HLA-DR, TdT, CD22, iMPO were positive in majority of B + My cases. CD45, iMPO, iCD3, CD7, CD2 and CD5 were positive in majority of T + My cases. HLA-DR, TdT, CD10, CD22, iCD79a, iMPO, CD45, iCD3, CD7, CD3, CD2, CD5 were positive in majority of B + T + My cases. BCR/ABL1 fusion was positive for 3 cases (11.1%) of p190 fusion and 2 cases (7.4%) of p210 fusion in B + My cases. WHO 2016 criteria are the current standard for diagnosing MPAL. Also, evaluation of TdT, CD2, CD5, CD7 expressions by flow cytometry in EGIL criteria is useful for the better diagnosis of MPAL subsets. In addition, evaluation of BCR/ABL1 and MLL rearrangements in patients should be part of standard work-up in MPAL.

Abstract 44: Characterization and targeting of a temporal micro-metastatic signature in human brain metastases

Abstract Metastases to the brain (BM) are the most common neoplasms to affect the adult central nervous system, arising in 40% of cancer patients and at a rate 10 times greater than primary brain tumors. Despite the prevalence and poor survival rates, therapeutic strategies for BM remain limited, and a substantial cause is the lack of proper preclinical models available to interrogate the intricacies of BM development. Previous work in our lab utilized BM samples from patient-derived lung-to-brain metastases to successfully establish clinically relevant in vivo models of BM. Here we further characterized the cells responsible for BM, termed brain metastasis initiation cells (BMIC). Patient-derived BMICs were injected via intracranial (BT), intracardiac (IC) and intrathoracic (IT) routes into NOD/SCID mice and re-isolated at different stages of metastatic progression. We isolated cells from primary lung tumors (LT), cells injected via intra-thoracic route that crossed the blood brain barrier to seed the brain forming micro-metastases (BMIT), and cells injected via intracardiac route that seeded large macro-metastases (BMIC). Through RNA sequencing we determined cells from BMIT (micro-metastasis stage) to retain a vastly different genetic profile compared to BMICs isolated at other stages of metastasis. Several of these genes belonged to pathways implicated in autonomic central nervous system neoplasms and neural development. Through connectivity mapping of BMIT profiles we discovered drugs that could inhibit the micro-metatsasis signature, and further in vitro validation revealed apomorphine to reduce BMIC sphere formation and proliferation. In vivo treatment with apomorphine blocked both micro- and macro-metastatic stages of our BMIC model. Of 5 BMIT genes identified to be specifically targeted by apomorphine, KIF16B, TESK2 and SEPW1 were shown to have significant value when applied as an independent prognostic signature in a cohort of lung adenocarcinoma patients. Future work will further validate the efficacy of apomorphine in targeting BMICs in primary lung cancer patient samples. With this work we present a possible new avenue for therapeutic targeting toward the prevention of BM development, where it is anticipated to transform a uniformly fatal disease into one that is eminently more treatable. Citation Format: Mohini Singh, Chitra Venugopal, Tomas Tokar, Nicole McFarlane, Minomi Subhapanditha, David Bakhshinyan, Maleeha Qazi, Parvez Vora, Neil Savage, Naresh K. Murty, Igor Jurisica, Sheila K. Singh. Characterization and targeting of a temporal micro-metastatic signature in human brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 44.

Evaluation by Flow Cytometry of Mature Monocyte Subpopulations for the Diagnosis and Follow-Up of Chronic Myelomonocytic Leukemia.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis and dysplasia in at least one myeloid cell lineage. This persistent monocytosis should be distinguished from the reactive monocytosis which is sometimes observed in a context of infections or solid tumors. In 2015, Selimoglu-Buet et al. observed an increased percentage of classical monocytes (CD14+/CD16− >94%) in the peripheral blood (PB) of CMML patients. In this study, using multiparametric flow cytometry (MFC), we assessed the monocytic distribution in PB samples and in bone marrow aspirates from 63 patients with monocytosis or CMML suspicion, and in seven follow-up blood samples from CMML patients treated with hypomethylating agents (HMA). A control group of 12 healthy age-matched donors was evaluated in parallel in order to validate the analysis template. The CMML diagnosis was established in 15 cases in correlation with other clinical manifestations and biological tests. The MFC test for the evaluation of the repartition of monocyte subsets, as previously described by Selimoglu-Buet et al. showed a specificity of 97% in blood and 100% in marrow samples. Additional information regarding the expression of intermediate MO2 monocytes percentage improved the specificity to 100% in blood samples allowing the screening of abnormal monocytosis. The indicative thresholds of CMML monocytosis were different in PB compared to BM samples (classical monocytes >95% for PB and >93% for BM). A decrease of monocyte levels in PB and BM, along with a normalization of monocytes distribution, was observed after treatment in 4/7 CMML patients with favorable evolution. No significant changes were observed in 3/7 patients who did not respond to HMA therapy and also presented unfavorable molecular prognostic factors at diagnosis (ASXL1, TET2, and IDH2 mutations). Considering its simplicity and robustness, the monocyte subsets evaluation by MFC provides relevant information for CMML diagnosis.

ABO and D typing and alloantibody screening in marrow samples: relevance to intraosseous blood transfusion

Blood transfusion through the intraosseous route is gaining popularity in emergency medicine. Pretransfusion peripheral blood (PB) samples are usually not available in these patients, leading to discrepancies in blood group typing and a possible delay in transferring to group-specific blood products. The aim of this study was to assess the feasibility of ABO and D typing and red blood cell alloantibody screening in marrow (BM) samples. Direct and reverse ABO typing, D typing, and a two-cell alloantibody screen were performed in EDTA-anticoagulated BM samples with standard manual column agglutination techniques. EDTA-anticoagulated PB samples were used as controls. The mean age of the study subjects (n=71) was 47 years (range, 1-82 years). All ABO groups and both D+ and D- types were represented. In all subjects, concordant results were observed for all analyses in BM and PB samples. In 15 (21%) of the samples, a discrepancy of one reaction strength step (1+) was observed in at least one of the analyses (Cohen's weighted κ=0.993); this did not affect interpretation of the results. Blood group typing and alloantibody screening are feasible in BM samples, providing proof-of-concept that intraosseous samples for blood group serologic analyses can be collected from emergency patients before intraosseous blood transfusion. This will enable a timely transfer to group-specific blood products and enable conservation of the valuable universal-donor blood products.

Corrosion properties of low carbon steel prepared by submerged friction stir processing

Submerged friction stir processing (SFSP), that is, the entire process was carried out underwater, was successfully conducted on low carbon steel. The microstructure, electrochemical corrosion, and salt fog corrosion properties of SFSP low carbon steel were investigated. The results indicated that phase transformation and dynamic recrystallization occurred during SFSP and finally resulted in a dual phase fine grain microstructure in the processed zone (PZ), which consisted of ferrite and homogenous martensite phase. The grain size of ferrite was refined to 5.7 µm, and the width of martensite phase was 650 nm, respectively. In addition to changing the microstructure of low carbon steel, SFSP could also affect its corrosion property. SFSP samples exhibited better resistance against electrochemical corrosion and salt fog corrosion than BM samples. The reason was that, grain refinement and martensite formation induced by SFSP were conducive to forming dense protective layer, which effectively hindered the electrochemical corrosion and salt fog corrosion of the SFSP low carbon steel.

Knockdown of Long Noncoding RNA HOXA-AS2 Suppresses Chemoresistance of Acute Myeloid Leukemia via the miR-520c-3p/S100A4 Axis

Background/Aims: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells. Methods: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML. Results: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3’-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. Conclusion: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.

Reduction of safrole content of Cinnamomum porrectum leaves by blanching and the effect on the antioxidant and anti-inflammatory activities of its herbal tea

Background: Cinnamomum porrectum (C. porrectum) is an aromatic medicinal plant from southern Thailand. While many Cinnamomum species demonstrate medicinal properties such as anti-inflammatory effects, some species are known to contain safrole, a group 2B carcinogen. Although the leaves of C. porrectum are used to produce herbal tea, there is no scientific data to support its properties as a functional food or any guarantee of its safety.Objective: To determine the effect of blanching before drying of C. porrectum leaves on their safrole content, phenolic profile, total phenolic content (TPC), and total flavonoid content (TFC), antioxidant and anti-inflammatory activities were compared to leaves dried with hot air.Methods: In the first part of the study, the leaves were dried in three conditions: freeze dried (F), hot air dried at 60 oC (C), and blanched for 60 seconds at 100 oC (B) before being dried at 60 oC until the moisture content reached 5-7% and then were ground and sieved to make powders. The F, C, and B were extracted by methanol and redefined as FM, CM, and BM, for the determination of their chemical constituents by GC-MS using C. porrectum wood oil (CWO) as the safrole standard. In the second part of the study, the C and B powders were analyzed for their proximate composition and were also used to make herbal tea infusions (CT and BT respectively) which were analyzed for their phenolic profile, TPC, TFC, antioxidant activities, cytotoxicity, and anti-inflammatory activity in RAW 264.7 cells.Results: Significantly decreased safrole contents of 89% and 82% were found in the BM sample when compared with CM and FM respectively. The CT and BT had similar phenolic profiles but different contents. The TPC, TFC, ABTS, and FRAP activities, in addition to cell viability and the nitric oxide inhibition of BT were higher than that of CT.Conclusion: The results demonstrate how blanching C. porrectum leaves before drying can decrease the safrole content when compared to freeze dried and hot air-dried leaves, thereby leading to a decrease of cytotoxicity in RAW 264.7 cells. Additionally, BT demonstrated a more intense phenolic profile, TPC, ABTS, and FRAP compared with the un-blanched control sample.Keywords: C. porrectum, freeze drying, hot air drying, blanching before drying, safrole, RAW 264.7 cells.

FANCD2 and HES1 Synergistically Regulate the Immunometabolism of Hematopoietic Stem Cells through Transcriptional Suppression of Pparg- mediated Fatty Acid Oxidation and Oxidative Phosphorylation

Emerging evidence has revealed that resting quiescent hematopoietic stem cells (HSCs) possess a distinct metabolic profile with a preference for anaerobic glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS). Recent few scattered studies on the relationship between metabolism and HSCs raise a completely different perspective on the immunological/metabolic interface between HSC defect and human diseases. How immunometabolism influences HSC function, and what signaling cascades help drive cell-intrinsic immunometabolic modes in HSCs, has begun to emerge as an area of intense interest. Here we have investigated the immnometabolic regulation of HSCs using a model of Fanconi anemia (FA), a cancer-prone disease known to impact the immune system and promote inflammation. We and others have demonstrated that inflammation in FA hematopoietic stem and progenitor cells (HSPCs) plays a crucial role in FA pathophysiology. Recently, we have shown that FA HSCs are more dependent on OXPHOS relative to glycolysis for energy metabolism. However, the mechanism underpinning the link between inflammation and the altered metabolic program in FA HSCs has not been defined. More recently, we exploited the repopulating defect of FA HSCs to conduct an unbiased in vivo shRNA screen in transplant recipients, and found enrichment of shRNAs targeting genes involved in the Pparg pathway. Pparg is a central transcriptional factor regulating adipocyte differentiation and energy metabolism but has not been linked to HSC homeostasis. Pparg inhibition by shRNA or chemical compounds significantly improved the repopulating ability of Fancd2-KO HSCs. Conversely, activation of Pparg in wild-type HSCs impaired hematopoietic repopulation. Suppression of inflammation-induced expression of Pparg required both Fancd2 and the Notch target Hes1. Deletion of Hes1 de-repressed Pparg expression in wild-type LSK cells, which are enriched for HSPCs, but had no further effect on Fancd2-KO LSK cells. Mechanistic studies showed that Fancd2 and Hes1 acted synergistically to repress the Pparg promoter. Furthermore, transcriptomic study revealed that inflammation orchestrated an overlapping transcriptional program in HSPCs deficient for Fancd2 and Hes1, featuring upregulated genes in fatty acid oxidation (FAO) and OXPHOS. Consistently, we observed a marked increase in FAO and OXPHOS in mouse HSPCs deficient for Fancd2 or Hes1 and in the BM samples of FA patients. Using a newly developed Fancd2-KI mouse model, we identified a novel and inflammation-responsive interaction between Fancd2 and Hes1. This inflammation-responsive FANCD2-HES1 interaction was abolished in FA-D2 patient-derived lymphoblast cells but rescued in those constituted with a functional FANCD2 (with a 3×FLAG tag) gene. Finally, a Fancd2 mutation lacking the Hes1-interacting domain abolished the activity of the repression of Pparg expression, FAO and OXPHOS. Our results indicate that the FA pathway may constitute a key component of a novel immunometabolism axis, connecting inflammation, cellular metabolism and HSC function. DisclosuresNo relevant conflicts of interest to declare.

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

Abstract 1758: A more mature immunophenotypic makeup of multiple myeloma clone(s) at diagnosis correlates with a higher genomic instability

Abstract Intro The sequence of events underlying the process of Multiple Myeloma (MM) plasma cells (PC) differentiation have not yet fully elucidated, even if recent findings suggest that different cell subpopulations, with distinct phenotype, compose the MM clone(s), whose plasticity has emerged as a typical feature. Aim To evaluate the phenotypic plasticity and genomic background of MM clone(s) at diagnosis in order to stratify patients (pts) according to the PC differentiation stages and possibly correlate with disease outcome. P&amp;M Phenotypic characterization of both CD138+/CD38+ and CD19+ populations was carried out on 44 newly diagnosed MM. Fresh BM samples was analysed by 6color multiparametric flow citometry analysis, combining CD138PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC and SHH-PE, as a functional marker of Hedgehog pathway activation (Miltenyi Biotech). Cytoscan HD array were carried out in order to detect genomic copy number alterations (CNAs). Results According to the CD19 and CD81 markers co-expression, pts were stratified in 3 subgroups, recapitulating a progressive PC maturation process: the most immature one, including pts with CD19+/CD81+ PC (11/44 = 25%); the intermediate CD19-/CD81+ phenotype one (19/44 = 43%), and the CD19-/CD81- PC one, whose clone was mainly composed by most mature PCs. The two extreme subgroups were characterized by a differential expression of maturation markers: the more mature PCs displayed a higher expression of CD28 and CD44, which usually characterized advanced disease stages, as well as a reduced expression of CD20, CD27 and CD45, commonly associated to preceding PC differentiation stages (p&amp;lt;.05). Any differential expression of SHH pathway’s ligand was observed. On the contrary, a higher Hedgehog pathway activation was detected in the immature CD19+ compartment of pts with immature plasma cells (median SHH expression CD19+/CD81+ vs. CD19-/CD81-: 98,1% vs. 11,3%; p&amp;lt;.05), which probably highlighted a less quiescent immature reservoir pool. The CNAs analysis showed that mature PCs were characterized by a higher genomic instability, as compared to the more immature ones (total CNA: 306.9 vs. 171.5, p 0.08; genome changed: 36.96 vs. 13.55, p 0.01), including several alterations commonly associated to worse prognosis (e.g. del17p). Finally, a higher frequency of baseline clinical characteristics associated to bad prognosis was observed in the more mature, as compared to the more immature subgroups of pts (e.g. n. PET lesions, k/l ratio; p&amp;lt;.05). Conclusion MM clone(s) is a mixture of different cell populations endowed with an inner phenotypic plasticity. Various PC differentiation stages were appreciable already at diagnosis and genomic features associated to bad prognosis characterize pts carrying more mature clones. Acknowledgements: AIRC, Berlucchi, Prog. Bolondi, FUV Citation Format: Marina Martello, Rosalinda Termini, Barbara Santacroce, Enrica Borsi, Vincenza Solli, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Francesca Ulbar, Gabriella Chirumbolo, Mario Arpinati, Giovanni Martinelli, Michele Cavo, Carolina Terragna. A more mature immunophenotypic makeup of multiple myeloma clone(s) at diagnosis correlates with a higher genomic instability [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 1758. doi:10.1158/1538-7445.AM2017-1758

Abstract 3936: A branching evolution model at relapse characterizes multiple myeloma patients who responded to upfront combination therapy including new drugs

Abstract Intro: Multiple Myeloma (MM) is a biologically complex disease, whose genetic plasticity favors the coexistence of genetically heterogeneous subclones, selected in a Darwinian fashion throughout the disease course. Therapy might represent a major selective pressure over the different subclones, thus supporting an evolutionary model of the disease. Aim: To explore the existence of different clonal evolution patterns in MM, eventually driven by therapeutic selective pressure. P&amp;M The study included 33 pts with symptomatic MM, up-front treated either with combination regimens including a proteasome inhibitor (28), or with cyclophosphamide. For each pts, paired BM samples were collected both at diagnosis and at relapse. SNPs array analyses were performed on the CD138+ enriched cell fractions. Results: Two approaches were applied: a) monitoring the variations of macro CNAs; b) focusing on changes of CNAs frequencies, as observed in 27 genes of interest. Both approaches were consistent in highlighting three major evolution patterns: in 7/33 (21%) pts, the genomic background at relapse was almost identical to that of diagnosis. In 13/33 (39%) pts, an overall increase in the frequencies of the same CNAs as observed at diagnosis was detected at relapse. Finally, in 13/33 (39%) pts, either increased or decreased frequencies of several CNAs, as well as several differences in the CNAs type’s prevalence were observed at relapse, as compared to diagnosis. Of interest, even if an overall CNAs median number increase was observed from diagnosis to relapse (226 vs 507, respectively) - supported by acquisition of CNAs either commonly described as secondary genomic events (i.e. del17p13, amp1q21, del1p23), or associated to the resistance to bortezomib (i.e. del8p21) - any peculiar CNAs resulted significantly prevalent in the 3 identified subgroups of pts. A high rate (92%) of achievement of VGPR or better quality of response to upfront therapy characterized the third subgroup of pts, whereas the rate of VGPR in the remaining pts was only 20% and PR or SD were observed in 9 and 7 pts, respectively. Finally, the median time to first progression of this subgroup of pts was significantly shorter as compared to that of pts with branching evolution (24 vs 35 months, range 4-41 and 7-123 months, respectively, p=0,01). Conclusion: The genomic architecture of a subgroup of relapsed MM pts, up-front responsive to new drugs-based combination therapies, resulted overall different from that of diagnosis, suggesting a branching evolution of the disease, sustained by the shrinking of the most prevalent clone (therapy-sensitive), as well as by the expansion of subclones (therapy-resistant) not already evident at diagnosis. This observation raises the question whether re-treatment of relapsed pts should be appropriate in the case of branching evolution. Acknowledgements: AIRC (MC), Fondazione Berlucchi (CT), FUV (EB). Citation Format: Carolina Terragna, Marina Martello, Barbara Santacroce, Vincenza Solli, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Beatrice Zannetti, Katia Mancuso, Giulia Marzocchi, Nicoletta Testoni, Gaia Ameli, Rosalinda Termini, Angela Flores Dico, Enrica Borsi, Giovanni Martinelli, Michele Cavo. A branching evolution model at relapse characterizes multiple myeloma patients who responded to upfront combination therapy including new drugs [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 3936. doi:10.1158/1538-7445.AM2017-3936

Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). We measured specific IgA to a series of overlapping peptides in major CM allergens (αs1 -, αs2 -, β- and κ-caseins and β-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in αs1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated.

Flow cytometry minimal residual disease assessment in peripheral blood of adult acute lymphoblastic leukemia patients.

e18517 Background: Multiparametric flow cytometry (FC) of bone marrow aspirate (BM) is a widely used method of minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL). In practice, ALL with hematogone-like phenotype and patients with recurrence of isolated extramedullary disease present challenges in accurate determination of MRD status. We hypothesized that addition of FC MRD of peripheral blood would aid in the interpretation of MRD status. Methods: 76 matched BM and PB specimens were analyzed independently for presence of ALL MRD by 6-color FC. Results: The overall rate of BM MRD-positivity was 24% (18/76) and PB was also MRD-positive in 22% (4/18) of BM-positive cases. PB MRD sensitivity and specificity relative to BM MRD was 13% [95% confident interval (CI) 2%-42%] and 98% [88-100%] for B-ALL samples (n = 65) and 67% [13%-98%] and 75% [36%-95%] for T-ALL samples (n = 11), respectively. We identified 2 cases with evidence of leukemic cells in PB at the time of the extramedullary relapse that were interpreted as MRD-negative in BM. Conclusions: PB MRD demonstrates high specificity with relatively low sensitivity, especially in B-ALL when compared to T-ALL. The use of combined PB and BM samples in MRD assessment by current FC methods may have added clinical and diagnostic value in patients with high risk of extramedullary relapse, including PB MRD as a non-invasive method for monitoring of systemic relapse.