Bone Marrow Collection Research Articles

Quiescence/Mobilization of Hematopoietic Immature Cells Induced By Polyphenols through Modulation of APC/EPCR/PAR-1 Axis

The mechanism underlying quiescence and/or mobilization of hematopoietic stem cells and their bone marrow progenitors (HSPC) into circulation are tightly regulated for the continuous supply of peripheral blood cells; however, non-physiological or stress conditions, such as infections, can accelerate these mechanisms. Our results have shown that polyphenols modulate quiescence/mobilization of HSPC, but do not affect mature populations. Thrombin has been reported to induce the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). Our objective was to investigate the effect of polyphenols on thrombin/PAR-1 and APC/EPCR/PAR-1 axis. C57BL/6J mice (6-8 weeks old) were treated with polyphenols from green tea extract (250 mg/kg body weight) orally (gavage) once every seven days and injected (i.p.) at day 7 with lipopolysaccharide (LPS) (100μg;Sigma) (n=6). The control group received vehicle and was injected with LPS (n=6). After 24h of LPS injection, mice were anesthetized for blood collection, and then sacrificed for bone marrow collection. PAR-1 and EPCR expression, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) phosphorylation were evaluated in HSPC by flow cytometry. The functional ability of HSC was assessed by competitive repopulation assay. Vascular permeability was studied using Evans blue. After LPS injection, mice showed reduced expression of EPCR in bone marrow LSK parallel to an increase of PAR-1 expression in circulating immature and mature cells. Treatment of these mice with polyphenols partially prevented the reduced expression of EPCR in bone marrow LSK (13±3 vs 54±12; p<0.05), but did not affect the increased PAR-1 expression in circulating immature and mature cells. Evans blue assay revealed a reduction in the vascular permeability of the bone marrow of LPS-injected mice treated with polyphenols (3.9±0.5 vs 2.1±0.1; p<0.05). To assess whether polyphenols altered NO production, we measured NO levels and eNOS phosphorylation in immature LSK EPCRhigh (or LT-HSC) cells. NO production is activated by eNOS phosphorylation at Ser1177 and negatively regulated by eNOS phosphorylation at Thr495. LPS injection rapidly increased NO levels and eNOS phosphorylation at Ser1177 in bone marrow LSK of mice. Treatment of these mice with polyphenols reduced the percentage of bone marrow LSK EPCRhigh cells with higher intracellular NO (52±2.8 vs 28±5.6; p<0.01) and increased eNOS phosphorylation at Thr495 in immature LSK. In order to evaluate the action of polyphenols on the functional ability of HSC, a competitive bone marrow repopulation assay was performed. Donor mice (C57BL/6J) received or not polyphenols followed by LPS injection (treated group: Polyphenols+LPS; control group: LPS), and bone marrow cells were transplanted (1:1) together with bone marrow cells of competitors (B6.SJL-PtprcaPepcb/BoyJ) in lethally irradiated recipients (B6.SJL-PtprcaPepcb/BoyJ). Mice were followed for 16 weeks and hematological analysis revealed no difference in circulating leukocytes, platelets or hemoglobin levels. Transplanted mice (recipients) presented a higher percentage of CD45+ cells from Polyphenols+LPS donors (33.7±13 vs 78.6±0.9; p<0.05) in the peripheral blood, as well as increased number of T lymphocytes (6.7±4.5 vs 37.2±2.9; p<0.05) and myeloid cells (68.5±1.7 vs 82.5±3.5; p<0.05) from Polyphenols+LPS group. After 16 weeks, mice were euthanized and a higher percentage of LSK (or HSC) and LSK EPCRhigh (or LT-HSC) cells from Polyphenols+LPS donors were detected in the bone marrow, although only the percentage of LSK EPCRhigh was statistically different (0.0014±0.0001 vs 0.0032±0.001; p<0.05). Taken together, our results indicate that polyphenols increased the functional ability of HSC in LPS-injected mice showing increased percentage of bone marrow LSK EPCRhigh cells, which are the most quiescent stem cells with strong self-renewal ability. Polyphenols reduced EPCR expression and NO production in immature cells of LPS-injected mice, exhibiting an anti-inflammatory effect that leads to the maintenance of barrier integrity and quiescence of cells, which was corroborated by reducing vascular permeability in the bone marrow. Thus, polyphenols appear to modulate quiescence/mobilization of HSPC through APC/EPCR/PAR-1 axis. Disclosures No relevant conflicts of interest to declare.

Associations between Microrna Expression, Disease Progression and Ex Vivo Drug Response in Multiple Myeloma

Targeted drug treatment strategies have significantly prolonged the overall survival rate among multiple myeloma (MM) patients. However, high relapse rates and multiple drug resistance still pose major challenges. Although, the underlying molecular features of the disease have been explored both at the genomic and transcriptomic levels, the functional role of microRNAs (miRNA) in MM disease progression and prognosis is yet to be investigated at a personalized level. In earlier studies, microRNAs have been implicated to regulate gene expression and were determined to play crucial roles in the biology of MM by acting as oncogenes or tumor suppressors. Nevertheless, considering the heterogeneity of MM, little is known about the roles of miRNAs in controlling MM disease progression and drug response at an individualized systems level. We collected bone marrow aspirates from MM patients at diagnosis (n=20) and relapse (n=25) after informed consent and following approved protocols in accordance with the Declaration of Helsinki. CD138+ plasma cells were enriched from the bone marrow samples and used for miRNA-sequencing and drug sensitivity and resistance testing (DSRT). The miRNA was prepared from the CD138+ cells and subjected to sequencing using Illumina compatible technologies. DSRT was performed and responses to 83 clinically approved drugs and investigational compounds were measured as drug sensitivity scores (DSS) as described previously (Majumder et al., Oncotarget 2017). The pairwise comparative analysis of miRNA expression and drug responses was performed using Spearman's rank-order correlations, to elucidate significant associations of miRNA expression with drug sensitivity and resistance. Additionally, using DEseq2 the differential miRNA expression was determined for the newly diagnosed and relapse samples to deconvolute the role of miRNAs in MM disease progression. The comparative analysis of the miRNA expression and drug sensitivity scores revealed statistically significant associations between miRNA expression and drug sensitivity measures with the Spearman coefficient (r) ranging from -0.71 to 0.64 (adjusted p-value ≤ 0.05) (Figure 1A). Negative associations were more prevalent, with 40 miRNAs negatively associated with ≥1 drug response from the total of 30 predicted drugs. miR-486, which is known to be an effective biomarker in diagnosis and prognosis of multiple cancer types (Jiang et al., Oncotarget 2018), was found to have significant negative correlation (r= -0.71 to -0.52, p-value ≤ 0.01) with the responses of 14 drugs. Similarly, negative correlation was observed for miR-144 with 12 drugs and miR-584 with 9 drugs. We observed that PI3K/mTOR inhibitors and HDAC inhibitors were common amongst all the significant negative correlations predicted. Specifically, the PI3K/mTOR inhibitors apitosilib, omipalisib and buparlisib were found to be negatively associated with the expression of 18, 14 and 7 miRNAs respectively. These observations can lead to the understanding of miRNA mediated regulation of molecular pathways involved in drug resistance. Differential miRNA expression analysis between newly diagnosed and relapse MM samples revealed the involvement of miRNAs in disease progression. The analysis resulted in total of 31 significant differentially expressed miRNAs with fold change ≥2 and adjusted p-value ≤ 0.1 (Figure 1B). Several miRNAs known to play crucial roles in cancer diagnosis and prognosis were found to be significantly upregulated in the relapse samples. In particular, 25 miRNAs were upregulated, including following miR-17/92 cluster members: miR-18b, miR-20a, miR-92b and miR-106a, which are known to have an oncogenic role in various cancer types (Mogilyansky & Rigoutsos, Cell Death and Differentiation 2013). Interestingly, 12/31 differentially regulated miRNAs were located on chromosome X. Although cytogenetic alteration data predicted that chromosome 1q gain is significantly prominent in the relapse samples (p-value = 0.009), only 3/31 differentially regulated miRNAs were located on chromosome 1. These results demonstrate the role of miRNAs in regulating drug response and disease progression in multiple myeloma. Monitoring miRNA expression profiles in MM patients can facilitate the assessment of treatment outcome and prognosis, and miRNAs could potentially be useful prognostic and treatment biomarkers for MM. Disclosures Silvennoinen: Amgen: Research Funding; Bristol-Myers Squibb (BMS): Research Funding; Takeda: Research Funding; Celgene: Research Funding. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

The MarrowMiner: A Novel Minimally Invasive and Effective Device for the Harvest of Bone Marrow.

Bone marrow (BM) is a rich source of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and other important stem/progenitor cells. It is the traditional source of cells used in hematopoietic cell transplantation, which is a proven curative treatment for many blood and immune diseases. BM-derived cells have also been shown to have other diverse clinical uses and are increasingly being used in orthopedic medicine, regenerative medicine, and gene therapy applications. Traditional methods for harvesting BM are crude, tedious, time-consuming, and expensive, requiring multiple bone punctures under general anesthesia with serial small-volume aspirates often diluted with peripheral blood. The MarrowMiner (MM) is a novel device designed for rapid and minimally invasive BM harvest. Here we show the safety and efficacy of the MM in both preclinical and clinical settings. In a large-animal porcine model, the MM enabled effective BM collection with similar total nucleated cell collection and increased colony formation compared with standard methods. The MM was subsequently evaluated in a clinical study showing effective and complication-free anterior and posterior BM collection of 20 patients under only local anesthesia or light sedation. Increased total nucleated and mononucleated cell collection was achieved with the MM compared with standard methods in the same patients. Importantly, stem cell content was high with trends toward increased HSC, MSC, and endothelial progenitor cells with similar T cell content. Given the MM is a novel device approved by the US Food and Drug Administration, enabling safe, effective, and minimally invasive harvest of BM, we anticipate rapid adoption for various applications.

Preoperative autologous blood donation in adult bone marrow donors: reappraisal of a single-centre experience.

To avoid risk for allogeneic transfusions in healthy bone marrow (BM) donors, 1-2 preoperative autologous blood donations (PAD) are usually collected before the BM harvest. We analysed the haematological parameters in BM donors before and after the harvest, to assess the efficacy of this practice in limiting the postharvest anaemia. Overall, 102 consecutive donors underwent BM harvest preceded by one (26 cases) or two PAD (76 cases), which were infused during BM collection. We analysed the parameters related to donors, PAD timing and BM graft characteristics. PAD induced a significant decrease in Hb (from 14·6g/dl, IQ range 13·3-15·5 to12·9g/dl, IQ range 11·8-13·9; P<0·0001) in all donors, with a median Hb loss at day -1 of 10·9% (IQ range 6·8-14·2). The PAD-related Hb decrease was independent of sex or number of PAD, and was inversely related to the time elapsed from first or last PAD. In comparison with values recorded at day-1, BM harvest produced an additional Hb decrease, accounting for a median Hb loss of 18·9% (IQ range 14·9-24·4). Overall, in comparison with pre-PAD values, Hb levels at day +1 were reduced of 28·9% (IQ range 23·6-32·2), independently if donors had 1 or 2 PAD reinfused. In conclusion, these data show that two PAD do not carry any advantage over one PAD. An eventual benefit of PAD can be achieved only if an adequate interval between PAD and BM harvest elapses. Prospective randomized studies could be worth to establish if any role for PAD does exist in BM donors.

NAT10 upregulation indicates a poor prognosis in acute myeloid leukemia

Background: N-acetyltransferase 10 (NAT10) is considered as an oncogene in many tumors. This study investigated the NAT10 expression in Chinese acute myeloid leukemia (AML) patients and evaluated the predictive significance of NAT10 with a single-center retrospective study. Methods: The Oncomine was used to analyze NAT10 expression in AML. We also collected bone marrow samples of 48 newly diagnosed AML patients and 20 benign individuals in our center. NAT10 mRNA expression levels were detected by real-time qPCR. Clinical data was obtained from inpatient medical records. Results: Two microarrays in Oncomine showed that NAT10 was upregulated in AML. Our data revealed that AML patients had higher NAT10 expression levels than the normal controls (P < 0.01). NPM1-mutant patients had higher NAT10 mRNA levels than NPM1-wt patients. NAT10 expression level was higher in nonremission group than in overall remission group (P < 0.05). High NAT10 expression indicated a poor progression-free survival and overall survival. Conclusions: The results support NAT10 as a potential prognostic and therapeutic biomarker for AML.

Safety of Intrathecal Administration of Autologous Mesenchymal Stem Cells (MSCs) in Patients with Minimally Conscious State

Background: Patients who are in a minimally consciousness state (MCS) have often remained in the MCS for a long time. At the moment, the only chance for these patients seems to be the development of regenerative medicine based on stem cells as the studies on laboratory animals have demonstrated promising results. The favourable outcomes of these experiments insentivize to check the safety of using similar tests in humans. The aim of this study was to evaluate the safety of intrathecal administration of autologous bone marrow derived-mesenchymal stem cells (BM-MSCs) in patients with minimally conscious state. Methods: The study involved 28 patients diagnosed with persistent consciousness disorder who had completed an intensive care unit therapy at University Clinical Hospital in Olsztyn, Poland. The patients were referred for an in-patient care and admitted to hospital in a regular mode without prior verification of the diagnosis. All patients were evaluated according to a uniform test protocol. Diagnostics included a neurological, internal, cardiological and ENT examinations as well as anesthesiological, and rehabilitation assessment, and logopedic and psychological tests. The next stage of the experiment covered 9 selected MCS patients. This stage aimed at establishing a colony of autologous stem cells derived from the bone marrow and then three intrathecal administrations of stem cells at 2 month intervals. All the patients underwent a thorough virological examination before the stem cell cultures were started. The BM-MSC transplantation procedure safety assessment relied on a thorough analysis of the immediate and delayed adverse events. The immediate adverse events comprised an allergic reaction, respiratory failure, local complications (hematoma, local infection), and paralysis. Among the delayed adverse events an intraspinal tumor formation, permanent loss of sensation and paralysis, unrelated to the progress of the disease, were determined. During the hospitalization, a daily assessment procedure of the patient's condition was adhered to and the data recorded in the patient's medical chart. Findings: No severe side effects were documented during the procedure. of the patients demonstrated any symptoms of infection. In 2 patients, an increased psychomotor activity was observed, more increased when placed on the side from which the bone marrow had been collected. In one patient it was necessary to repeat the bone marrow collection due to the detection of bacteria in the stem cell culture. Out of the 9 patients, 3 were administered 3 intrathecal doses. In 2 patients, there were 2 intrathecal stem cell administrations completed, and in 1 patient, after the first administration, the subsequent doses were abandoned. Moreover, in a group of 6 patients, who had undergone 3 administrations, 1 patient had to have the treatment postponed due to an upper respiratory tract infection. In 1 patient, after two intrathecal administrations of stem cells without any evidence of adverse effects, the third dose could not have been given on account of a necessary urological treatment in another medical center. Thus, this patient had to be excluded from the experiment. In 1 patient, an epileptic status occurred after the first injection of stem cells. Due to the unclear coincidence of the epileptic status with the administration of stem cells, any subsequent administrations were abandoned. Interpretation: The study showed more difficulties occurring in patients with consciousness disorders than in patients treated for other diseases. Moreover, data collected based on a small and changeable group of patient require to be confirmed in a study encompassing a larger group of patients. However, in the course of any further research, the risks of this treatment should be borne in mind and proper precautions employed. Funding Statement: This work was supported by the National Centre for Research and Development (No: STRATEGMED I/234261/2/2014). Declaration of Interests: The authors state: None to declare. Ethics Approval Statement: The study was approved by the Bioethical Committee of the Faculty of Medicine, University of Warmia and Mazury in Olsztyn, Poland (ethical approval No. 7/2013 of February 2013, and No. 46/2013 of December 2013). The study was carried out in accordance with the provisions of the Declaration of Helsinki.

PF212 NEXT‐GENERATION SEQUENCING OF CIRCULATING CELL‐FREE DNA PROVIDES COMPLEMENTARY INFORMATION TO GENOMIC PROFILING OF BONE MARROW IN PATIENTS WITH ACUTE LEUKEMIAS

Background:Circulating cell‐free DNA (ccfDNA) allows for non‐invasive peripheral blood sampling of cancer‐associated mutations and has established clinical utility in several solid tumors. The ability of ccfDNA to detect and track leukemia‐associated mutations may also be useful in leukemias, both by identifying prognostic or targetable mutations at diagnosis or as a non‐invasive marker of measurable residual disease (MRD).Aims:To evaluate the feasibility of genomic analysis of ccfDNA to detect somatic mutations in patients (pts) with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).Methods:Plasma ccfDNA was collected and analyzed using a capture‐based next‐generation sequencing (NGS) assay (Qia275) targeting 275 genes. NGS was performed on DNA extracted from the bone marrow (BM) in a CLIA‐certified molecular diagnostics laboratory for the detection of mutations in the coding sequence of 28 genes, all of which were also assessed in the ccfDNA panel. An established bioinformatics pipeline was used to identify somatic variants.Results:A total of 24 pts (AML, n = 22; ALL, n = 2) underwent paired ccfDNA and BM sequencing at diagnosis. ccfDNA was also collected in 11 pts at ≥1 time point during remission. Among baseline samples, ccfDNA was collected a median of 6 days after BM collection (range, 0 to 27 days) and 0.5 days after the start of induction (range, −7 to 7 days). Using the 28 overlapping genes between the two assays, 55 somatic mutations were detected. 12 mutations (22%) were detected in ccfDNA but not BM. The median number of mutations detected in the BM and in ccfDNA per pt were 1.5 (range, 0–5) and 1 (range, 0–4), respectively (P = 0.31). Time from the start of chemotherapy until ccfDNA collection did not impact the concordance with BM mutation analysis. Among mutations detected in baseline ccfDNA samples, the median variant allelic frequency (VAF) was 33.7% (range, 2.7% to 90.8%). ccfDNA VAF was higher in mutations also detected in BM compared to those detected only in ccfDNA (median 38.3% vs 25.4%; P = 0.01), suggesting that these may be small subclonal populations more likely to be missed with BM sampling. Conversely, BM VAF was higher in those also detected in ccfDNA compared to those only found in BM (39.7% vs 21.0%; P &lt; 0.05). Mutations detected with ccfDNA but not BM included FLT3‐ITD in 4 pts, ASXL1 in 2 pts, WT1 in 1 pt, IDH1 in 1 pt, and BRAF and two EGFR mutations in 1 pt. In the 4 pts with FLT3‐ITD, the mutation was detected by PCR of the BM but not by BM sequencing. Among the other 5 pts with mutations detected in ccfDNA but not BM, 2 pts eventually relapsed; in both pts, the discordant mutation (IDH1 and ASXL1) was detected in the relapse BM. Using ccfDNA, the persistence of leukemia‐associated mutations during remission or the emergence of new mutations appeared to herald relapse. Two pts with t(8;21) AML developed new RUNX1 mutations detected by ccfDNA while in remission and relapsed 3 months and 14 months later; in both pts the emergent RUNX1 mutation was also detected in the relapse BM. Another pt with AML in morphological remission on day +30 after stem cell transplant had persistent TP53 and TET2 mutations detected by ccfDNA and relapsed 1 month later.Summary/Conclusion:This study demonstrates the feasibility of ccfDNA monitoring in pts with acute leukemias. ccfDNA can identify clinically significant prognostic or targetable mutations not detected by BM sequencing. The potential use of ccfDNA as a non‐invasive method of MRD assessment needs to be evaluated in larger, prospective cohorts.

PF275 NAT10 UPREGULATION INDICATES A POOR PROGNOSIS IN ACUTE MYELOID LEUKEMIA

Background:Acute myeloid leukemia (AML) is a series of hematologic malignancies with high morbidity and mortality. It is characterized by impaired differentiation, excessive proliferation, and apoptosis escape. N‐acetyltransferase 10 (NAT10) is a transcriptional activator of telomerase reverse transcriptase (hTERT)[1]. It has acyltransferase domain and participates in microtubule acetylation, rRNA modification, DNA damage response, and cytokinesis[2–5]. NAT10 was upregulated in various kinds of tumors such as hepatocellular carcinoma, colorectal cancer, ovarian cancer and melanoma[6–9]. NAT10 upregulation is associated with poor prognosis. This study investigated NAT10 expression in AML patients and its correlation with clinical characteristics, chemotherapy response and prognosis.Aims:N‐acetyltransferase 10 (NAT10) is considered as an oncogene in many tumors. This study investigated the NAT10 expression in Chinese acute myeloid leukemia patients and evaluated the predictive significance of NAT10 with a single‐center retrospective study.Methods:We collected bone marrow samples of 48 newly diagnosed acute myeloid leukemia patients and 20 benign controls in our center. NAT10 mRNA expression levels were detected by Real‐time qPCR. Clinical data was obtained from inpatient medical records.Results:AML patients had higher NAT10 expression level than the normal controls (P &lt; 0.01). NPM1‐mutant patients had higher NAT10 expression level than NPM1‐wt patients. NAT10 expression level was higher in non‐remission group than that of overall remission group (P &lt; 0.05). High NAT10 expression indicated a poor progress free survival and overall survival.Summary/Conclusion:The results support NAT10 as a potential prognostic and therapeutic biomarker for acute myeloid leukemia.image

Bone marrow donation perceptions among healthcare workers: A follow-up study at University of Cincinnati Medical Center.

e18032 Background: It has been well-documented that there is a severe lack of participants enrolled in the National Bone Marrow Donor registry. Even those who are registered often decline to donate at a critical juncture in the donation process due to a wide variety of misconceptions. Health care professionals should be among the most educated about the bone marrow donation process as they will often be giving advice to potential donors. Methods: We conducted an anonymous online survey of medical students, residents, fellows, and attending physicians at the University of Cincinnati Medical Center to determine the common misconceptions surrounding bone marrow donation. Results: There were a total of 187 participants (73% medical students, 23% residents/fellows, 4% attending physicians). Fifty-one percent were already bone marrow donors, while 49% were not. Forty percent of respondents believe bone marrow biopsy is necessary for donation and 10% believe CT scans are done prior to donation. Twenty-eight percent responded that bone marrow is extracted under general anesthesia, 43% responded that it is extracted not under general anesthesia, 5% responded that it is extracted via central intravenous line, and 24% responded that it is extracted via peripheral intravenous line. Thirty percent responded that they are not bone marrow donors because bone marrow donation is painful, 12% because it involves surgery, 2% because it weakens the donor, 13% because it involves a lengthy recovery process, 3% because donors have to pay for donation, 1% because of ethnic/religious/cultural beliefs, while 39% have not had the opportunity to donate but would or are planning on it. Conclusions: The majority of allogeneic transplants are performed with peripherally collected bone marrow stem cells, however, most potential donors including health care professionals do not know this. We conclude that a great deal of education surrounding bone marrow donation is still needed among healthcare workers today. Educating health care workers is of the utmost importance as they will impart this knowledge to those considering donation. Future aims of this project are to develop an educational curriculum to address the most common misconceptions, particularly in medical students, as they are in the age group most targeted in bone marrow donation and they will be the future generation of physicians to educate patients on the process.

Bone marrow response to multiple filgrastim stimulations in patients with non hematological disorders

Background & Aim Introduction Filgrastim is used to mobilize CD34+ cells to peripheral blood (PB) or to enhance bone marrow (BM) collection for autologous and allogeneic hematopoietic cell transplantation (HCT) in hematological disorders. Filgrastim has also been used to improve bone marrow collection of total nucleated cells (TNC), which were then administered intrathecally (IT) to treat neurological diseases. The impact in the BM functioning in non-hematological patients, after multiple stimulations with filgrastim, is unknown. Purpose To establish the BM capacity of response after multiple stimulation procedures with filgrastim in children with neurological disorders. Methods, Results & Conclusion Methods Retrospective analysis of 13 patients with no BM diseases who underwent more than one cellular therapy (CT) since 2013, in our center. In each CT, patients received 10μg/kg/day of subcutaneous filgrastim for three days. Bone marrow collection was done on the fourth. The patients’ count of peripheral blood WBC before and after stimulation was analyzed, as well as the number of TNC, mononuclear cells (MNC), CD34+, and CD133+ cells per microliter contained in the collected BM. Every patient had received 3 or more CT procedures with at least 6 months in between each procedure. Wilcoxon's test was used to compare the count of CD34+ cells of the first CT against the subsequent CT. The standard p≤0.05 boundary was defined as a statistically significant value. Results From the thirteen patients included, 4 patients (31%) had undergone four CT, and the rest (69%) only three CT. The medians of the absolute MNC obtained from bone marrow showed minimal variations between the different collections (14.4, 18.06, 15.3, 13.8 k /µL, but the Wilcoxon's test did not show any statistically significant differences between the count of CD34+ and MNC cells in each CT. (Table 1) Conclusions BM performance in children without BM diseases, was effective in each stimulation. No significant differences in several cell counts can be observed between multiple CT. It does not seem that the use of filgrastim affects BM functioning after multiple stimulations.

Excellent outcomes of combined umbilical cord blood and bone marrow grafts for allogeneic transplantation of pediatric patients from HLA-matched siblings. a single institution experience.

Background & Aim We hypothesized that combining cord blood (CB) and bone marrow (BM) grafts from HLA-identical siblings would improve the outcome of allogeneic transplantation in pediatric patients: the increased number of infused BM progenitor cells would expedite engraftment and the CB naive T cells would lower the risk of GvHD. Methods, Results & Conclusion Methods We reviewed the data of consecutive patients who received combined CB and BM grafts at our institution (MSKCC). Outcomes of interest were patient neutrophil engraftment, GVHD, donor chimerism and overall survival (OS) and donor safety. Results 15 patients (10 females, 5 males), with median age 5.9 (3.1-17.2) years, were included. Diagnoses were beta-thalassemia (N=8), sickle cell disease (N=1), Diamond Blackfan anemia (N=1), Fanconi anemia (N=2), acute lymphoblastic leukemia (N=2) and myelodysplastic syndrome (N=1). 12 patients had genetic diseases, and, in 6 cases, donors were via preimplantation genetic diagnosis. All sibling CB units were stored in private family banks. Donors’ age at transplant was 0.8-6.8 years (median 1.6 years). Patients’ weights were greater than donors’ in all cases with a ratio of 1.1-7.9:1 (median 1.7:1). All donors underwent bone marrow collection (median: 175ml; range: 100-375ml) and were discharged home on the same day. Most CB units underwent washing prior to infusion. No infusion related serious adverse events were noted. Median BM CD34+ cell dose was 7.3 × 10^6 (range: 1.6-11.4). Median CB infused viable CD34+ cell dose was low 0.34 × 10^5 (range: 0.01-2.0). Viable infused CB CD3 doses ranged 0.5-8.3 × 10^6 (median: 2.5). All 15 patients engrafted with median neutrophil engraftment (ANC ≥500) at day +14 (range: 11-19 days) and median platelet engraftment (Plts ≥50,000) at day +35 (range: 27-43 days). No patient developed acute GvHD, while one patient developed limited chronic GvHD that resolved. T-cell reconstitution occurred at 1-7 months post-transplant in 13 evaluable patients. Donor chimerism was 100% (n=10) and 81-99% (n=5). With a median follow-up of 5.2 years (range: 5-16.9 months), all 15 patients are alive, disease-free. SUMMARY When CB units from HLA identical siblings are available and donors are able to undergo BM collection, this combination of grafts for transplantation has shown excellent outcomes, and this approach is our current practice. A retrospective review is underway comparing this approach with BM or CB only grafts to confirm advantage of a combined grafting strategy.

Osteogenic Differentiation of Normotensive Rats Depends on the β‐adrenergic Receptor

Bone remodeling is a complex process that depends on the balance between formation and resorption bone, a process which is regulated by bone cells and systemic factors, like the Sympathetic Nervous system (SNS). The mediators of these system are able to regulate bone metabolism through adrenergic receptors on the surface of the osteoblasts. However, the role of β‐adrenergic receptors is not clear in the osteogenic differentiation process. Thus, in this study we aimed to evaluate the role of B‐adrenergic receptor on osteoblastic differentiation of bone marrow mesenchymal stem cells from normotensive and Spontaneously Hypertensive Rats (SHR).Methods70‐days‐old male Wistar and SHR rats were used for bone marrow collection from femurs, which was placed in cell culture flasks and after in to 24‐well plates, where they received osteogenic medium (OM: MEM, plus 50 μg/mL ascorbic acid, 10 mM β glycerophosphate, and 10−8 M dexamethasone) and the treatment with Carvedilol (1μM), non‐selective adrenergic receptor antagonist, or Nebivolol (0,1 μM), β1‐adrenergic receptor antagonist. Cell proliferation (MTT assay) and alkaline phosphatase specific activity (Alp) were analyzed at day 7, 10 and 14. Mineralization was evaluated at day 14, by Alizarin Red S. Gene expression of osteogenic markers and B1 and B2‐adrenergic receptor were evaluated at day 7, by real time‐RT‐PCR. The proteolytic activity of matrix metalloproteinase 2 and 9 (MMP‐2 and MMP‐9) were evaluated at day 7 using gelatin zymography. The protocol was approved by Institutional Animal Care and Use Committees (School of Dentistry of Araçatuba; Process 00686‐2016).ResultsAlp activity increased in OM group from Wistar rats at day 7 and the Nebivolol group reduced those activity at day 7 and at day 10. SHR OM group did not present Alp increase, and the treatment with both drugs did not change it. Nebivolol treatment decreased Runx2, Osterix and β‐catenin in Wistar compared to OM. In SHR, only Osterix was reduced. Alp, Osteocalcin and Bone sialoprotein have significative decrease in cells from Wistar treated with Nebivolol compared to OM. Pro and active MMP‐2 was reduced by Nebivolol treatment just at Wistar cells. Besides that, Nebivolol reduced Adrb1 gene expression at day 7 in Wistar group compared to OM. Mineralization showed that Nebivolol reduced mineral deposition in Wistar compared to OM.Conclusionβ1 adrenergic receptor seems to be involved in the osteogenic differentiation of cells from Wistar rats.Support or Funding InformationFAPESP (Grant: #2015/03965‐2) and CAPES.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Capture Rate of the Adaptive Next Generation Sequencing VDJ Assay in Multiple Myeloma

BackgroundMinimal residual disease (MRD) negativity is a strong predictor for outcome in multiple myeloma. Next generation sequencing (NGS) for immunoglobulin heavy chain and kappa light chain VDJ rearrangements has become increasingly more common for MRD assessment. One of the known challenges with NGS for VDJ rearrangements is the vast diversity of sequences that are present, resulting in a need for a multiplex approach as common primers cannot be used to amplify all rearrangements. Also, somatic hypermutation may affect the annealing of primers and decrease the capture rate. The NGS VDJ assay developed by Adaptive Biotechnologies targets all theoretical combinations of VDJ sequences and has been used in several recent large randomized trials in multiple myeloma. The reported ~80% capture rate of the first version of the Sequenta/Adaptive 1.3 assay limited the ability to track MRD status post therapy. The assay has recently been updated and validated to increase resilience to somatic hypermutation. As there is no published reference data using this assay, we were motivated to assess VDJ capture in the clinical setting.MethodsIn total, 147 patients with newly diagnosed multiple myeloma (NDMM, n=101) or relapse/refractory multiple myeloma (RRMM, n=46) seen at Memorial Sloan Kettering Cancer Center were identified and included in the study. At bone marrow collection, patient samples were sorted for mononuclear cells and a subset of samples were sorted for CD138+ plasma cells. Stored bone marrow samples from these patients underwent DNA extraction and were sequenced with the Adaptive NGS VDJ assay. The same samples were also sequenced for genomic events using our internal NGS panel myTYPE. myTYPE is a custom capture panel targeting the most frequent multiple myeloma associated-somatic mutations, copy number alterations, and IGH translocations. Logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs) of detection success in relation to clinical parameters such as age, gender, percent bone marrow plasma cells, as well as immunoglobulin heavy and light chain types, and myTYPE positivity.ResultsThere overall capture rate for a unique VDJ sequence was 80%, 75% in NDMM samples and 89% in RRMM samples, respectively. The VDJ capture rate in samples that were myTYPE positive, e.g. samples with at least one genomic aberration detected by myTYPE, was 94%. In univariate analysis, the ORs of detecting a clonal VDJ sequence was 1.8 (95% CI 1.3-2.5) and 1.5 (1.2-1.9) for every 10% increase in plasma cells on bone marrow aspirate and biopsy, respectively. For every 1g/dL increase in M-spike, the OR of VDJ capture was 1.6 (1.2-2.2). Samples with at least one genomic aberration detected by myTYPE had a significantly higher detection rate of VDJ sequence, the OR of VDJ capture in myTYPE positive samples was 8.8 (3.2-31.3). Conversely, age, gender, type of immunoglobulin heavy chain (IgG or IgA), or light chain type (kappa or lambda) had no significant effect on the VDJ detection rate (Table). In multivariate analysis, myTYPE positivity was found to be an independent predictor of VDJ capture, with an OR of 4.9 (1.6-18.4, p=0.009) for myTYPE positive samples. The ORs were 1.4 (1.1-2.2, p=0.052) for an increase in 10% plasma cells on bone marrow aspirate and 1.5 (0.97-2.3, p=0.083) every 1g/dL increase in M-spike.ConclusionThe VDJ capture rate using the updated Adaptive NGS VDJ assay was 94% in multiple myeloma samples of high quality as indicated by myTYPE positivity. The capture success rate was higher in samples with a greater disease burden. As expected, the assay was less sensitive in samples with insufficient DNA content. Our results are supportive of the use of this NGS VDJ in multiple myeloma, but also illustrate the importance of optimal sample ascertainment and processing. [Display omitted] DisclosuresJacob:Adaptive Biotechnologies: Employment, Equity Ownership. Korde:Amgen: Research Funding. Mailankody:Juno: Research Funding; Physician Education Resource: Honoraria; Janssen: Research Funding; Takeda: Research Funding. Lesokhin:Serametrix, inc.: Patents & Royalties: Royalties; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Genentech: Research Funding. Hassoun:Oncopeptides AB: Research Funding. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Landgren:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding.

Robust Cell Selections and Depletions across Various Hematopoietic Cell Fractions on the Clinimacs Plus Instrument

Introduction: Cell enrichment and/or depletion by selection is critical for graft engineering in cellular and gene therapies. The semi-automated CliniMACS Plus Instrument (Miltenyi Biotec) is used in clinical cell processing to enrich or deplete a variety of hematopoietic cells including T, B, monocytes, NK cells, and/or CD34+ hematopoietic stem/progenitor cells (HSPCs). Antibody-conjugated paramagnetic beads attach to the desired cells which are separated from the rest of the apheresis or bone marrow product in a closed-system magnetic separation column. Either the selected cells retained in the separation column (positive selection) or those in the “flow-through” fraction (negative selection) may be used for downstream processing. There is sparse data comparing cell selection efficiency and purity across the various selection methods on the CliniMACS Plus Instrument. Further, the effect of donor demographics and other upstream processing events on final viable cell recovery (VCR)/ cell purity (CP) are unknown. This study systematically compared these parameters across various selection methods.Methods: From 2008 to 2018, data were obtained from 45 clinical processing protocols involving 991 consecutive adult and pediatric donor/patient apheresis products or bone marrow harvests, which underwent cell selections on the Miltenyi CliniMACS Plus device. Cell selections included 1.CD34+ (n=669), 2.CD4+ (n=179), 3.CD3-/56+ (n=79), 4.CD3+/19- (n=6), 5.CD4+/8+ (n=21), and 6.CD25- (n=37) cells. Based on protocol design or logistical needs, apheresis or bone marrow collections were processed/selected on the same day or held overnight for next day processing. Products were also either washed to remove platelets (PW) or loaded onto the selection device directly. Data on donor age, BMI, donor type (autologous vs. allogeneic), sex, race, pre-apheresis peripheral blood cell counts and concentrations, as well as apheresis bag blood counts were evaluated. Outcome variables included post-selection VCR and CP. Correlations and multivariate regression analyses were performed for the largest selection type, i.e CD34+ cell selections.Results: Summary data (means ± 1SD) on method-specific enrichments and depletions are shown in the Table. Median post-selection VCR and CP varied by selection method. VCR was the highest for the CD34+ cell selections (Fig. a). CP was the highest and most consistent for CD4+ selections (Fig. b). Platelet and red cell depletions also varied by selection method and averaged 3.9 ± 1.63 and 2.06 ± 0.74 logs, respectively. Selection procedures used for cell depletion resulted in near complete removal of the undesired cell fractions (Table).In multivariate regression analyses, products processed on the same day (compared to those held overnight) (Fig. c), those that underwent a PW (Fig. d), and had higher pre-apheresis peripheral blood CD34+ absolute cell counts and concentrations (Fig. e) were associated significantly with higher CD34+ CP (adjusted r2=0.2; p<0.0001). Higher pre-apheresis CD34+ concentrations (Fig. f) were associated with a lower CD34+ VCR (15%, CI: 5.6-25.3). Conversely, PW (Fig. g) correlated with higher CD34+ VCR (3.5%; CI: 0.8-6.28). Trends across the study period demonstrated a significant improvement in CD34+ VCR from 2008 to 2017 (Fig. h).Conclusions: This is the largest study, to our knowledge, to systematically summarize and analyze Miltenyi CliniMACS Plus selections for clinical graft engineering. Cell selections on the Miltenyi CliniMACS Plus resulted in robust enrichment with near complete depletion of the undesired cell fractions. CD34+ CP was higher in patients with higher CD34+ cells counts following mobilization. Same day processing, as well as PW further improved CD34+ CP. PW also improved CD34+ VCR. This was likely due to a reduction in non-specific platelet binding to the paramagnetic beads. Higher pre-selection CD34+ cell concentrations in the product marginally worsened VCR. This was possibly due to bead saturation in each selection method. Improved CD34+ VCR over time was likely due to increased platelet washes in the latter years. Further studies are ongoing to assess impact of cell selection variations on downstream manufacturing steps (cell transduction, expansion) and clinical (cell engraftment, GVHD incidence) outcomes. [Display omitted] DisclosuresLarochelle:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.

Validation and Clinical Impact of the Oncomine Myeloid Targeted DNA and RNA Ion Semiconductor Sequencing Assay

Introduction: The expanding diagnostic and clinical significance of genes associated with myeloid malignancies necessitates higher throughput testing. Targeted next-generation sequencing (NGS) of DNA and RNA may be an ideal approach to capture these variants and eventually streamline, or replace, conventional cytogenetic testing. We present our clinical validation of the OncomineTM Myeloid NGS assay and early experience regarding the clinical impact in patients with suspected or known myeloid malignancies.Methods: OncomineTM Myeloid (Thermo Fisher Scientific) panel (OMP) validation was conducted at Kingston Health Sciences Centre (KHSC) on anonymized and commercial samples according to IQMH and ACMG standards for NGS (PMID: 23887774). As of April 2018 the test has been formally validated and offered at KHSC. Prior to this, patients were consented at the time of blood (PB) or bone marrow (BM) collection for research-based testing. BM and PB samples were collected, gDNA and RNA extracted, and libraries prepared using the Ion ChefTM System. Sequencing was completed using the OMP on Ion PGM (research phase) and S5XL (clinical) sequencers. The OMP uses amplicon-based semiconductor sequencing technology to cover 40 DNA genes recurrently mutated in myeloid malignancies (23 hotspots and 17 full coding), 29 RNA fusion driver genes and 10 gene expression targets. Sequencing data were analyzed using Torrent Suite, Torrent Variant Caller, Ion Reporter (v.5.2-5.6) and Alamut software. The OMP includes a Torrent Suite FLT3-ITD “plug-in” and Ion Reporter “Oncomine filter”. Orthogonal clinical assays were performed where necessary for additional confirmation. Variants were interpreted based on a 4-tier classification system developed by Li et al (PMID: 27993330). Variants in the top 3 tiers were reported after assessment by at least one clinician and one certified medical geneticist. Actionability was assessed in the context of electronic diagnostic and clinical information and molecular board review.Results: Validation involved 24 paired DNA-RNA samples previously run at KHSC on an early access OMP version and also at an external site (kind exchange by Dr. N. Carson, Saint John, NB), along with SeraCareTM DNA and RNA controls. Validation run metrics were as follows: DNA means (mapped reads 1306216, read length 226bp, uniformity 98%, depth 2518), RNA means (mapped reads 260350, read length 114bp). Complete concordance was found for RNA variants while the DNA filter chain was only modified to better capture a JAK2 exon 12 variant in a SeraCare control. The minimum depth of coverage was subsequently set to 400X for somatic DNA variants and sensitivity set at 5% variant allele fraction (VAF) for clinical reporting. To date, we have performed sequencing for 38 samples from 35 unique patients (avg age = 64.5y). Of these, 12/35 (34%) involved suspected or known myelodysplastic syndromes (MDS), 12/35 (34%) with acute myeloid leukemia (AML), 4/35 (12%) with query myeloproliferative neoplasms (MPNs), 2/35 (5%) with MDS/MPN, and 5/35 (14%) other (2 B-ALL, 1 aplastic, 1 hairy cell + ?MDS, 1 ?CHIP). OMP detected 56 variants (in 80% of cases), including 43% tier 1, 48% tier 2, and 9% tier 3 variants. The most frequently mutated genes included TET2 (21%), SRSF2 (14%), SF3B1 (9%), TP53 (5%), and RUNX1 (5%). The average VAF was 36% for all samples, with an average of 1.5 variants per sample. Of detected variants, 73% were potentially actionable. Specifically, 51% of actionable variants facilitated or clarified diagnosis (38% of total variants), 32% (23%) affected prognosis, and 17% (13%) had the potential to affect treatment decisions. Among 10 patients with suspected (but subthreshold for) MDS or MPN, clonality was detected in 5, while 5 had no variants, prompting further investigation of reversible cytopenia or cytosis. We are rapidly accruing patients and expect to have OMP profiles for at least 100 patients at the time of the ASH 2018 meeting.Conclusions: Oncomine Myeloid DNA and RNA molecular profiling has found greatest clinical use at our centre in the realm of MDS and AML. In the majority of patients, OMP permits the identification of actionable variants that may aid clinicians in achieving or clarifying diagnoses, inform follow-up and prognosis, and guide treatment decisions. The OMP represents a promising strategy to address the challenge of expanding, important molecular features of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Cyclophosphamide, Pomalidomide and Dexamethasone Significantly Improves Response over Poma/Dex in Relapsed/Refractory Myeloma Patients Previously Treated with Cyclophosphamide Combination Therapy - Initial Results of the Randomised Multicentre Mukseven Trial

Cyclophosphamide, Pomalidomide and Dexamethasone Significantly Improves Response over Poma/Dex in Relapsed/Refractory Myeloma Patients Previously Treated with Cyclophosphamide Combination Therapy - Initial Results of the Randomised Multicentre Mukseven Trial

Cytogenetic Abnormalities in Systemic Mastocytosis: Who Subcategory-Specific Incidence and Prognostic Impact Among 348 Informative Cases

Background:The World Health Organization (WHO) system lists five morphological categories of systemic mastocytosis (SM): indolent (ISM), smouldering (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive (ASM) and mast cell leukemia (MCL) (Blood. 2016;127:2391). Recent studies have highlighted the prognostic importance of mutations in SM, including ASXL1, RUNX1 and SRSF2 (AJH 2016;91:888;Leukemia 2016;30:2342). In the current study, we reviewed the cytogenetic findings in 348 cases of SM, in order to clarify incidence and prognostic impact of cytogenetic abnormalities, stratified by WHO morphologic subcategories.Methods:Study patients were selected, based on availability of cytogenetic information. Diagnoses of SM and its morphological subcategories were confirmed by both clinical and bone marrow examinations, in line with WHO criteria (Blood. 2016;127:2391). Next-generation sequencing was performed in a subset of the study population. Abnormal karyotype was further distinguished into favorable and unfavorable class based on previously published criteria for myelodysplastic syndromes (MDS) (Blood 2012;120:2454) and myeloproliferative neoplasms (MPN) (Leukemia 208;32:1189). Statistical analyses considered clinical and laboratory data collected at the time of initial diagnosis at the Mayo Clinic, which coincided with collection of bone marrow for cytogenetic studies. Conventional statistics was used for calculation of overall survival and determination of risk factors. JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations.Results:348 SM patients were included in the current study (median age 59 years; range 18-88 years; 53% males); 142 (41%) constituted ISM and 206 (59%) advanced SM; the latter included 155 (45%) SM-AHN, 49 (14%) ASM and 2 MCL cases. The SM-AHN cases included 39 (11%) SM-MPN, 36 (10%) SM-CMML, 22 (6%) SM-MDS, 22 (6%) SM-myeloid unclassified, 14 (4%) SM-MDS/MPN, 12 (3%) SM-lymphoid malignancy and 10 (3%) SM-acute leukemia. Adverse mutations, including ASXL1 (19% mutated), RUNX1 (3% mutated) and NRAS (3% mutated) were detected in 30 (23%) of 129 cases screened. After a median follow-up of 21 months, 139 (40%) deaths and 6 (5%) leukemic transformations were documented.Karyotype was abnormal in 53 (15%) cases and included unfavorable (n=29; 8%) and favorable (n=24; 7%) abnormalities. Abnormal karyotype incidences were 6% for ISM and 22% for advanced SM (p<0.001). Among advanced SM cases, abnormal karyotype incidences were 26% for SM-AHN and 8% for ASM (p<0.001); one of 2 MCL cases displayed abnormal karyotype. Among SM-AHN cases, abnormal karyotype incidences were 0% for SM-AHN-lymphoid, 28% for SM-AHN-myeloid (p<0.001); the latter included 19% for SM-CMML, 21% for SM-MDS/MPN, 23% for SM-MPN, 36% for SM-MDS and 41% of SM-myeloid-unclassified (p<0.001). Clinical correlative studies disclosed significant associations between abnormal karyotype and male sex (p=0.002), age >60 years (p=0.04), thrombocytopenia (p<0.001; 27% vs 10%) and anemia (p<0.001; 25% vs 6%), but not with the presence of adverse mutations (p=0.19).In univariate analysis, abnormal karyotype was associated with inferior survival (HR 3.0, 95% CI 2.0-4.3) and significance was sustained when analysis was adjusted for two-tiered (advanced vs indolent SM; p<0.01) or multi-tiered (ISM vs ASM vs SM-AHN; p<0.01) WHO subcategories. WHO category-specific analysis clarified prognostic relevance of abnormal karyotype in ASM (HR 4.4, 95% CI 1.0-14.4; p=0.05; figure 1a) and SM-AHN-myeloid (HR 1.9, 95% CI 1.2-2.9; p=0.005; figure 1b); however, the near-significance in ASM was fully accounted for by thrombocytopenia (p value corrected to 0.35) and for SM-AHN-myeloid by thrombocytopenia and anemia (p value corrected to 0.06); further stratification of abnormal karyotype into favorable vs unfavorable categories did not affect the results in ASM but revealed an independent prognostic effect for unfavorable karyotype in SM=AHN-myeloid (p=0.009).Conclusions:Abnormal karyotype in SM clusters with SM-AHN-myeloid. We found no correlation between abnormal karyotype and adverse mutations. Anemia and thrombocytopenia were significantly associated with abnormal karyotype and accounted for the apparent prognostic relevance of the latter in ASM. Unfavorable karyotype carries independent prognostic effect in SM-AHN-myeloid. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Genomic Analysis of IDH Inhibitor-Treated AML Samples Delineates Molecular Mechanisms of Differentiation and Heterogeneous Patterns of Acquired Resistance

Allosteric inhibitors of mutant IDH1 or IDH2 induce differentiation of IDH-mutant AML myeloblasts, which in some patients (pts) can lead to a life-threatening differentiation syndrome (DS). The in vivo mechanism of how IDH inhibitors induce differentiation and occasionally DS is not fully understood. Furthermore, responders to the inhibitors often lose the differentiation effect, after median of 7 months. Although a recent study identified secondary mutations in the dimer-interface of IDH1/2 as a mechanism for acquired IDH-inhibitor resistance, given the rarity of this event, it is unlikely to represent the primary mechanism in the majority of pts.We conducted a comprehensive genomic analysis (DNA sequencing, single-cell DNA sequencing, RNA sequencing and cytosine methylation array) on longitudinally collected bone marrow specimens from IDH1/2-mutant AML pts treated with one of the IDH1/2 inhibitors (ivosidenib, enasidenib, IDH305 or AG881). In total, 146 samples from 39 pts collected at different time points (median 4 [IQR 3-5] samples per patient) were analyzed. 24 pts were IDH1-mutated receiving IDH1 inhibitor and 15 were IDH2-mutated receiving IDH2 inhibitor. 29 pts responded (CR, CRi, CRp or MLFS) and 10 had no response to the inhibitors. 8 of 29 responders developed DS. 22 of 29 responders relapsed after the median of 5 months.Cytosine methylation analysis revealed significant decrease in global methylation at response than baseline (mean beta value 0.54 at baseline vs. 0.45 at response, P < 0.001). This corroborated with a decrease in serum 2HG level, supporting the role of IDH inhibitors in reversing CpG island methylator phenotype (CIMP) likely through TET restoration. Motif enrichment analysis of differentially methylated promoter regions showed significant de-methylation of PU.1(SPI1) and C/EBP binding motifs at response. Consistent with this, RNA sequencing revealed that PU.1 and C/EBP target genes such as CSF2RA, CSF3R, IL34, TREM2, IL6R, CEBPE, GFI1, and KLF5 had significant upregulation at response. Expression of PU.1 itself was also significantly up-regulated upon response, along with other key hematopoietic differentiation genes and pathways such as GM-CSF, IL8-CXCR1 and IL3 pathways. Pts who developed DS had significantly higher upregulation of PU.1 and GM-CSF pathway compared to other DS-free responders, implicating the potential activation of these pathways in the mechanism of DS. At relapse, these pathways that mediated differentiation were all down-regulated when compared to that at response. Interestingly, methylation level remained suppressed at relapse (mean beta value 0.45 at response vs. 0.47 at relapse, P = 0.566), suggesting that the down-regulation of pro-differentiation signals at relapse is independent of methylation.Combined whole exome and targeted deep sequencing revealed the heterogeneous patterns of acquired mutations at relapse. Of 22 cases with baseline and relapse pairs, 18 acquired new driver mutations at relapse (Fig 1). The most frequently acquired mutations at relapse were in BCOR/BCORL1 (N = 6), hematopoietic differentiation genes (CEBPA [N=2] and RUNX1[N=4]), and RTK pathways (NRAS/KRAS [N=5] and PTPN11 [N=1]). We did not identify acquisition of dimer-interface IDH1/2 mutations at relapse. However, in 1 case, relapse was associated with acquisition of mutation in reciprocal IDH gene (IDH2-mutated case acquired IDH1 mutation). Singe-cell DNA sequencing of the double-mutant revealed that the reciprocal acquisition occurred in a different clone.Analysis of differentially expressed genes between baseline and relapse showed significant upregulation of genes in DNA damage response (DDR) pathways at relapse. Consistent with this, relapse samples showed significantly higher mutation burden than baseline, suggesting that accumulation of mutations are upregulating DDR pathway at relapse.In summary, IDH inhibitors induce differentiation and DS through upregulation of PU.1 and C/EBP target genes, which is associated with de-methylation of their binding motifs. The mechanisms of acquired resistance to IDH inhibitors are heterogeneous and involves various genes and pathways including BCOR/BCORL1, hematopoietic differentiation transcription factors (CEBPA, RUNX1), and RTK pathways (RAS, PTPN11). In a rare case, acquired mutation in reciprocal IDH gene was detected, occurring in a different clone. [Display omitted] DisclosuresDiNardo:Agios: Consultancy; Abbvie: Honoraria; Medimmune: Honoraria; Bayer: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria. MacBeth:Celgene Corporation: Employment, Equity Ownership. Tosolini:Celgene Corporation: Employment. Frattini:Celgene Corporation: Employment, Equity Ownership. Kadia:Abbvie: Consultancy; Celgene: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Celgene: Research Funding; Takeda: Consultancy; Abbvie: Consultancy. Ravandi:Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Sunesis: Honoraria; Bristol-Myers Squibb: Research Funding; Xencor: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Jazz: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Macrogenix: Honoraria, Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria. Konopleva:Stemline Therapeutics: Research Funding.

Biology of Acute Myeloid Leukemia (AML) with Monosomy of Chromosome 7 or Loss of 7q. a Study on 487 Patients Analyzed By Gene Expression Profile (GEP), Single Nucleotide Polymorphism (SNP) Arrays and Metabolomics

Biology of Acute Myeloid Leukemia (AML) with Monosomy of Chromosome 7 or Loss of 7q. a Study on 487 Patients Analyzed By Gene Expression Profile (GEP), Single Nucleotide Polymorphism (SNP) Arrays and Metabolomics

Bone Marrow–Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration

Background: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. Hypothesis: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. Study Design: Controlled laboratory study. Methods: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques—BM aspiration and BM collection—after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. Results: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45–CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56– cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. Conclusion: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. Clinical Relevance: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.