Low Bone Marrow Research Articles

MICROENVIRONMENT IMMUNE CELLS IN BONE MARROW AND LYMPH NODE MEASURED BY FLOW CYTOMETRY CAN PREDICT SURVIVAL IN DIFFUSE LARGE B CELL LYMPHOMA

Introduction: Tumor microenvironment (TME) in diffuse large B-cell lymphoma (DLBCL) has recently been in the spotlight. The predictive role of immune cells (IC) in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) has been individually assessed, often using techniques not widely available. However, data regarding the relative prognostic impact of TME in each compartment evaluated simultaneously in DLBCL is still lacking. Aims: To determine the prognostic impact on survival of the Monocyte-Lymphocyte prognostic score (MLS) in PB and the percentage (%) of IC in the BM and LN measured by flow cytometry (FC) in DLBCL. Methods: We retrospectively collected information of patients (pts) with DLBCL and available BM aspiration and LN FC data at diagnosis, treated at our center between 2012 and 2019. Pts were stratified according to the MLS (Wilcox et al, leukemia 2011) in two groups: low (PB monocyte counts <630/ml and lymphocyte counts ≥1000/ml) and intermediate-high. FC analysis was performed with 8-color panels according to Euroflow protocols. % of BM and LN IC by FC were compared to normal values determined by Matarraz (Cytometry part B 2010) and Battaglia et al (Immunology 2003), and analyzed in 3 categories: low, normal and high. Survival analysis was performed with Kaplan-meier (variables compared with log-rank) and Cox regression. Results: 71 pts were included with a median age of 59 years. 49.3% had poor R-IPI. All pts received immunochemotherapy. Complete remission rate was 82%. Median overall survival (OS) was 120.5 months (m), with a median follow up time of 34.7 m. On univariate analysis TME variables from all the compartments showed prognostic impact on OS. BM IC levels did not statistically differ in involved vs not involved BM. Both low LN TL % and high and low BM Mo % remained independent predictors of survival on multivariate analysis. Moreover, pts with both of these unfavorable variables had a HR for death of 40.22 (CI 95% 5.16-313.04, p < 0.001) with a median OS of only 22.19 m. Variables Univariate analysis Multivariate analysis HR (95% CI) P-value HR (95% CI) P-value LN T lymphocytes (TL) low 3.98 (1.31-12.06) 0.014 5.44 (1.48-20.04) 0.011 BM B lymphocytes low 4.73 (1.62-13.79) 0.004 3.36 (0.99-11,34) 0.0501 BM Monocytes (Mo) High 10.53 (3.66-30.26) <0.001 4.66 (1.34-16.25) 0.015 BM Monocytes Low 3.87 (1.28-11.7) 0.016 3.40 (1.05-10.99) 0.04 PB MLS intermediate-high 2.59 (1.12-6) 0.02 1.05 (0.4-2.77) 0.91 Conclusion: Abnormal BM Mo % and low LN TL % measured by FC were associated with inferior OS in our cohort of DLBCL pts. These two TME variables presented at diagnosis could be easily translated into prognostic biomarkers in DLBCL, identifying a subgroup of pts with a dismal short term prognosis. In the future, therapies that target the crosstalk between lymphoma cells and the LN and BM ME might represent an encouraging strategy to improve outcomes in this high risk subset of pts. Kaplan-meler estimates of overall survival stratified by number of adverse prognosis tumor microenviroment varaibles present(low lymph node T-lymphocytes and high low bone marrow monocytes) Keywords: Diagnostic and Prognostic Biomarkers No conflicts of interests pertinent to the abstract. On univariate analysis TME variables from all the compartments showed prognostic impact on OS. BM IC levels did not statistically differ in involved vs not involved BM. Both low LN TL % and high and low BM Mo % remained independent predictors of survival on multivariate analysis. Moreover, pts with both of these unfavorable variables had a HR for death of 40.22 (CI 95% 5.16-313.04, p<0.001) with a median OS of only 22.19 m. Variables Univariate analysis Multivariate analysis HR (95% CI) P-value HR (95% CI) P-value LN T lymphocytes (TL) low 3.98 (1.31-12.06) 0.014 5.44 (1.48-20.04) 0.011 BM B lymphocytes low 4.73 (1.62-13.79) 0.004 3.36 (0.99-11,34) 0.0501 BM Monocytes (Mo) High 10.53 (3.66-30.26) <0.001 4.66 (1.34-16.25) 0.015 BM Monocytes Low 3.87 (1.28-11.7) 0.016 3.40 (1.05-10.99) 0.04 PB MLS intermediate-high 2.59 (1.12-6) 0.02 1.05 (0.4-2.77) 0.91

Bone Marrow Mastocytosis: A Diagnostic Challenge.

Bone marrow mastocytosis (BMM) represents a provisional, indolent subvariant of systemic mastocytosis (SM). Utilizing WHO criteria, BMM requires bone marrow (BM) involvement and the absence of mastocytosis skin lesions. BMM is characterized by male sex prevalence, a slight increase of serum tryptase levels, low BM mast cells (MC) burden, and an indolent clinical course. BMM shows a strong correlation with severe anaphylaxis, mainly due to an IgE-mediated allergy to bee or wasp venom and, less frequently, to unexplained (idiopathic) anaphylaxis. Furthermore, BMM is often associated with osteoporosis which could be the only presenting symptom of the disease. BMM is an undervalued disease as serum tryptase levels are not routinely measured in the presence of unexplained osteoporosis or anaphylaxis. Moreover, BMM patients are often symptom-free except for severe allergic reactions. These factors, along with typical low BM MCs infiltration, may contribute to physicians overlooking BMM diagnosis, especially in medical centers that lack appropriately sensitive diagnostic techniques. This review highlights the need for a correct diagnostic pathway to diagnose BMM in patients with suspected symptoms but lacking typical skin lesions, even in the case of normal serum tryptase levels. Early diagnosis may prevent potential life-threatening anaphylaxis or severe skeletal complications.

Bone marrow involvement and subclonal diversity impairs detection of mutated CXCR4 by diagnostic next-generation sequencing in Waldenström macroglobulinaemia.

CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinaemia (WM). Non-uniform testing for CXCR4 mutations may account for discordant findings in WM clinical trials. We compared two approaches used in these trials for detection of the most common CXCR4 (S338X) variant: targeted next-generation sequencing (NGS) using unselected bone marrow (BM) samples, and combined allele-specific polymerase chain reaction (AS-PCR) and Sanger sequencing with unselected and CD19-selected BM samples. Our findings showed that targeted NGS frequently yielded false-negative results. Both CD19 selection and AS-PCR markedly improved detection of CXCR4S338X mutations. Sensitivity was adversely impacted by low BM involvement and CXCR4 mutation clonality.

Prodromal / Pre-Leukemic Acute Leukemia of B-Lymphoblasts in 7 Adult Patients

Background: Prodromal/pre-leukemic acute leukemia is rare, mostly described in childhood B-lymphoblastic leukemia (B-ALL), with its initial presentation confused with aplastic anemia (AA). Findings reported in pediatric prodromal/pre-leukemic B-ALL include female preponderance, bone marrow (BM) fibrosis and uniform hypocellularity, and transient recovery. Reports in adults are rarer still. We conducted a retrospective review of adult patients we diagnosed with B-ALL and mixed phenotype acute leukemia with B-lymphoid and myeloid components (MPAL-B/myeloid) with prodromal/pre-leukemic manifestations. We sought to identify common clinical features, peripheral blood (PB) and BM morphology, diagnostic considerations, and clinical follow-up (whenever available) to aid in improved recognition in the adult setting. Methods: Patients with unusual presentations preceding overt acute leukemia were identified from pathology and clinical records from our institution from 2010-2020. These include (a) incidental PB blasts and partial BM-based disease, followed by spontaneous, transient regression (STR); (b) pancytopenia with rare to absent PB blasts and unexpected detection of BM-based blasts (10% or lower), compatible with aleukemic prodrome (AP) cases; (c) pancytopenia without PB blasts yet unexpected excess BM-based blasts (50% or greater) followed by marked reduction of BM-based blasts (compared to initial BM) despite absence of treatment (AP--&amp;gt;SR). BM pathology during prodromal/pre-leukemic phases were reviewed to identify key morphologic features. Results: Seven patients were identified (4 M, 3 F; age range 20-75 years), including one previously described by our group (Table 1). All were of Hispanic ethnicity. They displayed a broad range of symptoms including fatigue, fevers, throat pain, and weight loss. Initial presentations were STR in 3, AP in 2, and AP--&amp;gt;SR in 2. Although variably cellular, all BM trephine biopsies in prodromal/pre-leukemic phase had at least focal hypocellularity with stromal damage, characterized by granular to fibrillary stroma, striking cellular dropout, and multiloculated fat cells with fibrinoid appearance. Relative erythroid hyperplasia was seen in 4, while dyserythropoiesis and dysmegakaryocytopoiesis were both observed in 2 cases, raising concern for myelodysplastic syndrome (MDS). AA and MDS were, in some cases, seriously considered in the initial differential diagnosis. Because of low level BM blasts at initial presentation in most (5/7), definitive lineage designation was sometimes challenging. Furthermore, flow cytometric data was limited and IHC stained only a sparse number of immature B-cells; thus normal precursor B-cell (hematogone) hyperplasia was a strong possibility. Workup in 2 cases during low numbers of BM-based blasts or spontaneous blast reduction (cases #6 and #7, respectively) revealed T-cell clones on PCR/NGS testing. In STR and AP cases, the disease proceeded from initial presentation to overt leukemia within a fairly short interval (range 1.5 - 7.0 months; median 2.5). Final diagnosis in the majority of cases (6/7) was B-ALL, with one case of MPAL-B/myeloid. Conclusion: Herein we describe a series of 7 adult prodromal/pre-leukemic cases of B-lymphoblasts (6 B-ALL; 1 MPAL-B/myeloid) with a variety of clinical presentations. In contrast to the female-predominant pediatric prodromal B-ALLs, most of our adult cases occurred in males. Given pancytopenia, relatively low numbers, and difficulty in assigning blast lineage, diagnostic considerations initially included MDS, AA, and hematogone hyperplasia with marrow regeneration. Key morphologic features that favored a prodromal/pre-leukemic case of B-lymphoblasts over MDS or AA were profound stromal damage and expansion (albeit low numbers) of immature B-cells - findings that would be unusual in low-grade MDS and AA. Intriguingly, in 2 of our cases, T-cell clones detected on PCR testing during low blast levels may indicate an immunologic role of T-cells in transiently reducing blasts in prodromal acute leukemia, as previously suggested by Zimmermannova and colleagues (Haematol2017;102:e225-228). Regardless of whether patients experienced STR, AP, or AP--&amp;gt;SR, close clinical follow-up is advised, as progression to overt leukemia occurs within a relatively short time frame (median of 2.5 months in our series). Disclosures No relevant conflicts of interest to declare.

Azacitidine (AZA) with Nivolumab (Nivo), and AZA with Nivo + Ipilimumab (Ipi) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia: Clinical and Immune Biomarkers of Response

Azacitidine (AZA) with Nivolumab (Nivo), and AZA with Nivo + Ipilimumab (Ipi) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia: Clinical and Immune Biomarkers of Response

Bone Marrow Microenvironment Immune Cells Measured By Flow Cytometry Can Predict Survival and Identify a Subgroup in Risk of Early Relapse in Diffuse Large B Cell Lymphoma Patients

INTRODUCTION: Several studies have disclosed the predictive role of tumor microenvironment (ME) in diffuse large B cell lymphoma (DLBCL). However, there is limited data regarding the prognostic impact of immune cells (IC) in the bone marrow (BM) of DLBCL patients (pts). Regulatory molecules secreted from primary tumor sites may induce a pro-tumorigenic ME within BM, resulting in an impaired systemic immune response that could promote lymphoma survival. AIMS: -To determine the prognostic impact on survival and risk of early relapse (ER) of the % of T lymphocytes (TL), monocytes (Mo), neutrophils, NK cells and polyclonal B lymphocytes (BL) measured by flow cytometry (FC) in pre-treatment BM aspirates of DLBCL pts. METHODS: We selected pts with DLBCL and available BM aspiration FC data at the time of diagnosis who received treatment at our institution between 2012 and 2019. Clinical information was collected from medical records. FC analysis was performed with 8-color FC panels according to international Euroflow protocols. % of TL, Mo, neutrophils, NK cells and polyclonal BL by FC were compared to the normal values determined by Matarraz et al. (Cytometry part B, 2010). All parameters were analyzed as ordinal variables in 3 categories: low, normal and high. Odds ratio for ER (relapse within a year) was calculated using logistic regression. The survival analysis was estimated with Kaplan-Meier method. The comparison between variables was performed through log-rank test and multivariate analysis with Cox regression. RESULTS: A total of 119 pts were included in this retrospective study. Pts characteristics are summarized in Table 1. All pts were treated with immunochemotherapy regimens. Median PFS and OS were not reached, 75th percentile of 12.7 and 27.8 months (m), respectively; with a median follow up time of 30.7 m (range: 3.4-60). ER was documented in 25 pts. Regarding BM IC, pts with normal TL, polyclonal BL and Mo % were significantly associated with superior PFS and OS (figure 1A and table 2). No correlation between BM NK cells or neutrophils levels and outcome was observed. Moreover, BM IC levels did not statistically differ in involved vs not involved BM. In our cohort, the R-IPI was able to discriminate outcomes in poor and good-very good (G-VG) risk (median OS of 48 m vs. not reached, p &amp;lt;0.001; and median PFS of 34.8 m vs. not reached, p: 0.001, respectively). Histopathologic involved BM also predicted inferior survival (median OS of 48.3 vs not reached, p:0.037; and median PFS of 16.9 m vs not reached, p:0.028). R-IPI, BM involvement and BM IC were included in a multivariate analysis. G-VG R-IPI and low BM TL % remained independent prognostic factor of PFS on multivariate analysis with HR of 0.43 (95% CI 0.21-0.89, p: 0.023), and 3.77 (95% CI 1.8-7.92, p &amp;lt;0.001) respectively (Table 3). The odds of ER was 4 times higher in pts with low BM TL % (95% CI 1.47-10.84, p: 0.006) and 2.8 times higher in pts with low polyclonal BL (95% CI 1.07-7.58, p: 0.036). On the contrary, G-VG R-IPI showed a protective effect with an odds ratio for ER of 0.22 (95% CI 0.08-0.61, p: 0.003). On multivariate analysis both the low BM TL % [OR 5.18 (95% CI 1.45-18.53, p: 0.011)] and the R-IPI subgroup [OR 0.21 in G-VG R-IPI (95% CI 0.06-0.71, p: 0.012)] were predictive of ER. Pts with poor R-IPI were subsequently stratified according to BM TL categories. Pts with low and high TL % had a median PFS of only 9.4 vs 17 m respectively. However, it was unexpectedly not reached in the normal TL subgroup, p: 0.005 (Figure 1B). There was also a trend towards inferior OS in pts with low and high TL (median of 23.1 and 27.8 m respectively vs not reached for the normal subgroup, p: 0.09). CONCLUSIONS: Normal BM % of TL, BL and Mo in DLBCL pts measured by FC was associated with better outcomes in our cohort irrespective of BM involvement. Furthermore, concomitant low BM TL% and poor R-IPI identified a subgroup of pts with extremely poor results. These two variables presented at diagnosis might be used as prognostic factors of early relapse in DLBCL. In the future, therapies that could target the crosstalk between lymphoma cells and the BM ME might represent an encouraging strategy to improve outcomes in DLBCL. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Chemo-Resistance Is Mediated by E-selectin Receptor CD162 in Bone Marrow Niches.

The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-(endothelial)-selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance in vitro. Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.

Analysis of bone marrow supernatant neutrophil gelatinase-associated lipocalin and hematological parameters in hematological malignancy.

BackgroundNeutrophil gelatinase‐associated lipocalin (NGAL) is a urine biomarker related to acute renal injury. Whereas several studies have evaluated NGAL levels in hematological malignancy, using peripheral blood (PB). Recently, bone marrow (BM) NGAL level was reported to be higher than PB NGAL level in individuals with hematological malignancy, suggesting that BM NGAL would reflect BM microenvironment better than PB NGAL. We measured BM NGAL levels in patients with hematological malignancy, comparing those with NGAL levels in normal BM. We evaluated the association of BM NGAL with hematological parameters including neutrophil counts.MethodsBM samples were collected from 107 patients who underwent BM examination. Immunoassays were used to assess NGAL levels. Data on hematological parameters were collected from medical records. Intergroup comparisons were performed using the Kruskal‐Wallis H test and Pearson chi‐square test. Single and multiple regression analyses were performed to analyze the relationships.ResultsThe independent factors that affected the BM NGAL level were neutrophil counts and BM band neutrophil%, while neutrophil count was the main influencing factor. The acute myeloid leukemia (n = 18) and myelodysplastic syndrome (n = 25) groups showed statistically lower BM NGAL levels than patients with normal BM. The myeloproliferative neoplasm group (n = 34) showed higher BM NGAL levels than patients with normal BM, but this difference was not statistically significant. Neutrophil counts and BM band neutrophil% showed intergroup patterns similar to those of BM NGAL levels.ConclusionBM NGAL was related to neutrophil count and BM band neutrophil%, showing different levels according to hematological malignant disease entities.

Atypical infectious spondylitis with reduced bone marrow enhancement: "black vertebra sign".

A few patients suspected of having infectious spondylitis exhibited a reduced enhancement pattern on postcontrast T1-weighted (T1W) magnetic resonance imaging (MRI). To investigate the characteristics of infectious spondylitis patients who exhibited reduced vertebral enhancement. From January 2010 to November 2017, 456 patients with findings suspicious for infectious spondylitis on 706 postcontrast T1W imaging were retrospectively evaluated. When an affected vertebra exhibited markedly reduced enhancement compared to normal bone marrow (BM), the vertebra was termed a "black vertebra." MRI and computed tomography (CT) imaging findings within two-week intervals and the patients' clinical characteristics were reviewed. Ten patients (5 men, 5 women; mean age 66.4 years) whose MRI scans revealed the black vertebra sign were included. Among the 10 patients with black vertebrae, six patients exhibited signal voids in or around the affected vertebral bodies on T2-weighted (T2W) images. Eight patients showed air bubbles on CT images, suggestive of emphysematous infection. However, the typical image findings of infectious spondylitis were uncommon, namely, low BM signal intensity (SI) on T1W images (n=1) and intradiscal fluid-equivalent SI on T2W images (n=5) at an early stage. On follow-up MRI (average interval 7.2 weeks from initial MRI), available for five patients, marked progression of infection was evidenced by increased numbers of affected segments (n=3), low T1 SIs of the affected vertebrae (n=5), and increased contrast enhancement (n=3). Five patients underwent surgery to treat infections not controlled by antibiotics. A black vertebra sign on postcontrast T1W imaging seems to reflect an early emphysematous infection.

Azacitidine (AZA) with Nivolumab (Nivo), and AZA with Nivo + Ipilimumab (Ipi) in Relapsed/Refractory Acute Myeloid Leukemia: A Non-Randomized, Prospective, Phase 2 Study

Azacitidine (AZA) with Nivolumab (Nivo), and AZA with Nivo + Ipilimumab (Ipi) in Relapsed/Refractory Acute Myeloid Leukemia: A Non-Randomized, Prospective, Phase 2 Study

Therapy of Paediatric B-ALL with a Fast Off Rate CD19 CAR Leads to Enhanced Expansion and Prolonged CAR T Cell Persistence in Patients with Low Bone Marrow Tumour Burden, and Is Associated with a Favourable Toxicity Profile

Introduction: The CARPALL study (NCT02443831) employed a novel CD19CAR (CAT-41BBz CAR) with a faster off rate than the Kymriah FMC63-41BBz CAR (CAT 3.1x10-3s-1, FMC 6.8 x 10-5s-1), with equivalent on-rate (CAT 2.2 x 105, FMC 2.1 x 105). We herein report updated outcomes and CAR T cell persistence with an additional 6 months follow up from a submitted manuscript (Ghorashian et al., Nat Med, submitted) Methods: Patients aged &amp;lt;25 years with high risk, relapsed CD19+ B-ALL were eligible on this multi-centre, open label, non-randomised phase I study of autologous CAT-41BBz CAR T cells. Patients were followed to a data cut-off of 07/18/2019. CAT-41BBz CAR T cells were generated by magnetic bead activation of leucapheresed PBMCs, lentiviral transduction, followed by bioreactor expansion and magnetic bead removal prior to cryopreservation. All patients received lymphodepletion (fludarabine + cyclophosphamide) followed by 1x106/kg CAR T cells. Presence of CAR T cells in the blood and bone marrow (BM) was assessed (flow cytometry and qPCR) monthly for 6 months, then 6 weekly to 1 year and then 3 monthly. BM MRD was assessed (IgH qPCR, flow cytometry) at the same time-points up to 2 years to establish durability of responses as a stand-alone therapy. Primary end-points were incidence of grade 3-5 toxicity and the proportion of patients achieving molecular remission. Results: Of 17 patients recruited, 14 were treated due to manufacturing failure in 3 patients.The median age was 9 years (range 1-19 years). All patients had advanced ALL with a median of 4 prior therapy lines. 10 of 14 patients (71%) had relapsed post allogeneic SCT. Prior to lymphodepletion, 4 patients had &amp;gt;5% BM disease, 6 had disease between 5x10-2and 1x10-5, 4 were BM MRD negative having had recurrent isolated CNS disease. Median transduction efficiency was 31% (range 16.5 to 96.4%). 12/14 treated patients received the anticipated dose of 1x106CAR T cells/kg (2 received 0.9x106/kg). Considering all evaluable patients, (n=14 for CAR T cell persistence by qPCR, n=13 by flow) the geometric mean of Cmax was 128 912/µg DNA and of the area under the curve between D0 and D28 was 1,721,355 copies/ µg DNA (Table 1). At the point of maximal expansion, a median of 35% of circulating T cells were CAR+. Median half-life was 34 days (range 3-102). CAR T cells continued to be detectable by qPCR in 11 of 14 (79%) patients at last assessment and by flow cytometry up to 30 months post infusion in 8 of 13(61%). Median duration of CAR T persistence by flow was 261 days (range 7-917). 3 patients failed to have persistence of CAR T cells beyond 1 month. T cell mediated anti-CAR specific cytotoxic activity was detected in 2/2 evaluable patients. Updated persistence data will be presented at the meeting Cytokine release syndrome (CRS) occurred in 13 (93%, grade 1 n=9, grade 2 n=4). None developed ≥grade 3 CRS, had CRS-related ICU admission, or received Tocilizumab. CRS was associated with modest elevations of IL-6, IFN-γand IL-10. Grade 2 neurotoxicity was observed in 3 patients and resolved spontaneously. One patient had grade 4 leucoencephalopathy presumed due to chemotherapy as well as grade 5 sepsis. Ten patients (71%) had grade 3-4 cytopenia persisting beyond day 28 or recurring afterthis. 12/14 (86%) patients achieved molecular complete or continuing complete remission at a median of 30 days post infusion (range 30-90 days, Table 2). At a median follow-up of 20.3 months, 4/14 (29%) evaluable patients remain MRD negative. 5 relapsed with CD19-disease, 1 with CD19+ disease. The median duration of EFS (based on death or morphological relapse) has not been reached, 12 month EFS = 52%, OS = 70% (Figures 1, 2 and Table 3). Conclusion: We noted excellent CAR T cell expansion and persistence in a ALL cohort treated with the fast off-rate CAT-41BBz CAR despite their lower BM disease at treatment compared to other studies. The kinetics documented for all evaluable patients showed a 5-fold greater CAR T cell expansion and 2-fold longer half-life than responders in published series utilising tisagenlecleucel in a similar ALL cohort (Mueller et al., Blood 2017). Patients had a favourable toxicity profile with no severe (grade 3-4) CRS and equivalent disease outcomes to the ELIANA study despite having similarly advanced disease (Maude et al., NEJM 2018292). These data suggest long lived CAR T cell persistence supports stand-alone therapy for ALL with durable responses. Disclosures Ghorashian: Celgene: Honoraria; novartis: Honoraria; UCLB: Patents &amp; Royalties: UCLB. Kramer:UCLB: Patents &amp; Royalties. Ciocarlie:Servier: Other: Financial Support. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Pule:Autolus: Employment, Equity Ownership, Patents &amp; Royalties. Amrolia:UCLB: Patents &amp; Royalties.

Functional Interrogation of Variants of Undetermined Significance of the Isocitrate Dehydrogenase 1 and 2 Genes in Myeloid Neoplasms

Introduction: Isocitrate dehydrogenase 1 and 2 (IDH1/2) are metabolic enzymes in the citric acid cycle, producing alpha-ketoglutarate (αKG). Mutations in specific regions of these genes have been characterized in gliomas, AML and chronic myeloid neoplasms such as MDS and MPN. These mutations produce an oncometabolite 2-hydroxyglutarate (2HG) that disrupts the epigenetic phenotype of myeloid cells promoting oncogenesis. While this process has been characterized for arginine hot spot (R132/R140) mutations, there exist variants of unclear significance (IDHVUS), with potential pathogenic predictions using in silico assessment models. With the availability of mutant IDH inhibitors in practice, it has become important to characterize these VUS. We carried out this study to functionally interrogate potentially pathogenic IDHVUS in MDS and MPN. Methods: After institutional approval, bone marrow (BM) DNA of patients with MDS and MPN, including overlap syndromes were subjected to next generation sequencing (29-gene, including the entire coding region of IDH1/2) by previously described methods (Patnaik et al., BCJ 2016). A sub-set of patients with available plasma [IDH wild type (IDHWT), bona fide IDH mutations (IDHMT) and IDHVUS with predicted pathogenicity (≤0.01% minor allele frequency, and predicted damaging in silico scores)] were submitted for mass spectrometry (LC-MS) based detection of IDH-related metabolites; D and L isomers of 2HG, αKG, and total 2HG. The impact of variations on protein secondary structure was assessed by the GorIV prediction algorithm. Potential alterations within the tertiary structure of the proteins was evaluated by in silico mutagenesis. Results: One hundred and seventeen patients were included in the study; median age 68 years (range, 39-94), 68% male. Of these, 40 (34%) had MDS, 42 (36%) MPN, and 35 (30%) MDS/MPN overlap syndromes. Eighty six (74%) had IDHMT, including n=28 (24%) with IDH1MT (R132C/G/H/S) (11 MDS, 8 MPN, 9 MDS/MPN) and n=59 (50%) with IDH2MT (R140G/Q/W) (22 MDS, 20 MPN, 17 MDS/MPN). IDHVUS were detected in 32 (27%) patients, including 1 IDH1MT patient, and involved IDH1 in 14 (12%) cases and IDH2 in 18 (15%) cases. All IDHVUS were missense, except for a 2 amino acid insertion in IDH2, with the most frequent VUS being IDH1Y183C (7%) and IDH2T352P (7%). While there were no phenotypic differences between IDH1/2MUT and IDH1/2VUS, in comparison to patients with IDH1/2MT, those with IDH1VUS were more likely to have higher WBC counts (median 8.1 vs 3.6 x109/L, p=0.0322), higher neutrophil counts (median 3.3 vs 1.3 x109/L, p=0.0203), and lower BM blast % (median 1 vs 3%, p=0.02061). Compared to IDH1/2MT patients, those with sole IDH1/2VUS more frequently had mutations in SF3B1 (p=0.0167), while they were associated with a lower frequency of SRSF2 mutations (p=0.0002). Interestingly, TET2 mutations were detected in only 5% of patients with IDHMT and 16% of IDHVUS patients, although without reaching statistical significance. LC-MS analysis on 75 plasma samples (n=43 IDHMT, n=9 IDHVUS, n=20 IDHWT, 3 normal) revealed a significant increase in total 2HG levels in IDHMT vs IDHVUS (p=0.0089), and IDHWT (p=0.0020) cases (Figure 2A-D). While there were no significant differences in αKG levels between the groups, the D-2HG enantiomer levels were higher in IDHMT samples vs IDHVUS (p=0.0199) and IDHWT (p=0.0055). On examination of the crystal structures of the IDH genes using PDB, while we observed a direct interaction between R132/R140/R172 and the catalytic core, the IDHVUS were located away from this active center and were hence thought to be allosteric in nature (Figure 2F). These VUS however, did demonstrate predictive steric clashes in the tertiary structure context of IDH1 using the PyMol artificial mutagenesis tool. The absence of wild-type crystal structure for IDH2precluded a similar analysis for IDH2. Median overall survival (OS) of the entire cohort (Figure 1) was 36 (range, 0-190) months, with OS being inferior in IDH1/2MT patients (34 months) vs those with IDH1/2VUS (50 months, p=0.0255). Conclusion: Our study demonstrates that in spite of predictive pathogenicity of IDH1/2VUS using in silico predictive tools and crystal studies, the lack of involvement of the arginine hot spot precludes IDH neomorphic activity, as shown by normal 2HG levels. In addition, in comparison to IDHMT patients, those with IDHVUS had fewer BM blasts and a better survival. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Microvesicles expressing adenosinergic ectoenzymes and their potential role in modulating bone marrow infiltration by neuroblastoma cells

ABSTRACTMetastatic diffusion of Neuroblastoma (NB) cells in the bone marrow (BM) represents the most negative prognostic factors for NB patients. Multiple immune escape mechanisms are postulated as responsible. Our working hypothesis is that adenosine (ADO), an immunosuppressive molecule along with the ectoenzymatic pathways (CD39-CD73 and CD38-CD203a/PC-1-CD73) controlling its production, are involved in the dynamics of NB cells in the BM. The results indicate that ectonucleotidases are expressed by i) NB cell lines, ii) metastatic NB cells isolated from NB patients’ BM, iii) microvesicles (MV) derived from both NB cell types and iv) resident BM cell populations. BM infiltration by NB cells increased CD203a/PC-1 and CD73 expression on lymphoid and myeloid cells, respectively. Expressions of ectoenzymes and GD2 (NB-associated marker) were higher on MV from NB patients’ BM than in controls. Moreover, CD203a/PC-1 expression on BM-derived MV provide a basis for distinguishing NB patients with high or low BM infiltration. ADO production and consumption of related by-products were significantly higher when assessed on NB patients’ MV than those from controls. MV isolated from NB patients’ BM significantly downregulated in vitro T cell proliferation. Lastly, NB patients with worse prognosis are identified by a high percentage of CD38+ or CD73+ MV in the BM. In conclusion, ectonucleotidases are present and functional on NB cells, as well as in NB-infiltrated BM and in MV derived from BM. It is reasonable that MV are involved in BM infiltration by NB cells. Therefore, targeting these molecules may widen the therapeutic armamentarium for metastatic NB patients.

Molecular Characterization By Immune Profiling of Paired Blood and Bone Marrow in Multiple Myeloma and Its Precursor States

INTRODUCTION: Progression from precursor states, MGUS and smoldering multiple myeloma (SMM), to multiple myeloma (MM) is dependent upon adaptive and innate immune contexture shaped by cross-talk between malignant plasma cells and bone marrow (BM) milieu. The complexity and heterogeneity of interactions between the immune system and plasma cells in BM triggers alterations in peripheral blood (PB) immune cell subsets. The advantage of using PB as a surrogate is that dynamic changes in the immune cells can be measured at various time points during disease progression or therapeutic intervention. Here, we performed a comprehensive analysis of immune repertoire to identify immune signatures in PB and BM associated with MM or its precursor states. We also performed T cell receptor (TCR) clonotyping to quantify clonal expansion specific to each immunotype.METHODS: Paired PB and BM specimens were collected from patients with MGUS/SMM (n=12) and MM (n=16) through an IRB-approved biospecimen protocol. PB mononuclear cells and BM mononuclear cells were isolated for immune profiling. A total of 59 immune variables were analyzed by flow cytometry surveying 6 cell lineages' [NK, NK-T, Th, CTL, Treg and ɣδ T cells] distribution and functional status [activation, differentiation and anergy]. In addition, ArcherDx Immunoverse TCR αδ-βɣ CDR3 targeted NGS assay was performed to study clonal distributions of Vα24Jα18 NK-T, βα and ɣδ T cell. Univariate analyses (ANOVA) were performed using p<0.15 cutoff. Each set of variables (PB or BM) was then validated by multivariate analyses (Wilk's lambda) and used for unsupervised hierarchical analysis by WPGMA methods. Innate (NK-T, ɣδ T) and adaptive (βα T) mobilization for each cluster were finally confirmed by calculating Shannon's TCR clonal diversity index (SI).RESULTS: PB immunotyping identified 1 marker of innate inflammation and 9 markers of adaptive T mobilization that differentiated precursor states and MM (p=0.005). This model generated 3 PB immune clusters (Figure): cluster #1 [8 precursor states, 1 MM] showed a lack of innate inflammation and low Th/CTL mobilization, cluster #2 [2MGUS, 5MM] showed low innate inflammation and, cluster #3 [2SMM, 10MM] showed strong innate inflammation (Vɣ9-Vδ2-NKG2D+), Th terminal differentiation (central memory phenotype) and CTL anergy (Tim3+). TCR clonotyping confirmed increased innate inflammation (TCRδ SI 3.99±0.3 vs 4.75±0.15, p<0.05) and T cell mobilization (TCRα SI 7.12±0.3 vs. 8.20± 0.2, p<0.05) in PB cluster #3 compared with PB cluster #1.BM immunotyping identified 3 markers of innate inflammation and 2 markers of adaptive T mobilization (p=0.0274) distinguishing precursor states from MM. This model generated 3 BM immune clusters: cluster #1 [6 precursor states, 6 MM] showed innate inflammation (ɣδ T) and CTL terminal differentiation (central memory phenotype); cluster #2 [4 SMM, 8 MM] showed innate inflammation (NK-T, ɣδ T) and CTL effector anergy; and cluster #3 [2 MGUS, 2 MM] showed low NK cell cytotoxicity (KIR3DL1+) and CTL terminal differentiation. TCR clonotyping confirmed qualitative differences in innate inflammation between BM cluster #1 and #2 with higher NK-T (%Vα24Jα18 p<0.01) but lower ɣδ T (TCRδ SI 3.36±0.2 vs 4.57±0.2, p<0.05). In addition, CTL mobilization whether resulting in terminal differentiation or anergy in BM cluster #1 and #2, respectively was associated with similar clonal expansion of T cells (TCRα SI 7.21±0.26 vs. 7.87± 0.4, ns).Comparisons showed associations between PB and BM ɣδ T cell involvement in 13/13 patients. High PB Th/CTL mobilization (terminal differentiation) was associated with high T cell anergy in BM in 9/12 patients; conversely low PB Th/CTL mobilization was associated with low BM T cell involvement in 6/7 patients.CONCLUSION: This pilot study shows immune clustering of MGUS, SMM and MM patients based on BM and PB immunotypes. This is the first study to demonstrate two very distinct MM immunotypes based on low vs. high inflammatory states. We also show a high correlation between innate immune inflammation status in both PB and BM, specifically pertaining to ɣδ T cell, conventional T cell mobilization or lack thereof. Additional studies including a larger cohort for validation and longer follow up to establish correlation with clinical outcomes are currently underway. [Display omitted] DisclosuresFoureau:Teneobio Inc.: Research Funding. Berlin:ArcherDx: Employment. Johnson:ArcherDx: Employment. Williams:ArcherDx: Employment. Voorhees:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Other: served on an IRC; Amgen Inc.: Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: served on an IRC; TeneoBio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Usmani:Amgen, BMS, Celgene, Janssen, Merck, Pharmacyclics,Sanofi, Seattle Genetics, Takeda: Research Funding; Abbvie, Amgen, Celgene, Genmab, Merck, MundiPharma, Janssen, Seattle Genetics: Consultancy.

Association between Immunoglobulin Isotypes and Cytogenetic Risk Groups in Multiple Myeloma

Introduction:Cytogenetic abnormalities (CAs) in multiple myeloma (MM) have prognostic significance and predict the risk of clonal evolution, disease progression, and response to treatment. The Revised International Scoring System (R-ISS) was recently updated to incorporate high risk CAs, including del(17p), t(14:16), and t(14:20), the presence of which confer a poorer prognosis. Prior studies have demonstrated the prognostic value of specific CAs and MM immunoglobulin isotypes, for example, non-IgG isotypes having been associated with poorer prognosis. However, data showing a link between cytogenetic risk groups and MM isotypes are limited. Thus, we determined whether a relationship exists between higher-risk CAs and MM isotypes, as well as the degree of malignant plasma cell infiltration of the bone marrow (BM).Methods:We performed a retrospective analysis of a multi-institutional commercial pathology repository of 442 MM patients identified according to the International Myeloma Working Group criteria that included assessment of BM infiltration (BM%), MM isotype, CA, and cytogenetic risk group. The study protocol was approved in accordance with IRB standards. As per the R-ISS, the high risk CAs included del(17p), t(14:16), and t(14:20), intermediate risk CAs included t(4;14), del(13q), dup(1q), and hypodiploidy, and standard risk CAs included t(6;14), t(11;14), and hyperdiploidy. Patients with CAs that included more than one risk group were considered to be in the higher risk category. Associations between categorical variables were made using the chi-squared test or Fisher's exact test. The Mann-Whitney test was used to compare groups (MM isoytpe, CAs, or cytogenetic risk group). Statistical significance was considered at p<0.05. Analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC).Results:Among the 442 MM patients, isotype groups included 42% IgG, 16% IgA and 42% light chain-only (LCO). There were 184 patients (42%) who had CAs associated with known MM cytogenetic risk groups, of which 34% were standard-risk, 61% were intermediate-risk, and 5% were high-risk. The median BM% of clonal plasma cells was 50% (range 1-95%). When examining the relationship between any CA and BM%, del(13q14), dup(1p32), dup(1q21), t(4;14), trisomy(11q13), and monosomy(16q23) were associated with a significantly higher BM% than patients who were lacking those respective CAs. For example, the median BM% for patients with t(4;14) was 60% compared with 15% in patients lacking this translocation (p<0.0001). LCO isotype was associated with a lower BM% (p<0.04), however there was no significant correlation between IgG/IgA isotypes and BM%. There was a higher median BM% seen in the intermediate risk group (50%, p<0.0001) compared to standard risk group (20%, p<0.04). The high risk group was not significantly associated with BM%. When comparing isotypes to cytogenetic risk groups, IgA isotype was significantly associated with intermediate risk cytogenetics (p<0.03). Conversely, there was a significantly lower rate of standard risk CAs among IgA isotypes (p<0.01). There was no significant correlation between IgG/LCO and cytogenetic risk. IgA isotype correlated with specific CAs, including del(13q) (p<0.02) and del(16q23) (p<0.04). There was a higher rate of t(11:14) in the non-IgA isotype groups. Additionally, IgG isotype was associated with a trisomy(11q13) (p<0.03). LCO isotype also corresponded with a higher rate of del(17p) (p<0.03), t(11:14) (p<0.03), and a lower rate of t(4:14) (p<0.04) and trisomy (11q13) (p<0.01).Conclusions:In MM, CAs are valuable in risk stratifying patients and predicting treatment responses. In this study, data suggest that the IgA isotype is significantly associated with intermediate-risk cytogenetics, including del(13q) and hypodiploidy, and LCO disease correlates with high risk cytogenetics, including del(17p). When taken together this may explain the previously known association of these isotypes with poorer prognosis, and suggests divergent clonal evolution among MM isotypes. Associations between clinical parameters and disease responses remain ongoing. DisclosuresNo relevant conflicts of interest to declare.

Deregulated Expression of Long Noncoding RNAs H19, LEF1-AS1, TCL6, and WT1-AS1 Predicts Poor Outcome of Patients with Myelodysplastic Syndromes

INTRODUCTIONMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by dysplastic and ineffective blood cell production and a tendency for transformation to acute myeloid leukemia (AML). Because of the heterogeneity of MDS, the development of additional molecular tools able to refine the Revised International Prognosis Scoring System (IPSS-R), predict outcome and monitor the response to treatment would be highly beneficial.Long noncoding RNAs (lncRNAs) are protein-noncoding RNAs that play multiple roles in hematopoietic differentiation. They contribute to the pathogenesis of hematologic malignancies, representing a new class of potential biomarkers and therapeutic targets. AIMSIn this study, we aimed to identify lncRNAs with deregulated expression in MDS that could serve as novel potential molecular markers of poor outcome. METHODSThe discovery cohort included 54 MDS patients, 14 AML patients with myelodysplasia-related changes (AML-MRC), and 9 controls. Agilent microarrays were used to profile lncRNAs expression in CD34+ bone marrow (BM) cells. Data analyses were focused on the identification of lncRNAs with altered levels between MDS patients (i) with (very) low vs. (very) high risk IPSS-R, and (ii) with progression-free survival (PFS) shorter vs. longer than 2 years. RT-qPCR was applied to validate expression levels of selected deregulated lncRNAs (H19, TCL6, LEF1-AS1, and WT1-AS) and levels of their protein-coding counterparts (LEF1 and WT1) in an independent validation cohort (79 MDS and 14 AML-MRC patients, and 13 controls). Further, we examined whether the expression of H19, TCL6, LEF1, and WT1 changed in patients treated with azacitidine (AZA). For this purpose, we retrieved data from our previous microarray analysis (GEO GSE77750) performed on a cohort of 19 MDS, 3 chronic myelomonocytic leukemia (CMML), 10 AML-MRC patients and 10 controls. RESULTSIn the MDS patients with higher risk IPSS-R, 23 lncRNAs were downregulated (e.g., CXADRP3, LEF1-AS1, PCED1B-AS1, SLC8A-AS1, and TCL6) and 35 lncRNAs were upregulated (e.g., BCAR4, FAM225A, FARP1-AS1, H19, and RBPMS-AS1) vs. lower risk IPSS-R (|logFC| ≥ 1, p ≤ 0.01). Furthermore, we identified significant upregulation of e.g. H19, SATB2-AS1, and WT1-AS in patients with PFS < 2 years vs. those with longer survival.For subsequent study, we selected H19, LEF1-AS1, TCL6, and WT1-AS lncRNAs based on their known association with hematopoiesis. In the validation cohort, we proved that downregulation of LEF1-AS1 and TCL6 and upregulation of H19 and WT1-AS were associated with poor PFS and OS (p < 0.01, Figure 1). Further, we correlated lncRNA expression levels with multiple clinical parameters (sex, age, BM blasts, hemoglobin, neutrophils, platelets, and IPSS-R-based cytogenetics) and found out that only level of H19 was independent from all these variables whereas expression of other lncRNAs was associated with BM blast count. Multivariate analysis confirmed that H19 level, BM blasts, and platelets remained independent factors for OS and PFS in MDS (p < 0.05). Importantly, WT1-AS level, platelets, and hemoglobin were identified as independent variables for survival of MDS patients with BM blast counts < 5 % (p < 0.05). Additionally, we measured levels of appropriate protein-coding counterparts of LEF1-AS1 (i.e. LEF1) and WT1-AS (i.e. WT1) and found strong positive correlation between these pairs (p < 0.001), indicating their co-transcriptional regulation.Because expression of the studied lncRNAs significantly varied between lower and higher risk MDS patients, we further investigated impact of AZA treatment on the expression of H19, TCL6, LEF1, and WT1 in higher risk disease. However, we detected neither any difference in their expression between responders and nonresponders nor any significant return to normal expression levels caused by AZA therapy. CONLUSIONSOur data proved that particular lncRNAs are specifically deregulated in higher risk MDS. Altered transcript levels of these lncRNAs may serve as independent markers of poor survival, even in the patients with low BM blast counts, providing additional information about prognosis of MDS patients beyond the currently used scoring system.The work was supported by grants 17-31398A, 16-33617A, 16-33485A, and 00023736 from the Ministry of Health of the Czech Republic. DisclosuresNo relevant conflicts of interest to declare.

The plasma lipidome in acute myeloid leukemia at diagnosis in relation to clinical disease features

BackgroundEarly studies established that certain lipids were lower in acute myeloid leukemia (AML) cells than normal leukocytes. Because lipids are now known to play an important role in cell signaling and regulation of homeostasis, and are often perturbed in malignancies, we undertook a comprehensive lipidomic survey of plasma from AML patients at time of diagnosis and also healthy blood donors. MethodsPlasma lipid profiles were measured using three mass spectrometry platforms in 20 AML patients and 20 healthy blood donors. Data were collected on total cholesterol and fatty acids, fatty acid amides, glycerolipids, phospholipids, sphingolipids, cholesterol esters, coenzyme Q10 and eicosanoids. ResultsWe observed a depletion of plasma total fatty acids and cholesterol, but an increase in certain free fatty acids with the observed decline in sphingolipids, phosphocholines, triglycerides and cholesterol esters probably driven by enhanced fatty acid oxidation in AML cells. Arachidonic acid and precursors were elevated in AML, particularly in patients with high bone marrow (BM) or peripheral blasts and unfavorable prognostic risk. PGF2α was also elevated, in patients with low BM or peripheral blasts and with a favorable prognostic risk. A broad panoply of lipid classes is altered in AML plasma, pointing to disturbances of several lipid metabolic interconversions, in particular in relation to blast cell counts and prognostic risk. ConclusionsThese data indicate potential roles played by lipids in AML heterogeneity and disease outcome. General significanceEnhanced catabolism of several lipid classes increases prognostic risk while plasma PGF2α may be a marker for reduced prognostic risk in AML.

Safety and Efficacy Evaluation of 4SCAR19 Chimeric Antigen Receptor-Modified T Cells Targeting B Cell Acute Lymphoblastic Leukemia - Three-Year Follow-up of a Multicenter Phase I/II Study

Background: CD19 chimeric antigen receptor (CAR)-modified T cell therapy has demonstrated clinical efficacy but often associated with severe adverse effects manifested by cytokine release syndrome (CRS). To increase safety and efficacy of CAR T therapy, a 4thgeneration CAR design has been developed and investigated in a multi-center trial in China.Patients and Methods: From July 2013 to July 2016, the 4SCAR19 phase I/II multi-center trial has enrolled 125 patients (pts) with chemo-resistant, CD19-positive, acute B cell lymphoblastic leukemia (B-ALL) eligible for CAR T cell preparation and infusion. Laboratory data and clinical records were carefully evaluated and 102 pts were qualified for statistical evaluation, including 55 children and 47 adults; 27 had received allo-HSCT prior to CAR T therapy. The median age is 9 (2 to 17) and 37 (19 to 70) for pediatric and adult pts, respectively. The median leukemia blast count in the bone marrow (BM) is 14.5%, with BM blast >50% accounting for nearly one third (33 pts). Autologous/donor T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentivector CAR containing four intracellular signaling domains: CD19-scFv//CD28/CD137/CD27/CD3ζ-iCasp9 (4SCAR19). Pts received conditioning regimens of cyclophosphamide (17), cyclophosphamide/fludarabine (54), other chemotherapy (29) or none (2), followed by CAR-T cell infusion (average 1.05x106cells/kg). The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR T infusion dose were quantitatively monitored. Statistical analysis used COX proportional hazard model involving categorical or continuous covariates, univariates, or multivariates analyses, and survival analysis was based on right-censored data and Kaplan-Meier estimation (KM curve).Results: The compiled data indicate that the quality of CAR T cells positively correlated with overall survival (OS). The median follow-up time was 7 months (range from 1~35 months). Patient (Pt) cohort 1 (<50% BM blast count, 69 pts) and cohort 2 (≥50% BM blast count, 33 pts) achieved complete response (CR) at 91.3% and 75.8%, respectively. The median OS time of cohort 1 and cohort 2 are 485 days (CI: [387, NA] days) and 317 days (CI: [135, NA]), respectively (P=0.03). The average 4SCAR19 lentivector transduction efficiency was 37.3%. While the infusion dose of CAR T cells positively correlated with OS in pediatric pts (p=0.041), it lacked significant correlation in adults (p=0.95), suggesting that other factors rather than CAR T infusion dose play an important role in CAR T therapy in adults. When pts were analyzed based on low (< 5%) versus high (> 5%) BM blasts, the CRS grade showed no significant correlation with disease burden (P = 0.45 for low burden group, and P = 0.06 for high burden group). Of note that total 73 of the 102 pts experienced 0-1 grade CRS and 8 of them had very high BM leukemia load (>80%), suggesting a very low toxicity of the 4SCAR19 T cells. In addition, of the 17 high (> 80%) BM blast pts, only 3 experienced grade 3-4 CRS. For 38 pts with BM blast ≥ 50%, most had grade 1 (30) or grade 2 (13) CRS, and only 5 pts had grade 3, and 3 pts had grade 4 CRS. For low burden pts (0-5% BM blasts), 86% (42 pts) developed low grade CRS (0 or 1), and even pts with BM blasts above 5%, 53% experienced low grade CRS (0 or 1). Further analysis of inflammatory genetic profile reveals that high CRS might correlate with high inflammatory profile, as several pts with high inflammatory gene patterns, while only had residual disease or no detectable leukemia cells (BM blasts 0-0.005%), developed grade 3-4 CRS.Conclusion: The three-year follow-up of the 4SCAR19 T cell therapy further supports that CAR T immunotherapy could benefit not only low leukemia burden pts, but also late-stage, chemo-resistant, very high-burden leukemia pts. Importantly, our study demonstrates a good safety profile of the 4SCAR19 T cells even under high disease burden. While the multicenter trial involves 22 clinical centers, the variable clinical settings do not seem to impact patient outcomes due to the highly standardized CAR T cell preparation protocol and manageable CRS in most. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow (BM) Hematopoietic Dysfunction in Chronic Lymphocytic Leukemia (CLL) - Association with Leukemic Burden and Reversibility with Therapeutic Responses.

Introduction: CLL is the most prevalent leukemia in the United States (≥120,000 cases) with approximately 5000 deaths per year. The dominant cause of mortality in CLL patients is serious infection. Indeed, about 80% of CLL patients will sustain infectious complications at some point during their disease course and infection risk increases with length of the disease. Steady-state hematopoiesis in BM is required to sustain a peripheral repertoire of blood cells and maintain immune competence. There have been few studies evaluating the hematopoietic capacity of CLL BM and how it may relate to immune competence. Thus, the goal of this study was to determine the status of BM hematopoiesis in untreated and treated CLL patients.Methods and findings: The hematopoietic stem (HSC) and progenitor cell pool (HSPC) in human BM is defined by expression of CD34 and lack of surface markers (Lin-) expressed on their mature progeny. Frequencies of Lin- CD34+ progenitors in BM between age matched healthy subjects (HS) (n=22) and untreated CLL patients (n=10) were compared. We note that the Lin+ cocktail used included an antibody to CD19, eliminating dilution of CD34+ HSPC frequency due to variation in leukemic B cell infiltration. CD34+ HSPCs were significantly reduced in untreated CLL patients (6.33±3.01% vs. 2.04±2.33% in HS vs. CLL, p=0.0004). Next we evaluated total CFU responding cells within the residual CD34+ cells to determine if they were functionally compromised. Numbers of total CFU-responsive CD34+ progenitors (CFU-GM, CFU-E, and CFU-GEMM) were reduced when assayed from purified CLL patient CD34+ cells compared to HS. Thus, while HSPC are present in CLL patient BM, they are functionally compromised even after removal from the tumor microenvironment. To determine if reductions in CFUs were accompanied by decreased myeloid and erythroid lineage differentiated progeny, we evaluated frequencies and absolute numbers of CD14+ monocytes and CD71+ erythroid progenitors by flow cytometry and found both were severely reduced in CLL patient BM. To determine if the reductions in myeloid/erythroid lineage cells was due to deficiencies in their precursor HSPCs, we evaluated frequencies and absolute numbers of HSC, multipotent progenitors (MPP), and common myeloid progenitors (CMP). Proportions, but not absolute numbers, of HSC, MPP, and CMP were significantly reduced in CLL patient BM. Frequencies of CD14+ monocytes and CD71+ erythroid precursors were also significantly reduced in patients with low BM tumor burden (designated as 20-40% BM involvement), suggesting that the hematopoietic dysfunction is not solely due to tumor burden. Comparative analysis of frequencies of CD14+ monocytes in BM to frequencies of monocytes in peripheral blood revealed a strong association, suggesting that for some immune cell types the blood immune repertoire may be reflective of BM dysfunction. In addition to BM evaluation of untreated CLL patients, we also studied a cohort of post-therapy patients (n=23). Not surprisingly, we found considerable heterogeneity in the extent of BM hematopoietic reconstitution. Nevertheless, we observed robust BM hematopoiesis post therapy in a subset of treated patients (n=9 of 23).Summary: Our findings reveal BM hematopoietic dysfunction (HD) in CLL not solely due to the degree of tumor burden. Importantly, the BM HD is reversible by effective therapies. The HD is manifested by reduced numbers and function of HSPCs and production of their mature progeny. Of significance is the severe reduction in monocytic and granulocytic cells found in CLL BM. This is an important finding as these two innate cell types provide critical first responder protection against invading pathogens. Our study also uncovered that levels of blood monocytes can mirror their diminished production in BM. Future studies will focus on determining the mechanism(s) that reduce the ability of CLL BM to sustain immune cell production in addition to determining if restoration of hematopoiesis is a necessary outcome for more effective therapies. DisclosuresDing:Merck: Research Funding. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Mitochondria Transfer from Hematopoietic Stem and Progenitor Cells to Pdgfrα+/Sca-1-/CD48dim BM Stromal Cells Via CX43 Gap Junctions and AMPK Signaling Inversely Regulate ROS Generation in Both Cell Populations

Modulation of reactive oxygen species (ROS) levels in hematopoietic stem cells (HSC) is crucial to control HSC quiescence and blood formation. High ROS levels are required for leukocyte formation while low ROS levels are essential to maintain HSC quiescence. However, regulation of ROS content in HSC is poorly understood. Adhesion interactions between HSC and their bone marrow (BM) stromal cells (BMSC) via CXCL12/CXCR4 maintain HSC in a quiescence non-motile state, protecting them from 5-FU chemotherapy insult (Sugiyama, Immunity, 2006). Surface CXCL12 expression by BMSC is dependent on connexin-43 (Cx43) gap junctions mediated cell contact (Schajnovitz, Nat. Immunol., 2011) and BM hematopoietic stem and progenitor cells (HSPC) survive and eliminate excess ROS levels post 5-FU chemotherapy treatment, by transferring ROS to BMSC in a Cx43 dependent manner (Taniguchi, PNAS 2012).Here, we report that ROS content of BM HSPC inversely correlates with ROS levels in adjacent BMSC. Administration of the pro inflammatory cytokine G-CSF results in decreased HSPC Cx43 expression, elevated ROS levels and increased glucose uptake. Conversely, in the BM stromal microenvironment, G-CSF administration generated lower ROS level and reduced glucose uptake. Up-regulation of BM Sphingosine 1-Phosphate (S1P), a downstream target of G-CSF required for ROS production in HSPC, reduced stromal ROS content and proliferation. Accordingly, mice with reduced BM S1P levels (S1Plow) have lower BM content of HSPC, accompanied by reduced ROS, glucose uptake and lactate production in these cells. More importantly, BM from S1Plow mice has a 3 fold increased frequency of primitive ROSlow/ EPCR+ long-term repopulating cells, as evident by immunophenotypic analysis and long-term competitive repopulation assays. Concomitantly, S1Plow mice have increased content of BMSC with higher ROS levels and glucose uptake, leading to higher BM content of colony-forming unit fibroblasts. Our results reveal a dynamic and inverse metabolic relationship between BM HSC and the stroma microenvironment. We hypothesized that the opposite metabolic state of HSPC and BMSC is due to mitochondrial transfer between the two populations. Therefore, we created chimeric mice by transplanting mitochondria labeled GFP (mito-GFP) HSPC to wild type (WT) mice and detected 88% of the host BMSC to contain donor-derived mitochondria, indicating the existence of mitochondria transfer from hematopoietic cells to BMSC in vivo. This transfer is bidirectional, albeit at a lesser degree, as determined in reverse chimeric mice where up to 26% of the donor-derived HSPCs acquired recipient mitochondria. Mitochondrial transfer can be recapitulated also in vitro in an overnight co-culture system of mito-GFP HSPC and primary BMSC, resulting in mitochondrial transfer and increased ROS content in a subpopulation of osteogenic BM PDGFRα+/ Sca-1-/CD48dim stromal cells. Mitochondrial transfer is cell contact dependent and mediated by Cx43 gap junctions. In vitro co-culture of mito-GFPHSPC from Cx43 deficient (KO) mice with WT or Cx43 KO BMSC reduced 50% mitochondrial transfer to PDGFRα+/Sca-1-/CD48dim stromal cells. Contrarily, the mitochondrial transfer from WT HSPC to Cx43 KO stromal cells was not affected, revealing that Cx43 expression on HSPC, but not on BM stromal cells, is specifically required for mitochondrial transfer. Interestingly, in vitro inhibition of AMP-activated protein kinase (AMPK), a crucial metabolic regulator, dramatically increased mitochondrial transfer from HSPC to BMSC. Administration of the AMPK inhibitor BML in vivo increased ROS content of PDGFRα+/Sca-1- BMSC while decreasing it in HSPC, further suggesting that AMPK inhibition regulates mitochondrial transfer and ROS production. Our results imply that mitochondria are scavenged by the BM osteogenic microenvironment to prevent excessive ROS levels in the HSC pool and in parallel to activate bone formation. Altogether, we have discovered a dynamic, inverse metabolic state between BM HSPC and their supporting stromal microenvironment during quiescence, proliferationand differentiation of these two populations. Thus, blood cell production and bone generation take place at the expense of the other. This metabolic seesaw is mediated by mitochondrial transfer from HSPC to osteogenic BM stroma in a HSPC Cx43 gap-junction dependent manner and regulated through AMPK signaling. DisclosuresNo relevant conflicts of interest to declare.