Myeloid Antigens Research Articles

Prognostic value of myeloid antigens expression in childhood acute lymphoblastic leukemia

Background: Leukemic blasts in acute lymphoblastic leukemia (ALL) may have immunological features of both lymphoid and myeloid lineages known as aberrant myeloid antigens expression in ALL, which are explained as abnormal genetic program of leukemic cells that lead to lineage infidelity. In this study, CD13, CD14, and CD33 (which are the most frequent myeloid antigens associated with aberrant antigens expression in ALL) were investigated. Objectives: To evaluate the occurrence of aberrant myeloid antigens expression in childhood ALL and its effects on complete remission and other parameters. Materials and Methods: This study was conducted on 31 pediatric patients with newly diagnosed de novo ALL (27 B-ALL, 4 T-ALL) in Children Welfare Teaching Hospital/Medical City of Baghdad; diagnosis of ALL based on morphology and cytochemistry, CD13, CD14, CD33 were investigated as myeloid antigens using four-color flow cytometry. Results: Five cases of ALL (16.13%) out of 31 cases were confirmed to have aberrant myeloid antigens expression (CD13 was expressed in all five cases, CD33 was expressed in three cases, and CD14 was not expressed in any of these five cases). Complete remission was achieved in 90.30% (28 patients) and all cases with aberrant myeloid antigens expression achieved complete remission; however, despite this, there was no significant difference in complete remission between myeloid-positive (MY+) and myeloid-negative (MY−) cases, P > 0.05. Regarding other parameters, there were significant statistical differences in lactate dehydrogenase (LDH), hemoglobin value, and bone marrow (B.M.) blasts percent at diagnosis between MY+ and MY− cases, P 0.05. Conclusions: The most frequent aberrant myeloid antigens expression in childhood ALL is CD13 and less frequently is CD33 while CD14 showed no expression; myeloid antigens expression in ALL may have better prognosis as they have lower B.M. blast percent and LDH value.

Immunophenotypic Profile in Adult Patients with Acute Leukemia Association with Clinical Feature: Fluorescence Cytometry Quantitative Analysis

Antigen surface markers represent as the new prognostic tool for detection of acute leukemia. To investigate the prevalence expression of lymphoid and myeloid antigen lineage in acute leukemias. This study included 100 acute leukemias patients. Specimens were selected from consecutive patients who had sufficient material available. Among the 100 patients in which a detailed history, hematological, clinical and immunophenotyping analysis were performed. This study was showed distribution of immunophenotyping characters between studied AML and ALL cases. The most abundant immunophenotyping features in acute myeloid leukemia were cMPO, CD33, CD117, CD13, CD14 and CD64, while the most abundant immunophenotyping features in acute lymphoblastic leukemia were CD19, CD79a, TdT, CD20, CD10 and CD34. cMPO which act as independent prognostic factor for AML, CD10 and TdT can used as independent prognostic factor for differentiate between ALL and AML.

Clusters of Childhood Mixed Phenotype Acute Leukemia Diagnosed By Nation-Wide Immunophenotyping in Japan

[Background] Acute leukemia of ambiguous lineage is a very rare subtype defined by WHO 2008 classification. It is classified as mixed phenotype acute leukemia (MPAL), acute undifferentiated leukemia (AUL) and natural killer lymphoblastic leukemia. Basically, these category of leukemia consist of heterogenous pathogenesis. Determination of the lineage were usually decided by major lineage markers such as myeloperoxidase (MPO), CD19, and cytoplasmic CD3. However, more detailed immunophenotyping is necessary to confirm the cell lineages, especially in case of MPAL. Japanese pediatric leukemia/lymphoma study group (JPLSG) started nation-wide immunophenotyping with unified panel including more than 50 antigens for childhood hematological malignancy. Throughout this study, we found 32 acute leukemia of ambiguous lineage, including 3 AUL and 29 MPAL cases. MPAL cases were divided into several peculiar groups by our detailed phenotyping. We report here the characteristics of pediatric AUL/MPAL cases, including clinical manifestations and peculiar immunophenotype.[Result] In 3,229 case from January 2012 to December 2015, 32 cases were diagnosed as acute leukemia of ambiguous lineage. Gender ratio (M:F) was 18:14 and median age were 8.4 years (0.3 to 18.1) respectively. These cases were consisted of 3 AUL and 29 MPAL. According to detailed immunophenotyping, MPAL cases could be divided into 4 distinct clusters, (1) 7 T cell (T-) acute lymphoblastic leukemia (T-ALL)/acute myelogenous leukemia (AML)-M1 biphenotypic, (2) 4 early T-cell precursor (ETP)-like, (3) 8 B cell precursor (BCP-) ALL/Myelomonocytic bilineal, and (4) 10 BCP-ALL with MPO expression subtypes. T-ALL associated cases indicated relatively older age; (1) 12.3 and (2) 10.2 vs (3) 5.9 and (4) 7.5 respectively. 3 of (4) cases indicated characteristic genotypes; 1 minor bcr-abl, 1 major bcr-abl, and 1 AML1-ETO/Flt3-ITD. 4 out of 8 bilineal cases (3) were infantile ALL with MLL rearrangement, but other 4 older cases did not indicate MLL rearrangement.[Discussion] Acute leukemia of ambiguous lineage is a rare subtype of leukemia. According to WHO 2008 classification, it is further classified as MPAL, AUL and natural killer lymphoblastic leukemia. In this study, we evaluate 32 patients of acute leukemia of ambiguous lineage. It is of note that we could not identify no natural killer lymphoblastic leukemia in this childhood leukemia cohort. We also evaluate 29 MPAL cases, which showed 4 distinct subgroups. T-ALL/AML-M1 biphenotypic cases indicated relatively older age, usually diagnosed as AML-M1 with morphologic FAB classification, and received mostly myeloid oriented therapy. ETP-ALL like cases were positive for T cell antigens such as CD2 and/or CD7 and also some myeloid antigens but lacked cytoplasmic CD3 expression. BCP-ALL related cases were also divided into two groups, BCP-ALL/Myelomonocytic bilineal and BCP-ALL with MPO expression. Bilineal leukemia often found in infantile ALL with MLL gene rearrangement. In this study, we also found 4 non-infantile bilineal leukemia without MLL gene rearrangement. These cases occasionally cause lineage-switch relapse, so diagnosis of bilineal leukemia seems to be very significant. Recent studies showed EP300-ZNF384 fusion indicate CD10 negative or low BCP-ALL with MPO expression. Our cases also tended to indicate negative or low CD10 expression. In conclusion, we identified a spectrum of immunophenotype in MPAL patients. In childhood cases, most of MPAL cases were divided into 4 distinct categories. It must be useful information before examination of genetic abnormality. DisclosuresNo relevant conflicts of interest to declare.

CD97 Is a Critical Regulator of Acute Myeloid Leukemia Stem Cell Function

Despite recent advances in our understanding of the genetic origins of acute myeloid leukemia (AML), clinical outcomes remain poor. While standard induction chemotherapy induces remission in most patients, the majority of patients eventually relapses and dies from progressive disease. A number of cell surface proteins have been shown to be expressed at high levels on AML stem cells compared to normal HSCs including CD47, CD44, CD96, TIM3, CD123, CD25 and IL1RAP. Despite the attention these antigens have received, data supporting their roles as cell-intrinsic regulators of LSCs are more limited. We have identified CD97, a member of the adhesion class of GPCR, as a frequently upregulated antigen expressed on AML blasts that is a critical regulator of LSC function.In order to determine whether CD97 is expressed on LSCs, we assessed CD97 expression in human AML primary samples by flow cytometry. This study revealed that CD97 was 10 fold increased on LSC-enriched (CD34+CD38-) blasts compared to cord blood HSCs in all 30 patients tested. In contrast, the expression of other previously reported LSC was more variable and less frequently increased over HSCs. To test if CD97 may regulate leukemic cell function, we knocked-down (KD) CD97 in human AML cell lines. CD97-KD in HL60 and MOLM-13 AML cell lines increased apoptosis, reduced cell growth 4-fold, and delayed mortality once injected into NSG mice (20 vs. 28 days, p=0.0012). As CD97 expression increases during normal myelomonocytic differentiation, we cultured control and CD97-KD HL60 or U937 cells (which can be induced to differentiate along the myelomonocytic lineage following all-trans-retinoic acid (ATRA) treatment) for 4 days in the presence of 10nM ATRA or vehicle control. Flow cytometric analysis revealed that CD97-KD cells expressed higher levels of the myeloid differentiation antigens CD11b and CD15 both before and after ATRA treatment. In addition, mRNA expression of CD11b and PU.1, key genes associated with myeloid differentiation, were increased in CD97-KD cells compared to controls.To investigate whether CD97 regulates leukemic initiation, purified HSPCs from WT and CD97-/- mice were transduced with the MLL-AF9 oncogene (MA9). CD97-/--MA9 cells (GFP+) showed a 3-fold reduction in colony formation in serial replating assays in methylcellulose, consistent with a reduction in self-renewal. Mice transplanted with 5,000 CD97-/--MA9 blasts survived significantly longer than those receiving WT-MA9 blasts (70 vs 100 days, p=0.0039). Consistent with reduced disease aggressiveness, at the time of sacrifice mice injected with WT-MA9 cells contained a higher percentage of total GFP+ cells, L-GMPs and CD11b+Gr1+ cells in the BM than those receiving CD97-/--MA9 grafts. Secondary transplant experiments revealed that CD97-/--MA9 blasts exhibit a 25-fold lower LIC frequency than WT-MA9 blasts. Similar results were obtained using the AML1-ETO9a mice model.To better understand the molecular pathways underlying CD97 regulation of AML cells, we performed a combination of RNA-sequencing and protein phospho-array analyses in CD97-KD and scramble control cells. Gene Ontology and GSEA analyses revealed enrichment of transcripts encoding genes that regulate apoptosis as well as downregulation of cell cycle regulators in CD97-KD cells. Consistent with our flow cytometry data, CD97-KD induced differentiation, as evidenced by increased expression of myeloid differentiation genes and global increases in granulocyte and monocyte fingerprint signatures. Phosphoproteome analysis revealed downregulation of several mediators of the MAPK cascade and MTOR signaling pathways in CD97-KD cells. These signaling changes were confirmed by western blot, as activating phosphorylation events of ERK 1/2, c-Jun, and STAT3 were decreased upon CD97-KD. Together, these data indicate that functional alterations in CD97-KD AML cells are due to the modulation of signaling cascades including MAPK and mTOR.Our studies indicate that CD97 is expressed at a higher frequency in AML patient samples than previously identified LSC antigens and that its expression is elevated, irrespective of genetic subtype. Studies using human cell lines and mouse models of AML demonstrate that CD97 regulates the proliferation and survival of leukemic blasts as well as disease initiation and self-renewal. Collectively, these data credential CD97 as a promising therapeutic target in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD16+ Dendritic Cells Are a Unique Myeloid Antigen Presenting Cell Population

Dendritic cells (DC) are phenotypically identified in human blood as HLA-DR+ cells, which lack major cell surface lineage markers. We demonstrated that myeloid antigen presenting cells, including monocytes and DC display a continuum of CD14 and CD16 expression (10th Human Leucocyte Differentiation Antigen Workshop). The robustness of DC and monocyte identification, particularly when identifying cell subsets with little or no surface CD14 is limited by subjective gating strategies for determining rare cell populations. Application of Poisson counting statistics established that rare cell types such as "CD14- CD16+ DC" are often overlooked in analyses powered to detect the much larger populations of "classical" and "non-classical" monocytes.We used fluorescent and mass cytometry, in conjunction with unsupervised high dimensional clustering, to show that the continuum of CD14 expression separates CD14lo CD16+ non-classical monocytes and CD14- CD16+ DC. We have defined the CD14-CD16+ DC using a broad panel of cell surface markers and established a CD14-CD16+ DC phenotypic signature that is distinct from both classical and non-classical monocytes in healthy donor blood. The CD14-CD16+ DC differ in both size to CD14lo CD16+ monocytes and functional antigen uptake, with slower kinetics of soluble antigen uptake into lysozymes. Their proteasome processing and presentation of influenza matrix protein by MHC I was comparable to other primary blood monocytes and DC antigen presenting cell populations. The CD14-CD16+ DC had limited capacity for further in vitro differentiation. The recovery of CD14-CD16+ DC after autologous and allogeneic myeloablative hematopoietic cell transplants (HCT) followed similar kinetics to other monocytic and DC populations. CD14lo CD16+ monocytes expressed CCR5 as did other myeloid DC but CD14-CD16+ DC lacked CCR5, although interferon induced CCR5. The early differentiation and induction of CCR5 on circulating CD16+ DC after allogeneic HCT predicted for the onset of acute graft versus host disease.These data demonstrate that "CD14- CD16+ DC" represents a distinct clinically relevant human white blood cell population, whose ontogeny and function are under further investigation. DisclosuresFromm:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Papadimitrious:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hsu:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Larsen:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gibson:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Bradstock:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Kupresanin:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Clark:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hart:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd.

Immunophenotypic Profiling of Childhood B-Cell Precursor Acute Lymphoblastic Helps Identifying Genetic Subtypes, Including Recently Identified Ones

Background B-cell precursor acute lymphoblastic leukemia (B-ALL) comprises distinct entities characterized by recurring genetic alterations. These classifying alterations are associated with gene expression profiles, reflecting the specific biology of each genetic subgroup. In patients with none of these classifying alterations, referred as "B Other", RNAseq recently led to the identification of a huge number of in frame fusions which are likely to define novel genetic subgroups. Leukemic blasts often show aberrant antigen expression, giving rise to leukemia-associated immunophenotypic profiles (LAIP) distinct from their normal immature B-lineage counterparts. These variations in protein expression are likely to reflect the specific biology of B-ALL. We evaluated how LAIP were associated with the various classifying genetic lesions, including recently identified ones.Materials and Methods A cohort of 299 childhood B-ALL was studied prospectively. Genetic typing by cytogenetics, MLPA and RT-PCR identified high hyperdiploidy (Heh, n=77), ETV6-RUNX1 (n=81), TCF3-PBX1 (n=21), TCF3-HLF (n=3), MLL rearrangement (n=25), BCR-ABL/Phie-like fusions (n=10), ERGdel (n=8), TSLPR (n=8), iAMP21 (n=6), Hypodiploidy (n=6). The remaining patients were considered "B-Other" (n=54). Immunophenotyping was performed by 6-8-colors flow cytometry with a panel of 29 antibodies targeting surface proteins routinely used for ALL diagnosis and MRD assessment. The log of Ratio Fluorescence Intensity (RFI) of B-ALL/normal B precursor cells was calculated for each marker. RNAseq was performed in 18 B-Other and 8 BCR-ABL/Phie-like. Fusions were detected using TopHat2. Samples were classified using hierarchical clustering and/or principal component analyses (R software).Results Unsupervised clustering identified 6 groups with shared pattern of LAIP. Four groups were associated with known classifying lesions: ETV6-RUNX1 (of which 87% co-clustered), Heh (82%), TCF3-PBX1 (100%) and MLL (96%). ALL that did not cluster according to their genetic group mostly clustered (23/26; 89%) in two groups, hereafter named "B Other-1" and "B Other-2", which mainly contained ALL lacking any previously known cytogenetic lesion. Interestingly, 3 of 5 ETV6-RUNX1 with IKZF1del clustered in the "B Other 2", suggesting that misclassifications may not be due to poor LAIP specificity but rather reveal distinct biological features in some ALL.Although comprising too few cases to define specific groups, 8/10 B-ALL with BCR-ABL/Phie-like, 3/3 TCF3-HLF, and 8/8 TSLPR co-clustered in "B Other 2", and 7/8 ERGdel in "B Other 1". No clear clustering was observed for Hypodiploid and iAMP21. B-Other ALL were allocated either to oncogenic groups, e.g. TCF3-PBX1 (n=7) or MLL (n=7), or to the "B Other" groups (n=35). Only one B Other, harboring an IGH-IL3 fusion, co-clustered with BCR-ABL/Phie-like ALL. RNAseq of 18 B-Other identified no additional Phie-like fusion and none of these B-Other co-clustered with a group of 8 controls BCR-ABL/Phie-like. This makes unlikely that some Phie-like cases remained unidentified. However, other fusions involving MEF2D (n=3), PAX5 (n=5) or ZNF384 (n=2) were evidenced in B Other ALL. Cases with MEF2D or ZNF384 clustered both at the transcriptome and protein level. Using LAIP clustering, MEF2D ALL layed close to those with TCF3 fusions. ZNF384 cases clustered in the MLL group, consistent with a lack of CD10 expression combined to myeloid antigen expression. PAX5 ALL were allocated to the "B Other 2" group with no clear clustering and, more surprisingly, no consistent effect on CD19 expression. Secondary aberrations such as deletions in IKFZ1 or CDKN2A/B genes did not significantly affect clustering.Conclusion Unsupervised clustering based on surface protein expression allowed to classify most childhood ALL genetic groups, including Phie-like, MEF2D or ZNF384, which are challenging to identify due to the diversity of the underlying gene fusions. LAIP can thus rapidly orientate diagnosis, in particular for Phie-like which are candidates to targeted therapies. Interestingly, B-Other that clustered in known genetic groups may present biologically equivalent alterations undetected in our routine workup. Further studies will allow to assess whether cases co-clustering with ETV6-RUNX1 (n=2) or ERGdel (n=6) correspond to the recently described ETV6-RUNX1-like or DUX4 group respectively. DisclosuresBaruchel:Servier: Consultancy; Novartis: Consultancy; Jazz: Consultancy; Baxalta: Research Funding; Celgene: Consultancy.

In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia.

BackgroundApproximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia.Methods and findingsWe developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85–90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination.ConclusionsThis strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a ‘bridge” therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft.

Interleukin-4 induced 1 (IL4I1) promotes central nervous system remyelination.

This scientific commentary refers to ‘IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven inflammation’, by Psachoulia et al. (doi:10.1093/brain/aww254) . Multiple sclerosis is an inflammatory demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin sheaths and axons (Lorscheider et al. , 2016). Most patients initially develop relapsing-remitting disease (RRMS), in which acute neurological deficits are interspersed with periods of partial or complete recovery. However, up to 65% of patients with RRMS eventually develop secondary progressive multiple sclerosis (SPMS), in which disability increases continually. Conversion to SPMS is associated with a relatively poor prognosis, in part due to the limited effectiveness of disease-modifying therapies in the progressive phase (Lorscheider et al. , 2016). This in turn partly reflects the complex interactions between the multiple pathophysiological mechanisms responsible for demyelination and neurodegeneration. However, there is also compelling evidence that a decline in regenerative capacity occurs in the brain in chronic multiple sclerosis, possibly due to dysregulation of inflammation (Fitzner and Simons, 2010). Unravelling the mechanisms that link the immune system with CNS damage and repair therefore represents both a challenge and an opportunity for multiple sclerosis research. In this issue of Brain , Psachoulia et al. (2016) exploit a published remyelination transcriptome obtained from laser capture microdissected CNS lesions induced in the rodent CNS by lysolecithin/lysophosphatidylcholine (LPC) (Huang et al. , 2011), to identify novel markers of remyelination. Of the more than 8000 differentially regulated genes, Il4i1 , a phenylalanine oxidase secreted mainly by myeloid antigen presenting cells (APCs), was among the most strongly upregulated (4-fold increase at 14 days versus 5 days post-lesion). In situ hybridization demonstrated that Il4i1 was predominantly expressed in cells with morphology reminiscent of foamy macrophages, beginning as early as 10–14 days post-lesion, a …

Morphology and flow cytometry analysis of acute B-cell leukemia with high expression of myeloid antigen CD13

目的 分析伴异常表达CD13急性B细胞白血病形态学及流式细胞学特点，提高诊断此类疾病的诊断水平。 方法 对确诊的4例患者的形态学及流式细胞术散点图进行分析，研究并复习相关文献，总结特点。 结果 急性B细胞白血病伴异常表达CD13的患者形态学上多数具有高度异质性与分化发育的不协调性，且此类细胞均高表达CD34。 结论 急性淋巴细胞白血病伴异常表达髓系抗原的确诊必须依靠免疫学分型，同时该类原始细胞既具有典型B细胞的免疫标志又有髓系细胞的特有标志，具有髓系原始细胞及原始淋巴细胞的双重特点，是形态学误诊的最主要原因。

Abstract 494: Evaluation of soluble FLT3 ligand as a marker of bone marrow stress and recovery

Abstract Depletion of bone marrow (BM)-resident myeloid progenitor cells is a consequence of various anti-cancer treatments, including bispecific T-cell engager (BiTE®) antibodies targeting myeloid cell antigens. Currently, aspirates are required to determine the extent of myeloid cell depletion in the BM. This procedure is painful and potentially invasive for patients. As a surrogate marker for BM myeloid cell depletion, we evaluated soluble FLT3 ligand (FLT3L) in non-human primates treated with myeloid cell-depleting BiTE® antibodies. While FLT3L is not detectable in serum during steady-state hematopoiesis, it is detectable in response to hematopoietic deficiency. The goal of this study was to evaluate the relationship of serum FLT3L with depletion and recovery of BM myeloid cells in non-human primates treated with BiTE® antibodies against CD33. Serum FLT3L levels were determined using an anti-human FLT3L ELISA kit. Validation studies demonstrated that both the capture and detection antibodies recognized cynomolgus FLT3L. Serum FLT3L concentrations were determined using a standard curve with rhesus FLT3L (99.6% identical to cynomolgus FLT3L). Serum samples were taken from cynomolgus monkeys before, during and after treatment with anti-CD33 BiTE® antibodies and compared with the number of BM-resident target cells. In the first study, cynomolgus monkeys were dosed once with different dose levels of anti-CD33 BiTE® antibody ranging from 1- 15 μg/kg. BM-resident myeloid cells were transiently reduced in some, but not all, cohorts. Soluble FLT3L was detected only when there was a reduction in BM myeloid cells. When FLT3L was detectable, the serum concentration correlated with the extent of target cell reduction and FLT3L levels decreased as the myeloid cells recovered. In the second study, cynomolgus monkeys were dosed three times with anti-CD33 BiTE® antibody. In these studies target cell reduction was greater and persisted longer compared to the single dose studies. Similarly, the magnitude and duration of increased FLT3L in the multidose studies was also greater. Within the multi-dose study, FLT3L levels continued to increase as more BM-resident myeloid cells were depleted, suggesting that it was possible to detect cumulative stress to the BM. In summary, serum FLT3L levels are correlated with the extent and duration of myeloid cell depletion in the BM suggesting that serum FLT3L levels reflect depletion and subsequent myeloid cell replenishment. All studies to date have involved treatment with myeloid antigen-specific BiTE® antibodies; future studies will evaluate FLT3L levels following treatment with anti-lymphoid or solid tumor antigen-specific BiTE® antibodies to determine if FLT3L increases are associated with myeloid cell depletion or indicative of general BM stress or inflammation. The potential benefit of this marker is that it could allow for repeat non-invasive BM monitoring in patients on myelosuppressive therapies. Citation Format: Christine Sastri, Mercedesz Balazs, Priya Koppikar, James R. Lipford, Angela Coxon, Tara L. Arvedson. Evaluation of soluble FLT3 ligand as a marker of bone marrow stress and recovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 494.

Abstract 2692: ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor

Abstract Childhood acute lymphoblastic leukemia (cALL) is distinct from that in adults with higher incidence, better prognosis and a distinct mutational spectrum. One hypothesis for this difference is that cALL arises in transient cells unique to early human development. We explored this in ETV6-RUNX1 cALL where evidence from twins and neonatal heel prick testing has shown that this mutation arises in utero and is an initiating event. We characterized B cell development in first trimester human fetal liver (FL) to identify compartments vulnerable to ETV6-RUNX1. Using CD19 as a marker of B lineage commitment we found the first CD19+ B cells emerge in the human FL at Carnegie Stage (CS) 17 and were distinct from adult in that the majority expressed surface IL7 receptor. We used IL7R to identify a CD19-IL7R+ B progenitor compartment that produced B cells in vitro, possessed DJH recombination, but also had monocytic potential. Single cell analysis of CS20 IL7R+ progenitors revealed co-expression of lymphoid and myeloid programmes, whereas at CS17 they were strongly myeloid primed indicating that IL7R+ progenitors acquire lymphoid potential in this developmental window. Some co-expression of lymphoid and myeloid programmes also persisted in CS20 FL B cells. We tested whether FL B cell development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing pro and preB cells as well as an IL7R+ progenitor that switched from myeloid to B-myeloid priming during culture. At the global transcriptional level the hPSC lymphoid hierarchy mapped closely with FL, with both separating from adult suggesting that hPSCs provide a developmentally relevant model of early FL B lymphopoiesis. We next used CRIPSR-directed homologous recombination to engineer the expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B cell differentiation at the level of the IL7R+ progenitor. ETV6-RUNX1 expressing B cells co-expressed an abnormal B-myeloid gene expression signature akin to that seen in the IL7R+ progenitor. Both the transcriptional and differentiation phenotypes were dependent on ETV6-RUNX1 as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model where expression of ETV6-RUNX1 inhibits lymphoid specification in an early FL IL7R+ lymphomyeloid progenitor, arresting B lineage differentiation and resulting in the production of myeloid-primed B cells. This may explain the relatively high levels of myeloid antigen expression lineage promiscuity seen in cALL. ETV6-RUNX1 hPSCs will afford the systematic evaluation of the contribution of additional mutations seen in cALL and may offer a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lymphomyeloid progenitor in the human FL is a candidate target cell for in utero pre-leukemic initiation in cALL. Citation Format: Simon E. Richardson, Charlotta Böiers, Alya Zriwil, Virginia Turati, John Brown, Dapeng Wang, Javier Herrero, Stefan Karlsson, Andrew J. H. Smith, Sten Erik Jacobsen, Tariq Enver. ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2692.

Gr-1intCD11b+ myeloid-derived suppressor cells accumulate in corneal allograft and improve corneal allograft survival.

We identified the characteristics of myeloid-derived suppressor cells (MDSCs) and investigated their mechanism of induction and their functional role in allograft rejection using a murine corneal allograft model. In mice, MDSCs coexpress CD11b and myeloid differentiation antigen Gr-1. Gr-1+CD11b+ cells infiltrated allografted corneas between 4 d and 4 wk after surgery; however, the frequencies of Gr-1+CD11b+ cells were not different between accepted and rejected allografts or in peripheral blood or BM. Of interest, Gr-1intCD11b+ cells, but not Gr-1hiCD11b+ cells, infiltrated the accepted graft early after surgery and expressed high levels of immunosuppressive cytokines, including IL-10, TGF-β, and TNF-related apoptosis-inducing ligand. This population remained until 4 wk after surgery. In vitro, only high dose (>100 ng/ml) of IFN-γ plus GM-CSF could induce immunosuppressive cytokine expression in Gr-1intCD11b+ cells. Furthermore, adoptive transfer of Gr-1intCD11b+ cells reduced T cell infiltration, which improved graft survival. In conclusion, high-dose IFN-γ in allograft areas is essential for development of Gr-1intCD11b+ MDSCs in corneal allografts, and subtle environmental changes in the early period of the allograft can result in a large difference in graft survival.

Meta-Analysis from Gene Expression Profiles of Lupus Affected Tissues Reveals Novel Immune Cell Contributions

Abstract Immunologic mechanisms causing tissue damage in autoimmune diseases such as SLE are not fully understood. To gain additional insight, gene expression profiles obtained from lupus affected skin, synovium and kidney were obtained and compared to meta-analyzed data obtained from active lupus B, T and myeloid cells. More than 300 arrays from lupus patients and appropriate controls were analyzed to determine differentially expressed (DE) genes (8279 discoid lupus skin, 5465 synovium, 6381 glomerulus, 5587 tubulointerstitum). Notably, the majority of lupus affected tissue DE genes were detected in more than one tissue and 439 were differentially expressed in all tissues. A variety of approaches assessed the molecular pathways and cellular phenotypes accounting for these common lupus affected tissue DE genes. Curated STRING-based interaction analysis identified a number of pathways including co-stimulation of T cells, activation of B and myeloid cells, antigen presentation, TLR signaling and p38 activation; from a total of 193 IPA-documented pathways, 59 were common to all tissues including p38 signaling, TLR signaling, maturation of dendritic cells, B cell activation and ICOS-ICOSL in T cells. Novel bioinformatics approaches documented that more than 50% of the DE genes in the tissues were associated with immune cell function and 11–18% were unique to the immune system. Further analysis using LINCS and additional novel software highlighted specific molecular pathways of immune cell activation including the JAK/STAT and the IL12 pathways. These results demonstrate the value of comprehensive application of orthogonal curated bioinformatics tools in identifying the role of immune cells in lupus pathogenesis and tissue damage.

TGFBR1M318R+/− Knock-In mice recapitulate the immune phenotypes of Loeys-Dietz syndrome.

Abstract Loeys-Dietz syndrome (LDS) is an autosomal dominant disorder caused by mutations in genes encoding proteins in the transforming growth factor beta (TGFβ) signaling pathway. Patients heterozygous for a point mutation in the kinase domain of TGFβR1 (TGFBR1M318R) exhibit classic manifestations of LDS. Mice with this mutation were made with the hope that an animal model of LDS will lead to better understanding of the mechanisms underlying LDS symptoms. TGFBR1M318R+/− mice exhibit the hallmark skeletal and cardiovascular phenotypes of LDS. We sought to determine if TGFBR1M318R+/− mice also recapitulate the immune phenotypes characteristic of LDS. We find that like their human counterparts, TGFBR1M318R+/− mice spontaneously develop elevated serum IgE and eosinophilic esophagitis (EoE), characterized by esophageal dilation, increased collagen, food impaction, and increased infiltration of eosinophils, T cells, mast cells, and myeloid antigen presenting cells. We made bone marrow (BM) chimeras to determine whether altered TGFβR signaling in hematopoietic and/or stromal cells led to EoE. Both irradiated TGFBR1M318R+/− mice reconstituted with WT BM, and WT mice reconstituted with TGFBR1M318R+/− BM had EoE 8 weeks post-reconstitution, indicating that aberrant TGFβ signaling in either BM-derived or non-hematopoietic cells can be associated with EoE, but that neither is absolutely required. To determine which hematopoietic cells are essential to the development of EoE we generated RAG2−/− TGFBR1M318R+/− mice and found that they too developed EoE, demonstrating that neither T or B cells were essential. Using TGFBR1M318R+/− mice to study the immune pathology of LDS should lead to better therapeutic strategies for the treatment of EoE.

Early T-cell precursor acute lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) in adolescents and adults: a high-risk subtype

AbstractEarly T-cell precursor (ETP) acute lymphoblastic leukemia/lymphoma (ALL/LBL) is a recently recognized high-risk T lymphoblastic leukemia/lymphoma (T-ALL/LBL) subgroup. The optimal therapeutic approaches to adult patients with ETP-ALL/LBL are poorly characterized. In this study, we compared the outcomes of adults with ETP-ALL/LBL who received treatment on frontline regimens with those of patients with other T-ALL/LBL immunophenotypic subtypes. Patients with newly diagnosed T-ALL/LBL who received frontline chemotherapy between the years 2000 and 2014 at The University of Texas MD Anderson Cancer Center were identified and immunophenotypically categorized into early, thymic, and mature per the World Health Organization (WHO) classification using CD1a and surface CD3 status. Patients with ETP-ALL/LBL were identified on the basis of the following immunophenotypes: CD1a−, CD8−, CD5− (dim), and positivity for 1 or more stem cell or myeloid antigens. A total of 111 patients with T-ALL/LBL (68% T-ALL; 32% T-LBL) with adequate immunophenotype data were identified. The median age was 30 years (range, 13-79). There was no difference in the outcomes of patients based on the WHO subtypes. Nineteen patients (17%) had ETP-ALL/LBL. The complete remission rate /complete remission with incomplete platelet recovery rate in patients with ETP-ALL/LBL was significantly lower than that of non–ETP-ALL/LBL patients (73% vs 91%; P = .03). The median overall survival for patients with ETP-ALL/LBL was 20 months vs not reached for the non–ETP-ALL/LBL patients (P = .008). ETP-ALL/LBL represents a high-risk disease subtype of adult ALL. Novel treatment strategies are needed to improve treatment outcomes in this T-ALL/LBL subset.

Small, abnormal B lymphoid blast populations in chronic myelogenous leukemia at diagnosis: Does this finding indicate an accelerated course?

The 2008 WHO is not specific regarding subclassification of chronic myelogenous leukemia (CML) with less than 20% abnormal B lymphoid blasts (ABLB), and suggests patients with ABLB often show rapid progression (Swerdlow, 2008). Recent studies have shown variable outcomes when small abnormal B cell populations are seen by flow cytometry (El Rassi et al., Cancer 2015; 121:872-875; Vrotsos et al., Cytometry B Clin Cytom 2015). The hematopathology database was searched (7.4-year period), and patients identified through routine clinical study, who were BCR-ABL1 positive CML and had an ABLB of less than 20%. Flow cytometric (FC) and histologic data was evaluated to determine immunophenotypic abnormalities, immunohistochemical patterns, and percentage of ABLB, hematogones, and mature B cells. Seven patients with CML and ABLB identified by FC studies were found, five of which also had available histologic material to review. ABLB by FC ranged from 0.006% to 3.4%, typically demonstrated an immunophenotype with increased CD10, increased CD19, and decreased CD38, without myeloid antigens, abnormalities similar to that reported previously (Vrotsos et al., Cytometry B Clin Cytom 2015). The ABLB population was found only in diagnostic samples and always with more numerous hematogones. By immunohistochemistry, three of five patients showed ≥10% TdT positive cells. All patients showed a response to tyrosine kinase inhibitor therapy, and no patients progressed to clinical lymphoid blast phase. Immature B cells, occasionally including a small subset with an abnormal phenotype can be observed in chronic phase CML at diagnosis. We believe an approach incorporating clinical data, cytogenetic/molecular findings, and morphologic evaluation may be helpful when determining the best management of CML patients at diagnosis if small ABLB populations are identified by FC. © 2016 Clinical Cytometry Society.

A Phase 1b Study of Neoadjuvant Immune Biomarker Modulation With Cetuximab and Motolimod in Head and Neck Cancer (HNC)

Inflamed tumors are known to generate a better response to immunotherapy. HNC has many immunosuppressive features, including impaired natural killer cell (NK) activity. The monoclonal antibody cetuximab (CTX) is effective in a subset of HNC patients, with clinical activity linked to NK-mediated antibody-dependent cellular cytotoxicity (ADCC). Recent data show that CTX increases the frequency of intratumoral CTLA-4+FoxP3+ regulatory T cells (Treg), which suppress ADCC and are associated with poor clinical outcome. In ex vivo experiments, this effect could be attenuated by targeting CTLA-4 on Tregs. It is possible that the clinical efficacy of CTX may be improved by enhancing tumor inflammation, activating the immune system, and/or inhibiting the Treg suppressive effects. Motolimod (VTX-2337), a novel Toll-like receptor 8 (TLR8) agonist. Preclinical data show enhanced CTX and NK-mediated lysis of HNC cells and dendritic cross-priming of EGFR-specific CD8+ T cells. CTX and motolimod in HNC patients was tolerable and active in a phase 1b study, with enhanced NK cell activity. The central hypothesis in this study is that NK and monocyte/mDC activation by CTX is enhanced by concomitant administration of motolimod, thereby amplifying the innate and adaptive immune response in the circulation and in the tumor microenvironment (TM). In this prospective phase 1b clinical trial (NCT02124850) of preoperative treatment with CTX and motolimod, the primary objective is to evaluate how neoadjuvant CTX plus motolimod modulates innate and adaptive immune biomarkers. An exploratory objective of the study is to assess whether this modulation of biomarkers can predict antitumor response. Subjects (n=12) with stage II–IVA HNC received weekly doses of CTX and motolimod followed by definitive surgical resection. Biomarker modulation in tumor and blood are correlated with clinical response by CT or MRI. Tumor apoptosis/proliferation is assessed by biopsy pre- and posttreatment. Phenotypic markers of suppression in Treg were reduced when motolimod was combined with CTX, including lower levels of CTLA-4 (P=.01), CD73 (P=.04), and membrane-bound TGF-β (P=.05). These mediators were induced in nonresponders in our single-agent cetuximab therapy study (P=.005). Myeloid antigen–presenting cells achieved strong maturation and expression of stimulatory markers in the peripheral blood. CTX plus motolimod induces inflammatory changes in the TM and peripheral blood. Treg-suppressive mediators are reduced with CTX/motolimod, overcoming a negative prognostic biomarker (Treg induction) of CTX therapy alone. A pending amendment will assess the impact of adding checkpoint inhibition to the CTX/motolimod combination.

Myeloid Cell Nuclear Differentiation Antigen (MNDA) Expression Distinguishes Extramedullary Presentations of Myeloid Leukemia From Blastic Plasmacytoid Dendritic Cell Neoplasm.

Myeloid neoplasms constitute one of the most common malignancies in adults. In most cases these proliferations initially manifest in the blood and marrow; however, extramedullary involvement may precede blood or marrow involvement in a subset of cases, making a definitive diagnosis challenging by morphologic and immunohistochemical assessment alone. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive entity that frequently presents in extramedullary sites and can show morphologic and immunophenotypic overlap with myeloid neoplasms. Given that BPDCN and myeloid neoplasms may both initially present in extramedullary sites and that novel targeted therapies may be developed that exploit the unique molecular signature of BPDCN, new immunophenotypic markers that can reliably separate myeloid neoplasms from BPDCN are desirable. We evaluated the utility of myeloid cell nuclear differentiation antigen (MNDA) expression in a series of extramedullary myeloid leukemias (EMLs) and BPDCN. Forty biopsies containing EML and 19 biopsies containing BPDCN were studied by MNDA immunohistochemistry. The majority of myeloid neoplasms showed nuclear expression of MNDA (65%). In contrast, all cases of BPDCN lacked MNDA expression. These findings show that MNDA is expressed in the majority of EMLs and support the inclusion of MNDA immunohistochemistry in the diagnostic evaluation of blastic hematopoietic infiltrates, particularly when the differential diagnosis is between myeloid leukemia and BPDCN.

Small-lymphoid cells and myeloid antigen expression in a patient with IgG myeloma: A case report.

Multiple myeloma is defined as a malignant proliferation of a single clone of plasma cells resulting in monoclonal immunoglobulin production. Due to the number of plasma cell morphological variants, difficulty is often faced during morphological diagnosis. The current study describes the case of a 49-year-old woman presenting with atypical plasma cell morphology detected by a bone marrow examination. Flow cytometric immunophenotyping determined the nature of the neoplastic cells as monoclonal myeloma cells with myeloid antigen expression. Serum electrophoresis with immunofixation and subsequent clinical findings confirmed this diagnosis. Therefore, the immunophenotyping of plasma cells in myelomas may be useful for the diagnosis of cases with atypical plasma cell morphology.

Value of Quantitative assessment of Myeloid Nuclear Differentiation Antigen expression and other flow cytometric parameters in the diagnosis of Myelodysplastic syndrome.

The diagnosis of myelodysplastic syndrome (MDS) based on morphology is particularly difficult in low-grade MDS. Thus, the role of myeloid nuclear differentiation antigen (MNDA) and other flow cytometric (FCM) parameters in MDS was evaluated. Bone marrow aspirates (BMA) collected from 52 patients with unexplained persistent cytopenias were divided into three groups: (i) proven MDS (n = 12) based on morphology and/or cytogenetics; (ii) suspected MDS (n = 6), noncontributory morphology, and cytogenetics; and (iii) non-MDS (n = 34). Sixteen control BMA were studied. Cases were analyzed for MNDA expression (on granulocytes, blasts, monocytes, and lymphocytes) and for seven quantitative parameters: CD34(+) myeloblasts % in nucleated cells, CD34(+) B-cell progenitor% in CD34(+) cells, lymphocyte/myeloblast CD45 MFI ratio, granulocyte/lymphocyte SSC peak channel ratio and the proportion of CD34(+) myeloblasts expressing CD15, CD11b, and CD56. A score of 1 was given to each parameter beyond the cutoff, and score ≥3 was considered FCM positive. MNDA expression on granulocytes and blasts was significantly lower in proven MDS and suspected MDS vs. non-MDS. Quantitative FCM parameters successfully distinguished MDS and suspected MDS from non-MDS. MNDA expression is an independent marker for the evaluation of dyspoiesis and may be added to the standard panel for quantitative assessment by FCM.