Bone Marrow Specimens Of Patients Research Articles

Abstract 580: MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma

Abstract Novel strategies to target tumors are needed to enhance the efficacy of immune redirection therapies. The detection of tumors based on mammalian immunoglobulin scFvs has been a significant innovation in immunotherapy but is impacted by limits of immune tolerance and immune system evolution. The discovery of unique variable lymphocyte receptors (VLRs) - analogues to antibodies in evolutionarily distant jawless vertebrates - in the Cooper lab (Pancer, Nature) provides an opportunity to target antigens beyond those recognized by conventional antibodies. A particular VLR, termed MM3, was found to identify a distinct epitope specific to human plasma cells (PCs) and plasmablasts (PBs), created by CD38 dimerization with activity as an NAD+ glycohydrolase (Yu, JCI Insight). CD38 is an extensively explored target for treating multiple myeloma (MM); however, specific scFv-based targeting of CD38 on malignant plasma cells is difficult, as the protein is expressed on a variety of other immune cell lineages. Therefore, MM3 provides a unique, novel mechanism to target CD38 in a tumor-specific manner in MM and other plasma cell dyscrasias. Here, we present a CAR T cell with an MM3 binding domain replacing the standard scFv. Our CAR construct consists of the MM3 binding domain, a CD8 hinge and transmembrane domain, and 4-1BB and CD3zeta intracellular domains. Staining of MM patient bone marrow specimens revealed that the strong specificity for PCs and PBs previously observed in healthy donors was maintained in MM bone marrow, illustrating the ability of MM3 to target the malignant PC population. To assess in vitro function of the MM3 CAR T cells, flow cytometry analysis of co-cultures using healthy donor-derived MM3 CAR T cells and target cell lines revealed specific increased degranulation (CD107a expression), activation (CD69 expression), and killing (target cell Annexin V binding) over untransduced control T cells only in cell lines sensitive to MM3 binding, with no differences in negative control cell lines, demonstrating specific recognition and killing of target cells. Preliminary in vivo studies in the MISTRG6 myeloma xenograft mouse model were performed using intrafemorally injected INA-6, an IL-6-dependent MM cell line, which MM3 binds after growth in vivo. After treatment with MM3 CAR T cells via intravenous injection, we observed that the MM3 CAR T cells specifically homed to and were retained in the injected femur along with the tumor cells, suggesting tumor recognition and potential for in vivo efficacy. In summary, these data demonstrate that VLR-based targeting may allow design of novel CAR constructs. Specifically, the VLR MM3 is a promising novel, non-scFv mechanism for targeting CD38 in a malignant PC-specific manner, and the MM3 CAR T cells have shown promising preliminary activity. Therefore, the VLR-based MM3 CAR T cell platform may provide a novel strategy to improve T cell redirection to target tumors in MM. Citation Format: Samuel S. McCachren, Julia Manalo, Katherine Pendleton, Mary E. Ballestas, Kavita M. Dhodapkar, Melissa L. Kemp, Max D. Cooper, Madhav V. Dhodapkar. MM3 CAR T: A novel tumor targeting and T cell redirection platform for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 580.

Bone marrow necrosis in pediatric malignancies: 10-Year retrospective review and review of literature.

Bone marrow necrosis (BMN) is a rare pathologic finding, but when encountered is most often associated with malignancy. In adults, its presence correlates with an inferior prognosis, however in children the prognostic implication is unclear. We performed a retrospective review of 3,760 bone marrow specimens in patients ≤18 years over a 10-year period. BMN was identified in less than 1% of specimens and only in patients with leukemia, lymphoma, or neuroblastoma. BMN contributed to a delay in diagnosis; however, advanced medical imaging may serve as a tool to localize nonnecrotic areas for bone marrow sampling, facilitating an expedited diagnosis.

Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress

AbstractWe investigated and modeled the mesenchymal stromal cell (MSC) niche in adult acute lymphoblastic leukemia (ALL). We used gene expression profiling, cytokine/chemokine quantification, flow cytometry, and a variety of imaging techniques to show that MSCs, directly isolated from the primary bone marrow specimens of patients with ALL, frequently adopted an activated, cancer-associated fibroblast phenotype. Normal, primary human MSCs and the MSC cell line HS27a both were activated de novo, when exposed to the reactive oxygen species (ROS)–inducing chemotherapy agents cytarabine (AraC) and daunorubicin (DNR), a phenomenon blocked by the antioxidant N-acetyl cysteine. Chemotherapy-activated HS27a cells were functionally evaluated in a coculture model with ALL targets. Activated MSCs prevented therapy-induced apoptosis and death in ALL targets, via mitochondrial transfer through tunneling nanotubes (TNTs). Reduction of mitochondrial transfer by selective mitochondrial depletion or interference with TNT formation by microtubule inhibitors, such as vincristine (VCR), prevented the “rescue” function of activated MSCs. Corticosteroids, also a mainstay of ALL therapy, prevented the activation of MSCs. We also demonstrated that AraC (but not VCR) induced activation of MSCs, mitochondrial transfer, and mitochondrial mass increase in a murine NSG model of disseminated SEM cell–derived ALL, wherein CD19+ cells closely associated with nestin+ MSCs after AraC, but not in the other conditions. Our data propose a readily clinically exploitable mechanism for improving treatment of ALL, in which traditional ROS-inducing chemotherapies are often ineffective at eradicating residual disease, despite efficiently killing the bulk population.

MiR‐215‐5p is an anticancer gene in multiple myeloma by targeting RUNX1 and deactivating the PI3K/AKT/mTOR pathway

This study examined the underlying mechanism of miR-215-5p in multiple myeloma (MM) at the molecular level. miR-215-5p was upregulated in bone marrow specimens of patients with MM and MM cell lines through real-time polymerase chain reaction, and downregulation of miR-215-5p was related to poor survival of patients with MM. The results of Western blot assay showed that the PI3K/AKT/mTOR signaling pathway was activated in bone marrow specimens of patients with MM. miR-215-5p was found to negatively correlate with runt-related transcription factor 1 (RUNX1) expression in MM clinical bone marrow samples. Therefore, to test the probable mechanisms of the regulation of MM cell proliferation and apoptosis, a miR-215-5p mimics/inhibitors/negative control was transfected into MM cells. The targets of miR-215-5p were estimated using four bioinformatics databases (including miRDB, miRTarBase, Starbase, and Targetscan). Furthermore, enrichment analyses of gene ontology and Kyoto Encyclopedia of Genomes pathway were carried out at the Enrichr website. Cell viability was detected using the MTT method, while apoptosis and cell cycle were measured using flow cytometry. RUNX1, CDK2, CDC25B, cleaved-caspase-3, cleaved-PARP, p-PI3K, PI3K, p-AKT, AKT, p-mTOR as well as mTOR protein were also investigated using Western blot analysis. According to our findings, miR-215-5p overexpression could induce apoptosis while rendering cell cycle arrest at G1 phase and reducing the proliferation of cells. Meanwhile, miRNA-215-5p could negatively regulate messengerRNA and protein levels of RUNX1 in MM cells. In addition, there was a similar effect in cell proliferation and apoptosis after miR-215-5p mimics or siRNA-RUNX1 transfection. miR-215-5p inhibition inhibited apoptosis while increased the phosphorylation levels of PI3K, AKT, and mTOR proteins, whereas, miR-215-5p overexpression increased cell proliferation. Therefore, miR-215-5p inhibited MM cell apoptosis via targeting RUNX1 and suppressing the activation of the PI3K/AKT/mTOR pathway.

EBV-encoded RNA1-positive cells in the bone marrow specimens of patients with EBV-negative lymphomas and sarcomas.

Epstein-Barr virus (EBV) infection is associated with pathogenesis of various cancers, including extranodal natural killer/T-cell lymphoma, nasal type (ENKL). ENKL tumor cells are positive for EBV-encoded RNA1 (EBER1), which is the most useful marker to identify ENKL tumor cells in histopathology. Currently, EBER1 in situ hybridization (ISH) is recommended to evaluate bone marrow (BM) involvement of ENKL. However, the actual burden of EBER1-positive cells in normal BM specimens remains unclear. In the present study, we performed EBER1 ISH on 111 BM specimens, which were obtained during an initial staging procedure in patients with EBV-negative cancers and were also negative for BM involvement. One or more EBER1-positive cells per whole specimen were observed in 38 specimens (34%). The number of EBER1-positive cells was distributed as follows: single positive cell, n = 17; two positive cells, n = 13; three positive cells, n = 3; and four positive cells, n = 5. These findings suggest that four or fewer EBER1-positive cells can be observed in BM specimens of patients with non-EBV-related cancers. The clinical implications of a small number of EBER1-positive cells in BM specimens of patients with ENKL should be evaluated in further studies.

Persistent IDH1/2 mutations in remission can predict relapse in patients with acute myeloid leukemia.

Persistence of IDH1 or IDH2 mutations in remission bone marrow specimens of patients with acute myeloid leukemia has been observed, but the clinical impact of these mutations is not well known. In this study, we evaluated 80 acute myeloid leukemia patients with known IDH1 R132 or IDH2 R140/R172 mutations and assessed their bone marrow at the time of remission to determine the potential impact of persistent IDH1/2 mutations. Approximately 40% of acute myeloid leukemia patients given standard treatment in this cohort had persistent mutations in IDH1/2. Patients with an IDH1/2 mutation had an increased risk of relapse after 1 year of follow-up compared to patients without a detectable IDH1/2 mutation (59% versus 24%; P<0.01). However, a persistent mutation was not associated with a shorter time to relapse. High IDH1/2 mutation burden (mutant allelic frequency ≥10%) did not correlate with relapse rate (77% versus 86% for patients with a low burden, i.e., mutant allelic frequency <10%; P=0.66). Persistent mutations were also observed in NPM1, DNMT3A and FLT3 during remission, but IDH1/2 mutations remained significant in predicting relapse by multivariate analysis. Flow cytometry was comparable and complementary to next-generation sequencing-based assay for predicting relapse. Monitoring for persistent IDH1/2 mutations in patients with acute myeloid leukemia in remission can provide information that could be used to justify early interventions, with the hope of facilitating longer remissions and better outcomes in these patients.

Abstract 3791: TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents

Abstract Background: Multiple myeloma and its associated bone disease are generally incurable. Hence, better therapies are needed, ideally targeting biomolecules implicated in the aberrant biology of the disease. Overexpression of transient receptor potential cation channel TRPV6, a highly selective calcium channel has been observed in breast, colon, thyroid, ovary and prostate cancer tissues, and in several tumour cell lines. TRPV6 expression has also been seen in osteoclasts, however its role in bone metabolism remains unclear. The reciprocal interaction between osteoclasts and myeloma cells is pivotal to the generation of bone lesions that characterize myeloma. While myeloma cells secrete factors promoting osteoclast activity, osteoclasts in turn are known to induce myeloma cell growth and survival. The TRPV6 peptide antagonist SOR-C13 is currently in phase I clinical trials as an anti-cancer therapy for advanced cancers. We investigated the expression and potential therapeutic significance of TRPV6 in human osteoclasts and myeloma cells using shrew venom derived peptide antagonists. Methods: High affinity TRPV6 peptide antagonists SOR-C13 and SOR-C27 derived from the C-terminus of venom from the short-tailed shrew, Blarina brevicauda were used. Human primary osteoclasts were generated in vitro from human bone marrow (BM) aspirates; characterized by Hoechst-phalloidin staining, Tartrate resistant acid phosphatase (TRAP) staining, TRAP enzyme activity and Cathepsin K expression. CD138 positive myeloma cells were isolated from patient bone marrow specimens by EasySep immunomagnetic separation, or examined in tissue microarrays of patient BM core biopsies. TRPV6 expression in primary human osteoclasts, myeloma cell lines and myeloma patient BM microarray was checked by qPCR, immunohistochemistry or immunoblotting. Anti-resorptive potential of SOR peptides using human osteoclasts was evaluated in Osteoassay plates that mimic bone, and myeloma cell growth inhibition was determined by prestoblue cell viability assays. Results: We found strong expression of TRPV6 protein in human myeloma cells and osteoclasts by immunohistochemical staining on myeloma patient bone marrow sections. Human osteoclasts generated in vitro and CD138 positive myeloma patient bone marrow plasma cells were found to express TRPV6. We saw dose-dependent inhibition of osteoclast activity in vitro by SOR-C13 and SOR-C27, including markedly reduced osteoclast formation, decreased TRAP activity and reduced osteoassay surface resorption. TRPV6 peptide antagonists were also found to inhibit the growth of human myeloma cell lines U266 and KMM-1. Conclusion: Anti-myeloma and anti-osteoclast activity of human TRPV6 antagonist peptides was seen in the current study. Taken together, our findings suggest a novel therapeutic approach for multiple myeloma involving TRPV6 inhibition to target both myeloma cells and osteoclasts. Citation Format: Alli Murugesan, Philippe Tremblay, Ming Han, Bithika Ray, Tyler Lutes, Tony Reiman. TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents. [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 3791.

Abstract PR14: In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches

Abstract Background. Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF1) may represent a target for preventing transition from MGUS (micrometastatic-stage) to active-MM (macrometastatic-stage). We therefore evaluated SDF1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity L-oligonucleotide binder to SDF1, looking at its ability to modulate MM cell tumor growth and homing to the BM in vivo and in vitro. Methods. SDF1 levels were evaluated by immunohistochemistry on BM specimens of patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells (BM-MSCs) from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also evaluated. Co-localization of SDF1 with MM cell (MM.1S-GFP+)-enriched BM niches was evaluated using in vivo confocal microscopy. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo MM metastasis model. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) was assessed by using in vivo confocal microscopy and bioluminescence, in mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on explanted femurs. DNA synthesis and adhesion of MM cells in the context of NOX-A12 treated primary MM BM-MSCs in presence or absence of bortezomib were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. Results. Patients with active-MM present with higher BM SDF1 expression vs MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary tumors had higher SDF1 levels compared to healthy subjects, thus suggesting the importance of SDF1 in favoring tumor cell metastasis to BM niches. SDF1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. Importantly, NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo by reduction of MM cells in the BM and increased number of MM cells within the PB compared to control mice (P&amp;lt;.05). This was supported by inhibited homing and dissemination of MM cells to the BM of those mice pre-treated with NOX-A12. NOX-A12 enhanced MM cell sensitivity to bortezomib, in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor growth vs. control (P&amp;lt;.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib-treated mice (P &amp;lt;.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy. In vitro studies confirmed synergism between NOX-A12 and bortezomib in modulating MM cell survival and adhesion to BM-MSCs. Conclusion. SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF1 inhibiting spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of macrometastatic MM. This abstract is also presented as Poster B80. Citation Format: Aldo M. Roccaro, Antonio Sacco, Michele Moschetta, Patricia Maiso, Yuji Mishima, Yosra Aljawai, Fabio Facchetti, Anna Kruschinski, Giuseppe Rossi, Irene M. Ghobrial. In vivo targeting of stromal-derived factor-1 as a strategy to prevent myeloma cell dissemination to distant bone marrow niches. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR14. doi:10.1158/1538-7445.CHTME14-PR14

Evidence of an Epigenetic Origin for High-Risk 1q21 Copy Number Aberrations in Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy stratified in part by cytogenetic aberrations including the high-risk copy number alterations (CNAs) of 17p- and +1q21. CNAs of 1q21 are known to occur as a result of jumping translocations of 1q (JT1q12) which increase the copy number (CN) of 1q21, while at the same time causing collateral genomic damage including deletions and amplifications in some receptor chromosomes (RC). We have previously reported the decondensation of the 1q12 region and triradial chromosomes in the bone marrow specimens of patients with aggressive disease, and have speculated that hypomethylation of the pericentromeric region of 1q12 may play a role in the origin of 1q21 CNAs. To test the hypothesis that 1q12 hypomethylation induces CN gains of 1q21, we used the hypomethylating agent 5-azacytidine to treat blood lymphocyte cultures of five myeloma patients with balanced constitutional 1q12 aberrations as well as five normal control subjects. The patients with constitutional aberrations of 1q12 were selected based on their potential to show either novel CN gains of regions translocated distal to 1q12 or CN gains of 1q21 when 1q12 was translocated to a non-homologous chromosome. The constitutional aberrations included two patients with pericentromeric inversions of chromosome 1 inv(1)(p11q12), and one patient each with a balanced translocation of t(1;2)(q12;p11), t(1;2)(q12;q34), or t(1;9)(q12;q11). These particular aberrations should provide a unique model for testing the association between the hypomethylation of 1q12 and the origin of CN gains of 1q21. In vitro blood lymphocyte cultures of patients and controls were treated for 72 hours with 5-azacytidine at a final concentration of 10uM. 5-azacytidine is known to cause site-specific hypomethylation and chromatin decondensation in the 1q12 region. Utilizing FISH probes for 1q12 (satII/III) and 1q21(CKS1B), we analyzed 100 metaphase cells from each patient and each control for CN gains for 1q21 and any novel region translocated distal to 1q12. To fully characterize the induced rearrangements of 1q12, we also applied inverse G-banding and spectral karyotyping in selected cells. Remarkably, all patients with the balanced constitutional 1q12 rearrangements showed CN gains of chromosome regions on the derivative chromosomes distal to the inverted or translocated 1q12 regions. Recurring aberrations of 1q12 included pericentromeric decondensation and triradials of chromosome arms 1q, 1p, and 2p, in addition to localized pulverizations distal to 1q12 of these same arms. Specifically, in both patients with inv(1), whole-arm CN gains of 1p were identified in 2-3% of cells, while in the patient with t(1;2)(q12;p11), whole-arm gains of 2p (MYCN) were identified in 3% of cells. In these same three patients, on the derivative chromosomes where the 1q12 region was separated from 1q21 by an inversion or translocation, no CN gains of the 1q21 occurred. In the patients with 1q12 translocated to a non-homologous chromosome, 1q21 was amplified on the RC in 4% of the cells in the patient with t(1;2)(q12;q34), and in 3% in the patient with t(1;9)(q12;q11). Control subjects showed CN gains of 1q21on both chromosomes 1 in the range of 3-13%. Whole-arm JT1q12s to RC16q were identified in three patients and three controls. This JT1q12 results in a CN gain of 1q21, and a whole-arm CN loss in RC16q, identical to those found in the bone marrow specimens of MM patients. Interestingly, one control showed JT1q12s to two different RCs, 9q and 12q, causing whole-arm deletions in the long arms of both. The key structural aberration responsible for the generation of CN gains of 1q21 and subsequent JT1q12s is the formation of a triradial involving the 1q12 region. The novel finding in this study is the induction of triradials and CN gains for chromosome arms of 1p and 2p, which demonstrates that epigenetic modifications to 1q12 can amplify non-homologous chromosome regions juxtaposed to it. Furthermore, the CN gains induced here occurred only distal to 1q12 on all normal and derivative chromosomes and mimic the triradials and JT1q12s reported in the bone marrow of patients with aggressive disease. These findings demonstrate for the first time that the hypomethylation of the 1q12 region can potentially amplify any genomic region distal to it, and provide evidence for an epigenetic origin of the high-risk 1q21 CNAs found in the progression of MM. DisclosuresNo relevant conflicts of interest to declare.

Association Between Downregulation of POT1 Expression and Chromosome 7 Deletion, Response to Hypomethylation Agent Treatment, and Patient Survival in Myelodysplastic Syndromes

The POT1 gene is located in chromosome 7 and encodes a key component of the shelterin complex, which is essential for the maintenance of telomere and chromosome integrity. Somatic mutations of POT1 have been identified in chronic lymphocytic leukemia, which indicates that POT1 dysfunction is involved in the pathogenesis of hematological neoplasms. At the same time, abnormal telomere shortening has been observed in MDS/AML and a spectrum of bone marrow failure syndromes. We therefore sought to study the potential role of POT1 in MDS by sequencing the gene and characterizing its expression in primary bone marrow specimens of patients with MDS.We first sequenced all POT1 coding regions that are known to have mutations in CLL. PCR-Sanger sequencing was performed in bone marrow mononuclear cells (BM-MNNC) of a cohort of 30 patients with MDS (15 with RAEB/RAEBT, 11 with RA/RARS/RCMD/MDS-U, 2 with CMML, and 2 with 5q- syndromes). No genetic mutations in the POT1 gene were detected. This result suggests that genetic alteration of POT1 is rare in MDS.We then evaluated the expression of POT1 using cDNA arrays (n=183) or RT-PCR (n=58) in a cohort of 241 patients with MDS from two centers. The median age of our patients was 71 years (32-95). Diagnoses included RAEB in 108 (45%), 5q- syndrome in 18 (8%), and other syndromes (RA, RCMD, and MDS-U) in 115 (47%) cases. In this cohort, 140 (58%) patients were diploid, 22 (9%) had chromosome 7 alterations, 21 (9%) had 5q deletion, and 58 (24%) had other cytogenetic abnormalities.Results indicate that POT1 was underexpressed (less than 50% of the POT1 level in normal controls) in the bone marrow CD34+ hematopoietic progenitor cell population in 138 patients (57%). However, no significant difference was observed between the whole MDS cohort and control BM CD34+ cells from healthy donors (n=25). Further subset analysis based on karyotypes revealed that 81% of patients with chromosome 7 alterations (7- and 7q-) had lower expression of POT1 versus 38% of diploid patients, 35% of 5q patients, and 42% of patients with other cytogenetic alterations (p=0.001). ANOVA testing indicated that expression of POT1 was significantly downregulated (less than 50% of control) only in patients with chromosome 7 alteration (p<0.000) but not in other cytogenetic subsets.When we compared the survival of patients with POT1 downregulation to other groups, we observed a strong tendency toward shorter overall survival in patients with POT1 downregulation (median OS of 37 months [95% CI: 21-52] vs 53 months [95%CI: 30-75]; p=0.139). This tendency toward poorer OS was also observed when we excluded cases with chromosome 7 alterations (37 months [95% CI: 17-57] vs 53 months [95%CI: 25-80]; p=0.186).Next, we evaluated the potential impact of POT1 expression on responses to therapies. In the subgroup of patients with available treatment records for analysis (n=58), a total of 42 patients received hypomethylating agents (HMA), and 47% of them achieved responses. When comparing POT1 expression levels to HMA response, we observed significantly lower POT1 expression in HMA non-responders than in responders (U Mann-Whitney test p= 0.028). In a regression model for response to HMA, we also observed that downregulation of POT1 was associated with a poorer response to HMA (OR 4.96 [1.01-24.37]; p=0.049). However, when we introduced chromosome 7 alterations into the model, POT1 expression lost its effect, which suggests that the impact of POT1 on response to HMA is due to its interaction with chromosome 7 alterations.Taken together, the results of this study indicate that the downregulation of POT1 gene expression, which is related to chromosome 7 deletions, may play a role in the pathogenesis and prognosis of MDS, including response to HMA-based therapies. DisclosuresNo relevant conflicts of interest to declare.

MLL-Rearranged Infant ALL, but Not Standard-Risk ALL, Induces Inflammasome Signaling and Immune Activation

T cell-based therapy for infants with MLL-rearranged B-ALL (MLL-ALL) represents a promising approach in this high-risk, poor-prognosis subset. Factors in the immune microenvironment that influence T-cell function, however, are poorly understood in MLL-ALL. We have observed complete inhibition of healthy donor T-cell proliferation by ALL cells from MLL-ALL, but not standard risk ALL (SR-ALL) patients. T-cell function is restored in the presence of bone marrow macrophages. In this study, we hypothesized that MLL-ALL cells selectively induce the inflammasome- a cytokine signaling complex formed by macrophages in response to damage-associated molecular patterns (DAMPs) released by leukemic cells. In the canonical inflammasome pathway, DAMPs engage macrophage Toll-like receptors, which activate caspase-1 and drive production of T-cell activating cytokines such as IL1-β.First, we determined whether ALL cells obtained from MLL-ALL vs. SR-ALL patients could produce high mobility group box 1 (HGMB1), a prototypical DAMP associated with tumor cell stress and macrophage PRR activation, particularly after anthracycline therapy. CD19+ cells were isolated from the diagnostic bone marrow specimens of patients with MLL-ALL (n=1), SR-ALL (n=4), and healthy donor PBMC (n=2) and treated with either control media or 2uM doxorubicin. Prior to treatment both MLL-ALL and SR-ALL, but not healthy donor cells, expressed HMGB1 protein as measured by Western immunoblot (29 kDa, GADPH internal control). Following doxorubicin treatment, HMGB1 protein was increased in MLL-ALL but not SR-ALL. Soluble HMGB1, measured by ELISA, was significantly increased in MLL-ALL (12.6 + 3.7 ng/ml untreated vs. 51.4 + 1.9 treated) compared to SR-ALL cells (5.0 + 0.8 ng/ml untreated, 4.3 + 1.5 treated, p<0.05). Soluble HMGB1 was not detectable in healthy donor cells before or after treatment.Next, we determined whether the increased HMGB1 secretion observed in MLL-ALL vs. SR-ALL cells was associated with macrophage caspase-1 activation. Control media and 2 uM doxorubicin treated-and-washed MLL-ALL, SR-ALL and healthy control CD19+ cells were cultured alone or 1:1 with allogeneic primary human macrophages for 24 hours. The active isoform of caspase-1 (20 kD tetramer) was detectable by immunoblot only in macrophages exposed to doxorubicin-treated MLL-ALL. Mean levels of IL1-β, measured by ELISA, were significantly increased in cocultures of doxorubicin-treated MLL-ALL + macrophages (1255 pg/ml + 322) compared to untreated MLL-ALL (820 pg/ml + 183, p=<0.05) and doxorubicin-treated SR-ALL + macrophages (494 pg/ml + 85 p<0.01). Macrophages cultured alone did not produce significant levels of IL1-β before or after doxorubicin treatment.Finally, we wished to determine whether expression of Toll-like receptor 4, a surface receptor for HMGB1 known to signal caspase-1, was increased on macrophages exposed to doxorubicin-treated MLL-ALL vs. SR-ALL. Folowing coculture with untreated and doxorubicin treated MLL-ALL and SR-ALL cells, macrophage TLR4 mRNA and surface expression was quantified by qPCR and flow cytometry, respectively. Both mRNA and surface expression of TLR4 was high at baseline, and did not change significantly after coculture. Unexpectedly, however, the MLL-ALL cells themselves displayed a significant increase in TLR4 mRNA (20-fold compared to endogenous beta-glucuronidase, p<0.0001; 12-fold compared to untreated MLL-ALL, p<0.05 by ΔΔCt) and surface expression (median fluorescence intensity 1005 + 89 vs. 367 + 32 respectively, p<0.05) following doxorubicin treatment. This phenomenon was not observed in SR-ALL or healthy CD19+ cells and is consistent with other tumor models of autocrine immune activation by HMGB1.Taken together, these data indicate that primary MLL-ALL cells, but not SR-ALL cells, are capable of inducing a caspase-1 associated inflammasome that enhances both innate and T-cell immune activation following anthracycline therapy. Studies are currently underway with additional primary MLL-ALL samples to determine how strategic augmentation of this pathway may impact the anti-leukemia killing capacity of therapeutic T-cells. DisclosuresNo relevant conflicts of interest to declare.

Clonal chromosomal abnormalities in Philadelphia-negative cells in chronic myeloid leukemia patients treated with nilotinib used in first-line therapy

Nilotinib is an effective option for the first-line treatment of chronic myeloid leukemia (CML) patients in chronic phase (CP). In CML patients, clonal cytogenetic abnormalities (CAs) in Philadelphia-negative (Ph-) metaphases have been widely observed after treatment with imatinib, or dasatinib/nilotinib following failure with imatinib. However, such abnormalities in CML patients treated with nilotinib as the first-line therapy have not been reported. Thirteen CML CP patients with Philadelphia-positive (Ph+) cells were initially diagnosed in our hospital from December 2010 to July 2011. Patients were followed up by clinical assessment, cytogenetic analysis, and BCR-ABL transcriptional level every 3 to 6 months. Retrospective fluorescence in situ hybridization was performed on stored bone marrow specimens of patients when the cytogenetic analysis showed CAs. During nilotinib therapy, 12 (92.3 %), 5 (38.5 %), and 2 (15.4 %) patients achieved complete cytogenetic response, major molecular response, and complete molecular response at 18 months, respectively. Two patients developed CAs in Ph- cells, including trisomy 8 and monosomies 20 and 21. Monosomies 20 and 21 appeared in the same patient simultaneously. Our data confirmed that clonal CAs in Ph- cells is a general phenomenon in Ph+ CML patient treated with tyrosine kinase inhibitors (TKIs), including nilotinib. The clinical significance of these CAs that arise in Ph+ CML patient treated with TKIs and whether these CAs exist before or after treatment of TKIs are not clear.

Spectra of Chromosomal Aberrations in 325 Leukemia Patients and Implications for the Development of New Molecular Detection Systems

This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.

Dose Dependent Effect of Deferasirox on Hematopoietic Progeneitors of Myelodysplastic Syndrome

AbstractAbstract 3994Defarasirox (DFX) is an orally available iron chelator used for management of iron overload in myelodysplastic syndrome (MDS). Given the importance of iron in generation of reactive oxygen species (ROS) and the importance of ROS in hematopoietic progenitor regulation, we sought to examine the effect of varying doses of deferasirox on CD34+ hematopoietic progenitors from MDS patients and compare to control CD34+ progenitors from human umbilical cord blood. CD34+ hematopoietic progenitors were isolated from bone marrow specimens of patients with MDS (who had below 10% bone marrow blasts) and human umbilical cord blood. The proliferation of these progenitors cultured in growth factor-containing medium with iron chelators deferasirox (5, 10 and 20 μM) or deferoxamine (DFO) 10 μM was examined on days 3, 7, and 14 of culture. Differentiation along granulocytic, monocytic and erythroid lineages was examined by flow cytometry. Apoptosis was determined by Annexin-V surface expression. Colony formation assays were performed in methylcellulose after varying periods of exposure to DFX. Total and mitochondrial ROS levels were determined by flow cytometry after exposure to DFX at varying concentrations. Progressive dose-dependent suppression of MDS progenitor proliferation in culture was observed with DFX at dose range of 5μM to 20μM [inhibition at day 14 compared to controls with DFX 5μM 4%, 10μM 60% (p<0.01), 20μM 90% (p<0.001), n=7]. This suppression was more pronounced in MDS compared to cord blood progenitors (DFX 20μM 22% inhibition at day 14 compared to controls, n=5). There was reduced viability of MDS progenitors at 20μM concentration of DFX which increased with duration of exposure in culture (58% viability with DFX 20μM at day 14 compared with 90% for controls, p<0.001, n=7). Viability was not reduced in normal progenitors at this concentration of DFX (83% viability with DFX 20μM at day 14 compared with 85% for controls, n=5). The percentage of CD34+ cells as well as granulocytic and monocytic lineage cells were reduced after day 14 of culture at 10 and 20μM concentrations of DFX compared to 5μM in MDS specimens. Such an effect was not observed in normal cord blood progenitors. 20μM DFX as well as DFO markedly suppressed colony growth when progenitors were plated after 14 days of exposure to chelators, suggestion depletion of primitive hematopoietic progenitors. ROS levels were suppressed by 5μM DFX, but were increased above control at 20μM. DFO (10μM), also had marked suppressive effect on proliferation of MDS progenitors at days 7 and 14 of culture compared to cord blood. We conclude that exposure to the iron chelators DFX and DFO results in enhanced dose-dependent inhibition of proliferation, differentiation and survival in MDS progenitors compared to cord blood progenitors. MDS progenitors are particularly sensitive to the anti proliferative and apoptotic effects of iron chelators at concentrations above 5μM of DFX. Our data have important implications for use of DFX in MDS patients, may explain worsening of cytopenias in some MDS patients receiving DFX therapy, and suggest that lower doses of DFX started earlier in disease course may be the preferable strategy to manage iron overload. Such doses could have favorable impact on hematopoiesis and ROS- induced genomic instability in MDS. Disclosures:Pullarkat:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau. Bhatia:Novartis Pharma: Membership on an entity's Board of Directors or advisory committees.

Effect of heat shock protein-90 (HSP90) and vascular endothelial growth factor (VEGF) on survival in acute lymphoblastic leukemia: an immunohistochemical study

The significance of vascular endothelial growth factor (VEGF) and heat shock protein-90 (HSP90) has received only limited attention especially in acute lymphoblastic leukemia (ALL). In this study, we assessed expressions of HSP90 and VEGF in bone marrow samples of patients with ALL and effect of these expression quantities on the mean overall survival. Using immunohistochemical methods, we assessed expression of HSP90 and VEGF in 22 cases of ALL. Expression of HSP90 was detected in 19/22 (86.4%) and 3/22 (13.6%) of patients with ALL, for strongly positive and moderate-weakly positive, respectively. Negative HSP90 expression was not detected in patients with ALL. Expression of HSP90 in patients with ALL and in control group were statistically significant (P<0.001), however, did not reflect the mean overall survival (P=0.910). Mean OS was evaluated 992±181 and 724.8±88.2 days for moderate-weak and high HSP90 expression, respectively. VEGF expressions were not significantly different between ALL and control groups (P<0.087). We did not find any relationship between HSP90 and VEGF expressions in bone marrow specimens of patients with ALL. This study demonstrated that HSP90 expression grades in patients with ALL were significantly higher than that in controls and presence of strong HSP90 expression was associated with worse overall survival. VEGF expression in patients with ALL was not different from that in control samples. Determination HSP90 with immunohistochemical method in bone marrow can provide information about prognosis.

High avidity myeloid leukemia-associated antigen-specific CD8+ T cells preferentially reside in the bone marrow

AbstractThe activity of allogeneic CD8+ T cells specific for leukemia-associated antigens (LAAs) is thought to mediate, at least in part, the curative effects of hematopoietic stem cell transplantation (HSCT) in myeloid malignancies. However, the identity and nature of clinically relevant LAA-specific CD8+ T-cell populations have proven difficult to define. Here, we used a combination of coreceptor-mutated peptide-major histocompatibility complex class I (pMHCI) tetramers and polychromatic flow cytometry to examine the avidity profiles, phenotypic characteristics, and anatomical distribution of HLA A*0201-restricted CD8+ T-cell populations specific for LAAs that are over-expressed in myeloid leukemias. Remarkably, LAA-specific CD8+ T-cell populations, regardless of fine specificity, were confined almost exclusively to the bone marrow; in contrast, CD8+ T-cell populations specific for the HLA A*0201-restricted cytomegalovirus (CMV) pp65495-503 epitope were phenotypically distinct and evenly distributed between bone marrow and peripheral blood. Furthermore, bone marrow-resident LAA-specific CD8+ T cells frequently engaged cognate antigen with high avidity; notably, this was the case in all tested bone marrow samples derived from patients who achieved clinical remission after HSCT. These data suggest that concomitant examination of bone marrow specimens in patients with myeloid leukemias might yield more definitive information in the search for immunologic prognosticators of clinical outcome.

Serum levels, and bone marrow immunohistochemical expression of, vascular endothelial growth factor in patients with chronic myeloproliferative diseases

Current data suggest that angiogenesis plays a significant role in the pathogenesis and progression of chronic myeloproliferative diseases (cMPDs). In the present study, we evaluated serum levels of vascular endothelial growth factor (VEGF) in 83 patients with cMPDs [myelofibrosis with myeloid metaplasia (MMM, n = 25), essential thrombocythaemia (ET, n = 40), polycythaemia vera (PV, n = 8) and chronic myeloid leukemia (CML, n = 10)] and in 27 healthy individuals. Serum VEGF levels were significantly increased in patients with cMPDs compared to healthy individuals (all p values were ≤ 0.05) and were significantly correlated with bone marrow microvessel density (MVD) (p = 0.0013). In addition, the immunohistochemical expression of VEGF protein in bone marrow biopsy specimens were analyzed in 61 patients with cMPDs, (ET, n = 36 and MMM, n = 25) and in 27 healthy individuals. The cellular distribution of VEGF expression was similar in bone marrow specimens of patients and healthy individuals. VEGF protein was detected mainly in erythroid cells, whereas myeloid cells and megakaryocytes exhibited a variable expression of the protein. The percentage of bone marrow VEGF positive cells was positively correlated with serum levels of VEGF (p = 0.001). The results of the present study suggest that, VEGF is a major angiogenetic factor in patients with cMPDs and contributes to the pathogenesis of these diseases.

CD8+ T cells Reactive to Survivin Antigen in Patients with Multiple Myeloma

Survivin is a member of the inhibitors of apoptosis family and is overexpressed in different types of malignancies. Cytotoxic T cells recognizing survivin epitopes can be elicited in vitro and by vaccination in patients with leukemia, breast cancer, and melanoma. We did this study to investigate whether survivin-specific CD8+ T cells occur in patients with multiple myeloma. An HLA-A2.1-binding survivin peptide was used to detect peptide-specific T cells by a quantitative real-time PCR to measure antigen-specific IFN-gamma mRNA expression in 23 patients with myeloma and 21 healthy volunteers. T cells producing IFN-gamma in response to survivin were further analyzed for expression of CD45RA and CCR7 to determine phenotypic characterization. Additional immunohistochemical analyses of survivin antigen expression in bone marrow specimens of patients was done. T cells recognizing HLA-A2.1-binding survivin peptide were detected in 9 of 23 patients and in 1 of 21 healthy volunteers. Survivin-reactive T cells were identified as terminally differentiated effector T cells (CD8+, CD45RA+, and CCR7-). Positive survivin expression of myeloma cells in bone marrow specimens was shown in 7 of 11 patients. We provide, for the first time, evidence of T cell reactivity against survivin antigen in patients with multiple myeloma. Our data suggest the immunogenicity of survivin antigen in multiple myeloma and that immunotherapeutic strategies using survivin as a target antigen might be an option for patients with this disease.

Experimental Study Enhancing the Chemosensitivity of Multiple Myeloma to Melphalan by Using a Tissue-Specific APE1-Silencing RNA Expression Vector

Purpose:Because of a developing resistance to chemotherapy agents, multiple myeloma (MM) has been an incurable disease until now. As a means to overcome MM tumor cell resistance and/or sensitize tumor cells to chemotherapeutic treatments currently used, we examined the role of human apurinic/apyrimidinic endonuclease 1 (APE1) in resistance and prognosis in patients with MM. Patients and Methods:Multiple myeloma cells were analyzed by using bone marrow specimens from 32 patients with MM and 10 normal volunteers. Results:The positive rate of APE1 protein expression was 65.6% in the bone marrow specimens of patients with MM with known clinical outcome. Positive rate of APE1 expression beyond grade 2 in the relapsed/refractory group was significantly higher than that in the untreated group. No positive results of grade > 2 were detected in bone marrow specimens from patients with noncancerous disease. It was also confirmed that the amount of APE1 protein in KM3 cells was positively correlated with the dose and action time of melphalan. Because APE1 was overexpressed in refractory/relapsed MM cells, siRNA-targeted technology was used to decrease APE1 levels in KM3 cells, with protein levels deceasing to 80%-90% within 24 hours and continuing to decease for 72 hours. The best dose and time of inhibiting expression of APE1 protein were 3 μg and 2 days long. A decrease in APE1 levels in siRNA-treated KM3 cells led to enhanced cell sensitization to melphalan. Conclusion:The findings herein present prognostic and therapeutic implications for treating relapsed/refractory MM. The APE1-silencing RNA results demonstrate the feasibility of the therapeutic modulation of APE1 using a variety of molecules and approaches.

The use of monoclonal antibody R92F6 and polymerase chain reaction to confirm the presence of parvovirus B19 in bone marrow specimens of patients with acquired immunodeficiency syndrome.

Abstract Background.—Parvovirus B19 infection is a cause of chronic anemia and red cell aplasia in patients with acquired immunodeficiency syndrome (AIDS) and in other immunocompromised hosts. Anemia in AIDS patients has a multifactorial etiology, with parvovirus B19 infection being an infrequent but nevertheless treatable cause. Therapy with intravenous immune globulin can result in rapid improvement of parvovirus-induced anemia. This treatment is expensive, therefore accurate and rapid confirmation of parvovirus infection is important in providing appropriate and cost-effective therapy. Methods.—Bone marrow samples from 2 AIDS patients with severe anemia and reticulocytopenia were studied. Bone marrow morphology and serologic studies were evaluated for parvovirus B19 infection. An immunohistochemical method using a monoclonal antibody, R92F6, to B19 capsid proteins was utilized on decalcified, B5-fixed, paraffin-embedded bone marrow biopsies. Bone marrow aspirate cells were examined by electron microsco...