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Current murine models of myeloproliferative neoplasms (MPN) cannot address how MPN can invade hematopoietic organs from a defined single anatomical origin as observed in patients. Murakami et al. developed an immunocompetent wild-type mouse model of Polycythemia Vera (PV) spreading from a single bone marrow site. PV development was abrogated in mice with splenectomy or lacking carbonic anhydrase 1 (CA1) in mutant hematopoietic progenitors. Increased expression of CA1 was found in CD34+ cells from PV patients, and its pharmacologic inhibition decreased variant allele frequency in colony-forming assays, suggesting CA1 as a potential therapeutic target for PV.See article, p. 285.Approximately every fifth patient with myeloproliferative neoplasms (MPN) harbors a CALR mutation of either type I or type II, which differ in clinical features and outcomes. Ibarra et al. show that type I, but not type II, mutant CALR proteins lose Ca2+ binding, causing Ca2+ depletion from the endoplasmic reticulum and activation of the IRE1α/XBP1 pathway. IRE1α inhibition prevented type I-mutant CALR cell survival in culture and thrombocythemia development in mice. These results identify IRE1α/XBP1 as a potentially targetable dependency specific to type I CALR-mutated MPNs.See article, p. 298.By single-cell DNA, RNA and proteogenomic sequencing of matched patient samples, Guess et al. uncovered three clonal evolution patterns associated with clinical progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML). The Static pattern, characterized by minimal clonal change, had founding clones with mutations in DNA methylation genes and transitioned to sAML in the absence of further genomic aberrations, suggesting nongenetic evolution. Clonal architecture changed dramatically in Dynamic groups, often involving acquisition of mutations in signaling pathways (Dynamic-S) or progressive structural aberrations along with TP53 mutations (Dynamic-C). Collectively, these findings elucidate the interplay of genetic and nongenetic factors in leukemic transformation.See article, p. 316.To investigate patterns of subclone evolution during progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML), Menssen et al. sequenced paired MDS and sAML samples from a cohort of patients. Signaling gene mutations were found to occur universally in subclones and undergo complex patterns of clonal evolution, including acquisition, expansion, persistence, and contraction of mutations during progression, with multiple patterns often coexisting in the same patient. The presence of at least one signaling gene mutation at MDS was associated with future progression to sAML in patients with lower risk MDS.See article, p. 330.Nakauchi et al. report the genome-wide landscape of DNA hydroxymethylation (5hmC) in human hematopoietic lineages. The authors map TET2-dependent 5hmC, epigenetic and transcriptional programs matched to competitive advantage, myeloid skewing, and reduced erythroid output in TET2-deficient hematopoietic stem and progenitor cells (HSPC). Vitamin C and azacitidine restore the 5hmC landscape and phenotypes in TET2-mutant HSPCs. These findings offer a comprehensive resource for TET-dependent transcriptional regulation of human hematopoiesis and shed light on the potential mechanisms by which TET deficiency contributes to clonal hematopoiesis and malignancies.See article, p. 346.

P1270: IMMUNE REGULATORY CELLS IN B-CELL LYMPHOMAS: SIGNIFICANT HETEROGENEITY ACROSS HISTOLOGIC TYPES.

Background: Immune regulatory cell populations play a fundamental role in tumor development and tumor immune evasion. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) considered as dominant suppressor cells which are significantly enriched in blood, bone marrow (BM), or tumor sites of patients with hematologic malignancies and often associate with high tumor burden, adverse clinical features, and outcome. Studies in lymphoma patients however, have generated conflicting results, regarding the function and phenotype of MDSC and Tregs mainly due to large heterogeneity of the disease as well as due to the lack of a unanimous nomenclature of these cell subsets in human samples. Furthermore, the molecular mechanisms via which MDSCs and Tregs contribute to clinical outcomes of disease and how they interfere with response to therapy remain elusive. Aims: To investigate the prevalence of immune suppressive population in B-cell lymphomas according to histologic, subtype providing the basis for further analysis of their biologic properties and association with clinical features and outcome of specific lymphoma subtypes. Methods: Peripheral blood (PB) and BM samples were collected from healthy individuals (n= 22) and lymphoma patients (n=50). Specifically, lymphoma subtypes were as follows:Marginal Zone (MZL, N=19), Diffuse Large B-cell Lymphoma (DLBCL, N=18) and Follicular Lymphoma (FL, N=13). Peripheral blood mononuclear cells (PBMCs) were isolated with ficoll gradient and subjected to flow cytometry analysis. MDSC characterization was based on HLADR-/lowCD11b+CD33- expression with CD14+CD15- for M-MDSCs and CD14-CD15+ for PMN-MDSCs. In addition, Treg cells were denoted as CD4+CD25highCD127-Foxp3+. Expression of PDL1 and PD1 on MDSCs and Tregs respectively, was also determined by flow cytometry. M-MDSCs and PMN-MDSCs were sorted from BM samples, RNA was extracted in order to subjected to transcriptomic analysis (RNAseq). Informed consent was obtained. Results: Frequencies of MDSCs were significantly increased in PB of all lymphoma patients compared to healthy individuals, with MZL subtype exhibiting the most prominent increase of MDSCs (p<.0001). Notably numbers of M-MDSCs were increased in FL and MZL patients and to a lesser extend in DLBCL patients while PMN-MDSCs were elevated in all subtypes with MZL displaying the higher frequencies. Regarding the Treg cells, although all lymphoma patients exhibited increased absolute numbers of CD4+CD25highCD127- Treg cells, Foxp3-expressing Tregs were significantly increased in the PB of DLBCL and MZL patients, but not in FL patients. Of interest, CD4+ and CD8+ T cells in the PB of DLBCL patients exhibited increase expression of PD1 compared to respective cell subsets from healthy individuals. Summary/Conclusion: Our findings comprehensively demonstrate enhanced numbers of MDSC and Treg immune suppressive cells in the PB of B-cell lymphoma patients while an increased heterogeneity was observed among the different histological lymphoma types. Functional characterization of MDSCs and Tregs by transcriptomic (RNAseq) and proteomic analysis are ongoing.

Abstract 6290: Microenvironment begets phenotype: Tumor specific CD8+ T cells derived from bone marrow and other lymphoid sites exhibit differential exhaustion profiles

Abstract Current immunotherapy approaches aim to mitigate or reverse CD8+ T cell exhaustion to generate meaningful anti-tumor immune responses. However, the multi-step process of exhaustion fixes epigenetic programs that permanently scar T cells, limiting immunotherapeutic efficacy. Rather than reversing T cell exhaustion, we hypothesize that exploiting tumor-specific T cells from protected niches could yield therapies with improved anti-tumor benefit. A subset of TCF1-expressing precursor exhausted CD8+ T cells (TPEX) drives the anti-tumor immune response. The tumor draining lymph nodes (dLN) maintain a reservoir of TPEX cells which replenishes intra-tumoral effector T cells. Similarly, the bone marrow (BM) houses long-lived memory T cells with specificity against a broad spectrum of antigens, including tumor-associated antigens. Unlike T cells from the dLN or tumor infiltrating lymphocytes (TILs), BM T cells can be easily acquired from all patients at any disease stage. We used the B16.OVA-OT-I model to delineate how exhaustion affects tumor-specific T cells within the BM, dLN, spleen (SP), and tumor (TUM) during tumor progression. This model uses adoptively transferred transgenic, OVA-specific OT-I cells as a source of tumor-specific CD8+ T cells. High-dimensional immune profiling using spectral flow cytometry enabled us to characterize in vitro and in vivo anti-tumor functionality and the phenotype of OT-I cells derived from different compartments. Interestingly, upon adoptive transfer, OT-I cells resident in the TUM, dLN, BM, and SP or found in circulation were heterogenous in terms of function, persistence, and stemness. These findings suggest that discrete tissues impart location-specific T cell adaptations, and that tissue microenvironment markedly influences the exhaustion profile of tumor-specific T cells. OT-I cells derived from distinct lymphoid organs exhibited differential capacity for cytokine production and response to cognate antigen, especially compared to their TIL counterparts. Importantly, we identified BM- and dLN-derived CD8+ T cells as less exhausted and more functional than TILs. Moreover, BM resident T cells appear phenotypically more stem-like and similar to TPEX. Further studies to characterize this population are ongoing. This work implies that, in the context of localized disease, the kinetics of T cell exhaustion differ in lymphoid organs as compared to TILs. Leveraging a less-exhausted pool of tumor-specific cells may lead to greater anti-tumor benefit with new approaches to adoptive cell therapy. As the BM is accessible in all patients, understanding where tumor-specific BM T cells fall on the spectrum of exhaustion and memory could justify their use for such applications. Ongoing work aims to transcriptionally characterize tumor specific BM CD8+ T cells and evaluate their role in anti-tumor immunity. Citation Format: Elizabeth Maria Zawidzka, Luca Biavati, Amy Thomas, Megan Heimann, Ervin Griffin, Ivan Borrello. Microenvironment begets phenotype: Tumor specific CD8+ T cells derived from bone marrow and other lymphoid sites exhibit differential exhaustion profiles [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 6290.

Abstract LB100: Dickkopf-1 induces breast cancer progression by limiting T cell infiltration into the tumor microenvironment

Abstract Activation of Wnt/β-catenin signaling pathway in the tumor cells has been shown to promote cell proliferation and correlate with cancer progression. Therefore, dickkopf-1 (Dkk1), a soluble Wnt/β-catenin inhibitor, has been considered as a tumor suppressor. However, contradicting evidence demonstrated that high levels of circulating Dkk1 correlate with poor prognosis in various cancers. Although Dkk1 is best known to modulate bone remodeling, we recently reported that Dkk1 supports the growth of subcutaneous B16 melanomas by creating an immune-suppressive environment. However, the detailed mechanism by which Dkk1 alters anti-tumor immune responses remains to be elucidated. In this study, we used mice orthotopically injected with hormone-resistant luminal B, ER+ PyMT, EO771 and triple-negative 4T1 cell lines. We find that Dkk1 is upregulated in all models and its neutralization reduces tumor growth. To our surprise, Dkk1 was barely expressed by each tumor lines tested. However, we found elevated expression of Dkk1 in the bone by the osteoblasts (OBs) and in the breast tumor stroma by cancer-associated fibroblasts (CAFs), although at a much lower level compared to the OBs. To address the role of bone-derived versus tumor stroma-derived Dkk1 during tumor progression, we generated mouse models specifically deleting Dkk1 from OBs (OsxCre;Dkk1fl/fl), and from CAFs (Fsp1Cre;Dkk1fl/fl and aSMACreERT2;Dkk1fl/fl). To our surprise, all our mouse models reduced orthotopic PyMT tumor growth significantly, suggesting that Dkk1 could act both systemically and locally to modulate tumor progression. Interestingly, however, deletion of Dkk1 in the bone or in the CAFs did not appear to change the immune composition in the bone marrow, spleen, or at the tumor site. Next, we orthotopically injected PyMT cells into the severely immune-compromised NSG mice treated with anti-Dkk1 or IgG. Strikingly, Dkk1 neutralization did not reduce tumor growth in NSG mice, suggesting that Dkk1 modulates breast cancer growth by altering anti-tumor immune responses. Similar to mice with deletion of Dkk1 in the bone or in the CAFs, Dkk1 neutralization did not alter the percentage of the myeloid and lymphoid cell subsets or the activation status of T cells in bone marrow, spleen, or tumor site compared to IgG control mice. However, by IHC we observed that tumors from anti-Dkk1-treated mice or with deletion of Dkk1 in the CAFs had increased numbers of CD4+ and CD8+ T cells infiltrating the center of tumor mass while these immune populations remained at the edges of the tumors in control mice. In summary, we provide evidence that Dkk1 exerts local and systemic effects to promote tumor progression by modulating the infiltration of immune cells into the TME. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Dickkopf-1 induces breast cancer progression by limiting T cell infiltration into the tumor microenvironment [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 LB100.

Characterization of Erythroferrone in a Teleost Fish (Dicentrarchus labrax) With Two Functional Hepcidin Types: More Than an Erythroid Regulator.

Erythroferrone is a recently identified erythroid regulator produced by erythroblasts in the mammalian bone marrow and extramedullary sites, known to be induced in conditions of anemia or blood loss. Iron metabolism is affected by erythroferrone through its capacity to inhibit hepcidin production, leading to the increase of iron availability required for erythropoiesis. However, little is known about erythroferrone function in other vertebrates, in particular teleost fish, that unlike mammals, present two different functional types of hepcidin, one type mostly involved in iron metabolism and the other in antimicrobial response. The study of erythroferrone evolution and its biological role in teleost fish can give us valuably new insights into its function. To address these questions, we characterized erythroferrone in the European sea bass (Dicentrarchus labrax), a species presenting two hepcidin types, and evaluated variations in its expression levels in response to different experimental conditions. During experimental anemia, erythroferrone responds by increasing its expression and suppressing hepcidin production, following the pattern observed in mammals, but it is not influenced by iron overload. However, during bacterial infection, erythroferrone is downregulated and hepcidin levels increase. Furthermore, administration of Hamp1 but not of Hamp2 peptides suppresses erythroferrone expression. In conclusion, in dual hepcidin teleost fish erythroferrone seems to only interact with type 1 hepcidin, known to be involved in iron homeostasis, but not with type 2, which has an almost exclusive antimicrobial role.

Abstract 14092: Arachidonate 5 Lipoxygenase Deficiency Drives Age-Related Obesity, Macrophage Dysfunction in Cardiac Repair, and Omnipresence of Inflammation

Introduction: Age-related inflammation is the primary hallmark of heart failure with reduced and preserved ejection fraction; however, the fundamental source of inflammation is unclear. Arachidonate 5 lipoxygenase (ALOX5) is known to direct the biosynthesis of leukotrienes and proinflammatory mediators (PIMs) that orchestrate the initiation of inflammation following myocardial infarction (MI). Hypothesis: Thus, we set out to test the role of ALOX5 to understand inflammation-resolution signaling in acute HF in aging. Methods: Male C57Bl/6J and ALOX5 -/- mice were divided into two groups: 1) young (2-4 months) and 2) aging (18 months). Mice were subjected to MI surgery that develops acute heart failure maintaining no-MI as naïve controls. Cellular and molecular mechanisms are detemined. Results: The absence of ALOX5 leads to age-related obesity with series of metabolic defects, including fatty liver. Next-gen sequencing analysis revealed that immune regulating pathways of infarcted myocardium were dysregulated in young and aging ALOX5 -/- mice compared to respective age-matched controls. In response to cardiac injury, ALOX5 -/- mice fail to initiate an inflammatory response with reduced leukotrienes B 4 and PIMs, with marked obesity-related chronic inflammation in immune defense hubs: spleen bone marrow and LV infarcted site. At the molecular level, ALOX5 deletion also reduced resolution mediators (specialized proresolving mediators; SPMs), thereby making macrophage dysfunctional with the decrease in SIRP-α. Aging superimposed on ALOX5 deficiency triggers macrophage dysfunction with the increase in proinflammatory phenotype (Ly6C hi ) and notable upregulation of CXCR4 and downregulation of resolution receptor formyl peptide receptor 2; in infarcted heart post-MI. Conclusions: Presented age-related outcome provides translational insight that ALOX5 serves as a connector for initiating PIM (turn-on) and SPMs (turn-off) essential for macrophage function and cardiac repair processes. Thus, future research is warranted to validate the role of ALOX5 in cardiometabolic defects, PIMs, and resolution mediators (SPMs) in heart failure with reduced and preserved ejection fraction.

Discovering Myeloid Cell Heterogeneity in Mandibular Bone - Cell by Cell Analysis.

The myeloid-derived bone marrow progenitor populations from different anatomical locations are known to have diverse osteoclastogenesis potential. Specifically, myeloid progenitors from the tibia and femur have increased osteoclast differentiation potential compared to myeloid progenitors from the alveolar process. In this study, we explored the differences in the myeloid lineage progenitor cell populations in alveolar (mandibular) bone versus long (femur) bone using flow cytometry and high-throughput single cell RNA sequencing (scRNA-seq) to provide a comprehensive transcriptional landscape. Results indicate that mandibular bone marrow-derived cells exhibit consistent deficits in myeloid differentiation, including significantly fewer myeloid-derived suppressor cell (MDSC)-like populations (CD11b+Ly6C+, CD11b+Ly6G+), as well as macrophages (CD11b+F4/80+). Although significantly fewer in number, MDSCs from mandibular bone exhibited increased immunosuppressive activity compared to MDSCs isolated from long bone. Using flow cytometry panels specific for bone marrow progenitors, analysis of hematopoietic stem cells showed no defects in mandibular bone marrow in LSK (Lin–Sca1+cKit+) cell and LK (Lin–Sca1–cKit+) cell populations. While there was no significant difference in granulocyte progenitors, the granulocyte-monocyte progenitors and monocyte progenitor population were significantly decreased in the mandibular bone marrow. T-lymphocyte subsets were not significantly different between mandibular and femoral bone, except for CD4+CD25+Foxp3+ regulatory T lymphocytes, which were significantly increased in the mandible. In addition, B lymphocytes were significantly increased in mandible. Single cell RNA sequencing from mandible and femur BM revealed distinct differences in transcriptomic profiles in myeloid populations establishing previously unappreciated aspects of mandibular bone marrow populations. These analyses reveal site-specific differences in the myeloid progenitor cellular composition and transcriptional programs providing a deeper appreciation of the complex differences in myeloid cell heterogeneity from different anatomical bone marrow sites.

AML-137: Expression of Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit Gene (EZH2) In Acute Myeloid Leukemia

<h3>Background:</h3> Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of the chromatin-modifying enzyme polycomb group (PRC)2. As a complex, these proteins selectively silence target genes through trimethylation of histone H3 at lysine 27. EZH2 is strongly oncogenic. It has been observed in various malignancies, which makes it an interesting therapeutic target. Whether it function as a tumor suppressor or oncogene is not settled in acute myeloid leukemia (AML). <h3>Objective:</h3> This study was done to determine the relative expression level of <i>EZH2</i> in a cohort of Egyptian patients with adult newly diagnosed AML. <h3>Materials and Methods:</h3> The present study included 45 <i>de novo</i> AML and 40 healthy persons of matched age and sex as a control group. The following was performed on all patients: complete blood count, bone marrow examination, cytochemical and immunophenotyping studies, conventional cytogenetics, and hepatic and renal function tests. Detection of <i>EZH2</i> gene expression level by real-time quantitative polymerase chain reaction was done for all patients and controls. <h3>Results:</h3> The median age of our patients was 33 years. Males constituted 66.7% (30 patients) of our patients, and females constituted 33.3% (15 patients). The majority of our patients (71.1%, 32 patients) were M5. Cytogenetic studies revealed the presence of normal cytogenetic results in 28 (62.1%) patients. <i>EZH2</i> expression was lower in AML patients compared with controls (p <0.001). There was no difference in <i>EZH2</i> levels when considering age, sex, bone marrow blasts, cytogenetic study, or type or site of infection. Low <i>EZH2</i> expression was associated with higher mortality in 31(68.9%) patients and resistance to chemotherapy in 39 (86.6%) patients. <h3>Conclusions:</h3> From this study, we concluded that low <i>EZH2</i> is common in Egyptian AML patients. It functions as tumor suppressor gene, rather than an oncogene. Its presence is associated with resistance to chemotherapy and high mortality rate.

Remission, treatment failure, and relapse in pediatric ALL: an international consensus of the Ponte-di-Legno Consortium

Comparison of treatment strategies in de novo pediatric acute lymphoblastic leukemia (ALL) requires standardized measures of efficacy. Key parameters that define disease-related events, including complete remission (CR), treatment failure (TF; not achieving CR), and relapse (loss of CR) require an updated consensus incorporating modern diagnostics. We collected the definitions of CR, TF, and relapse from recent and current pediatric clinical trials for the treatment of ALL, including the key components of response evaluation (timing, anatomic sites, detection methods, and thresholds) and found significant heterogeneity, most notably in the definition of TF. Representatives of the major international ALL clinical trial groups convened to establish consensus definitions. CR should be defined at a time point no earlier than at the end of induction and should include the reduction of blasts below a specific threshold in bone marrow and extramedullary sites, incorporating minimal residual disease (MRD) techniques for marrow evaluations. TF should be defined as failure to achieve CR by a prespecified time point in therapy. Relapse can only be defined in patients who have achieved CR and must include a specific threshold of leukemic cells in the bone marrow confirmed by MRD, the detection of central nervous system leukemia, or documentation of extramedullary disease. Definitions of TF and relapse should harmonize with eligibility criteria for clinical trials in relapsed/refractory ALL. These consensus definitions will enhance the ability to compare outcomes across pediatric ALL trials and facilitate development of future international collaborative trials.

The Efficacy and Toxicity of Blinatumomab in Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia: Russian Multicenter Experience

Introduction. The prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (R/R ALL), especially after allogeneic hematopoietic stem cell transplantation (allo-HSCT), is very poor. Therapeutic options for these pts are limited. Blinatumomab is a bispecific T-cell engager (BiTE®) antibody construct with dual specificity for CD19 and CD3. BiTE® therapy may help to overcome the resistance to chemotherapy (CT) with minimal toxicity, and may be a bridge to allo-HSCT.Patients and Methods. We analyzed data of 52 pts from 7 hematologic centers in Russian Federation with CD19 positive R/R ALL, who received BiTE®. The median age was 21 (range 1-53) years, 18 (35%) pts <18 yrs, 34 (65%) pts ≥18 yrs. The diagnosis was ALL B-I (EGIL) subtype in 15 pts, B-II - in 29, B-III - in 5 pts, B-IV - in 2 pts, and 1 patient had mixed phenotype leukemia (M5 (FAB) and B-ALL). Five (10%) pts had Philadelphia positive (Ph+) ALL. Primary refractory in 1 pts. In 19 pts it was the 1st relapse of ALL, in 17 - 2nd, in 15 - 3rd. In 47 pts it was isolated bone marrow relapse, in 5 pts - combined relapse (bone marrow and extramedullary sites). The bone marrow blast infiltration was <50% in 27 pts, >50% in 25 pts. Disease relapse was revealed after CT in 31 pts. Allo-HSCT after BiTE® therapy was performed 12 (39%) pts (4 - from related, 5 - from unrelated, 3 - from haploidentical donors). Twenty one pts received BiTE® due to relapse after allo-HSCT (6 - from related, 12 - from unrelated, 3 - from haploidentical donors). Six pts received 2nd allo-HSCT after BiTE® therapy. In 10 pts with posttransplant relapse donor lymphocyte infusion (DLI) was used in combination with BiTE®. Every patient received from 1 to 7 cycles (median 2) of BiTE®.Results. Complete remission (CR) was achieved in 31/52 (60%) pts: in 18/31 (58%) pts with ALL relapse after CT, in 13/21 (62%) pts - after allo-HSCT. In 22/31 (71%) pts with CR after BiTE® therapy minimal residual disease was negative. Pts with less than 50% blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with 50% blasts or higher (70% (19/27) vs 48% (12/25)). Response rates were similar although the number of relapse - 58% (11/19) in 1st relapse, 59% (10/17) in 2ndrelapse and 60% (9/15) in 3drelapse. Pts with posttransplant ALL relapse who received BiTE® in combination with DLI had higher response rate than pts, who received BiTE® as monotherapy: 70% (7/10) vs 54% (6/11) respectively. One-year OS was 50% (95% CI 25-75%). One-year DFS was 42% (95% CI 18-66%). Grade ≥3 hematological toxicity (neutropenia, thrombocytopenia) was observed in 14 (27%) pts, grade ≥3 liver toxicity - in 6 (12%) pts. Ten pts (19%) developed toxic neuropathy during the therapy. Cytokine release syndrome occurred in 5 (9%) pts. One pts after allo-HSCT (but not after DLI) developed grade I aGVHD. One pts after allo-HSCT developed grade II aGVHD after BiTE® in combination with DLI. There were no fatal treatment related toxicity. Nine (29%) pts who responded to BiTE® had relapse, 6 of them bone marrow and 3 extramedullary sites. Nine (29%) pts died: 5 pts - due to R/R ALL, 2 pts due to infection, 1 case of transplant related mortality and 1 pts due to second solid tumor (synovial sarcoma).Conclusion. The treatment of R/R ALL with BiTE® is effective and has acceptable toxicity. We demonstrated high efficacy in therapy of posttransplant ALL relapses, especially when BiTE® was combined with DLI, perhaps, due to additional immunological effect of the transplant. DisclosuresNo relevant conflicts of interest to declare.

Deptor As a Target with Chemical Inhibitors; Alternative Therapy in Multiple Myeloma

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and/or extramedullary sites. The increase in PFS and overall survival (OS) has been achieved through the introduction of high dose therapy (HDT) with autologous stem cell transplantation (SCT), and the introduction of thalidolamide, bortezomib and lenalidomide. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. It is known that inhibition of deptor results in the inhibition of proliferation and induction of apoptosis in myeloma cells. In addition, high levels of deptor are predictive of a poor response to thalidomide in myeloma, indicating that levels of deptor expression is important as a prognostic marker for myeloma patients and would be a possible target for a novel therapeutic strategy, as using a small specific chemical inhibitor of deptor. In this study, we developed a novel small molecule chemical inhibitor (43 M) capable of inducing inhibition of the mTOR/Deptor interaction and results in negative regulation of the deptor that leads in the inhibition of proliferation and induces apoptosis in several cell lines of MM deptor-positive. This effect correlated with the Deptor expression level. The cytotoxic effect of 43 M depends on caspase activity. We analyzed potential mechanisms of 43 M, our findings showed that inhibition of Deptor/mTOR Pathway induces the activation of p70 and AKT. This leads to the induction of apoptosis. We analyzed whether the degradation of 43M-induced deptor-dependent proteosome complex using MG132, and this one prevent deptor degradation. Recent studies describe that the constitutive or inducible activation of the p38 MAPK pathway is considered an important molecular event contributing to the malignant phenotype of the tumor cell in MM. Therefore, these studies identify the interaction of p38 MAPK with Deptor as a new therapeutic target to improve the outcome of patients in MM. We analyzed the possible interaction of p38 MAPK with Debtor mediate a library in yeast. For confirmation of the possible interaction we analyzed the expression of p38 MAPK after treatment with 43 M, the data show that they induce an activation of p38 MAPK and that correlates inversely with the degradation of the deptor. However, simultaneous treatment of MM with 43M and SB203580 does not prevent the induction of 43M-mediated apoptosis. Additionally, the 43M effect was analyzed in a xenografic model of MM cell lines. 8226 MM cell lines were inoculated in 6-week Balb/C nu/ nu mice via s.c. When the tumor reached 500 mm3, they were inoculated on twice with 43 M at different concentrations per group (0, 20 or 40 mg / kg) and the tumor growth and the general state of the mice were subsequently analyzed. Tumor tissue was obtained and the expression of Deptor and 4E-BP1 by IHC was evaluated. The data showed that 43 M significantly inhibited tumor growth at the two doses tested 10 or 20 mg / kg and in tumors decreased the expression of Deptor and induction of 4E-BP1 activation. This study describes for the first time the possible role of Daptor as a therapeutic target using a chemical inhibitor capable of inducing a cytotoxic effect in cell lines positive for Deptor by inducing the degradation of this and activation p38 MAKP in the MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. DisclosuresNo relevant conflicts of interest to declare.

Dosimetric Evaluation Between the Conventional Volumetrically Modulated Arc Therapy (VMAT) Total Body Irradiation (TBI) and the Novel Simultaneous Integrated Total Marrow Approach (SIMBa) VMAT TBI.

PurposeThe purpose of this study was to assess the treatment planning feasibility of volumetrically modulated arc therapy total body irradiation (VMAT TBI) using a simultaneous integrated marrow and body approach (SIMBa). We also aimed to compare SIMBa TBI with the more conventional VMAT TBI approach using the entire body as the target. The goal of using an integrated approach like SIMBa is to balance the known clinical benefit of TBI with the toxicity decrease of Total Marrow Irradiation (TMI) using two prescription volumes. In anticipation of a clinical trial to investigate a novel conditioning regimen that uses SIMBa, our institution retrospectively analyzed the dosimetric differences between 20 clinical VMAT TBI which were re-planned using SIMBa.MethodsTwenty patients who previously received conventional VMAT TBI at our institution with a dose of 12 Gy in six fractions were re-planned using SIMBa with a planning aim of delivering a uniform dose of 12 Gy to at least 90% of the PTV_BodyEval. The planning aims of SIMBa were to deliver a uniform dose of 12 Gy to at least 90% of the PTV_Marrow and 8 Gy to at least 90% of the PTV_TotalBody while limiting the mean lung dose to less than 8 Gy. The plans were normalized so that 100% of the PTV_Marrow received at least 90% of the dose with the PTV_TotalBody optimized to stay as close to 100% at 90% as possible.ResultsAll 20 patient plans achieved 12 Gy/8 Gy to at least 90% of the PTV_Marrow and PTV_TotalBody, respectively, with max doses of <16 Gy (130%). As compared with the delivered TBI, the following reductions in mean dose were notable: small bowel 21.3±4.2%, lung 16.3±7.9%, heart 25.3±8.6%, and kidney 16.4±6.2%. Coverage of the sanctuary sites was maintained despite a significant reduction to sensitive organs at risk (OARs).ConclusionThis study supports that VMAT TBI treatment planning with SIMBa is feasible. In this sample, SIMBa provided dosimetrically similar doses to marrow and sanctuary site doses as TBI while achieving lower doses to OARs. A clinical trial is needed to investigate the clinical implications of VMAT TBI with SIMBa.

Assessment of medullary and extramedullary myelopoiesis in cardiovascular diseases

Recruitment of innate immune cells and their accumulation in the arterial wall and infarcted myocardium has been recognized as a central feature of atherosclerosis and cardiac ischemic injury, respectively. In both, steady state and under pathological conditions, majority of these cells have a finite life span and are continuously replenished from haematopoietic stem/progenitor cell pool residing in the bone marrow and extramedullary sites. While having a crucial role in the cardiovascular disease development, proliferation and differentiation of innate immune cells within haematopoietic compartments is greatly affected by the ongoing cardiovascular pathology. In the current review, we summarize key cells, processes and tissue compartments that are involved in myelopoiesis under the steady state, during atherosclerosis development and in myocardial infarction.

Progression signature underlies clonal evolution and dissemination of multiple myeloma

AbstractClonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.

Dental implants: a potential cause of bone marrow edema in the jaw\u2014preliminary report

BackgroundDifferent magnetic resonance imaging (MRI) sequences are frequently used to examine bone marrow in the jaw, including short tau inversion recovery (STIR). MRI is a sensitive method for detecting bone marrow lesions. Currently, pantomography and computed tomography (CT) are used frequently for preoperative dental implant treatment. However, no study has evaluated bone marrow edema around dental implants using MRI. This study aimed to assess bone marrow edema in the jaw around dental implants using brain magnetic resonance images.MethodsThis retrospective cohort study was approved by our university ethics committee (EC19-011). A total of 17 patients (170 sites) who underwent brain MRI between April 2010 and March 2016 were analyzed. All subjects underwent scanning more than 3 years after implant placement. This study investigated two bone marrow signals (with implant site and without implant site). These two groups were then compared using Fisher’s exact test. The Mann–Whitney U test was used to analyze bone marrow signal intensity as the dependent variable and the long and short-axis diameters of the implant as the independent variables.ResultsThe were 22/31 sites (71%) and 38/139 sites (27%) of bone marrow edema in the dental implants and without dental implants groups, respectively (p < 0.001). Furthermore, there was a significant correlation between bone marrow signal intensity and the short-axis diameter of the implant (p < 0.001).ConclusionThe signal intensity in the bone marrow sites in the jaw with dental implants was significantly higher than that in the sites without dental implants. The present study findings suggest that dental implants are a potential cause of bone marrow edema in the jaw.

Mechanisms of Immune Evasion in Acute Lymphoblastic Leukemia.

Simple SummaryStudies conducted in a recent decade revealed that acute lymphoblastic leukemia cells exploit various mechanisms to avoid immune recognition and destruction by the immune system. As a consequence, leukemia cells become more resistant to treatment, which results in poor patient outcomes. In this review, we describe the interactions between acute lymphoblastic leukemia (ALL) cells and selected populations of immune cells within the bone marrow microenvironment that contribute to leukemia development, promotion, and relapse. We also discuss how various elements of the bone marrow niche affect ALL chemo- and immunotherapy and present recently discovered treatment strategies that restore or stimulate the immune response to ALL.Acute lymphoblastic leukemia (ALL) results from a clonal expansion of abnormal lymphoid progenitors of B cell (BCP-ALL) or T cell (T-ALL) origin that invade bone marrow, peripheral blood, and extramedullary sites. Leukemic cells, apart from their oncogene-driven ability to proliferate and avoid differentiation, also change the phenotype and function of innate and adaptive immune cells, leading to escape from the immune surveillance. In this review, we provide an overview of the genetic heterogeneity and treatment of BCP- and T-ALL. We outline the interactions of leukemic cells in the bone marrow microenvironment, mainly with mesenchymal stem cells and immune cells. We describe the mechanisms by which ALL cells escape from immune recognition and elimination by the immune system. We focus on the alterations in ALL cells, such as overexpression of ligands for various inhibitory receptors, including anti-phagocytic receptors on macrophages, NK cell inhibitory receptors, as well as T cell immune checkpoints. In addition, we describe how developing leukemia shapes the bone marrow microenvironment and alters the function of immune cells. Finally, we emphasize that an immunosuppressive microenvironment can reduce the efficacy of chemo- and immunotherapy and provide examples of preclinical studies showing strategies for improving ALL treatment by targeting these immunosuppressive interactions.

A New PET Probe for Imaging Chimeric Antigen Receptor T Cells.

HomeRadiology: Imaging CancerVol. 3, No. 2 PreviousNext Research HighlightsFree AccessA New PET Probe for Imaging Chimeric Antigen Receptor T CellsGary D. LukerGary D. LukerGary D. LukerPublished Online:Mar 26 2021https://doi.org/10.1148/rycan.2021219004MoreSectionsPDF ToolsImage ViewerAdd to favoritesCiteTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinked In Take-Away Points■ Major Focus: To investigate the feasibility of imaging chimeric antigen receptor (CAR) T cells with a zirconium 89–labeled antibody targeted to inducible T-cell costimulator (ICOS or CD278).■ Key Result: ImmunoPET imaging of ICOS enabled the visualization of activated CAR T cells without affecting persistence or function of these cells in a mouse model of B-cell lymphoma.■ Impact: ImmunoPET with antibody detection of the endogenous activation marker ICOS provides a potentially general approach to image any CAR T-cell therapy.CAR T cells are a type of cancer therapy based on engineering a patient’s own T cells with a receptor for a specific protein target on cancer cells. After expansion of the cells in a laboratory, a patient receives an infusion of the engineered CAR T cells. For efficacy, the cells must migrate to tumors, proliferate, as well as become and remain activated to kill target cancer cells. CAR T cells have shown impressive successes in some blood cancers, such as B-cell malignancies, but efficacy remains limited in solid tumors. A major challenge in understanding successes and failures of CAR T-cell therapy is the ability to monitor these cells in vivo. Current methods rely on measuring numbers of cells in the circulation, but this approach does not reliably reflect actual migration of CAR T cells into a tumor.Simonetta and colleagues analyzed human and mouse CAR T cells and identified ICOS as a promising marker of activation to an antitumor state. The team labeled an antibody to ICOS with zirconium 89, providing a probe for PET imaging of CAR T cells in a mouse model of B-cell lymphoma. PET imaging showed greater accumulation of the immunoPET probe to ICOS in mouse bone marrow sites of lymphoma. Tracer doses of the immunoPET antibody did not affect persistence or function of CAR T cells as cancer immunotherapeutics. While the current study investigated therapy in mice, the study suggests that immunoPET imaging of ICOS could become a clinically translatable, general approach to monitor cancer therapy with a wide range of CAR T cells.Highlighted ArticleSimonetta F, Alam IS, Lohmeyer JK, et al. Molecular imaging of chimeric antigen receptor T cells by ICOS-ImmunoPET. Clin Cancer Res 2021;27:1058–1068. doi: https://doi.org/10.1158/1078-0432.CCR-20-2770Highlighted ArticleSimonetta F, Alam IS, Lohmeyer JK, et al. Molecular imaging of chimeric antigen receptor T cells by ICOS-ImmunoPET. Clin Cancer Res 2021;27:1058–1068. doi: https://doi.org/10.1158/1078-0432.CCR-20-2770 Crossref, Medline, Google ScholarArticle HistoryPublished online: Mar 26 2021 FiguresReferencesRelatedDetailsRecommended Articles CD38-targeted Immuno-PET of Multiple Myeloma: From Xenograft Models to First-in-Human ImagingRadiology2020Volume: 295Issue: 3pp. 606-615Nanotechnology for Cancer Imaging: Advances, Challenges, and Clinical OpportunitiesRadiology: Imaging Cancer2021Volume: 3Issue: 318F FDG PET/CT and Novel Molecular Imaging for Directing Immunotherapy in CancerRadiology2022Volume: 304Issue: 2pp. 246-264Identification of HER2-Positive Metastases in Patients with HER2-Negative Primary Breast Cancer by Using HER2-targeted 89Zr-Pertuzumab PET/CTRadiology2020Volume: 296Issue: 2pp. 370-378Radiotheranostics in Cancer Diagnosis and ManagementRadiology2018Volume: 286Issue: 2pp. 388-400See More RSNA Education Exhibits Immune Effector Cell Therapy for Solid Tumors: Opportunities for Interventional RadiologyDigital Posters2019When Things Go Wrong, Characteristic PET CT Patterns of Triple and Double-Hit Lymphoma; A Pictorial ReviewDigital Posters2018Imaging Biomarkers in Targeted Therapies: From Quantitative Imaging to RadiomicsDigital Posters2019 RSNA Case Collection Benign bone marrow stimulation RSNA Case Collection2020Primary Calvarial Lymphoma RSNA Case Collection2021Primary Bone LymphomaRSNA Case Collection2020 Vol. 3, No. 2 Metrics Altmetric Score PDF download

Abstract PD9-11: Identifying breast cancer survivors with dormant disseminated tumor cells: The PENN-SURMOUNT screening study

Abstract Background: Patients (pts) treated for early stage breast cancer have a 30% lifetime risk of developing incurable, distant metastatic disease. Current models suggest that this occurs through escape of cells from the primary tumor into the circulation and subsequent sequestration of “disseminated tumor cells” (DTCs), in the bone marrow and other sequestration sites, where they enter dormancy. DTCs identified by immunohistochemistry (IHC) are associated with poor prognosis in longitudinal studies and meta-analyses, increasing odds of recurrence by approximately 2 to 5-fold. However, little is known about the test characteristics of the DTC-IHC assay, clinical DTC detection rates over time, and patient and disease risk factors that can identify pts harboring these cells. Methods: The PENN-SURMOUNT Screening Study (NCT 02732171) is a prospective, longitudinal study examining bone marrow and blood biomarkers of recurrence among pts within 5 years of diagnosis who have completed therapy for primary breast cancer (with the exception of endocrine therapy). Pts with positive lymph nodes, triple negative receptors, ER-positivity with RS ≥ 25 and/or high-risk MammaPrint (MP), or residual disease after neoadjuvant chemotherapy were screened with bone marrow aspirate (BMA) for presence of DTCs. A positive DTC-IHC result is defined by the presence of at least one pancytokeratin-DAB positive cell utilizing the methods of Naume et al. Cytospin slides prepared from the BMA are independently reviewed by two pathologists with adjudication for the presence of DTCs; Pts who screen negative for DTCs are offered repeat screening annually. Pts who screen positive are referred to an interventional clinical trial (CLEVER, NCT 03032406). Results: A total of 194 pts screened eligible for enrollment on PENN-SURMOUNT between 6/2016 and 3/2020. Of these, 158 consented and 151 underwent BMA with successful IHC analysis on 100%. Pts came from 22 U.S. states; ≥ 1/3 traveled over 50 miles to the study center. At baseline BMA, 36/151 (24%) had at least 1 measurable DTC by IHC. Patient characteristics and DTC distribution among subpopulations are shown in Table 1. Of the 78/115 who were initially DTC negative and continued to be eligible for repeat screening, as of 3/2020, 46 (59%) returned for at least one repeat BMA. 13/46 (28%) had at least 1 detectable DTC on 1 of up to 3 subsequent follow up assessments for a total DTC positivity rate of 32.5% (49/151). 48 (98%) DTC+ pts have subsequently enrolled on the CLEVER trial. Conclusions: BMA assessment for DTCs is feasible in pts with high risk, early stage breast cancer. DTCs are detected in up to a third of breast cancer survivors with repeat assessment during the surveilance period. DTC positivity rates are relatively similar across all receptor subtypes, and after both neoadjuvant and adjuvant chemotherapy. Pts harboring DTCs are highly likely to enroll on interventional trials designed to reduce recurrence risk. Table 1. Patient characteristics and distribution of % DTC positivity among subpopulationsDTC+ (N=49)DTC- (N=102)Total (N=151)DTC+ Rate (Overall: 32.5%)DEMOGRAPHICSMedian Age at BMA (yrs)51.9 (43.9-60.6*)50.5 (42.9-58.1)50.5 (43.8-58.8)N/ARaceCaucasian44 (89.8%)91 (89.2%)135 (89.4%)32.6%African American5 (10.2%)9 (8.8%)14 (9.3%)35.7%Other0 (0%)2 (2.0%)2 (1.3%)0%Menopausal StatusPre-15 (30.6%)34 (33.3%)49 (32.5%)30.6%Post-34 (69.4%)68 (66.7%)102 (67.5%)33.3%BMI at BMA (kg/m2)24.2 (21.9-28.9*)26.9 (23.4-31.4)26.1 (22.8-30.4)N/ARECEPTOR STATUSER/PR+ HER2neg (by ASCO/CAP)24 (49.0%)51 (50.0%)75 (49.7%)32.0%HER2+ (any ER/PR)9 (18.4%)12 (11.8%)21 (13.9%)42.9%ER/PRneg HER2neg23 (46.9%)48 (47.1%)71 (47.0%)32.4%RISK CRITERIALymph Node Positive24 (49.0%)65 (63.7%)89 (58.9%)27.0%Triple Negative (ER/PR&amp;lt;10%)27 (55.1%)50 (49.0%)77 (51.0%)35.0%Non-pCR11 (22.4%)25 (24.5%)36 (23.8%)30.6%RS ≥ 25 and/or High Risk MP6 (12.2%)8 (7.8%)14 (9.3%)42.9%Median T size (cm) -excluding NACT2.1 (1.5-2.9*)1.8 (1.3-2.8)1.8 (1.3-2.9)N/APRIOR THERAPYAdjuvant Chemo25 (51.0%)60 (58.8%)85 (56.3%)29.4%Neoadjuvant Chemo22 (44.9%)41 (40.2%)63 (41.7%)34.9%Endocrine Therapy19 (38.8%)47 (46.1%)66 (43.7%)28.8%XRT29 (59.2%)75 (73.5%)104 (69.3%^)27.9%*Ranges represent interquartile range^ XRT data not available on 1 patient; n=150 was used to figure percentage Citation Format: Lauren Bayne, Isoris Nivar, Brooke Goodspeed, Shannon Deluca, E. Paul Wileyto, Natalie Shih, Anupma Nayak, Michael D Feldman, Joshua Edwards, Kevin Fox, Jennifer M. Matro, Susan Domchek, Hayley Knollman, Rachel Jankowitz, Angela Bradbury, Payal D. Shah, Jewell Graves, George Woodfield, Elizabeth Chislock, Jianping Wang, George Belka, Lewis A. Chodosh, Amy S. Clark, Angela DeMichele. Identifying breast cancer survivors with dormant disseminated tumor cells: The PENN-SURMOUNT screening study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-11.

Central Nervous System Infiltration in Acute Lymphoblastic Leukemia in a Dog

Background: Acute lymphoblastic leukemia (ALL) is a malignant neoplasia in which there is proliferation of lymphoid progenitor cells in the bone marrow, blood, and extramedullary sites. This disorder has a fast and progressive development; in dogs, cases of infiltration of ALL cells in the central nervous system (CNS) are uncommon and rare. Diagnosis can be achieved with the help of the clinical history and physical, radiographic, hematological, myelographic, and cerebrospinal fluid (CSF) tests in patients with or without neurological clinical signs. The present report aims to describe a case of ALL and the presence of lymphoblasts in the CSF of a dog with neurological clinical signs.Case: An 8-year-old Lhasa Apso dog was examined at the Veterinary Hospital of Universidade Federal do Paraná, Curitiba campus. At the physical examination, the animal exhibited apathy and paralysis of pelvic limbs, which progressed to tetraplegia. Abdominal palpation revealed presence of hepatosplenomegaly and absence of lymphadenomegaly. No alterations were observed in radiographs of the cervical, thoracic or lumbar spine. A complete blood count revealed presence of non-regenerative anemia (hematocrit = 22%), extreme lymphocytosis (185,229 cells/µL), lymphoblasts at a level of 72% (133,364 cells/µL), and thrombocytopenia (66,000 platelets/µL). The biochemical tests revealed increased alkaline phosphatase (859 IU/L). The levels of alanine aminotransferase, creatinine, urea, total protein, albumin, and globulin were normal. The diagnosis of ALL was achieved with the help of a myelogram. The myelogram findings included 39% of mature lymphocytes and 59% of lymphoblasts exhibiting large size, spherical shape, poorly delimited borders, with a high nucleus/cytoplasm ratio, marked cytoplasmic basophilia, and 2 to 3 evident nucleoli; metarubricytes (1%) and promyelocytes (0.6%) were also observed. The CSF contained an increased number of nucleated cells (27 cells/µL) comprising lymphocytes (43%), macrophages (33%), and segmented neutrophils (24%). Of the 11.6 lymphocytes per µL of CSF, 8.1 were lymphoblasts, which indicates infiltration of ALL cells in the CNS. The animal died one day after collection of bone barrow and CSF. Discussion: Relevant alterations observed in this case included the neurological signs caused by the infiltration of neoplastic cells in the CNS, severe leukocytosis and lymphocytosis, with large amounts of lymphoblasts in the blood and predominance of lymphoblasts in the bone marrow, which are alterations typically found in ALL. The animal also exhibited non-regenerative anemia and thrombocytopenia, which were secondary to infiltration of leukemic cells in the bone marrow. The CSF exhibited pleocytosis (27 cells/ µL), and 30% of the cells observed were lymphoblasts. Lymphoblast infiltration in the CNS of leukemic dogs is rare, and other studies have reported absence of neurological signs or neurological signs different from those observed in the present study. CSF analysis in indicated in cases of leukemia to assess leukemic cell infiltration in the CNS. In the case reported here, the plasma level of alkaline phosphatase was increased (859 IU/L) as a consequence of hepatomegaly and hepatic cholestasis. ALL is a very aggressive, proliferative neoplasia, and the resulting lymphoblasts infiltrated the CNS of the animal. In cases of ALL, performing complete blood count, myelogram, and CSF analysis is indicated whether the patients exhibit neurological signs or not.

Establishment of a Patient-Derived Acute B Lymphoblastic Leukemia (B-ALL)Ex Vivo and In Vivoplatform for Evaluation of Novel Therapeutic Agents

B-cell Acute Lymphoblastic Leukemia (B-ALL)is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors in bone marrow, blood and extramedullary sites. It is more prevalent in children and adolescent and is one of the top leading cause of death related to malignancies in this population. Majority of young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has a high relapse rate. Besides, prognosis in adult B-ALL patient is worse than younger individuals. Thus, there is an increasing need for development of novel and targeted therapeutic strategies to improve outcome in elderly and resistant individual. Lack of suitablein vitroculture conditions for primary B-ALL cells severely impairs their experimental accessibility and evaluation of new therapeutics. To corroborate theex vivostudies in a more natural microenvironment there is an unmet necessity of a robust preclinical patient derived xenograft model for B-ALL. We have established and optimized a short-term culture system that supports survival of primary B-ALL cellsex vivofor evaluation and/or screening of candidate agents. Our B-ALL bank comprise of patient-derived specimens with complete cytogenetics information including Philadelphia translocation positive and negative samples and surface marker expression. Different medias, supplements and culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL specimens. Cell growth/viability was assessed using Cell titer-Glo assay. Out of 13 primary B-ALL models studied 5 models proliferated in the optimized media and 5 remain stable and viable till day 3. Phenotypic characterization by flow cytometry showed that all the models stably expressed CD19 and CD20 throughout the assay. To verify the applicability of the 3-day ex vivo system for drug testing, models were tested for sensitivity towards chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC50 17nM to 0.3 µM doxorubicin), (IC50 3 nM to 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive, when treated with small molecule inhibitor imatinib. Next, to develop patient derived xenograft preclinical B-ALL models for drug efficacy study , sub lethally irradiated NCG mice were transplanted intravenously with two million PBMC from B-ALL patients(n=14). 100% engraftment(n=5/model P1) was observed in all the models, but the latency of engraftment(&amp;gt;3%) in peripheral blood varied from 15 days to 3.5 months depending on the aggressiveness of the models. The time of passage varied from 40 to 120 days at P1. Mice were sacrificed when 10-20% of hCD45+ve cells were observed in peripheral blood by flow cytometry. 30-95% of the bone marrow was occupied by hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells were lymphoid-restricted and matched the immunophenotype of the corresponding patient. On serial transplantation in secondary NCG mice (n=15/model P2,P3), these models showed similar engraftment pattern and immunophenotype characteristic. Thus , here we show that theex vivoculture condition optimized at Champions supports growth and proliferation of patient derived B-ALL specimen and is a good platform for screening new therapeutic drugs for B-ALL . We also demonstrate that NCG mice supports the engraftment and development of B-ALL disease initiating and leukemic cells in vivo, allowing to create a robust passagable patient derived preclinical xenograft model that recapitulates the disease and probably could be used for drug efficacy study. Disclosures No relevant conflicts of interest to declare.