Presence Of Leukemic Cells Research Articles

Microfluidic Affinity Selection of B-Lineage Cells from Peripheral Blood for Minimal Residual Disease Monitoring in Pediatric B-Type Acute Lymphoblastic Leukemia Patients.

Assessment of minimal residual disease (MRD) is the most powerful predictor of outcome in B-type acute lymphoblastic leukemia (B-ALL). MRD, defined as the presence of leukemic cells in the blood or bone marrow, is used for the evaluation of therapy efficacy. We report on a microfluidic-based MRD (MF-MRD) assay that allows for frequent evaluation of blood for the presence of circulating leukemia cells (CLCs). The microfluidic chip affinity selects B-lineage cells, including CLCs using anti-CD19 antibodies poised on the wall of the microfluidic chip. Affinity-selected cells are released from the capture surface and can be subjected to immunophenotyping to enumerate the CLCs, perform fluorescence in situ hybridization (FISH), and/or molecular analysis of the CLCs' mRNA/gDNA. During longitudinal testing of 20 patients throughout induction and consolidation therapy, the MF-MRD performed 116 tests, while only 41 were completed with multiparameter flow cytometry (MFC-MRD) using a bone marrow aspirate, as standard-of-care. Overall, 57% MF-MRD tests were MRD(+) as defined by CLC numbers exceeding a threshold of 5 × 10-4%, which was determined to be the limit of quantitation. Above a threshold of 0.01%, MFC-MRD was positive in 34% of patients. The MF offered the advantage of the opportunity for efficiently processing small volumes of blood (2 mL), which is important in the care of pediatric patients, especially infants. The minimally invasive means of blood collection are of high value when treating patients whose MRD is typically tested using an invasive bone marrow biopsy. MF-MRD detection can be useful for stratification of patients into risk groups and monitoring of patient well-being after completion of treatment for early recognition of potential impending disease recurrence.

Effect of CD22-directed CAR-T cells secreting anti-CD19 T cell engagers on control of leukemia progression compared to tandem anti-CD19/CD22 CAR-T cells.

2550 Background: Antigen-specific cancer immunotherapies, based on engineered T cells bearing chimeric antigen receptors (CARs) or the systemic administration of bispecific T cell-engagers (TCEs), have a significant impact on relapsed/refractory (R/R) B cell malignancies. However, a significant percentage of patients relapse following CAR-T or TCE therapy, with antigen loss accounting for up to one third of relapses/progressions. To avoid antigen loss after administration of single-targeted CAR-T cells and to minimize tumor escape, strategies targeting two antigens simultaneously have been developed and validated in both preclinical models and clinical trials. Nevertheless, despite lowering the risk of antigen loss, these strategies still have some limitations, mainly related to design and manufacturing challenges. Methods: In this context, we have generated the first dual-target strategy for hematological malignancies based on T cells bearing an anti-CD22 CAR and secreting an anti-CD19 T-cell engager antibody (CAR-STAb-T cells) and conducted a comprehensive preclinical study comparing its therapeutic potential with a previously validated anti-CD19/CD22 tandem CAR therapy (TanCAR-T) for B-cell acute lymphoblastic leukemia (B-ALL). Results: We have demonstrated in both short- and long-term assays, using contact and non-contact co-culture systems, that CAR-STAb-T cells efficiently redirect bystander T cells, resulting in enhanced cytotoxic activity compared to that exhibited by TanCAR-T cells at very low E:T ratios. In addition, CAR-STAb-T cells induce more potent and faster cytotoxic responses than TanCAR-T cells in both short- and long-term co-culture assays when reproducing antigen-downmodulated conditions in vitro. In vivo assays conducted in NSG mice transplanted with a B-ALL patient-derived xenograft (PDX), followed by intravenous injection of CAR-STAb-T or TanCAR-T cells under a T cell-limiting experimental setting, also showed that CAR-STAb-T cells maintained a tighter control of tumor progression than TanCAR-T cells in peripheral blood and bone marrow. Conclusions: In conclusion, we have demonstrated that the combination of a cell surface CAR and a soluble TCE recognizing different antigens may be advantageous over the use of conventional multi-targeted strategies based on cell surface-anchored receptors. Furthermore, we have proven that a small number of transduced CAR-STAb-T cells is sufficient to redirect non-transduced bystander T cells specifically and efficiently in the presence of leukemia cells, providing a significant advantage over current dual-targeted CAR-T cell therapies. CAR-STAb-T cells could therefore become an alternative to CAR-T therapies for the treatment of R/R B cell malignancies, especially in lymphodepleted patients with low T cell counts.

Contribution of the TIME in BCP-ALL: the basis for novel approaches therapeutics.

The bone marrow (BM) niche is a microenvironment where both immune and non-immune cells functionally interact with hematopoietic stem cells (HSC) and more differentiated progenitors, contributing to the regulation of hematopoiesis. It is regulated by various signaling molecules such as cytokines, chemokines, and adhesion molecules in its microenvironment. However, despite the strict regulation of BM signals to maintain their steady state, accumulating evidence in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) indicates that leukemic cells can disrupt the physiological hematopoietic niche in the BM, creating a new leukemia-supportive microenvironment. This environment favors immunological evasion mechanisms and the interaction of these cells with the development and progression of BCP-ALL. With a growing understanding of the tumor immune microenvironment (TIME) in the development and progression of BCP-ALL, current strategies focused on "re-editing" TIME to promote antitumor immunity have been developed. In this review, we summarize how TIME cells are disrupted by the presence of leukemic cells, evading immunosurveillance mechanisms in the BCP-ALL model. We also explore the crosstalk between TIME and leukemic cells that leads to treatment resistance, along with the most promising immuno-therapy strategies. Understanding and further research into the role of the BM microenvironment in leukemia progression and relapse are crucial for developing more effective treatments and reducing patient mortality.

Toll-like Receptor-9 Agonist Reverses T Cell Exhaustion in a Murine Model of Pediatric B-ALL

Despite significant treatment advances, the poor long-term prognosis for children with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) persists. The application of immune cell-based therapies, such as chimeric antigen receptor-expressing T cells and bispecific T-cell engagers, has improved progression-free survival for many B-ALL patients. However, the majority of these patients still experience disease relapse. An improved understanding of the mechanisms that impair T cell function in B-ALL patients could inform strategies to further improve outcomes. Recent studies have revealed T cell exhaustion, especially as indicated by PD-1 and Tim- 3 expression, to be predictive of disease relapse and overall survival in B-ALL. However, the precise contribution of T cell dysfunction to B-ALL development remains unclear. In this study, we explore the biology that leads to impaired T cell function and investigate potential approaches to restore it using both transgene-driven and syngeneic leukemia cell transplant models of B-ALL. First, we used the Emu-Ret transgenic mouse model of hyperdiploid B-ALL to investigate the induction of exhaustion-associated marker expression (PD-1, CTLA-4 and Tim-3) on CD4 and CD8 T cells. Longitudinal analysis of marker expression in various settings revealed that an upregulation of PD-1 and Tim-3, but not CTLA-4, on T cells occurred only in the presence of leukemia cells, not during the extended, nascent preleukemia phase. This suggests that T cell exhaustion occurs exclusively during the established malignancy phase, highlighting the dynamic nature of T cell responses as disease progresses. Next, B-ALL cells from moribund Emu-Ret mice were transplanted into 4-6-week-old wild type BALB/c mice and markers of T cell exhaustion were measured as disease progressed. Expression of PD-1 and Tim-3 on T cells correlated with disease progression. Notably, a significant increase in these markers was already detectable at low leukemia burden. These results indicate that T cell exhaustion is not simply a reflection of high disease burden and confirm the differing abilities of preleukemic and leukemic cells to affect T cell phenotype. Previously, we have demonstrated the ability of toll-like receptor (TLR) ligands to induce immune control over B-ALL cells. Thus, we investigated the impact of CpG oligodeoxynucleotides (CpG ODN), a ligand for TLR-9, on B-ALL depletion and T cell dysfunction. The treatment of leukemia bearing mice with CpG ODN led to a decrease in PD-1 and Tim-3 expression on T cells, which was also associated with a reduction in leukemia burden. We are currently investigating the functional implications of these expression changes using in vitro assays of T cell proliferation, cytokine production, and cytotoxicity. Additionally, we are also assessing if anti-tumour control by T cells can be revived in vivo, by comparing B-ALL progression and mouse survival after administration of antibodies against exhaustion markers, with or without CpG ODNs as adjuvant therapy. Overall, this study reveals that the functional impact of interactions between B-ALL cells and the immune system evolves during leukemia development. These insights could inform therapeutic strategies to overcome B-ALL immune evasion mechanisms and enhance patient outcomes.

Treatment of Acute Lymphoblastic Leukemia with PDT Regimen Improves the Prognosis of Newly Diagnosed Central Nervous System Leukemia

Objective To investigate whether pediatric-like regimen can improve the prognosis of newly diagnosed acute lymphoblastic leukemia (ALL) complicated with central nervous system leukemia (CNSL) and reduce the occurrence of CNSL during treatment compared with the adult regimen. Methods We collected the clinical data and survival data of 1023 patients with newly diagnosed ALL in the hematology department of Nanfang Hospital from January 2006 to December 2021. The adult regimen cohort included 612 patients (age≥14 years) diagnosed between January 2006 and December 2015, all of whom were treated with the Hyper-CVAD regimen. The pediatric-like regimen cohort included 411 patients diagnosed between January 2016 and December 2021, all of whom were treated with the PDT-ALL-2016 regimen (a GRAALL2003 backbone, pediatric-inspired regimen therapy). Newly dignosed central nervous system leukemia(Ndx CNSL) is defined as the presence of leukemia cells on cytological examination of the cerebrospinal fluid or significant neurological signs. Differences between OS and EFS in Kaplan-Meier curves were determined using the log-rank test in the R package “survival”. Results Among 1023 patients, a total of 59 patients had Ndx CNSL, including 35 patients (5.72%) in the adult regimen cohort and 24 patients (5.84%) in the PDT-ALL-2016 cohort. There were no statistically significant differences in baseline characteristics between the two groups. In the cohort of Ndx CNSL, after a median follow-up of 42.48 months, overall survival (OS) and event-free survival (EFS) of the adult cohort were significantly inferior than the PDT-ALL-2016 cohort (3-year OS, 14.26%vs38.2%, P=0.044; 3-year EFS, 12.42%vs37.1%, P=0.039). During the chemotherapy treatment, CNSL occurred in 69 patients (11.27%) in the adult regimen cohort and 29 patients (7.06%) in the PDT-ALL-2016 cohort, with a significant difference (P=0.032). Conclusion Compared with the adult regimen, the pediatric-like regimen PDT-ALL-2016 improves the prognosis of ALL patients with newly diagnosed CNSL and reduces the occurrence of CNSL during the chemotherapy treatment. This research was supported by the National Natural Science Foundation of China(NFSC82170163, 81970147), Clinical Study of Nanfang Hospital(LC2016ZD009/2019CR012).

Outcomes of Extramedullary Acute Myeloid Leukemia with Gemtuzumab Ozogamicin: Real-World Experience

Introduction: Extramedullary AML (EM AML) is characterized by the presence of leukemic cells in organs or tissues outside of the blood or bone marrow and remains a therapeutic challenge with dismal response rates to standard chemotherapy and short overall survival. Gemtuzumab Ozogamicin (GO) is an anti-CD33 antibody drug conjugate currently approved for single-agent therapy or in combination regimens for CD33+ relapsed/refractory (R/R) or newly diagnosed (ND) AML, respectively. Although GO is an active drug in AML, the optimal employment of this agent for treatment of EM AML at initial diagnosis, or at relapse, remains to be defined. Methods: A retrospective chart review on outcomes of GO in adults with EM AML at our institute from 2000 to 2023 was performed. Variables collected included age, sex, type of myeloid malignancy, and European LeukemiaNet (ELN) risk, bone marrow, peripheral blood and radiographic involvement, Response Evaluation Criteria in Solid Tumors (RECIST), prior lines of treatments, including stem cell transplant, next-generation sequencing (NGS), cytogenetics, mutational profile, and overall survival (OS). We screened patients with a diagnosis of EM AML who received GO at any point during their EM AML therapy. Patients with only CNS involvement as the extramedullary site were excluded. The aims of this study were to characterize responses and potential clinical benefit of EM AML to GO based regimens in the upfront and R/R settings. Results: 32 patients(pts) with EM AML received GO: 8 (25%) were ND and received GO-based induction regimen, while 22 (68.75%) received GO for R/R AML, and 2 (6.25%) patients received GO both at initial diagnosis and relapse. The median age of patient with ND EM AML was 66 years (y) (range: 28-84), and 60.5 (43-73) y in R/R EM AML. Twenty-four (75%) were men. One (12.5%) ND EM AML and 5 (10.1%) R/R EM AML had >1 extramedullary sites involved. The most frequent sites of EM-AML were skin (25%), lymph nodes (25%), soft tissues (25%), solid organs (25%), and visceral body cavity fluids (12.5%). Based on ELN 2022 risk group criteria, 17 patients were adverse, 8 with intermediate, and 3 with favorable risk. Next-Generation Sequencing (NGS) available for 21 patients identified mutations in TP53 (14.28%), FLT3 (19.04%), NPM1 (19.04%), KMT2A (19.04%), and ASXL1 (19.04%) as the most common mutations. Normal karyotype was noted in 6 (25%) R/R EM AML, while complex karyotype was present in 1 (12.5%) ND EM AML and 10 (41.67%) of R/R EM AML. While none of the ND pts had prior myeloid malignancy, R/R pts had prior AML (15), MDS (2), MDS-EB (1), CMML (1), therapy-related AML (1), myelofibrosis (1), and myeloid sarcoma (1). Concomitant bone marrow involvement at identification of EM AML was present in most ND cases (7/8, 88%) and R/R settings (16/23, 69.56%) (Table 1). Combination 7+3 plus GO was most frequently administered for ND EM AML (3/8) and R/R EM AML (8/24, 33%). Eleven patients had measurable radiologic findings prior to and following GO. Median OS was 10.91 months (m) (range: 0.2 to 27.1) for ND EM AML and 1.58 m (range: 0.7 to 7.5) for R/R EM-AML (Figure 1). 3 of 8 newly diagnosed EM-AML underwent allogeneic bone marrow transplant (AlloBMT) after GO based induction, followed by relapsed EM AML in 1 patient. Despite 50 percent of R/R EM AML pts undergoing prior AlloBMT, none had a durable disease-free response and received GO after relapse. Based on RECIST criteria, 7 of 11 (63.63%) pts achieved a response including 1 Complete Response (disappearance of all target lesions), 6 Partial Response (at least a 30% decrease in the sum of target lesions), and 2 Stable Disease (neither sufficient shrinkage nor increase). The 30-day mortality for ND and R/R were 12.5% and 8.33%, respectively. The 60-day mortality for ND and R/R were 25% and 50%, respectively. Conclusion: Our real-world single-institute experience in patients with EM AML suggests that GO combined with chemotherapy in the upfront setting results in promising outcomes with high overall response rates (CR 50%, PR 13%) by RECIST criteria, durable remissions, and prolonged overall survival in largely favorable and intermediate risk AML. However, despite high partial response rates (4% CR, 50% PR) following GO-based therapy in R/R EM AML, survival was less than 2 months likely due to the high proportion of patients in this cohort with poor risk disease. Further investigations and larger studies are warranted to establish the optimal use of GO in EM AML.

Cannabinoids Induce Cell Death in Leukemic Cells through Parthanatos and PARP-Related Metabolic Disruptions

BACKGROUNDWe have previously described the antitumor effect of the cannabinoid WIN-55,212-2 (WIN-55) and a set of cannabinoid derivatives (CNB) specific for CB2 in multiple myeloma (Barbado et al, 2018). In AML, we also observed a potent and selective antileukemic effect, affecting signaling and metabolic pathways essential for the viability of tumor cells. Among them, we found an increased stress on the endoplasmic reticulum, mitochondrial damage, and alteration of the metabolism of ceramides, although none of these events turned out to be the main trigger of cell death, since the inhibition of each of them did not prevent the antileukemic effect of CNB.On the other hand, disruption of the mecanisms of DNA repair have been identified as a key oncogeneic event in different solid tumors, and some studies have also suggested that it might be involved in leukemogenesis. More specifically, PARP1 is involved in DNA damage repair. Other functions include the regulation of glycolysis enzymes through the addition of Poly ADP-Ribose (PAR) and the execution of Parthanatos, that occurs whenever PARP-1 becomes over-activated in response to extreme damage inducing nuclear translocation of AIF and depletion of NAD +. OBJECTIVESIn this study we set out to identify the ultimate mechanism that justifies the aforementioned pleiotropic effect of CNB on the metabolism of leukemic cells and their viability. METHODSCell viability was determined by MTT and flow cytometry. The mRNA and / or protein expression profile of AML samples or healthy progenitor cells were studied by qPCR and / or Western blot. Glycolytic flux was studied with the XF Glycolytic Rate Assay (Seahorse Biosciences). NAD + levels and glycolytic enzyme activity were measured using quantification kits. Parylation of different enzymes were confirmed by Co-IP using the corresponding antibodies.Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60-Luciferase cell line. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. Also we tested the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. RESULTSPretreatment of leukemic cells with Olaparib, a PARP1 inhibitor, reversed WIN-55 induced apoptosis by almost 100%. WIN-55 affected the activity of most glycolysis enzymes, with a marked drop of the activity of GAPDH and pyruvate kinase which was reversed by Olaparib pretreatment. Also G6PDH activity was markedly affected upon culture with WIN-55. Co-IP confirmed parylation of these enzymes which was reversed with Olaparib.ECAR data detected by Seahorse also confirmed the drop in glycolytic capacity produced by WIN-55 in leukemic cells which again was reversed upon culture with Olaparib. In addition, the addition of nicotinamide mononucleotide (NAM), a precursor of NAD +, reversed the loss of viability produced by WIN-55. Next, we confirmed that PARP1 levels were significantly higher in leukemic cell lines and in a series of 40 AML patients as compared to healthy hematopoietic stem cells (HSC).Finally, we observed a translocation of AIF to the nucleus, confirming that WIN-55 produces PARTHANATOS.In a murine model we confirmed treatment with WIN-55,212-2 significantly prolonged survival in AML xenograft mice, with disappearance of the leukemic clone in a significant proportion of cases. By contrast, cannabinoids did not affect the viability of hematopoietic stem cells (HSC) in vivo, resulting in a lack of myeloid toxicity in healthy treated mice. CONCLUSIONSWIN-55 exerts a selective antileukemic effect through the overactivation of PARP1, affecting the levels of parylation in enzymes involved in glycolysis and pentose phosphate pathways, leading to the translocation of AIF to the nucleus and to depletion of NAD +, which were reversed through PARP1 inhibition. These effects are not observed in normal HSC. These data are confirmed in murine models in which we confirmed the antileukemic effect of WIN-55 withouth hampering normal hematopoiesis. [Display omitted] DisclosuresPérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Imbalance of Chemokines and Cytokines in the Bone Marrow Microenvironment of Children with B-Cell Acute Lymphoblastic Leukemia.

In the hematopoietic microenvironment, leukemic cells secrete factors that imbalanced chemokine and cytokine production. However, the network of soluble immunological molecules in the bone marrow microenvironment of acute lymphoblastic leukemia (ALL) remains underexplored. Herein, we evaluated the levels of the immunological molecules (CXCL8, CCL2, CXCL9, CCL5, CXCL10, IL-6, TNF, IFN-γ, IL-17A, IL-4, IL-10, and IL-2) in the bone marrow plasma of 47 recently diagnosed B-cell acute lymphoblastic leukemia (B-ALL) patients during induction therapy using cytometric beads arrays. The results demonstrated that B-ALL patients showed high levels of CXCL9, CXCL10, IL-6, and IL-10 at the time of diagnosis, while at the end of induction therapy, a decrease in the levels of these immunological molecules and an increase in CCL5, IFN-γ, and IL-17A levels were observed. These findings indicate that B-ALL patients have an imbalance in chemokines and cytokines in the bone marrow microenvironment that contributes to suppressing the immune response. This immune imbalance may be associated with the presence of leukemic cells since, at the end of the induction therapy, with the elimination and reduction to residual cells, the proinflammatory profile is reestablished, characterized by an increase in the cytokines of the Th1 and Th17 profiles.

Avaliação Citotóxica do Óleo Essencial de Siparuna cymosa Frente a Células de Leucemia Mielóide

This work presents the chemical characterization of the essential oil obtained from the Siparuna cymosa (Siparunaceae), an endemic species of the Atlantic Rainforest, as well as its cytotoxicity assessment in the presence of tumor cells. The oil was extracted from dry leaves by hydrodistillation and characterized (GC-MS); and its cytotoxicity was assessed by 3-(4,5-dimethyl-2-thyazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric method in AML, Acute Myeloid Leukemia (THP-1) and CML, Chronic Myeloid Leukemia (K652) cell lines. The average oil content was 2.11%, and the majority compound was a-bisabolol (68.9%) and, in significant amounts, p -cimen-9-ol (7.9%) and spathulenol (3.7%). The IC 50 obtained were the 25.44 ± 1.55 μg mL -1 (CML), and 30.88 ± 2.45 μg mL -1 (AML). The essential oil exhibits moderate cytotoxic action in the presence of leukemia cells, especially AML, for which the selectivity index was higher. The oil's cytotoxic action in the presence of the essential oil’s antitumor cells may be associated with the presence of the component a-bisabolol, a substance with recognized antitumor action. Keywords: Siparunaceae; GC-MS analysis; bisabolol; THP-1; K562

Investigation of Cell-Contact Dependent Alterations in Immune Synapse Formation between T Cells and BCP-ALL Cells

Cancer-development is often associated with a state of immune attenuation and impaired antigen-recognition, contributing to the failure of immune surveillance. Defective immunological synapse formation between T cells, B cells and leukemic cells has been demonstrated in patients with chronic lymphocytic leukemia (CLL) (Ramsay, A.G., et al., The Journal of Clinical Investigation, 2008. 118(7): p. 2427-37). Furthermore, CLL cells induce T cell dysfunction in a cell-contact dependent manner. Both actin cytoskeleton changes and alterations in RhoGTPase regulation and activation have been reported in T cells of CLL patients (Ramsay, A.G., et al., Blood, 2013. 121(14): p. 2704-14). Precursor B cell acute lymphoblastic leukemia (BCP-ALL) is the most prevalent immunophenotype of childhood leukemia. We therefore hypothesized that gene expression patterns and actin cytoskeleton reorganization, which is involved in immune synapse formation, may also be altered in T cells of children with BCP-ALL and result in modified T cell function. Comparison of global gene expression profiles of purified peripheral blood CD4+ and CD8+ T cells of untreated BCP-ALL patients and healthy donors using 3´-mRNA-seq technique revealed changes in pathways involved in cell communication, migration, adhesion, DNA damage, mitosis, cell cycle, cell death and signaling, suggesting an alteration of T cell function in the presence of leukemic cells. These results were stressed by in vitro experiments assessing the dynamics of immune synapse formation between allogeneic T cells from healthy donors and sAg activated precursor B-ALL cell lines (Nalm6 and REH) or activated, normal B cells using immunofluorescence staining and confocal microscopy. Cell-interactions were selected at random from imaging, counted and scored for accumulation of F-actin at the immune synapse. We found that CD8+ T cells from healthy donors show a significantly impaired ability to form an immunological synapse after direct short term contact with the cell line Nalm6 compared to normal B cells. Of note, this effect was less pronounced in the cell line REH, known to carry a t(12;21) and express the ETV6-RUNX1 fusion transcript. A decreased number of cell interactions was also seen between CD4+ T cells from healthy donors and the mentioned B-ALL cancer cell lines. However, this effect seemed to be time-dependent, as after slightly prolonged co-incubation of CD4+ T cells with the B-ALL cancer cell lines, synapse formation was improved. Interestingly, the F-actin polymerization pattern at the immunological synapse appeared also qualitatively different between allogeneic CD4+ T cells and normal B cells compared to allogeneic CD4+ T cells and B-ALL cancer cell lines. In normal T cell/B cell interactions improved antigen-recognition and T cell activation is mediated by costimulatory molecules. A lack of costimulatory signals on BCP-ALL cells may contribute to impaired immune recognition. Given the quantitative and qualitative differences in immune synapse formation, we analyzed surface expression of the costimulatory molecules CD70, CD80 and CD86 as well as CD40, one of their key regulators, on the BCP-ALL cell lines by flow cytometry and recognized differences similarly to those previously described in primary BCP-ALL cells, with higher TNFR expression in ETV6-RUNX1 positive leukemia cells compared to negative and more immature BCP-ALL cells (Troeger, A et al., Klin Padiatr., 2014 Nov 226(6-7): p. 332-7; Troeger, A et al., Blood 2008 Aug 15;112(4): p. 1028-34); Troeger A et al., Klin Padiatr., 2008 Nov-Dec 220(6): p. 353-7). These preliminary data suggest that immune synapse formation may be quantitatively altered in a time-dependent manner in T cell/BCP-ALL interactions and result in modified actin cytoskeleton rearrangement. More detailed analysis, also using primary BCP-ALL cells, is warranted to further investigate the underlying mechanism in order to identify novel immunomodulatory approaches that can be used to restore impaired T cell function in the presence of BCP-ALL with the aim to overcome immunosuppressive mechanisms and improve the outcome in high risk patients. Disclosures Williams: Novartis: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Overcoming Acute Lymphoblastic Leukemia Chemoresistance Induced By the Meninges

Central nervous system (CNS) relapse is a leading cause of treatment failure in acute lymphoblastic leukemia (ALL). We have shown that the meninges provide a unique leukemia niche in the CNS that enhances leukemia chemoresistance through effects on apoptosis and quiescence. We now describe our work to leverage this new knowledge of the CNS leukemia niche into novel CNS-directed leukemia therapies that 1) target vulnerabilities unique to leukemia cells in the meninges, 2) inhibit the meningeal pathways that contribute to leukemia chemoresistance, and 3) disrupt the interactions between leukemia and meningeal cells.First, we focused on identifying and targeting vulnerabilities unique to leukemia cells in the meninges. We used a leukemia-meningeal co-culture system and tested the ability of drugs targeting pathways important for leukemia biology to overcome meningeal-mediated leukemia resistance to cytarabine and methotrexate. Intriguingly, we found that pre-treatment of leukemia cells with ruxolitinib, a JAK1/2 inhibitor, prior to co-culture with primary meningeal cells diminished leukemia chemoresistance. Ruxolitinib had no effect on the chemosensitivity of leukemia cells in suspension. Other JAK1/2 inhibitors, such as CHZ868, yielded comparable results and we are currently testing the ability of JAK2 knockdown by shRNA in leukemia cells to overcome chemoresistance. As a potential explanation for the mechanism by which ruxolitinib overcomes meningeal-mediated leukemia resistance, we found that ruxolitinib decreases leukemia quiescence and enhances proliferation in co-culture, as measured by FxCycle and Ki67 staining. Finally, preliminary experiments in xenotransplanted mice showed that ruxolitinib significantly enhanced the efficacy of cytarabine in treating leukemia in the meninges.Second, we also identified and therapeutically targeted meningeal signaling pathways that contribute to leukemia chemoresistance. We used reverse phase protein arrays (RPPA; MD Anderson) to assess the effects of co-culture on protein expression and phosphorylation in primary meningeal cells. RPPA determines the levels of >300 proteins involved in multiple signaling pathways. Amongst other findings, we identified activation of the AKT pathway in primary meningeal cells in the presence of leukemia cells. Furthermore, pre-treatment of primary meningeal cells with INK128, an inhibitor that acts downstream of AKT and targets mTOR, prior to co-culture with leukemia cells diminished leukemia chemoresistance.Third, we tested whether disrupting meningeal-leukemia adhesion overcomes leukemia chemoresistance. Leukemia cells were dissociated from meningeal cells with trypsin and manual pipetting after co-culture for 48-72 hours. Leukemia cells were then purified with magnetic beads and placed back in suspension prior to further characterization and drug testing. We found leukemia cells placed back into suspension after co-culture with meningeal cells, or isolated from the meninges of mice, reverted back to baseline cell cycle, quiescence, and apoptosis balance characteristics. Moreover, leukemia cells removed from co-culture exhibited similar sensitivity to methotrexate and cytarabine as leukemia cells in suspension. These results suggest that drugs or biologic agents that disrupt adhesion between leukemia and meningeal cells may restore leukemia chemosensitivity in the CNS niche. Accordingly, we next identified several cell adhesion inhibitors that effectively disrupted leukemia-meningeal adhesion in co-culture. The more promising agents are currently being characterized more extensively in co-culture and tested in combination with chemotherapy in xenotransplanted mice.In summary, in order to address the need for more efficacious and less toxic therapies for CNS leukemia, we have identified mechanisms by which the meninges enhance leukemia chemoresistance. We are now testing novel strategies for overcoming leukemia chemoresistance that by targeting different components of the CNS leukemia niche (leukemia cells, meningeal cells, or the interactions between the two cell types) may be complementary or even synergistic. DisclosuresNo relevant conflicts of interest to declare.

Cannabinoids Derivatives Exert a Potent Antileukemic Effect By Modifying the Pattern of Membrane Sphingolipids

Endocannabinoid system is a set of ligands, receptors and endogenous enzymes which modulate a variety of physiological effects. There are two well-characterized cannabinoid receptors, CB1 (mainly expressed in Central Nervous System) and CB2 (mainly in hematopoietic cells).Here, we tested the effect of the cannabinoid WIN-55 212-2 in acute myeloid leukemia (AML) in vitro, ex vivo and in vivo and studied the molecular signaling pathways involved in this effect. Moreover, we synthesized a new family of twelve cannabinoids that are specific to CB2 receptor. For their design and synthesis, computational techniques of docking, analytical and spectroscopic techniques such as mass spectrometry (MS) were used.To assess the anti-leukemia effect of the different cannabinoids, we analyzed cell viability by MTT and flow cytometry using six human AML cell lines, primary cells from healthy donors (hematopoietic progenitor cells (HPC) and lymphocytes) and blasts from AML patients. Cannabinoids induced a potent proapoptotic effect on AML cell lines and on primary leukemic cells, which was not observed in normal HPC and lymphocytes from healthy donors.Fragmentation of PARP and activation of caspases 2, 3, 8 and 9 were confirmed by western-blot. Other proteins involved in the effect of cannabinoids were p-AKT, p-ERK 1/2, p-38 and p- JNK. Also studies on p-PERK, p-IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids.Mitochondrial damage was analyzed by flow cytometry using TMRE and by MitoSOXTMRed. These assays confirmed a very early mitochondrial damage in leukemic cells which was not observed in normal hematopoietic progenitor cells.Moreover, we analyzed the ceramide levels, a membrane lipid associated with death/survival cell processes by HPLC and immunohistochemistry. Remarkably, we observed significant differences in the amounts of certain subtypes of ceramides in untreated versus treated leukemic cells.The proapoptotic effect of cannabinoids on AML cells was abolished upon co-culture with either CB2 receptor antagonists or with pancaspase inhibitors.Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60 cell line. We confirmed disease infiltration in bone marrow (BM) by BM aspirates and flow cytometry assays. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered.We observed a significantly increased survival among mice treated with WIN-55 cannabinoid as compared to both the control group and the group treated with ARA-C. In addition, we tested in vivo the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. We confirmed that cannabinoids did not affect the viability of the different populations of hematopoietic progenitors (LK, GMP, CMP) and, moreover, an increased platelet count was observed in treated mice. Our findings indicate that cannabinoids display a highly selective proapoptotic effect against leukemic cells. Several pathways are involved in this effect, the modification in the ceramide pattern playing a main role. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adipose Tissue-Derived IGFBP1 Facilitates Progression of Leukemia

We recently reported that adipose tissue functions as a reservoir for leukemia stem cells (LSCs) using a murine model of leukemia (Ye et al., Cell Stem Cell, 2016). Intriguingly, the presence of leukemic cells in adipose tissue induces increased lipolysis and the release of free fatty acids (FFA) which in turn fuels the growth of LSCs. Further, adipose tissue protects resident LSCs from chemotherapy. These findings indicate that the unique characteristics of adipose tissue may provide key insights on the growth and survival of leukemic cells. Thus, in the present study we focus on the endocrine function of adipose tissue and explore its role in leukemia development.First, to evaluate secreted factors, we applied adipokine arrays which detect 38 adipokines to leukemia serum collected at different stages of leukemia pathogenesis. Interestingly, we observed a significantly elevated level of serum IGFBP1 as soon as leukemic disease became evident at low levels (~0.5%) in marrow. IGFBP1 increased to levels approximate 200X normal at peak stages of disease burden. ELISA analyses further confirmed these observations.IGFBP1 is normally considered as a liver-derived protein. However, we did not find any changes of IGFBP1 expression in leukemic liver. Rather, we observed a significant increase in the expression of IGFBP1 in adipose tissue. IGFBP1 in conditioned medium (CM) from leukemic gonadal adipose tissue (GAT) is approximate 100X higher than naive GAT. Together, these findings suggest adipose tissue-derived IGFBP1 contributes to the increased serum IGFBP1 we detected in leukemic animals.We next examined the role of IGFBP1 in leukemia development using a neutralizing antibody. Treatment of experimental animals with anti-IGFBP1 antibody significantly decreased leukemic burden in GAT (~40% IgG treated VS. 20% Anti-IGFBP1 treated) while bone marrow (BM) and spleen leukemic engraftment remained unchanged (~40%). Further, less atrophy of adipose tissue as well as less body weight loss was seen in the IGFBP1 neutralizing antibody treated group. Consistent with this observation, serum FFA level was also reduced, suggesting less lipolysis in the IGFBP1 antagonized group. Together, these results indicate that IGFBP1 is involved in the regulation of leukemic cells homing to adipose tissue and consequently leukemia-induced lipolysis.Next we explored the mechanisms for the increased expression of IGFBP1 in adipose tissue. Studies have shown that both FGF21 and hypoxia induce IGFBP1 expression. We did not find any changes of FGF21 expression in adipose tissue or in liver, suggesting FGF21 was not involved in IGFBP1 regulation in our system. In contrast, we observed a five-fold elevation in IGFBP1 mRNA in primary adipose tissue cultured under hypoxic conditions. Studies have shown that both BM and spleen in leukemia mice are already hypoxic in early stages of leukemic development (Benito et al., Plos One, 2011). Thus, we hypothesize that tissue hypoxia may at least partially regulate IGFBP1 in leukemia. Ongoing experiments are testing this hypothesis. Additionally, inflammatory cytokines have been reported to increase IGFBP1 expression. We previously reported significantly increased levels of inflammatory cytokines in the adipose tissue of leukemic mice including TNF-α, IL1 and CSF2 (1.5X normal adipose tissue). Therefore, we hypothesize the local inflammatory cytokine production may also contribute to increased expression of IGFBP1, a theory that is also currently under investigation.IGFBP1 has recently been reported to activate osteoclasts and thus promotes bone metabolism (Wang et al., Cell Metabolism, 2015). Studies have found an increased number of osteoclasts and thus bone loss in our leukemic model (Frisch et al., Blood, 2012). We hypothesize that adipose tissue-derived IGFBP1 contributes to bone loss in leukemic mice. Ongoing experiments are testing whether antagonization of IGFBP1 in leukemic mice will rescue bone loss.Collectively, these findings suggest that adipose tissue-derived IGFBP1 facilitates progression of leukemia through regulation of adipose tissue lipolysis and may promote bone marrow colonization by leukemia cells through activation of osteoclasts. DisclosuresNo relevant conflicts of interest to declare.

Inducible epithelial resistance protects mice against leukemia-associated pneumonia

AbstractDespite widespread infection prevention efforts, pneumonia remains the leading cause of death among patients with acute leukemia, due to complex disease- and treatment-dependent immune defects. We have reported that a single inhaled treatment with a synergistic combination of Toll-like receptor 2/6 (TLR 2/6) and TLR9 agonists (Pam2-ODN) induces protective mucosal defenses in mice against a broad range of pathogens. As Pam2-ODN–induced protection persists despite depletion of several leukocyte populations, we tested whether it could prevent pneumonia in a mouse model of acute myeloid leukemia (AML) remission induction therapy. Pam2-ODN prevented death due to pneumonia caused by Pseudomonas aeruginosa, Streptococcus pneumoniae, and Aspergillus fumigatus when mice were heavily engrafted with leukemia cells, had severe chemotherapy-induced neutropenia or both. Pam2-ODN also extended survival of pneumonia in NSG mice engrafted with primary human AML cells. Protection was associated with rapid pathogen killing in the lungs at the time of infection and with reduced pathogen burdens at distant sites at the end of observation. Pathogen killing was inducible directly from isolated lung epithelial cells and was not abrogated by the presence of leukemia cells or cytotoxic agents. Pam2-ODN had no discernible effect on replication rate, total tumor population, or killing by chemotherapy of mouse or human leukemia cells, either in vitro or in vivo. Taken together, we report that therapeutic stimulation of lung epithelial defenses robustly protects against otherwise lethal pneumonias despite the profound immune dysfunction associated with acute leukemia and its treatment. These findings may suggest an opportunity to protect this population during periods of peak vulnerability.

Technical Issues Behind Molecular Monitoring in Chronic Myeloid Leukemia.

Clinical papers frequently lack in-depth descriptions of analytical methods, triggering possible doubts regarding their conclusions. Through this short communication, we would like to stimulate a debate on the monitoring of chronic myeloid leukemia, a disorder that requires frequent laboratory analysis by RNA reverse transcription and realtime quantitative PCR (RQ-PCR). This is a challenging procedure influenced by a number of factors that are difficult to control [1, 2], and troubled by the need to use reference materials to standardize results against the socalled international scale (IS) [3]. The residual disease is evaluated through the amount of BCR-ABL1 transcript, and a patient’s response is reported in terms of IS logarithmic reductions, up to the possible condition of apparent undetectable disease [2]. Actually, the outcome of RQ-PCR represents only an estimation of the leukemic clone based on the assumed proportionality between chimeric RNA and leukemic cells. However, it is not clear if this proportionality is still valid at very high response levels, and negative results can be interpreted in a number of ways, e.g., the true absence of leukemic cells, the presence of leukemic cells that do not transcribe chimeric RNA [4], or measurements below the detection limit. Really, an accurate determination of the detection limit is not feasible since specimen handling, RNA extraction and retro-transcription are critical pre-analytical steps (due to in vitro RNA instability), and it is practically impossible to characterize the whole process. Even positive (but barely detectable) results are difficult to interpret because of the intrinsic variability of gene expression: patients with few leukemic cells can give significantly different results, depending on small variations in the mutual transcription levels of BCR-ABL1 and the reference gene used as a normalizer (usually ABL1, BCR, or GUSB [1]). As a consequence, a clear definition of molecular response is still under elaboration, though the potential for drug discontinuation makes this subject a pressing issue [2]. A survey conducted by the College of American Pathologists pointed out that diverse laboratories testing the same sample obtained different results [1]. Pilot methods based on DNA analysis have also been introduced. These methods require the sequence of the BCR-ABL1 junction for each patient, but have the advantage, of relying on a direct and univocal relationship between cell number and breakpoints (i.e., similar to cytogenetic, but with a higher sensitivity); thus an algorithm can be used to calculate the actual percentage of leukemic cells without the need for standards and reference materials [5]. However, the need to retrieve the breakpoint of each patient affects the routine application of this practice. Recent studies [6] also argue that chronic myeloid leukemia patients (even those with undetectable levels of chimeric RNA) maintain evidence of the BCR-ABL1 DNA, implying its limitations as a prognostic marker. The procedure used to prove this claim consisted of a nested PCR on genomic DNA and a high number of replicates to enhance the chance of detecting the target sequence. This method is frequently used [7, 8], but is prone to false positive results, and a thorough technical discussion would & Elia Mattarucchi elia.mattarucchi@alice.it

A case of type B lactic acidosis as a complication of chronic myelomonocytic leukaemia: a case report and review of the literature

IntroductionType B lactic acidosis represents a rare and often lethal complication of haematological malignancy. Here, we present a patient who developed a type B lactic acidosis presumably due to a concurrent chronic myelomonocytic leukaemia. Upon swift initiation of cytoreductive chemotherapy (doxorubicin), the lactic acidosis was rapidly brought under control. This case adds to the literature reporting other haematological malignancies that can cause a type B lactic acidosis and its successful treatment.Case presentationWe report the case of a 77-year-old Caucasian man brought to our Accident and Emergency department following an unwitnessed collapse; he was found surrounded by coffee-ground vomit. Although haemodynamically stable on admission, he rapidly deteriorated as his lactic acid rose. An initial arterial blood gas revealed a pH of 7.27 and lactate of 18mmol/L (peaking at 21mmol/L).ConclusionsA high degree of clinical suspicion for haematological malignancy should be held when presented with a patient with lactic acidosis in clinical practice, even without evidence of poor oxygenation or another cause. Treatment with emergency chemotherapy, in lieu of a definitive diagnosis, was rapidly successful at lowering lactate levels within 8 hours. This may suggest a causal and perhaps direct relationship between lactic acid production and the presence of leukemic cells. Veno-venous haemofiltration had no apparent effect on reducing the lactic acidosis and therefore its benefit is questioned in this setting, especially at the cost of delaying chemotherapy. In the face of a life-threatening lactic acidosis, pragmatic clinical judgement alone may justify the rapid initiation of chemotherapy.

Functional Inhibition of Mesenchymal Stem and Progenitor Cells (MSPC) Significantly Contributes to Hematopoietic Insufficiency with Acute Myeloid Leukemia (AML)

Pancytopenia is the most prominent clinical finding in patients with acute myeloid leukemia (AML) and represents a major cause for morbidity and mortality. So far, the underlying mechanisms leading to hematopoietic insufficiency in AML are poorly understood and therefore often mechanistically summarized as marrow replacement by infiltrating leukemic cells. Mesenchymal stem and progenitor cells (MSPC) are integral components of the bone marrow (BM) microenvironment and play an indispensable role for the regulation of normal hematopoiesis. Two AML mouse models have recently shown, that expansion of the leukemic clone leads to numeric changes and functional disturbances of niche components such as MSPC and osteoblasts, resulting in insufficient hematopoietic support (Hanoun et al. 2014; Frisch et al. 2012). As the knowledge about MSPC in human AML is limited so far, we conducted a detailed analysis of AML-derived MSPC in order to elucidate their contribution to hematopoietic failure.For this purpose we investigated the molecular and functional properties of BM-derived MSPC of 46 patients with AML covering all relevant subtypes according to WHO classification at diagnosis and/or during course of disease and compared them with MSPC functions of healthy controls. Hematopoietic insufficiency in the 31 patients at diagnosis was mirrored by 76% of the patients having bi- or pancytopenia and by median ANC of 1183/μl, median hemoglobin of 9.0 g/dl, and median platelet count of 67.000/μl. MSPC of these newly diagnosed patients exhibited significantly impaired growth capacities as shown by an altered morphology, reduced CFU-F activity, a lower number of passages and cumulative population doublings. While adipogenic differentiation potential was not affected, osteogenic differentiation potential of AML-derived MSPC was significantly reduced as indicated by cytochemical stainings, reduced Osterix and Osteocalcin (OC) mRNA levels as well as OC serum levels. Furthermore we detected altered mRNA and/or protein expression of key molecules involved in the regulation of hematopoietic stem and progenitor cells (HSPC), namely SCF, Angiopoietin-1, Jagged1 and Osteopontin. Functionally, this translated into a significantly diminished ability of AML-derived MSPC to support healthy CD34+ HSPC in LTC-IC assays.This insufficient stromal support was reversible and correlated with disease status, as LTC-IC frequency returned to normal values in patients in remission, but remained low in patients with refractory disease. Along with this we also observed a significant increase of OC serum levels in patients, who achieved complete remission. These data clearly suggests a direct causal relationship between the presence of leukemic cells and MSPC functionality. In further support of this idea we observed reduced proliferation and osteogenic differentiation of healthy MSPC following cultivation in conditioned media (CM) of 4 AML cell lines (THP-1, HL-60, MV4-11, MOLM-13). A comparable inhibitory effect of AML cells on healthy MSPC growth was seen in transwell-assays arguing in favor for a cell-contact independent mechanism.In summary, our data show that AML-derived MSPC are structurally and functionally altered resulting in an insufficient stromal support for normal hematopoiesis in AML. The correlation between clinical remission status and stromal support function together with the finding, that healthy MSC can adopt an AML-like phenotype when exposed to AML-CM suggest an instructive role of the leukemic precursor cells. DisclosuresDührsen:Celgene: Honoraria, Research Funding. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.

Primary granulocytic sarcoma: a rare presentation of an ovarian mass

Background: Primary granulocytic sarcoma (GS) only rarely presents with an ovarian mass classified as extramedullary myeloid progenitor tumor cells. Few patients with cases of primary GS survive after systemic chemotherapy. Objective: To present a case of primary GS treated with complete course of chemotherapy. The patient is currently in complete remission. Methods: Retrospective review of medical records and comparison with previous case reports. Results: A 41-year-old woman presented with an ovarian mass of 6 months duration. There was no previous history of hematologic disease or presence of leukemic cells in other organs at the time of diagnosis. Immunohistochemistry confirmed the diagnosis. She received complete course of chemotherapy, after which complete remission was achieved. Conclusion: A case of primary granulocytic sarcoma of the ovary was successfully treated with chemotherapy. This disease needs careful diagnosis and appropriate treatment for a good outcome.

HLA Analysis of CD15+ Granulocytes of Leukemic Patients and Assessment of its Implication in Leukemia Immunotherapy

It is of common knowledge that most leukemic patients succumb to infectious complications beside dysfunction in proliferation/differentiation of hematopoietic cells . Recently, it has been shown by several groups that there is a locus specific HLA class I downregulation in the leukemic cells. However, the HLA status of the phenotypically/morphologically normal granulocytes which can cope up with the infectious complications are not known. Therefore it may be worthwhile to study the HLA status in these cell types. We, therefore, investigated the status of HLA-ABC and HLA-DR in the CD15 + granulocytes and observed a higher expression of HLA-DR in several leukemic samples in comparison to normal volunteers (NV). Our data also suggest that only CD15 + granulocytes of myeloid leukemia, a clonal stem cell disorder, have a tendency of decreased HLA class Ia antigen expression. Moreover CD15 + granulocytes of NV showed an enhanced HLA-DR expression in presence of leukemic cells. Interestingly, CD15 + granulocytes collected from normal volunteers were observed to have phagocytic oxidative burst activity towards HLA class Ia downregulated primary leukemic cells. We therefore suggest that neutrophil transplantation may be used for the treatment of leukemia.

Clinical report. Primary granulocytic sarcoma: a rare presentation of an ovarian mass

Abstract Background: Primary granulocytic sarcoma (GS) only rarely presents with an ovarian mass classified as extramedullary myeloid progenitor tumor cells. Few patients with cases of primary GS survive after systemic chemotherapy. Objective: To present a case of primary GS treated with complete course of chemotherapy. The patient is currently in complete remission. Methods: Retrospective review of medical records and comparison with previous case reports. Results: A 41-year-old woman presented with an ovarian mass of 6 months duration. There was no previous history of hematologic disease or presence of leukemic cells in other organs at the time of diagnosis. Immunohistochemistry confirmed the diagnosis. She received complete course of chemotherapy, after which complete remission was achieved. Conclusion: A case of primary granulocytic sarcoma of the ovary was successfully treated with chemotherapy. This disease needs careful diagnosis and appropriate treatment for a good outcome.