Malignant Hematological Tumor Research Articles

Label-Free Identification of AML1-ETO Positive Acute Myeloid Leukemia Using Single-Cell Raman Spectroscopy.

Acute myeloid leukemia (AML) is a malignant hematological tumor disease. Chromosomal abnormality is an independent prognostic factor in AML. AML with t(8:21) (q22; q22)/AML1-ETO (AE) is an independent disease group. In this research, a new method based on Raman spectroscopy is reported for label-free single-cell identification and analysis of AE fusion genes in clinical AML patients. Raman spectroscopy reflects the intrinsic vibration information of molecules in a label-free and non-destructive manner, and the fingerprint Raman spectrum of cells characterizes intracellular molecular types and relative concentration information, so as to realize the identification and molecular metabolism analysis of different kinds of cells. We collected the Raman spectra of bone marrow cells from clinically diagnosed AML M2 patients with and without the AE fusion gene. Through comparison of the average spectra and identification analysis based on multivariate statistical methods such as principal component analysis and linear discriminant analysis, the distinction between AE positive and negative sample cells in M2 AML patients was successfully achieved, and the single-cell identification accuracy was more than 90%. At the same time, the Raman spectra of the two types of cells were analyzed by the multivariate curve resolution alternating least squares decomposition method. It was found that the presence of the AE fusion gene may lead to the metabolic changes of lipid and nucleic acid in AML cells, which was consistent with the results of genomic and metabolomic multi-omics studies. The above results indicate that single-cell Raman spectroscopy has the potential for early identification of AE-positive AML.

KIR-генетические факторы и ответ на терапию ингибиторами тирозинкиназ при хроническом миелоидном лейкозе

The development of tyrosine kinase inhibitors (TKIs) and their introduction into clinical practice considerably improved the prognosis for chronic myeloid leukemia (CML) patients. About 50 % of patients with achieved deep molecular response are eligible for safe TKI discontinuation. Despite these advances, no reliable biomarkers are known to predict a response and sustaining treatment-free remission after TKI withdrawal. As TKIs do not destroy leukemic stem cells, which can be responsible for relapse, critical importance in CML is attached to natural killers (NK-cells) having antitumor activity. Functional activity of NK-cells is evaluated by expression level and repertoire of killer cell immunoglobulin-like receptors (KIR). Current studies demonstrate that a patient’s KIR genotype affects the probability of achieving early and deep molecular responses to first- and second-generation TKIs, progression-free and overall survivals, and sustaining treatment-free remission. On that ground, KIR-genetic factors can be regarded as promising predictors of response to TKI therapy in CML. Early clinical studies, which dealt with monoclonal antibodies blocking the inhibitory KIR in order to increase NK-cell activity, revealed an acceptable safety profile and efficacy in some hematological diseases (such as acute myeloid leukemia, multiple myeloma, Т-cell lymphoma) if used in combination with cytostatic drugs or antitumor monoclonal antibodies. KIR genotype determination can contribute to the development of effective therapies of this malignant hematological tumor.

A novel ferroptosis-related gene signature for predicting prognosis in multiple myeloma.

Multiple myeloma (MM) is a highly malignant hematological tumor with a poor overall survival (OS). Due to the high heterogeneity of MM, it is necessary to explore novel markers for the prognosis prediction for MM patients. Ferroptosis is a form of regulated cell death, playing a critical role in tumorigenesis and cancer progression. However, the predictive role of ferroptosis-related genes (FRGs) in MM prognosis remains unknown. This study collected 107 FRGs previously reported and utilized the least absolute shrinkage and selection operator (LASSO) cox regression model to construct a multi-genes risk signature model upon FRGs. The ESTIMATE algorithm and immune-related single-sample gene set enrichment analysis (ssGSEA) were carried out to evaluate immune infiltration level. Drug sensitivity was assessed based on the Genomics of Drug Sensitivity in Cancer database (GDSC). Then the synergy effect was determined with Cell counting kit-8 (CCK-8) assay and SynergyFinder software. A 6-gene prognostic risk signature model was constructed, and MM patients were divided into high and low risk groups. Kaplan-Meier survival curves showed that patients in the high risk group had significantly reduced OS compared with patients in the low risk group. Besides, the risk score was an independent predictor for OS. Receiver operating characteristic (ROC) curve analysis confirmed the predictive capacity of the risk signature. Combination of risk score and ISS stage had better prediction performance. Enrichment analysis revealed immune response, MYC, mTOR, proteasome and oxidative phosphorylation were enriched in high risk MM patients. We found high risk MM patients had lower immune scores and immune infiltration levels. Moreover, further analysis found that MM patients in high risk group were sensitive to bortezomib and lenalidomide. At last, the results of the in vitro experiment showed that ferroptosis inducers (RSL3 and ML162) may synergistically enhance the cytotoxicity of bortezomib and lenalidomide against MM cell line RPMI-8226. This study provides novel insights into roles of ferroptosis in MM prognosis prediction, immune levels and drug sensitivity, which complements and improves current grading systems.

High levels of serum IL-10 indicate disease progression, extramedullary involvement, and poor prognosis in multiple myeloma.

Multiple myeloma (MM) is a common malignant hematological tumor in adults, which is characterized by clonal malignant proliferation of plasma cells in the bone marrow and secretion of a large number of abnormal monoclonal immunoglobulins (M protein), leading to bone destruction, hypercalcemia, anemia, and renal insufficiency (Alexandrakis et al., 2015; Yang et al., 2018). Since a large number of new drugs, represented by proteasome inhibitors and immunomodulators, have been successfully used to treat MM, treatment efficacy and survival of patients have been significantly improved. However, due to the high heterogeneity of this disease, patients have responded differently to treatments with these new drugs (Palumbo and Anderson, 2011; Wang et al., 2016; Huang et al., 2020). Growth and survival of MM cells depend on the bone marrow microenvironment, especially numerous inflammatory cytokines secreted by myeloma cells and bone marrow stromal cells, such as vascular endothelial growth factor (VEGF), interleukin (IL)‍-6, transforming growth factor-‍β (TGF‍‍-‍‍β), and IL-10. These cytokines can promote the growth of myeloma cells, induce angiogenesis, and inhibit antitumor immunity, and are often linked to patient prognosis (Kumar et al., 2017). In this era of new drugs, the prognostic values of the serum levels of these cytokines in MM need further evaluation.

CORRELATIONS BETWEEN PERIPHERAL BLOOD INFLAMMATORY MARKERS AND INTERLEUKIN-21 IN PATIENTS WITH HEMATOLOGIC MALIGNANCIES

Inflammation that occurs in the tumor microenvironment and in the systemic circulation correlates with disease progression and prognosis in a number of tumors. One way to assess the systemic immune response is to determine the cells/their ratio associated with inflammation, which can easily be measured with complete blood count. IL-21 has a variety of regulatory effects on both normal and tumor cells, leading to their proliferation, differentiation, and apoptosis.The aim of our study was to determine the level of IL-21 in patients with lymphoma-leukemia and to correlate it with the biomarkers of systemic inflammation involved in carcinogenesis - NLR, PLR, PMR, HLR, SII, dNLR.The study included patients with hematologic tumors who underwent splenectomy for therapeutic indications. The control group consisted of patients who also underwent splenectomy but not due to malignant tumor or autoimmune disease.According to our study analysis interleukin-21 levels did not differ significantly between the study and control groups. Serum levels of interleukin-21 in patients with malignant hematologic tumors are negatively correlated with dNLR.

Regimen of Azacitidine Combined with GemOx in the Treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm: A Case Report

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a highly malignant hematological tumor derived from the precursor cells of plasmacytoid dendritic cells. BPDCN primarily in the nasal cavity is clinically rare and easily misdiagnosed and missed. At present, there is no standard treatment for BPDCN at home and abroad. This article reports a patient with blastic plasmacytoid dendritic cell neoplasm primarily in the nasal cavity that received azacitidine combined with GemOx regimen has a good clinical effect, which can significantly control the condition and improve the clinical symptoms. Through the analysis and discussion on the clinical diagnosis and treatment of this patient, we aim to improve clinicians' understanding of rare BPDCN.

Tumor Microenvironmental Competitive Endogenous RNA Network and Immune Cells Act as Robust Prognostic Predictor of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is malignant hematologic tumors with frequent recurrence and cause high mortality. Its fate is determined by abnormal intracellular competitive endogenous RNA (ceRNA) network and extracellular tumor microenvironment (TME). This study aims to build a ceRNA network related to AML TME to explore new prognostic and therapeutic targets. The RNA expression data of AML were obtained from The Cancer Genome Atlas (TCGA) database. First, we used the ESTIMATE algorithm to calculate the immune cells and stromal cells infiltration scores in the TME and found that all scores were highly correlated with AML’s prognostic characteristics. Subsequently, differentially expressed mRNAs and lncRNAs between high and low score groups were identified to construct a TME-related ceRNA network. Further, the Cox-lasso survival model was employed to screen out the hub prognostic ceRNA network composed of two mRNAs (EPB41L3, COL2A1), three miRNAs (hsa-mir-26a-5p, hsa-mir-148b-3p, hsa-mir-148a-3p), and two lncRNAs (CYP1B1-AS1, C9orf106), and construct nomograms. Finally, we used CIBERSORT algorithm and Kaplan-Meier survival analysis to identify the prognostic TME immune cells and found that naive B cells, M2-type macrophages, and helper follicular T cells were related to prognosis, and the hub ceRNAs were highly correlated with immune cell infiltration. This study provided a new perspective to elucidate how TME regulates AML process and put forward the new therapy strategies combining targeting tumor cells with disintegrating TME.

Expression of Vav1-Myo1F Fusion Affects T-Cell Differentiation and Induces T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T cell lymphomas not otherwise specified (PTCL, NOS) account for >45% diagnoses, show limited response to intensified chemotherapy treatment and are associated with dismal survival. Genomic studies from our group have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS. Interestingly, mutation co-occurrence analysis in AITL and PTCL, NOS showed significant mutual exclusivity of VAV1 genomic alterations and the highly prevalent RHOA mutations (p-value 0.0142), supporting a common mechanism of action. Gene fusions involving VAV1 are characterized by the substitution of their auto-inhibitory C-terminal SH3 domains by different domains from their fusion partners, leading to increased activation of VAV1-dependent signaling pathways. Among them, the VAV1-MYO1F fusion shows the strongest increase in VAV1 activity and activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) pathways. To study the role of VAV1-MYO1F, we engineered a conditional knockin mouse that expresses the Vav1-myo1f fusion in CD4+ T-cells. Expression of Vav1-myo1f in CD4 T cells induces cell activation and alterations in T-cell specification, associated with up-regulation of master transcription factors involved in helper T-cell cell differentiation. Moreover, Vav1-Myo1f increased CD4+ T-cell survival upon cytokine withdrawal and enhanced Vav1 phosphorylation and activation of the MAPK pathway, resulting in increased cell activation and proliferation both in vivo and in vitro in response to TCR engagement. Notably, expression of Vav1-Myo1f fusion in CD4+ T-cells is sufficient to induce development of fatal malignant lymphomas with a latency of 6-14 months. Histological examination showed disrupted splenic architecture associated with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS phenotype. Similar results were obtained in a genetic model that combined the expression of Vav1-myo1f with the deletion of the Tet2 epigenetic regulator. In both genetic models, tumor cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features including increased expression of the transcription factors Gata3 and c-Maf and the IL4 and Il10 cytokines. Overall, these results demonstrate a direct oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, associated with deregulation of T-cell specification and of signaling programs critical for the control of T-cell proliferation. Disclosures Palomero: Kura Onclology: Research Funding.

Abstract PO-41: Expression of Vav1-Myo1F fusion affects T-cell differentiation and induces T-cell lymphoma

Abstract Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature T cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphomas not otherwise specified (PTCL, NOS) account for >45% diagnoses, show limited response to intensified chemotherapy treatment, and are associated with dismal survival. Genomic studies from our group have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS. Interestingly, mutation co-occurrence analysis in AITL and PTCL, NOS showed significant mutual exclusivity of VAV1 genomic alterations and the highly prevalent RHOA mutations (p-value 0.0142), supporting a common mechanism of action. Gene fusions involving VAV1 are characterized by the substitution of their auto-inhibitory C-terminal SH3 domains by different domains from their fusion partners, leading to increased activation of VAV1-dependent signaling pathways. Among them, the VAV1-MYO1F fusion shows the strongest increase in VAV1 activity and activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor of activated T-cells (NFAT) pathways. To study the role of VAV1-MYO1F, we engineered a conditional knock-in mouse that expresses the Vav1-myo1f fusion in CD4+ T-cells. Expression of Vav1-myo1f in CD4 T cells induces cell activation and alterations in T-cell specification, associated with upregulation of master transcription factors involved in helper T-cell cell differentiation. Moreover, Vav1-Myo1f increased CD4+ T-cell survival upon cytokine withdrawal and enhanced Vav1 phosphorylation and activation of the MAPK pathway, resulting in increased cell activation and proliferation both in vivo and in vitro in response to TCR engagement. Notably, expression of Vav1-Myo1f fusion in CD4+ T cells is sufficient to induce development of fatal malignant lymphomas with a latency of 6-14 months. Histologic examination showed disrupted splenic architecture associated with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS phenotype. Similar results were obtained in a genetic model that combined the expression of Vav1-myo1f with the deletion of the Tet2 epigenetic regulator. In both genetic models, tumor cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features including increased expression of the transcription factors Gata3 and c-Maf and the IL4 and Il10 cytokines. Overall, these results demonstrate a direct oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, associated with deregulation of T-cell specification and of signaling programs critical for the control of T-cell proliferation. Citation Format: Jose Rodriguez Cortes, Robert Albero, Ioan Filip, Juan Angel Patino, Anisha R. Cooke, Wen-Hsuan Lin, Anouchka P. Laurent, Bobby B. Shih, Aidan S. Quinn, Raul Rabadan, Adolfo Ferrando, Teresa Palomero. Expression of Vav1-Myo1F fusion affects T-cell differentiation and induces T-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-41.

Haploidentical Hematopoietic Stem Cell Transplantation Compared with HLA-Matched Stem Cell Transplantation for Refractory or Relapsed Aggressive Non-Hodgkin Lymphoma

Background: Non-Hodgkin's lymphoma (NHL) is a malignant hematological tumor that accounts for approximately 90% of lymphoma in China. About 2/3 of NHL patients can be treated or even cure, but still about one-third will die from refractory or relapsed disease, especially high-risk patients. In these cases, continued conventional chemotherapy will not benefit, and may even lead to significant chemo-related toxicity and reduce the quality of life. Allogeneic hematopoietic stem cell transplantation (allo-SCT) is an effective treatment for whom with chemoresistent, recurrence after autologous transplantation or without autologous transplantation indication. However, less than 30% of patients can find HLA-matched relative donors. hematopoietic stem cell transplantation (haplo-HSCT) enables more than 90% of patients to find suitable donors in the shortest time, bringing good news to patients without HLA-matched donors.Therefore, we compare the efficacy of haploidentical stem-cell transplantation (haplo-SCT) for patients with refractory relapsed(R/R) aggressive non-Hodgkin lymphoma (NHL) with those who contemporaneously receiving HLA-matched SCT in myeloablative conditioning settings. Methods: One hundred and fifty-one patients who had undergone haplo-SCT (n=81) or HLA-matched SCT (n=70, sibling or unrelated) in our center between January 2006 and December2018 were enrolled. A median age at alloSCT was 30(5-59) years old. All patients received a myeloablative conditioning (MAC) consisting of total body irradiation (12 Gy) combined cyclophosphamide or busulphan plus cyclophosphamide,and followed by infusion of granulocyte-colony stimulating factor-primed bone marrow (G-BM) and/or peripheral blood stem cells without in vitro T cell depletion. In the case of haplo-SCT and HLA-matched unrelated donor for SCT, the GVHD prophylaxis consisted of antithymocyte globulin, cyclosporine A, mycophenolate mofetil and a short course of methotrexate. The clinical effect, hematopoietic reconstitution, and transplant-related complications were retrospective analyzed. Results: One hundred and forty-five (96%) patients engrafted with a median time to neutrophil and platelet recovery of 12 and 15 days, respectively. At a median follow-up of 20 months, 66 of 151 patients were alive (43.7%) and 67 (44.4%) were dead (39 disease recurrence, 27 transplantation-related mortality (TRM)). Between the haplo-SCT and HLA-matched SCT group, the corresponding progress free survival (PFS) rate was 60.5% and 54.3% (P = .938), respectively; and overall survival (OS) rate were 67.9% and 55.7% (P = .460), respectively. The cumulative incidences of relapse (RI) were 36.6% and 37.7% (P = .836), and those of NRM were 20.0 % and 24.7% (P = .530), respectively. And cumulative incidences of chronic GVHD were 33.3% and 30.0%(P=.407), respectively. These data showed no difference in every major HSCT endpoint between two groups. Multivariate analysis suggested that occurrence of grade Ⅲ-Ⅳ aGVHD had a significant worse outcome, primary chemorefractory was the strongest factors for relapse. Conclusion: The haplo-SCT group obtained the OS and PFS similar to HLA-matched SCT. And there is no higher incidence of GVHD observed in haplo group. Our results indicate that the outcomes of haplo-SCT are equivalent to HLA-matched SCT, and that MAC followed by haplo-SCT can be an acceptable and feasible alternative for patients with R/R NHL who having no access to a HLA-matched donor. Figure Disclosures No relevant conflicts of interest to declare.

Role of Vav1 Genomic Alterations in T-Cell Differentiation and Transformation in Peripheral T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature post-thymic T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphomas not otherwise specified (PTCL, NOS) account for >45% diagnoses, show limited response to intensified chemotherapy treatment with dismal overall survival rate. Our genomic studies have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS, supporting a role for the VAV1 oncogene in the pathogenesis of PTCL. Genetic alterations in VAV1 found in PTCL included recurrent deletions resulting in the in-frame deletion of 9 codons (DV778_T786) in the linker region connecting the C-terminal SH2 domain to the downstream SH3 domain on VAV1 as well as recurrent different gene-fusion transcripts encoding proteins in which the C-terminal SH3 domain of VAV1 is replaced by different domains of the fusion partner. Consistent with the auto-inhibitory role of the C-SH3 domain, the PTCL-associated VAV1 alterations resulted in increased VAV1 activity and strong activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) pathways. Interestingly, mutation co-occurrence analysis shows significant mutual exclusivity of the highly prevalent AITL RHOA G17V mutation and VAV1 genomic alterations (p-value 0.0142) in AITL and PTCL, NOS. This observation supports that VAV1 genetic alterations and RHOA G17V could share a common mechanism of action. To study the role and mechanisms of VAV1 alterations in PTCL in vivo we have engineered a C57/BL6 mouse lines with conditional expression of the recurrent Vav1-Myo1f fusion. By crossing Vav1-myo1f conditional knockin with CD4-CreERT2 mouse deletor line, we have generated animals with CD4+ T cell-specific and tamoxifen-inducible expression of Vav1-myo1f. Preliminary studies indicate that expression of Vav1-myo1f in CD4+ cells induces expansion of TFH cells. This phenotype partially resembles that of animals with CD4 specific expression of Rhoa G17V, indicating a potential convergent effect and mechanisms for VAV1 alterations and RHOA G17V in T-cell development and transformation. However, and more interestingly, Vav1-myo1f expressing cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features that had not been observed in the Rhoa G17V model. Analysis of signaling pathways activated in control and Vav1-myo1f expressing CD4+ cells showed increased phosphorylation of Vav1 and increased activation of the MAPK pathway upon expression of Vav1-myo1f, supporting the role of Vav1 alterations as regulators of T-cell activation. Notably, single cell transcriptome analysis of bulk splenic cells isolated from CD4-CreERT2 control and CD4-CreERT2Vav1co-Vav1-myo1f/WT mice indicate that expression of Vav1-myo1f in CD4+ cells leads to a significant expansion of monocytes and TFH cells and a decrease in B-cell, both naïve and memory. These results support that expression of Vav1-myo1f in CD4+ cells can deeply modify the lymphoid microenvironment. Finally, preliminary analysis of a limited number of mice with targeted expression of the Vav1-Myo1f fusion in CD4+ cells (CD4-Cre Vav1co-Vav1-Myo1f /WT) showed development of a fatal CD4+ malignant lymphoproliferation with a latency of 6-14 months. Histological examination showed disrupted splenic architecture accompanied with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS characteristics and verified expression of both wild type Vav1 and Vav1-myo1f alleles. Interestingly, Vav1-myo1f lymphoma cells showed Th2 associated features including expression of Gata3, c-Maf and IL4 and Il10. These results support an important role for VAV1 alterations in T-cell differentiation, remodeling of the tumor microenvironment and T-cell lymphoma development and highlight the importance of our model as a new experimental system to study the evolution of PTCL and develop novel targeted therapies for this disease. Disclosures Palomero: Kura Oncology: Research Funding.

Preliminary Results from a Phase I, First-in-Human Study of YY-20394, a Highly Selective and Highly Potent PI3Kδ Inhibitor, in Patients with Relapsed or Refractory B-Cell Malignancies

Introduction: Aberrant activation of the PI3Kδ pathway is associated with increased malignant B-cell proliferation and survival. Recently, several PI3Kδ inhibitors have been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. YY-20394, a novel small molecule, is a highly selective and highly potent PI3Kδ inhibitor. The current study is a phase I, first-in-human, dose escalation study to assess the safety, tolerability, and efficacy of YY-20394 in patients with relapse or refractory B-cell malignant hematological tumor (Chinadrugtrials.org.cn ID: CTR20170995).Methods: Adult patients were eligible for study participation if they had relapsed or refractory lymphoid B-cell malignancies, ECOG ≤2, normal hepatic and renal function, and no autologous hematopoietic stem-cell transplant (HSCT) within 3 months of screening. YY-20394 was given orally once daily until disease progression, unacceptable toxicity, or withdrawal from the study. The protocol was initiated with a single-patient cohort, treated with oral YY-20394 20 mg once daily (QD). Subsequent cohorts used a 3+3 design and evaluated doses of 40-320mg QD. Adverse events (AEs) were graded by NCI-CTCAE v4.0. Efficacy was assessed according to IWG-NHL and CLL consensus response criteria.Results: The study enrolment is still ongoing. As of Jul. 14, 2018, total of 10 patients were enrolled. The patients including diffuse large B-cell lymphoma, n=1; follicular lymphoma, n=5; mantle cell lymphoma, n=1; lymphoplasmacytic lymphoma, n=1 and CLL , n=2, received YY-20394 20 mg (n=1), 40 mg (n=3), 80 mg (n=3) or 140 mg/day (n=3) respectively; All the patients had heavy treatment before participating in the current study including BTK inhibitor therapy (n=3) and CAR-T treatment (n=2). All the patient have completed cycle 1 safety observation and no dose-limiting toxicities occurred.The most common nonhematologic TEAEs (all grade [Gr]; Gr ≥3) were LDH elevation (30%; 0%), dizzy (10%; 0%), pneumonia (10%; 10%), alopecia (10%; 0%), and hyperuricemia (10%; 0%). Gr ≥3 hematologic TEAEs were neutropenia (10%) and lymphocythaemia (20%). 1 Serious AEs were reported as grade 3 pulmonary infection after the patient received 2 cycles treatment, it is still unknown if the SAE is related with the study drug. The pulmonary infection has been improved after anti-inflammation treatment.Of 10 patients treated, 71.4% of overall response rate and 100% of disease control rate were achieved in 7 patients who were available for efficacy evaluation, including 1 CR (80 mg, FL) , 4 PR (40 mg, n=2 with FL and CLL respectively; 80 mg, n=2 with FL and DLBCL respectively and 2 SD ( 20 mg with MCL, n=1 and 40 mg with CLL ,n=1 respectively. The PK results demonstrated dose-proportional increase in plasma concentrations.Conclusion: YY-20394 is well tolerated and with promised objective response rates in patients with relapsed or refractory B-cell malignancies. The current first-in-human study for dose escalation and dose expansion is still ongoing. DisclosuresSong:Peking University Cancer Hospital (Beijing Cancer Hospital): Employment. Zhu:Beijing Cancer Hospital: Employment.

Prophylactic Biosimilar Filgrastim Treatment during Cytotoxic Chemotherapy in Patients with Malignant Diseases: The Venice Observational Study

▪Introduction: Among other serious adverse events, febrile neutropenia (FN) is a serious, frequent complication associated with cytotoxic chemotherapy (CT). Biosimilar filgrastim (NivestimTM, Hospira Inc.) is a granulocyte-colony stimulating factor (G-CSF)approved for use in Europe since 2010 for the reduction in the duration of neutropenia and the incidence of FN in patients treated with cytotoxic CT for malignancy. The VENICE study was conducted to observe the tolerability, safety and efficacy of prophylactic treatment using biosimilar filgrastim among in-patients receiving cytotoxic CT for cancer outside the clinical trial setting in the context of real-life, day-to-day medical practice. This study is of particular relevancy due to the propensity for biosimilar G-CSF to lower overall costs of cancer treatment. ClinicalTrials.gov Identifier: NCT01627990Study Design: The study was conducted as a prospective, non-interventional study at 48 sites in Germany. Patients being treated for solid tumors or malignant hematologic tumors except for chronic myeloid leukemia (CML) or myelodysplastic syndrome (MDS) who had provided informed consent and for whom cytotoxic CT and biosimilar filgrastim treatment was planned were enrolled in the study. Data on demographics, clinical characteristics, treatment-related data on efficacy and safety (AEs including FN) and hospitalizations were collected. Three study visits were planned: at recruitment, at end of first biosimilar filgrastim administration and at end of CT or end of study.Results: The study included 386 adult patients undergoing cytotoxic CT who were treated with biosimilar filgrastim prophylactically to reduce the duration of neutropenia and to reduce incidence of chemotherapy-induced FN. Patients were 81% female with a mean age of 60 years. At baseline, 43% of the patients had already received CT and previous FN was reported by only 3% of patients. A total of 338 patients (88%) had solid tumors and 49 (13%) had hematological malignancies. Antibiotic prophylaxis was taken by 13% of patients at inclusion. Only 9% of the patients had received G-CSF from the first cycle. The majority (64%) of patients received biosimilar filgrastim as primary prophylaxis and 36% as secondary prophylaxis.At least one hospitalization was reported by 24 (6%) of 382 patients in the safety analysis set and the median hospital stay for these patients was 10 days. Hospitalization due to FN or infection was reported for 2% of patients.Only 1% of patients had a delay in their CT due to FN after the first biosimilar filgrastim treatment and for only 5% of patients, the CT in any subsequent CT cycle was delayed due to neutropenia. Reduction of CT dose after first biosimilar filgrastim treatment due to FN was reported for only 1% of patients and reduction of CT dose in any subsequent cycle due to neutropenia was reported for only 5% of patients.The median duration between the last dose of CT and treatment with biosimilar filgrastim was 5 days. The majority of patients were prescribed the 30 million units (MU) dose of biosimilar filgrastim administered SC. The median duration of treatment was 4 days. Most patients administered the biosimilar filgrastim injection themselves at both treatment visits (78% at first visit and 69% at last visit). Most of the self-applicators reported that the application was easy or very easy.During the study, 24% of patients experienced ≥1 AE that was at least possibly related to biosimilar filgrastim treatment. The treatment related AEs reported most frequently were musculoskeletal and connective tissue disorders (11%); general disorders and administration site conditions (5%); and blood and lymphatic system disorders (5%).Conclusions: The VENICE non-interventional study aimed to evaluate the use of biosimilar filgrastim according to the label approved usage of the biosimilar filgrastim in order to collect additional real world data on the tolerability, safety and efficacy in cancer patients. Biosimilar filgrastim was effective and well-tolerated in both the primary and secondary prophylactic settings in patients undergoing chemotherapy for solid tumors and hematological malignancies. DisclosuresErdlenbruch:Hospira Germany: Employment.

Diagnostic value of serum procalcitonin for infection in the immunocompromised critically ill patients with suspected infection

To evaluate the diagnostic and prognostic value of the serum procalcitonin (PCT) level in the non-acquired immune deficiency syndrome (AIDS) immunocompromised critically ill patients suspected to have infection. A retrospective study was conducted in the non-AIDS immunocompromised patients who were admitted to Department of Critical Care Medicine of Xiangya Hospital, Central South University during January 2011 to December 2014. Demographic characteristics, underlying disease, acute physiology and chronic health evaluation II (APACHEII) score at admission, and clinical records including baseline and peak levels of temperature, white blood count (WBC), PCT, and survival rate within 28 days, infection focus, infectious agents (bacterial, fungi or mixed infection), and the severity of infection (sepsis, severe sepsis, or septic shock) were recorded. Receiver operating characteristic (ROC) curve was plotted, and the diagnostic and protective value of above parameters was evaluated. A total of 98 patients (43 male and 55 female) were enrolled in the study with a median age of 44 (28, 52) years old and a median APACHEII score of 17 (11, 20); 47 with malignant hematological tumor, 45 with autoimmune diseases, and 6 post solid organ transplantation. Among them 53 patients (54.1%) died within 28 days. Twenty-seven patients were diagnosed as systemic inflammatory response syndrome (SIRS) without infection. Among 71 patients with infection, 45 were diagnosed as bacterial infection, 10 with fungal infection, and 16 with mixed infection. Sepsis was diagnosed in 7 patients, severe sepsis in 32 patients, and septic shock in 32 patients. (1) There was no statistical significance in the baseline and peak levels of PCT and WBC, or baseline level of temperature between the groups of SIRS patients without infection and infected patients. The peak level of temperature was significantly higher in the patients with infection as compared with that of the SIRS without infection patients [centigrade: 39.4 (38.9, 40.0) vs. 38.8 (37.8, 39.2), Z=-3.268, P=0.001]. It was showed by subgroup analysis that in patients with hematological malignant disease or autoimmune diseases, higher level of body temperature was found in infection group compared with non-infection SIRS group [centigrade: 39.5 (39.0, 40.0) vs. 39.0 (38.4, 39.4), Z=-2.349, P=0.019; 39.0 (38.4, 39.5) vs. 38.2 (37.0, 38.9), Z=-2.221, P=0.026]. (2) The baseline level of PCT (μg/L) were 0.54 (0.20, 4.19), 2.78 (0.50, 9.54), 1.00 (0.45, 6.89), and 0.22 (0.07, 1.86) in non-infection SIRS patients or the patients with bacterial, fungal, and mixed infection, respectively. The peak level of PCT (μg/L) were 4.19 (1.95, 13.42), 12.37 (3.82, 45.89), 1.82 (0.49, 17.86), and 5.14 (2.66, 12.62), respectively, in each subgroup. When the comparison was conducted among the patients with different infectious agent, the baseline level of PCT in patients with bacterial infection was significantly higher than that in SIRS patients without infection (P=0.026) and mixed infection patients (P=0.001), and the peak level of PCT was significantly higher than that in the SIRS patients without infection (P=0.009) and the patients with fungal infection (P=0.016). ROC curve showed that the higher value was found in the baseline and peak levels of PCT for diagnosis of septic shock in all patients [ area under ROC curve (AUC) of baseline level=0.681±0.054, P=0.001; AUC of peak level=0.690±0.054, P=0.002], and the same value was also found in the baseline and peak levels of PCT for diagnosis of bacterial infection in the patients with malignant hematological tumor (AUC of baseline level=0.687±0.080, P=0.008; AUC of peak level=0.697±0.079, P=0.021). (3) The peak level of PCT (μg/L) were 4.05 (0.53, 31.22), 5.78 (2.14, 16.68), and 11.64 (2.94, 58.14) in subgroup of patients with sepsis, severe sepsis and septic shock, respectively, and they showed no statistical significance among subgroups (P>0.05). A high serum level of peak PCT strongly indicated the presence of septic shock (AUC=0.646±0.060, P=0.019), especially in the subgroup of patients with systemic autoimmune disease (AUC=0.689±0.081, P=0.035). (4) The peak level of PCT (μg/L) in the APACHEII>18 group (38 cases) was significantly higher than that of APACHEII≤18 group [ 60 cases, PCT (μg/L): 11.64 (3.36, 39.39) vs. 4.42 (1.32, 14.70), P=0.016]; there was a certain correlation between the peak level of PCT and the severity of the disease. (5) The peak level of PCT in death group was significantly higher than that of the survival group [μg/L: 9.07 (3.05, 33.09) vs. 4.19 (1.26, 14.61), P=0.043]. ROC curve showed that the peak level of PCT might be valuable in predicting the prognosis in immunocompromised patients (AUC=0.619±0.057, P=0.043). The serum level of PCT is found to be a reliable marker for the diagnosis of bacterial infection in immunocompromised critical patients, especially in those with hematologic malignancy. Additionally, PCT provides a useful tool for evaluating the severity of infection and the prognosis of critically ill patients.

Cyclin-Dependent Kinase Inhibitors Enhance Sensitivity to Methotrexate In Human T-Cell Leukemia Jurkat Cells

AbstractAbstract 3976Methotrexate (MTX), a classical anti-folate drug, has been used in the treatment of various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. In fact, it is well known that elevated expression of DHFR is a direct factor in clinical therapeutic resistance to MTX. Therefore, small compounds that repress DHFR expression may be useful as chemosensitizers in combination with MTX.First of all, we demonstrated that the sensitivity to MTX was enhanced by the knockdown of the DHFR gene in Jurkat cells. Concretely, the expression of DHFR was efficiently knocked down by siRNA transiently transfected using electroporation. We subsequently confirmed that pretreatment with DHFR siRNA significantly enhanced sensitivity of Jurkat cells to MTX in vitro with colony formation assays. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX.On the other hand, the expression of DHFR has been assumed to be controlled by the transcription factor E2F. Unphosphorylated retinoblastoma (RB) protein is known to bind and inhibit the E2F family. Actually, it has been reported RB protein, which acts as a negative regulator of cell cycle transition, suppresses the expression of DHFR. When RB is phosphorylated by cyclin-dependent kinase (CDK)-cyclins, E2F is released from the complex and binds to a cis-element in the promoter region of various genes involved in cell proliferation and DNA synthesis. In other words, CDK inhibitors activate RB function and inhibit E2F function. Then we investigated whether CDK inhibitor acted as a suppressor of DHFR through the E2F site of the promoter, and whether it could be used to enhance sensitivity to MTX.We found that SU9516, a small chemical CDK inhibitor, showed the inhibition of cell growth without the significant increase of dead cells. Furthermore we also found that SU9516 decreased phospho-RB (Ser780) and reduced the expression of both DHFR mRNA and protein, suggesting that SU9516 activates the RB pathway. The DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. The results suggested that SU9516 inhibited DHFR expression at the promoter level. Furthermore, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay.To expand these results in Jurkat cells, we have also examined other leukemic cell lines (human T cell leukemia CCRF-CEM cells and human erythroleukemia K562 cells) and confirmed that SU9516 also repressed the levels of DHFR protein and phopho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in both of cell lines. These results might indicate that pretreatment of SU9516 is effective to enhance sensitivity to MTX in not only the T-cell leukemia cell lines but also erythroleukemia cell lines such as K562 cells.Finally, to expand these results by SU9516, we have examined other CDK inhibitors (Purvalanol A and Cdk4/6 Inhibitor IV) and confirmed that these agents also decreased DHFR protein and phospho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in Jurkat cells as similar as SU9516.In conclusion, we showed for the first time that SU9516 significantly enhanced sensitivity to MTX through the inhibition of DHFR expression in human T-cell leukemia Jurkat cells and CCRF-CEM cells, and human erythroleukemia K562 cells. Although further study is needed, these results raise the possibility that a combination of CDK inhibitors and MTX may be a suitable chemotherapeutic option for enhancing sensitivity to MTX when treating hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Abstract 2560: Cyclin-dependent kinase inhibitor SU9516 enhances sensitivity to methotrexate in human T-cell Jurkat cells

Abstract Methotrexate (MTX), a classical anti-folate drug, has been used to treat various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. In fact, it is well known that elevated expression of DHFR is a direct factor in clinical therapeutic resistance to MTX. First of all, we demonstrated that the sensitivity to MTX was enhanced by the knockdown of the DHFR gene in human T-cell leukemia cell. Concretely, the anti-proliferative effect of MTX was significantly enhanced by the knockdown of DHFR expression by siRNA in Jurkat cells. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX. On the other hand, the expression of DHFR is controlled by the transcription factor E2F. Unphosphorylated retinoblastoma (RB) protein is known to bind and inhibit the E2F. Cyclin-dependent kinase (CDK) inhibitors activate RB function and inhibit E2F function. Then we tested whether CDK inhibitor acted as a suppressor of DHFR through the E2F site of the promoter. We found that SU9516, a cyclin-dependent kinase inhibitor, decreased phospho-RB (Ser780) and reduced the expression of both DHFR mRNA and protein, and the DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. Furthermore, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay. To expand these results in Jurkat cells, we have also examined other leukemic cell lines (human T cell leukemia CCRF-CEM cells and human erythroleukemia K562 cells) and confirmed that SU9516 also repressed the levels of DHFR protein and phopho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in both of cell lines. To expand these results by SU9516, we have examined other CDK inhibitors (Purvalanol A and Cdk4/6 Inhibitor IV) and confirmed that these agents also decreased DHFR protein and phospho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in Jurkat cells as similar as SU9516. Based on these findings, we conclude that a combination of cyclin-dependent kinase inhibitors and MTX may be useful for overcoming resistance to MTX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2560.

Cyclin-dependent kinase inhibitor SU9516 enhances sensitivity to methotrexate in human T-cell leukemia Jurkat cells

Methotrexate (MTX) has been used to treat various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. The antiproliferative effect of MTX was significantly enhanced by the knockdown of DHFR expression by siRNA in Jurkat cells. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX. We found that SU9516, a cyclin-dependent kinase inhibitor, reduced the expression of both DHFR mRNA and protein. Moreover, we found that DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. Finally, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay. We conclude that a combination of cyclin-dependent kinase inhibitors and MTX may be useful for overcoming resistance to MTX.