Brief research report: in-depth immunophenotyping reveals stability of CD19 CAR T-cells over time.

Variability or stability might have an impact on treatment success and toxicity of CD19 CAR T-cells. We conducted a prospective observational study of 12 patients treated with Tisagenlecleucel for CD19+ B-cell malignancies. Using a 31-color spectral flow cytometry panel, we analyzed differentiation stages and exhaustion markers of CAR T-cell subsets prior to CAR T-cell infusion and longitudinally during 6 months of follow-up. The majority of activation markers on CAR T-cells showed stable expression patterns over time and were not associated with response to therapy or toxicity. Unsupervised cluster analysis revealed an immune signature of CAR T-cell products associated with the development of immune cell-associated neurotoxicity syndrome. Warranting validation in an independent patient cohort, in-depth phenotyping of CAR T-cell products as well as longitudinal monitoring post cell transfer might become a valuable tool to increase efficacy and safety of CAR T-cell therapy.

Vitamin D levels in patients of acute leukemia before and after remission-induction therapy.

OBJECTIVES: To determine the levels of 25-hydroxyvitamin [25(OH)D3] in patients with acute leukemia and the effect of remission-induction chemotherapy. METHODOLOGY: This study was case control, all newly diagnosed patients of acute leukemia between the age of one to sixty years and residents of Pakistan were enrolled and evaluated. Those who were unwilling or unable to provide written informed consent were excluded. All selected patients (n=86) were grouped in to acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). AML was further categorized as A1 before remission-induction (n=17) and B1 after remission induction (n=13), ALL was further categorized as A2 before remission-induction (n=31) and B2 after remission induction (n=25). The 25-hydroxyvitamin [25(OH)D3] levels were measured in the sera of all patients (before and after remission-induction) by one step delayed chemiluminescent micro particle immunoassay (CMIA).We compared 25(OH)D3 levels in all patients before and after the remission-induction chemotherapy. RESULTS: A total of 86 patients were analyzed, in which 60 patients were male. Mean age was 24.39 years (range, 1 to 60 years); the mean levels of 25(OH)D in group A1 (n=17) was 17.70±3.2 ng/ml, in group B1 (n=13) 14.06±2.4 ng/ml, 19.07±7.08 ng/ml in group A2 (n=31), while 10.59±3.9 ng/ml found in group B2 (n=25). CONCLUSION: 25(OH)D3 insufficiency was evident subnormal in majority of patients with acute leukemia and 25(OH)D3 were further reduced after remission-induction as compared to untreated group, difference was statistically significant when compared with each group.

Methotrexate-Induced Stroke-Like Syndrome: A Typical Presentation of a Rare Complication.

Methotrexate (MTX) is one of the mainstay drugs used in acute lymphoblastic leukemia (ALL) management; however, it can cause damage to the central nervous system (CNS), typically to the subcortical white matter. Stroke-like syndrome is one particular form of MTX-related neurotoxicity that occurs within 21 days of methotrexate administration (intrathecal or high-dose intravenous treatment). The clinical picture comprehends fluctuating neurological symptoms evoking acute cerebral ischemia or hemorrhage (paresis or paralysis, speech disorders - aphasia and/or dysarthria, altered mental status, and occasionally seizures), with spontaneous resolution in the majority of cases, without other identifiable cause. The typical neuroimage includes areas of restricted diffusion on diffusion-weighted imaging and non-enhancing T2 hyper-intense lesions in the white matter, on brain MRI. We report a 12-year-old boy with low-risk B-ALL without CNS involvement, who presented to the emergency department with complaints of sudden paresis of the four limbs (more severe on the right side), aphasia, and confusion. He had received one dose of intrathecal MTX 11 days prior to this episode. An angio-MRI of the brain revealed bilateral restricted diffusion areas in the centrum semiovale, and symptoms fluctuated until complete neurological recovery without any medical intervention, which is very suggestive of MTX-related neurotoxicity. This case illustrates a rare complication of MTX administration that presented with typical clinical and radiological characteristics, in an adolescent with hematological malignancy who experienced swift and full neurological recovery.

Neuropsychiatric disorders in adults undergoing chimeric antigen receptor T cells therapy for aggressive lymphomas and acute lymphoblastic leukemia.

Objective: To evaluate risk factors for neuropsychiatric disorders (NPD) in recipients of CART therapy. Methods: Patients ≥ 18 years with acute lymphoblastic leukemia (ALL), and aggressive B-cell lymphomas who received CART in 2018 were evaluated. Patients with and without NPD were compared. Results: NPD was diagnosed in 31.2% of patients. Compared to patients without NPD, patients with NPD were likely to be females (P = 0.035) and have ALL (P = 0.039). NPD was significantly associated with female gender (OR = 2.03) and diagnosis of ALL (OR = 2.76). No association between NPD and outcomes. Conclusions: Female gender and ALL were risk factors for NPD.

Involvement of Rho-Associated Coiled-Coil Containing Kinase (ROCK) in BCR-ABL1 Tyrosine Kinase Inhibitor Cardiovascular Toxicity.

Background: Second- and third-generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) are associated with cardiovascular adverse events (CVAEs) in patients with Philadelphia chromosome-positive (Ph+) leukemia. Objectives: We hypothesized that second- and third-generation BCR-ABL1 TKIs may cause CVAEs through the activation of Rho-associated coiled-coil containing kinase (ROCK). Methods: Peripheral blood mononuclear cells from 53 Ph+ patients on TKIs and 15 control patients without Ph+ leukemia were assessed for ROCK activity through capillary electrophoresis (median follow-up = 26 months [Q1-Q3: 5-37 months]). We also investigated the effects of TKIs and ROCK on endothelial dysfunction in vitro, which could contribute to CVAEs. Results: Patients receiving second- and third-generation TKIs had 1.6-fold greater ROCK activity compared with patients receiving imatinib and control patients. Elevated ROCK activity was associated with an increased incidence of CVAEs in Ph+ leukemia patients. In endothelial cells in vitro, we found that dasatinib and ponatinib treatment led to changes in actin intensity and endothelial permeability, which can be reversed by pharmacologic inhibition of ROCK. Ponatinib led to decreased cell proliferation, but this was not accompanied by senescence. Dasatinib and ponatinib treatment led to phosphor-inhibition of endothelial nitric oxide synthase and decreased nitric oxide production. ROCK inhibition reversed endothelial permeability and endothelial nitric oxide synthase-related endothelial dysfunction. Imatinib and nilotinib induce phosphorylation of p190RhoGAP. Conclusions: Our findings suggest ROCK activity may be a prognostic indicator of CVAEs in patients receiving BCR-ABL1 TKIs. With further study, ROCK inhibition may be a promising approach to reduce the incidence of CVAEs associated with second- and third-generation BCR-ABL1 TKIs.

Impact of Perioperative Absolute Neutrophil Count on Central Line-Associated Bloodstream Infection in Children With Acute Lymphoblastic and Myeloid Leukemia.

BACKGROUND: There is lack of evidence concerning safety of placement of tunneled central venous catheters (TCVCs) in neutropenic children with acute leukemias. Here, we evaluate the impact of absolute neutrophil count (ANC) at the time of TCVC placement on development of central line-associated bloodstream infections (CLABSI) in children with lymphoblastic (ALL) or myeloid leukemia (AML). MATERIALS AND METHODS: A retrospective observational study of children undergoing TCVC placement at a tertiary referral hospital between January 2000 and December 2019 was performed. Traditional and competing-risks regression models were used to estimate the effect of perioperative ANC on development of CLABSI. RESULTS: A total of 350 children (median age 6.4 [IQR: 3.1-10.9] years) underwent 498 consecutive TCVC implantations in neutropenic (n = 172, 34.5%) and non-neutropenic conditions (n = 326, 65.5%). The median length of observation per TCVC was 217.1 (IQR: 116.1-260.5) days with a total of 99,681 catheter days (CD). There were no differences in early (within first 30 days after TCVC placement) and overall CLABSI rates between neutropenic and non-neutropenic patients (HR 1.250, p = 0.502; HR 1.633, p = 0.143). We identified female sex (HR 2.640, p = 0.006) and the use of TCVC for treatment of relapsed leukemia (HR 4.347, p < 0.0001) as risk factors for early CLABSI and the use of double-lumen catheters (HR 2.607, p = 0.003) and use of TCVCs during leukemia relapse (HR 2.004, p = 0.005) for overall study period. CONCLUSION: The placement of TCVC in children with neutropenia undergoing anticancer therapy for acute leukemia is safe and not associated with an elevated rate of CLABSI.

Acute Myocardial Dysfunction and Hypereosinophilic Infiltrative Myocarditis Secondary to New-Onset Pediatric Acute Lymphoblastic Leukemia.

Myocardial infiltration by eosinophils leads to myocardial inflammation and fibrosis, resulting in restrictive hemodynamics. We describe an uncommon presentation of eosinophilic predominant acute lymphoblastic leukemia that manifested with hypereosinophilic infiltrative myocarditis. (Level of Difficulty: Advanced.).

Spontaneous bone infarction of the distal femur in a patient with Cushing's disease: a case report.

Avascular necrosis of the femoral head is a well-known complication of treatment with high dosage glucocorticoids and has been described in a few patients with Cushing's syndrome. In this case report, we describe the, to our knowledge, first case of a patient with endogenous Cushing's syndrome with a bone infarction located in the distal femur. In patients with Cushing's syndrome and bone pain, the diagnosis of bone infarction should be considered as it can occur as a rare complication of hypercortisolism.

Design, synthesis, and biological evaluation of Bcr-Abl PROTACs to overcome T315I mutation.

Bcr-Abl threonine 315 to isoleucine 315 (T315I) gatekeeper mutation induced drug resistance remains an unmet clinical challenge for the treatment of chronic myeloid leukemia (CML). Chemical degradation of Bcr-AblT315I protein has become a potential strategy to overcome drug resistance. Herein, we first described the design, synthesis, and evaluation of a new class of selective Bcr-AblT315I proteolysis-targeting chimeric (PROTAC) degraders based on GZD824 (reported as Bcr-AblT315I inhibitor by our group). One of the degrader 7o with 6-member carbon chain linkage with pomalidomide exhibits the most potent degradation efficacy with DR of 69.89% and 94.23% at 100 and 300 nmol/L, respectively, and has an IC50 value of 26.8 ± 9.7 nmol/L against Ba/F3T315I cells. Further, 7o also displays substantial tumor regression against Ba/F3-Bcr-AblT315I xenograft model in vivo.

Biological drug and drug delivery-mediated immunotherapy.

The initiation and development of major inflammatory diseases, i.e., cancer, vascular inflammation, and some autoimmune diseases are closely linked to the immune system. Biologics-based immunotherapy is exerting a critical role against these diseases, whereas the usage of the immunomodulators is always limited by various factors such as susceptibility to digestion by enzymes in vivo, poor penetration across biological barriers, and rapid clearance by the reticuloendothelial system. Drug delivery strategies are potent to promote their delivery. Herein, we reviewed the potential targets for immunotherapy against the major inflammatory diseases, discussed the biologics and drug delivery systems involved in the immunotherapy, particularly highlighted the approved therapy tactics, and finally offer perspectives in this field.

Identifying novel genetic alterations in pediatric acute lymphoblastic leukemia based on copy number analysis.

Copy number variations (CNVs) analysis may reveal molecular biomarkers and provide information on the pathogenesis of acute lymphoblastic leukemia (ALL). We investigated the gene copy number in childhood ALL by microarray and select three new recurrent CNVs to evaluate by real-time PCR assay: DMBT1, KIAA0125 and PRDM16 were selected due to high frequency of CNVs in ALL samples and based on their potential biological functions in carcinogenesis described in the literature. DBMT1 deletion was associated with patients with chromosomal translocations and is a potential tumor suppressor; KIAA0125 and PRDM16 may act as an oncogene despite having a paradoxical behavior in carcinogenesis. This study reinforces that microarrays/aCGH is it is a powerful tool for detection of genomic aberrations, which may be used in the risk stratification.

CAR T Cell Therapy-Related Cardiovascular Outcomes and Management: Systemic Disease or Direct Cardiotoxicity?

CD19-specific chimeric antigen receptor (CAR) T cell therapies have shown remarkable early success in highly refractory and relapsing hematological malignancies. However, this potent therapy is accompanied by significant toxicity. Cytokine release syndrome and neurotoxicity are the most widely reported, but the true extent and characteristics of cardiovascular toxicity remain poorly understood. Thus far, adverse cardiovascular effects observed include sinus tachycardia and other arrhythmias, left ventricular systolic dysfunction, profound hypotension, and shock requiring inotropic support. The literature regarding cardiovascular toxicities remains sparse; prospective studies are needed to define the cardiac safety of CAR T cell therapies to optimally harness their potential. This review summarizes the current understanding of the potential cardiovascular toxicities of CD19-specific CAR T cell therapies, outlines a proposed cardiac surveillance protocol for patients receiving this new treatment, and provides a roadmap of the future direction of cardio-oncology research in this area.

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells.

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.

Cytomegalovirus induced hemophagocytic lymphocytic histiocytosis in two pediatric patients with acute lymphoblastic leukemia.

Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory condition with tissue destruction due to abnormal immune activation. We present a series of 2 cases of cytomegalovirus-induced HLH in children during maintenance chemotherapy for acute lymphoblastic leukemia. These cases emphasize the importance of considering secondary HLH in this high-risk subset of pediatric patients.

The role of serratiopeptidase in the resolution of inflammation.

Inflammation remains a key event during most of the diseases and physiological imbalance. Acute inflammation is an essential physiological event by immune system for a protective measure to remove cause of inflammation and failure of resolution lead to chronic inflammation. Over a period of time, a number of drugs mostly chemical have been deployed to combat acute and chronic inflammation. Recently, enzyme based anti-inflammatory drugs became popular over conventional chemical based drugs. Serratiopeptidase, a proteolytic enzyme from trypsin family, possesses tremendous scope in combating inflammation. Serine protease possesses a higher affinity for cyclooxygenase (COX-I and COX-II), a key enzyme associated with production of different inflammatory mediators including interleukins (IL), prostaglandins (PGs) and thromboxane (TXs) etc. Currently, arthritis, sinusitis, bronchitis, fibrocystic breast disease, and carpal tunnel syndrome, etc. are the leading inflammatory disorders that affected the entire the globe. In order to conquer inflammation, both acute and chronic world, physician mostly relies on conventional drugs. The most common drugs to combat acute inflammation are Nonsteroidal anti-inflammatory drugs (NSAIDs) alone and or in combination with other drugs. However, during chronic inflammation, NSAIDs are often used with steroidal drugs such as autoimmune disorders. These drugs possess several limitations such as side effects, ADR, etc. In order to overcome these limitations and complications, enzyme based drugs (anti-inflammatory) emerged, and aim for a new high since the last decade. Serine protease, the largest proteolytic family has been reported for several therapeutic applications, including anti-inflammatory. Serratiopeptidase is a leading enzyme which has a very long history in medical as an effective anti-inflammatory drug. Current study emphasizes present scenario and future prospect of serratiopeptidase as an anti-inflammatory drug. The study also illustrates a comparative analysis of conventional drugs and enzyme based therapeutic to combat inflammation.

The plasma lipidome in acute myeloid leukemia at diagnosis in relation to clinical disease features.

BACKGROUND: Early studies established that certain lipids were lower in acute myeloid leukemia (AML) cells than normal leukocytes. Because lipids are now known to play an important role in cell signaling and regulation of homeostasis, and are often perturbed in malignancies, we undertook a comprehensive lipidomic survey of plasma from AML patients at time of diagnosis and also healthy blood donors. METHODS: Plasma lipid profiles were measured using three mass spectrometry platforms in 20 AML patients and 20 healthy blood donors. Data were collected on total cholesterol and fatty acids, fatty acid amides, glycerolipids, phospholipids, sphingolipids, cholesterol esters, coenzyme Q10 and eicosanoids. RESULTS: We observed a depletion of plasma total fatty acids and cholesterol, but an increase in certain free fatty acids with the observed decline in sphingolipids, phosphocholines, triglycerides and cholesterol esters probably driven by enhanced fatty acid oxidation in AML cells. Arachidonic acid and precursors were elevated in AML, particularly in patients with high bone marrow (BM) or peripheral blasts and unfavorable prognostic risk. PGF2α was also elevated, in patients with low BM or peripheral blasts and with a favorable prognostic risk. A broad panoply of lipid classes is altered in AML plasma, pointing to disturbances of several lipid metabolic interconversions, in particular in relation to blast cell counts and prognostic risk. CONCLUSIONS: These data indicate potential roles played by lipids in AML heterogeneity and disease outcome. GENERAL SIGNIFICANCE: Enhanced catabolism of several lipid classes increases prognostic risk while plasma PGF2α may be a marker for reduced prognostic risk in AML.

Design, synthesis, and biological evaluation of Bcr-Abl PROTACs to overcome T315I mutation

Bcr-Abl threonine 315 to isoleucine 315 (T315I) gatekeeper mutation induced drug resistance remains an unmet clinical challenge for the treatment of chronic myeloid leukemia (CML). Chemical degradation of Bcr-Abl

A case of Acute Myeloid Leukemia masquerading as unilateral exudative detachment.

PURPOSE: Leukemias can involve almost every part of the human eye. Ophthalmic manifestations of leukemias can be divided into direct infiltration, secondary vascular changes and neuro-ophthalmological changes. Our case presented with exudative retinal detachment mimicking Vogt Koyanagi Harada's disease (VKH). OBSERVATIONS: A 30-years old Asian (Indian) female presented with insidious onset of painless diminution of vision from her right eye for one month. She gave history of fever and severe headache at the time of onset of ocular symptoms. Fundus examination revealed exudative retinal detachment at the posterior pole of her right eye. Fundus fluorescein angiography showed early stippled pin point hyperfluorescence, placoid pooling of the dye and late disc staining in both the eyes. A provisional diagnosis of Vogt Koyanagi Harada disease was made and routine blood investigations and a physician check-up for fitness for systemic steroids was done. Peripheral blood smear showed the presence of blast cells. The patient was diagnosed to have Acute Myeloid Leukemia (AML) and was started on chemotherapy. CONCLUSIONS AND IMPORTANCE: Acute myeloid leukemia can present as an exudative retinal detachment and can mimic similarly presenting conditions like VKH. Hence, this very important differential diagnosis should be kept in mind and it stresses the importance of simple laboratory investigations like whole and differential blood counts.