Fas and Fas Ligand Expression In Children with Acute Lymphoblastic Leukemia, Benign Hematological Diseases and Solid Tumors

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4853-4853
Author(s):  
Eftichia Stiakaki ◽  
Georgia Martimianaki ◽  
Maria Kaparou ◽  
Chryssoula Perdikogianni ◽  
Maria Kalmanti
Keyword(s):  
Bone Marrow ◽  
Solid Tumors ◽  
Fas Ligand ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Response To Treatment ◽  
End Of Treatment ◽  
Significant Difference ◽  
Activated T Lymphocytes

Abstract Abstract 4853 Fas/FasL is a key pathway of cellular apoptosis. Fas receptor is expressed on membranes of both normal and neoplastic cells while, Fas ligand (FasL) mainly on activated T-lymphocytes. Fas-FasL abnormalities have been detected in malignancies and autoimmune diseases and implicated in resistance to treatment. The aim of the study was the determination of the levels of Fas, FasL and their coexpression in bone marrow cells of children with acute lymphoblastic leukaemia (ALL) at diagnosis, during the course of treatment and the comparison with relevant levels in other hematological and neoplastic diseases. Expression levels of Fas and FasL were determined with flow cytometry in children with ALL at diagnosis (ALLd, n=13), on day 15 of treatment (d15, n=6), on day33 (ALLd33, n=6), during consolidation (ALLhr, n=12) and at the end of therapy (ALLet, n=7) as well as in children with Langerhans Histiocytosis (LCH, n=4), cytopenias (Cytp, n=8) and solid tumors without bone marrow infiltration at diagnosis (STd, n=5) and on therapy (STther, n=6). Results: The lowest levels of Fas expression were detected at diagnosis of ALL and were gradually statistical significantly increased until remission of day 33 (ALLd vs ALLd33: 8.02±1.94 vs 24.04±6.11, p=0.035). At consolidation Fas levels were found to be decreased compared to day33 (16.1±4.18) and were again increased at the end of therapy (ALLd vs ALLet: 8.02±1.94 vs 24.96±7.95, p=0.024). On the contrary, FasL levels were gradually decreased and finally increased to similar to diagnosis levels at the end of treatment (ALLd: 4.59±1.41, ALLet: 5.89±1.99). In solid tumors at diagnosis Fas levels were similar to the ones while on chemotherapy (STd vs STth:16.04±2.2 vs 15.19±6.4). The highest FasL levels were detected in the group of STd with the relevant levels on treatment being lower in comparison (STd vs STther: 10.91±3.32 vs 2.92±0.79, p=0.052). In LCH both Fas and FasL levels were found to be as low as at ALL diagnosis. In cytopenias no significant difference was observed between groups for either Fas (11.05±4.49) or FasL (3.01±0.62). As for Fas+FasL+ coexpression no difference was evident between ALLd, ALLet and STd or STther [ALLd (0.73±0.38), ALLet (0.64±0.17), STd (0.68±0.095), STther (0.66±0.38)]. The lowest coexpreesion levels were observed in the group of cytopenias with statistical significant difference compared to STd (0.68±0.095 vs 0.26±0.08, p=0.031). In conclusion, at diagnosis of ALL Fas levels were expressed in lowest levels that were found to be gradually increased at remission and at the end of treatment. This finding probably correlates with the apoptotic process of the leukemia clone and possibly with response to treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clinical Utility of Morphological Evaluation of Day 14 Bone Marrow Biopsies in Acute Myeloid Leukemia Patients Undergoing Standard Induction Chemotherapy: Time to Change Practice?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1004-1004 ◽  
Author(s):  
Yehuda E. Deutsch ◽  
German Campuzano-Zuluaga ◽  
Matthew P Salzberg ◽  
Alexandra Gomez Arteaga ◽  
Justin M. Watts ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Induction Chemotherapy ◽  
Induction Therapy ◽  
Predictive Value ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Response To Treatment ◽  
Bone Marrow Biopsies ◽  
Count Recovery

Abstract Introduction Early bone marrow (BM) evaluation (during aplasia or at “day 14” (D14)) in patients with AML undergoing conventional induction therapy has been adopted from clinical practice in pediatric acute lymphoblastic leukemia (ALL). While it has been shown that early persistence of AML correlates with decreased complete remission (CR) rates and overall survival (OS), unlike in ALL, there are no data to indicate that early initiation of re-induction therapy based on these findings will positively influence outcome. In fact, early re-induction, especially in older patients, may increase morbidity and mortality from prolonged cytopenias, infectious complications, and longer hospital stays. Moreover, it can be challenging to determine the nature of scattered blasts identified in a hypocellular BM at D14, as they may represent normal recovering marrow elements or malignant blasts. Even if malignant, the chemosensitivityof these cells will only be fully determined by later assessment of the BM at the time of expected count recovery (“day 28”). For these reasons, early re-induction therapy may not be advisable. In this retrospective study, we sought to evaluate the validity of D14 BM assessments as post-therapy prognostication to guide treatment decisions in AML. Methods We conducted a retrospective institutional study (2006-2014) of AML patients undergoing routine induction chemotherapy where diagnostic, interim (around D14) and recovery (D21-42) BM evaluations were available for review. Clinical information and pathology data were retrieved from our institutional database. Responses at D14 were categorized morphologically into three categories: optimal response (OR, blasts ≤5%), indeterminate response (IR, blasts 6-19%), and residual leukemia (RL, blasts ≥20% or a relative decrease in blast count from baseline of <20%). Published response criteria were used to define responses at marrow recovery. Suboptimal response (SOR) at D14 was defined as either IR or RL during the assessment period. Mann-Whitney's U test was used to compare non-normally distributed variables. The Fisher's exact test was employed to assess for associations between response to treatment at D14 and likelihood of recovering in CR. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of a D14 BM to predict CR were calculated for patients that were observed without re-induction. Results Evaluable patients (n=98) had a mean age of 51 years (20-73), and 45% were male. The median BM blast percentages at diagnosis, D14 and recovery were respectively 54.5%, 0% and 2.5% (Figure 1). There was a significantly greater absolute decrease in blast percentage from diagnosis to D14 in patients who recovered in CR compared to those who did not achieve CR (median: 53.5% vs 23.0%, P = 0.001). In patients that got early re-induction therapy for SOR at D14, the relative differential in baseline and D14 BM blasts was significantly lower compared to patients with D14 SOR who did not get re-induction therapy (median: 21.8% vs 77.8%, P=0.004) Ninety patients did not receive early re-induction therapy. Of these, 86 (95.6%) achieved CR and 4 patients (4.4%) recovered counts with residual leukemia. Fourteen (14.3%) patients were classified as SOR at D14. Of these, 6 (6.7%) did not receive re-induction therapy and 4 of these 6 patients (67%) achieved a CR. Eight patients received early re-induction therapy based on SOR at D14 (IR = 2, RL = 6); of these, 4 patients (50%) achieved CR at count recovery. Achieving an OR at D14 was predictive of achieving CR at recovery (sensitivity = 95.3%, PPV = 97.6%). However, not achieving an OR at D14 had low specificity (50%) and NPV (33.3%) for achieving CR (P = 0.021). Conclusions Our results indicate that a SOR at the D14 BM evaluation does not uniformly identify patients with primary induction failure (low NPV) and should not be used to dictate the timing of re-induction therapy. We confirmed the PPV of achieving an OR at D14 as previously reported, but we argue that no additional prognostic data is provided by an OR at D14, beyond what can already be predicted by pre-treatment variables (e.g., age and chromosomal abnormalities). We suggest that the D14 BM should be omitted from the routine evaluation of AML patients during induction therapy outside the context of a clinical trial. Figure 1 Figure 1. Progression and percentage of blasts at diagnosis, day 14 and recovery assessments (n = 98). Disclosures No relevant conflicts of interest to declare.

Over-Expression of IGF-IR in Malignant Clonal Cells in Bone Marrow of Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4832-4832
Author(s):  
Qi He ◽  
Xiao Li ◽  
Zheng Zhang ◽  
Qingqiao Zhang ◽  
Feng Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Over Expression ◽  
Significant Difference ◽  
Clone Cells

Abstract Abstract 4832 To explore the expression level of insulin-like growth factor-1 receptor (IGF-IR) in malignant clone cells of myelodysplastic syndromes (MDS). Method Fluorescence in situ hybridization (FISH) and immunochemistry (APAAP, alkaline phosphatase anti-alkaline phosphatase) were used together to detect the expression of IGF-IR in the bone marrow cells of 26 MDS patients with known abnormal karyotypes. Result The average IGF-IR expression level on the surface of clone cells from the 26 MDS cases was markedly elevated compared to the corresponding level in normal cells (78.2±13.7% vs 14.1±14.0%,P <0.0001). The percentages of malignant clone cells in all 26 MDS cases were significantly correlated with the respective percentages of IGF-IR positive nucleated cells (r = 0.909; P <0.0001). No significant difference in the IGF-IR expression level on the clone cells were observed either between high- and low-risk MDS patients or among MDS patients with different abnormal karyotypes. Conclusion IGF-IR might be taken as a marker of clone cells in MDS because of its propensity to cause malignant proliferation. Disclosures No relevant conflicts of interest to declare.

Increased ADAM28 Levels In Plasma and Bone Marrow Cells of Patients With Adult Acute Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4990-4990
Author(s):  
Xiaohui Zhang ◽  
Qianming Wang ◽  
Hayley A Hanby ◽  
Shiyuan Zhou ◽  
Yi Zhou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Solid Tumors ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Transitional Cell ◽  
Acute Myelocytic Leukemia ◽  
Mrna Levels ◽  
Early Relapse ◽  
Marrow Cells

Abstract ADAM28, a member of the ADAM (A Disintegrin And Metalloproteinase) family, cleaves various substrates including von Willebrand factor. Two isoforms of ADAM28: the membrane associated (ADAM28m) and the secreted (ADAM28s) were identified. Both were overexpressed in solid tumors including breast carcinoma, non-small cell lung cancer, and bladder transitional cell carcinoma. Overexpression of ADAM28 has been shown to promote cell growth, invasion, and metastasis of solid tumors in humans. However, little is known about expression and potential role of ADAM28 in hematological malignancies. In this study, we determined ADAM28 protein and/or mRNA expression in plasma and/or bone marrow cells of patients with various acute and chronic leukemia. An informed consent was obtained from all participants. Plasma and bone marrow aspirates were obtained from a total of 65 patients including 24 patients with acute lymphoblastic leukemia (ALL), 23 patients acute myelocytic leukemia (AML), and 18 patients with other leukemia. Twenty one healthy volunteers were also included in the study (Table 1). By flow cytometry, we showed that mean fluorescent index ratios that are specific for ADAM28m were significantly <> <>increased in the bone marrow malignant cells in patients with ALL (2.9±0.13) and AML (2.1±0.17) compared with those in the control (0.4±0.06). The p values were <0.01 and <0.05, respectively. In addition, plasma levels of ADAM28s antigen assessed by a specific sandwich ELISA were significantly higher in patients with ALL (386.1±44.6 pg/ml) (p<0.01) and AML (291.3±24.0 pg/ml) (p<0.05) than those in the control (120.7±12.1 pg/ml). Furthermore, the mRNA levels encoding ADAM28m (1.7±0.0) and ADAM28s (2.9±0.1) in the bone marrow cells were significantly higher than those in the control (p< 0.05) (Table 1). Interestingly, cellular ADAM28 overexpression was inversely associated with cellular apoptosis in ALL patients. In four ALL patients with an early relapse, ADAM28 levels (both protein and mRNA) in bone marrow cells were significantly higher than those in patients with complete remission. In conclusion, our data indicate for the first time that a measurement of plasma and bone marrow ADAM28 levels may help identify patients with high risk of early relapse. Whether a treatment that targets ADAM28 has an effect in adult acute leukemia remains to be determined. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Igh-V(D)J NGS-MRD Measurement in Pediatric B-Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4923-4923
Author(s):  
Suying Lu ◽  
Jia Zhu ◽  
Junting Huang ◽  
Juan Wang ◽  
Feifei Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Induction Therapy ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Clinicopathological Characteristics ◽  
Marrow Cells

Abstract Introduction B-acute lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. Early response to induction chemotherapy is one of the important prognostic factors in B-ALL. However, the analytic sensitivity for flow cytometry (FC) is only 10 -4. The feasibility of using next-generation sequencing (NGS) of immunoglobulin for the determination of minimal residual disease (MRD) in B-ALL has been demonstrated. This study aimed to investigate the performance of NGS techniques measuring immunoglobulin heavy chain (IgH)-variable, diversity, and joining (V[D]J) clonal rearrangements compared with FC in detecting MRD for children with B-ALL and to predict the clinical outcome of B-ALL patients. Methods Newly diagnosed younger than 18 years old B-ALL patients who received the treatment strategy of South China children's leukemia Group (SCCLG)-ALL 2016 were recruited. DNA extracted from bone marrow cells at all available time points for each patient was submitted to Simcere diagnostics for sequencing using Illumina NovaSeq platform. We performed IgH V(D)J NGS and FCM on the bone marrow serially obtained at diagnosis (D0), 15 days at induction therapy (D15), 33 days at induction therapy (D33) and then at the end of induction therapy (EOI). We defined MRD positive (MRD +) by IgH V(D)J NGS and FCM as more than 1 blast cell among 10 4 and 10 6 bone marrow cells, respectively. The sensitivity of MRD detection by IgH V(D)J NGS and FCM, and the association of MRD status with clinicopathological characteristics were investigated. Statistical analysis was performed through SPSS Statistics 22. Enumeration data and correlation between MRD data and clinicopathological characteristics were compared by Chi-square test or Fisher's exact test. This trial was registered at www.clinicaltrials.gov as # NCT04977895. Results As of July 27, 2021, 22 patients (median age, 4.5 years; range, 3.0-7.3) were enrolled in the study. Three patients (13.6%) had a t (9;22) translocation consistent with Philadelphia chromosome positive disease. According to risk stratifications, 8 (36.4%), 8 (36.4%), and 2 (9.1%) patients were classified as low risk (LR), intermediate risk (IR), and high risk (HR) groups, respectively. The remaining 4 patients are still under treatment and have not been classified. We identified leukemic IgH clones in 100% of the diagnostic samples and 68.2% (15/22) of the patients were polyclonal. In 11 patients whose samples of all the four timepoints (D0, D15, D33, EOI) have been tested in parallel by FCM and IgH V(D)J NGS, the frequencies of patients with MRD + were 30.4% vs. 90.9% at D15 (P＜0.05) by FCM and IgH V(D)J NGS. IgH V(D)J NGS MRD monitoring could identify MRD + patients with frequency of 45.5% and 18.2% among patients achieved MRD negativity by FCM at D33 (P＜0.05) and EOI (P = 0.46). With an MRD detection limit of 10 -6, 90.9% (10/11), 36.4% (4/11) and 18.2% (2/11) patients were MRD negative by FCM but positive by the NGS test at D15, D33 and EOI, respectively. This suggested that the sensitivity of IgH V(D)J NGS was significantly higher than that of FCM. Correlation of the measured MRD between the two methods in the entire cohort (r = 0.7934, P &lt; 0.0001) as well as in the concordant cases (r = 0.5558, P = 0.0032) was very high. There was a high discordant rate with NGS identifying more patients MRD + at this threshold. Furthermore, NGS MRD was positive but the FCM MRD was negative in 13 samples (P &lt; 0.0001). In addition, positive MRD status of D33 by NGS was significantly associated with the age of B-ALL patients, patients under 6 years more frequently harbored detectable MRD compared with those ≥ 6 years old (87.5% vs. 11.1%, P &lt; 0.01). There was no patient relapsed after a medium follow-up of 10.5 months. Conclusions We demonstrated the higher sensitivity of IgH-V(D)J NGS in MRD detection of B-ALL, which implies that NGS MRD monitoring could be helpful for more accurate risk stratifications and more precise treatment according to risk stratifications. Further study with a larger sample size and a longer follow-up period is need. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 712-718 ◽  
Author(s):  
SD Smith ◽  
EM Uyeki ◽  
JT Lowman
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Assay System ◽  
Leukemic Cells ◽  
Specific Cell ◽  
Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

Evaluation of genotoxicity of pan masala employing chromosomal aberration and micronucleus assay in bone marrow cells of the mice

2009 ◽  
Vol 25 (7) ◽  
pp. 467-471 ◽  
Author(s):  
BN Mojidra ◽  
K. Archana ◽  
AK Gautam ◽  
Y. Verma ◽  
BC Lakkad ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromosome Aberration ◽  
Chromosomal Aberration ◽  
Bone Marrow Cells ◽  
Micronucleus Assay ◽  
Control Group ◽  
Genotoxic Potential ◽  
Polychromatic Erythrocytes ◽  
Significant Difference ◽  
Marrow Cells

Pan masala is commonly consumed in south-east Asian and other oriental countries as an alternate of tobacco chewing and smoking. Genotoxic potential of pan masala (pan masala plain and pan masala with tobacco known as gutkha) was evaluated employing chromosome aberration (CA) and micronucleus (MN) assay in vivo. Animals were exposed to three different doses (0.5%, 1.5% and 3%) of pan masala plain (PMP) and gutkha (PMT) through feed for a period of 6 months and micronucleus and chromosomal aberrations were studied in the bone marrow cells. Induction of mean micronuclei in polychromatic erythrocytes (MNPCE) and normochromatic erythrocyte (MNNCE) was higher in both types of pan masala treated groups with respect to control group. Both pan masala plain and gutkha treatment significantly induced the frequency of MNPCE and MNNCE in the bone marrow cells, indicating the genotoxic potential. Furthermore, slight decline in the ratio of polychromatic erythrocytes to normochromatic erythrocytes was also noticed, suggesting the cytotoxic potential even though the ratio was statistically non significant. A dose-dependent, significant increase in chromosome aberration was observed in both types of pan masala treated mice with respect to control. However, no significant difference in micronucleus and chromosomal aberration induction was noticed between two types of pan masala exposed (PMP and PMT) groups. Results suggest that both types of pan masala, i.e. plain and gutkha, have genotoxic potential.

scholarly journals INCIDENCE AND PREVALENCE OF ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) IN KASHMIRI POPULACE (NORTH INDIA)

2021 ◽  
Vol 9 (5) ◽  
pp. 1349-1354
Author(s):  
Fozia Mohammad ◽  
◽  
Arshad A. Pandith ◽  
Mithilesh Kumar ◽  
Aabid Koul ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rural Areas ◽  
Urban Areas ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
White Blood Cells ◽  
North India ◽  
Cell Phenotype ◽  
Age Group ◽  
Mixed Phenotype

Background: The most frequent cancer of the childhood is acute lymphoblastic leukaemia (ALL). It is the blood and bone marrow cancer affecting white blood cells. It is caused by errors in the DNA in the bone marrow cells. Our goal was to evaluate the prevalence of ALL in Kashmiri populace. Methods: The study in the hindsight was initiated for ALL patients registered between early 2018up to late 2019 to investigate its frequency and prevalence. Results: Overall from 74 ALL patients, based on gender, 44 (59%) were males and 30 (41%) were females. Based on age, 53 (72%) were in the age group of ≤18 years while 21 (28%) were in the age group of >18. Based on immunophenotypes 69 (93%) were of Pre B-cell phenotype, 3 (4%) belonged to T-cell phenotype while 2 (3%) were of mixed phenotype. Based on demography, 10 (14%) were from urban areas while as 64 (86%) were from rural areas of Kashmir region. Conclusions: Although the prevalence of ALL in this region is very high, but gender has no significance while age and dwelling has significance on its overall frequency and significance.

scholarly journals Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 49-49
Author(s):  
Qiuling Chen ◽  
Yuelong Jiang ◽  
Qinwei Chen ◽  
Long Liu ◽  
Bing Xu
Keyword(s):  
B Cell ◽  
Solid Tumors ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Lymphocytic Leukemia ◽  
Advanced Lung Cancer ◽  
Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

scholarly journals A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Sara M.A. Mohamed ◽  
Keith Sia ◽  
Karl-Heinz Friedrich ◽  
Andreas Wohlmann ◽  
Savvas Savvides ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Statistical Significance ◽  
Immunofluorescence Assay ◽  
Laser Scanning Microscopy ◽  
Indirect Immunofluorescence Assay ◽  
Single Chain ◽  
Genetic Characteristics ◽  
Significant Difference

Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells. Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate. Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed &gt;80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P &lt; 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site. Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL. Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document