Chronic Lymphoblastic Leukemia Research Articles

Role of microRNAs in Chronic Lymphocytic Leukemia Pathogenesis.

MicroRNAs (miRNAs) are a group of small endogenous non-coding RNAs involved in many cancers and various cellular processes such as cellular growth, DNA methylation, apoptosis, and differentiation. 13q14.3 chromosomal region contains miR-15 and miR-16 and deletion of this region is a commonly reported aberration in Chronic Lymphoblastic Leukemia (CLL), suggesting miRNAs involvement in CLL pathogenesis. MicroRNAs are known as oncogenes and tumor suppressors in CLL which may also serve as markers of onset and progression of the disease. The most prevalent form of leukemia diagnosed in adults in the western world, chronic lymphocytic leukemia, accounts for one-third of all leukemias. CLL is characterized by the presence of B Cell Malignant Clones in secondary lymphoid tissues, peripheral blood and bone marrow. The precise etiology of CLL is remained to be known, however, a number of Chromosomal Abnormalities such as deletions of 13q14.3, 11q and 17p and trisomy 12 have been detected. In this review, we offer our prospect on how miRNAs are involved in the CLL pathogenesis and disease progression. Further understanding of the underlying mechanisms and regulation of CLL pathogenesis has underscored the need for further research regarding their role in this disease.

Chronic lymphocytic leukemia complicated with multiple myeloma: report of one case and review of literature

Objective To investigate the clinical features, diagnosis, occurrence sequence and clonal origin of chronic lymphocytic leukemia complicated with multiple myeloma. Methods The diagnosis and treatment of one patient with multiple myeloma and chronic lymphocytic leukemia who was admitted to the First Hospital of Jilin University in May 2018 was retrospectively analyzed, and the related literatures were reviewed. Results This patient began with lumbosacral pain, and he was diagnosed as chronic lymphocytic leukemia complicated with multiple myeloma after bone marrow aspiration, flow cytometry, and blood and urine immunofixation electrophoresis. It is recommended that Rd (lenalidomide + dexamethasone) or MPV (melphalan + prednisone + bortezomib) regimen, but the patient did not receive chemotherapy and died of infectious diarrhea 1 month later. Conclusions The occurrence of multiple myeloma and chronic lymphoblastic leukemia may originate from the same clone or different new clone. It is very rare that multiple myeloma and chronic lymphoblastic leukemia can co-occur. Therapeutic options tend to be more aggressive multiple myeloma-based regimen. Key words: Leukemia, lymphocytic, chronic, B-cell; Multiple myeloma; Clone cells

Abstract ED01-02: Successes and failures of targeted drug development in pediatric tumors

Abstract Eighty percent of children diagnosed with cancer are cured with multimodality therapy including cytotoxic chemotherapy. However, up to 40% of survivors of childhood cancer have at least one serious chronic medical condition as a consequence of curative therapy. Thus, patients, families, clinicians, researchers, and regulatory agencies enthusiastically endorse the development of targeted therapy to improve survival, decrease life threatening acute toxicity, and late effects of therapy for childhood cancer. Genomic characterization demonstrates that the tumor mutational burden in childhood cancers is low. For children with cancer a small number of drugs, such as NTRK inhibitors have had major unequivocal successes including initial regulatory approvals that include specific dosing in children. Other molecularly targeted drugs have achieved moderate success, however, many have failed despite strong pre-clinical rationale. As of June 2019, 25 drugs have been FDA approved for use in children with cancer including five tyrosine kinase inhibitors (3 BCR-ABL inhibitors and 2 NTRK inhibitors), and one mTOR inhibitor. A single drug, dinutuximab, was approved for a pediatric specific indication in high-risk neuroblastoma. This parallels recent European Medicines Agency authorizations that include indications for treating childhood cancer in 10 of 177 anti-cancer drug approvals. Similar to adults, the era of targeted therapies in children with cancer began with imatinib. Drugs targeting BCR-ABL in chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) in children include imatinib, dasatinib, and nilotinib. Point mutations in the kinase domain of the BCR-ABL fusion protein result in resistance to imatinib. Dasatinib, nilotinib, and ponatinib were developed to overcome resistance. Each has a specific role in caring for children with CML or Ph+ ALL. Ph-like ALL, a high-risk acute leukemia in children and young adults, is characterized by genomic alterations and activation of signaling pathways including JAK/STAT, amenable to inhibition tyrosine kinase inhibitors. The landmark age and histology agnostic global approval of larotrectinib and entrectinib was based on durable objective responses in patients with tumors harboring NTRK fusions enrolled on early phase clinical trials. Larotrectinib has produced dramatic objective responses in children and adults with cancer harboring NTRK fusions including a very rare cancer, infantile fibrosarcoma which is NTRK fusion positive in at least 95% cases. In addition to potent inhibition NTRK fusions, initial clinical trials of larotrectinib included adults and children as well as a pediatric appropriate formulation. Children with brain tumors harboring NTRK1,2,3 or ROS1 fusions have had rapid, durable objective responses to entrectinib. MAP kinase pathway aberrations occur in childhood cancer including rhabdomyosarcoma and brain tumors. Everolimus is approved for patients with subependymal giant cell astrocytoma. Dabrafenib and vemurafenib have produced durable objective responses in patients with pediatric low-grade glioma harboring BRAF V600E mutations. Selumetinib has been FDA approved for the treatment of plexiform neurofibromas in children with neurofibromatosis type 1 and may have activity in children and adolescents with solid tumors harboring MAP kinase aberrations. Overall, the success of MAP kinase inhibition in children with cancer has been modest, leading to consideration for combinatorial approaches. Crizotinib is approved for the treatment of ALK-positive metastatic NSCLC and has activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and inflammatory myofibroblastic tumors, both associated with ALK fusions. Trials are ongoing to determine the activity of crizotinib and other ALK inhibitors in children with neuroblastoma with known translocations, mutations or amplifications in the ALK gene. Clinical trials of multi-targeted kinase inhibitors including cabozantinib, regorafenib, sorafenib and vandetanib have demonstrated activity in a variety of solid tumors. Clinical trials of these agents have been based on mechanistic rationale and pre-clinical evaluation rather than biomarker selection. Through large scale genomic sequencing efforts in the US and Europe actionable molecular alterations including mutations, amplifications and fusions have been identified in a variety of childhood cancers. Compared to cancers in adults, these molecular aberrations occur less frequently in most cancers in children. However, due to dramatic objective responses and the potential for improved response with decreased toxicity, efforts to evaluate and incorporate targeted agents into multimodality therapy for children with cancer remains a major focus. To date, multi-targeted kinase inhibitors and drugs that target fusion oncoproteins that are histology-agnostic oncogenic drivers in cancers that occur in both child and adults have been most successful. Understanding the characteristics of successful target- drug pairs is critically important to the prioritization of new drugs and clinical trials in children as well as the development of new therapies for pediatric specific targets. Citation Format: Elizabeth Fox. Successes and failures of targeted drug development in pediatric tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr ED01-02. doi:10.1158/1535-7163.TARG-19-ED01-02

Cellular Kinetics of CD19 Chimeric Antigen Receptor T Cells in Patients with Relapsed/Refractory Non-Hodgkin's Lymphoma

Background Chimeric antigen receptor T cell (CART) therapy against blood malignancies exhibits unique pharmacokinetic profiles in vivo. There are reports of CART kinetics against acute or chronic lymphoblastic leukemia (ALL/CLL), but reports on CD19-41BBζ CART against relapsed or refractory non-Hodgkin's lymphoma (r/r NHL) are rare. Here we present a summary of the cellular kinetics of CART cells from 35 patients of aggressive or indolent NHL, highlighting the potential correlations between CART kinetics and clinical outcomes Methods Thirty-five consecutive patients with r/r NHL from a CART trial (NCT03196830) were analyzed. CART cells were administrated at doses of 5-10x106/kg in 3 split doses following lymphodepleting chemotherapies of fludarabine and cyclophosphamide. CART kinetic profiles in peripheral blood were determined by CAR transgene copies measured by quantitative PCR at various time points, and correlation analyses between kinetic and clinical factors were performed. Kinetic parameters included peak CAR transgene (Cmax), time to reach Cmax (Tmax), and area under the curve at day 28 (AUC0-28d) and day 70 (AUC0-70d) describing short-term and long-term persistence, respectively. Clinical factors included responses, adverse events, and disease characteristics. Results Correlation analysis between kinetic and efficacy profiles revealed that responders exhibited significantly higher AUC0-70d (but not Cmax and AUC0-28d) compared with non-responders, indicating that AUC0-70d rather than Cmax and AUC0-28d was correlated with clinical efficacy. This result was partially inconsistent with previous reports of CD19-BBζ CART in ALL/CLL patients (Mueller KT, et al., 2017), suggesting that CART might exhibit distinct kinetic profiles in NHL compared with ALL/CLL. Additionally, AUC0-70d was 7-fold higher in patients who maintained in remission for > 6 months after prior therapies compared with those who did not, and 278% higher in patients receiving < 3 prior lines of therapies than those > 3 lines, both of which were correlated with better durations of remission (DOR) and/or overall survival (OS). These results indicated that longer remission periods after and fewer lines of prior therapies were favorable factors of long-term persistence of CART cells, which subsequently led to improved clinical efficacy. Again, Cmax or AUC0-28d did not show any differences between groups. No difference in Tmax were achieved between any two groups Correlation analysis between kinetic and safety profiles revealed that patients with grade 3-4 cytokine release syndrome (CRS) had significantly higher Cmax than those with grade 1-2 CRS, indicating that CART expansion correlated with the severity of CRS. No correlation was observed between neurotoxicity and Cmax. Neither neurotoxicity nor CRS was correlated with Tmax, AUC0-28d, or AUC0-70d. Lastly, transient drops in CAR copies early after infusions were observed in 78% patients (24/35). A transient drop usually happened around Day 5, divided the whole expansion phase into two peaks, and created a trough value of <10% of the earlier peak. Interestingly, AUC0-70 was significantly higher in patients with transient drops than in those without, while Cmax, Tmax and AUC0-28 were nearly identical. Moreover, 75% patients (18/24) with transient drops were responders, compared with only 36% patients (4/11) without transient drops. Patients with transient drops also exhibited better DOR. This unique kinetic feature tended to be a favorable factor of efficacy, leaving the mechanism to be further explored. Conclusion Overall, significantly higher levels of long-term persistence were observed in patients who successfully responded to CART therapy in r/r NHL patients, indicating that the kinetics of CART cells could be used as predictive factors in clinical practice. This is the first summary of CD19-41BBζ CART against r/r NHL emphasizing different kinetic profiles between NHL and ALL/CLL. These results also imply that means to enhance long-term persistence of CART cells may be potential strategies of to improve response rates. Disclosures No relevant conflicts of interest to declare.

Abstract 344: SJP1604, a novel targeted therapeutic agent for AML (acute myeloid leukemia) including standard therapy-resistant AML

Abstract Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are cancers characterized by the rapid growth of abnormal white blood cells. Generally, cytarabine (Ara-C), azacitidine, and decitabine are treated as the standard first-line chemotherapy for AML/MDS. Although the therapy leads to high rates of remission, approximately 30% of the treated patients are refractory and more than 50% of them face a relapse because of the occurrence of drug resistance and high toxicity. Therefore, there is a strong unmet need for the next-generation targeted therapy to overcome drug resistance and reduce toxicity in AML/MDS. Here, we designed an aptamer-nucleoside drug conjugate (SJP1604) via utilizing easy and continuous synthesis specific for nucleolin, which is highly expressed only on the cell membrane of cancer cells. In particular, AML, CLL (chronic lymphoblastic leukemia) and ALL (acute lymphoblastic leukemia) cells are known to express the highest mRNA level of nucleolin among various cancer cells, relatively. SJP1604 can be delivered into cancer cells with its high targeting ability and plasma stability via its unique conformational property. In this study, we verified that SJP1604 selectively targeted cancer cells by nucleolin-binding on the cancer cell membrane in the drug uptake assay. Owing to the selectivity, SJP1604 demonstrated less cytotoxicity on murine bone marrow cells in vitro and mouse white blood cells in vivo. SJP1604 also exhibited a long-lasting plasma stability in 50% human plasma up to 48 hours in contrast to unstability of general aptamers. Furthermore, SJP1604 inhibited the growth of various human AML/MDS cancer cells. Interestingly, SJP1604 showed the significantly reduced IC50 values with the decreased expression level of nucleolin in cytarabine-, azacitidine- and decitabine-resistant MOLM-13 while cytarabine caused no effect on the cell growth and the expression of nucleolin of the same resistant cell lines. Injection of SJP1604 led to tumor regression and improved survival rate in MOLM-13 xenograft mouse model and C1498 syngeneic mouse model. In ex vivo study, we figured out that the expression level of nucleolin is highly up-regulated in AML patient-derived bone marrow cells and the level is much higher in relapsed/refractory (R/R) AML patient-derived bone marrow cells. Moreover, SJP1604 also significantly reduced the colony forming unit (CFU) of R/R AML patient-derived bone marrow cells compared to that of AS1411 treatment. In conclusion, these findings suggest that SJP1604 could be developed as a first-in-class drug for novel targeted therapy of AML/MDS with less drug toxicity as well as as a remarkable orphan drug for overcoming drug resistance of AML/MDS in which distinctive solutions do not exist up until recently. Citation Format: Jihyun Um, Dohyeong Lee, Jisun Oh, Yongbin Park, Sung Hwan Moon, Su Jin Lee, Min-Hyo Ki, Eui Hwan Cho. SJP1604, a novel targeted therapeutic agent for AML (acute myeloid leukemia) including standard therapy-resistant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 344.

Distribution of Some Functional Molecules on T-Lymphocytes in Chronic Lymphoblastic Leukemia

T-lymphocytes, their subpopulations, and natural killer cells (NCs) were characterized according to the distribution of membrane molecules involved in intercellular interactions and signaling (CD45, CD3, CD4, CD8, CD16, CD56) in patients with chronic lymphocytic leukemia at the stage of disease diagnosis. Current methodological approaches to determine the studied parameters were used, taking into account the characteristics of the lymphoid pool of blood in patients with chronic lymphocytic leukemia. It has been established that the population of T-lymphocytes of the patient peripheral blood is characterized by decreased content of CD45 and CD3 surface receptors. The average level of the membrane CD4 receptor was reduced in lymphocytes belonging to the T-helper subpopulation. The CD8 receptor remained unchanged in subpopulation of cytotoxic T-lymphocytes. CD8 and CD16 molecules were distributed on the surface of NC cells of patients with chronic lymphocytic leukemia at a normal level, while the expression of CD56 was significantly less. The data facilitate understanding of the energy of immune system cells and can be used to predict the course of chronic lymphocytic leukemia.

Anticancer Nano Formulation of Imatinib with Chitosan Polymer

Objective: Drug targeting is the capacity of the dosage form. In which the therapeutic agent acts specifically to desired site of action in the non-targeted tissue with the help of Nano particles is called as the drug targeting. IMATINIB is a used to treat cancer by chemo therapy. Cancers like chronic myeloid leukemia cancer (CML) and acute lymphoblastic leukemia cancer (ALL) and other specific types of gastrointestinal stromal cell tumor (GIST) systemic mast cell disease and Bone marrow failure disorder. It is administered by oral root. For ATP, Tyrosine kinase is act as a binding site. Methodology: The drug IMATINIB is loaded in the polymer chitosan, poly-(D) glucosamine is a bio compactible, bio degradable, nontoxic, antimicrobial and soluble in solvents. This preparation is done by emulsion-droplet coalescence method. Content of the Drug, Size of the particle and Zeta potential, Encapsulation efficiency and Drug release testing are described for this formulation in this study. Results: The Imatinib Nano particles were formulated and evaluated for its invitro drug release profile. Based on the invitro drug release profile of Imatinib nano particles formulation (INP1 – INP5) formulation INP3 was selected as the best formulation in which the particle size was 285.9nm. The invitro % drug release of INP3 formulation was 99.76 ± 0.82 and it was found to be the suitable formulation to manage the cancer. Conclusion: Hence it is concluded that the newly formulated controlled release nanoparticle drug delivery system of Imatinib may be idol and effective by allowing the drug to release continuously for 24 hrs.

Fournier's gangrene: Review of 36 cases.

Fournier's gangrene (FG) is a very rare disease in daily urological practice. Despite medical improvements, mortality in FG is still high. Early diagnosis is very important to reduce additional instrumentations and mortality. In this study, we aimed to present the characteristics of the patients with Fournier's gangrene followed in two centers during ten years period. The medical records of patients with FG were reviewed retrospectively. The patient characteristics, causative pathogens, laboratory findings and treatment modalities were evaluated. A total of 36 FG cases admitted between January 2008 and February 2018 were included in this study, consisting of 35 male patients, and one female patient with a mean age of 59.27±12.91 years. The mean duration of hospital stay was 19±10.44 days. The most common predisposing factor was diabetes mellitus, which was found in 28 patients. Malignancy was detected in three patients; prostate cancer in two patients and chronic lymphoblastic leukemia in one patient. Two patients had liver cirrhosis, and one patient had Behcet's disease and psoriasis. The microbiological agent was isolated from a wound culture in nine patients. After urgent surgical debridement, daily dressing with nitrofurazone (Furacin) was done. Additional debridement was conducted when necessary. Orchiectomy was performed in 10 patients; two of them underwent bilateral orchiectomy. One patient died because of sepsis on the seventh day of hospital admission. FG is a life-threatening urological emergency with a high mortality rate. Treatment with broad-spectrum antibiotics and urgent surgery is pivotal for the prevention of mortality.

Identification of MiR-125a as a Novel Plasma Diagnostic Biomarker for Chronic Lymphoblastic Leukemia.

Background: Chronic lymphocytic leukemia (CLL) is a type of malignancy in which the bone marrow makes too many lymphocytes. MicroRNAs (miRNAs) are endogenous short (~22-nucleotides) non-protein-coding regulatory RNA molecules with key roles in cellular and molecular processes linked to different cancers including CLL. Re-cently, some investigations have demonstrated that miR-125a downregulation is correlated with the expression of P53, NRG1 and ERBB2. Methods: In this study, samples including 38 patients with CLL and 25 healthy individuals were collected. We used quantitative real-time PCR (qRT-PCR) to assess the expression of miR-125a in plasma of the CLL patients in comparison with healthy controls. Moreover, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis on miR-125a targets in the DAVID database in order to investigate the potential role of miR-125a in cancer pathways. MiR-125a exerted a variety of roles in the cancer pathway via downregulating target genes including ERBB2. Results: The expression of miR-125a dramatically decreased (~2-fold) in the patients with CLL compared with the healthy controls (p = 0.03). Furthermore, overexpression of miR-125a was associated with different CLL staging and B symptoms (all at p < 0.05). The KEGG pathway enrichment analysis demonstrated the eight statistically related KEGG signaling pathways with miR-125a targetome. Conclusions: The results suggested that the miR-125a expression level could be a novel potential biomarker for CLL prognosis.

Expression levels of the apoptosis genes FAS, TNFR2, TRAIL, DR3 and DR4/5 in patients with newly diagnosed chronic lymphatic leukemia before and after treatment with fludarabine, cyclophosphamide and rituximab (FCR)

Background: We have previously shown that the FAS, TNFR2, TRAIL, DR3, DR4/5 gene expression in patients with newly diagnosed chronic lymphoblastic leukemia (CLL) correlates with clinical manifestations of the disease: they are minimal in patients with high activity of the proapoptotic genes and low activity of the apoptosisinhibiting genes, and advanced in patients with high expression of the anti-apoptotic and low expression of the pro-apoptotic genes.Aim: To compare the levels of expression of the external apoptosis pathway genes in patients with newly diagnosed CLL before and after chemotherapy with fludarabine, cyclophosphamide and rituximab (FCR), taking into account baseline clinical data and the response to treatment.Materials and methods: This prospective one-center cohort study included 23 patients with newly diagnosed CLL, who underwent clinical and diagnostic assessments and treatment from November 2014 to December 2017. Immunophenotyping of peripheral blood lymphocytes for CLL diagnosis was done by fourcolor flow cytometry. Expression of the external apoptosis pathway genes was assessed by realtime reverse transcriptase polymerase chain reaction. All patients were treated with a standard FCR regimen with subsequent maintenance treatment with rituximab.Results: There were more men (n = 16) than women among our 23 CLL patients. Median age was 64 years (range, from 47 to 77 years). Sixteen (16) patients had CLL Rai Grade I and II, and 7 patients had CLL Grades III and IV. For convenience of analysis, all patients were divided into two groups depending on the FAS gene expression. At baseline, the patients with high FAS expression had higher TNFR2 (p &lt; 0.0015) and TRAIL (p &lt; 0.0053) expression levels. Before FCR therapy, the patients with low FAS expression had higher lymphocyte counts (р = 0.0016) and lower erythrocyte counts (р = 0.0159). At baseline, there were more Grade I and II patients in the group with higher FAS expression (р = 0.0205). At day 3 after the end of a four day FCR cycle, there was an increase only of the FAS (p = 0.0025) and TRAIL (p = 0.0045) expression. After the completion of the first FCR cycle, lymphocyte counts in the patients with low FAS expression decreased earlier than those in the patients with high FAS expression (p = 0.0019). After six FCR cycles, complete or partial remission was obtained in 82% (19/23) of the patients. The patients with high FAS expression had higher complete remission rate (р = 0.026). No adverse events related to FCR were registered.Conclusion: The external apoptosis pathway genes are one of the key factors of the tumor progression in CLL. Our data on the effect of FCR therapy on the FAS and TRAIL gene expression make it possible to consider them as a target for this combination regimen and may become the rationale to develop new pharmaceutical molecules.

Genome Wide Association Analyses Identify Pleiotropic Variants Associated with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) Susceptibility

The first two authors and last two authors contributed equally.Genome-wide association studies (GWAS) have identified risk loci for Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL) and Non-Hodgkin Lymphoma, however an Acute Myeloid Leukemia (AML) GWAS has not been published to date. We performed a GWAS to identify AML and Myelodysplastic Syndrome (MDS) risk loci using a nested case-control study design in the DISCOVeRY-BMT cohorts which includes almost 2000 AML and MDS patients as cases and 2813 unrelated donors as controls.Genotyping was performed using the Illumina Human OmniExpress BeadChip and imputed using the Haplotype Reference Consortium, yielding > 8 million high-quality variants for statistical analysis. Logistic regression models with AML (de novo AML with normal cytogenetics, de novo AML with abnormal cytogenetics and therapy-related AML) and MDS cases and European American healthy donor controls adjusted for age and sex were used to test the association of each SNP with disease status. To identify the strongest association signal with disease we conducted a summary statistic SNP-based association analysis (ASSET) using non-overlapping AML and MDS cases implemented in R statistical software. ASSET uses an exhaustive search for SNPs with small but common pleiotropic effects across groups of traits while accounting for the multiple tests required by the subset search, as well as any shared controls between groups. This approach allowed us to further investigate the heterogeneity within AML subtypes and to gain increased power by pooling subtypes that show pleiotropic effects. ASSET genome wide (GW) significance is defined as P<5.0x10-8, however, we present results of each subset analysis.ASSET analyses identified an association of T allele at rs12203592, Interferon Regulatory Factor 4 (IRF4), with increased risk of de novo AML and MDS (Figure 1) (OR=1.37; 95% CI, 1.25-1.51, Pmeta=1.59x10-10). The variant has been reported significant in GWAS of skin pigmentation, hair color, non-melanoma skin cancer, squamous cell carcinoma, actinic keratosis, and childhood ALL; GW case-control studies of neuroblastoma and breast cancer show suggestive association signals (P<5 x 10-6).IRF4 belongs to the IRF family of transcription factors and is a key regulator of differentiation stages in hematopoiesis. The T allele at rs12203592 is associated with significantly increased expression of IRF4 in whole blood and lung tissue and in vitro mouse studies have shown the T allele to be associated with higher levels of IRF4 transcription. rs12203592 is <80bp from an IRF4 transcription start site and in an important position within NF-κB motifs in multiple blood and immune cell lines, supporting the hypothesis that this SNP modulates NF-κB repression of IRF4 expression.Another variant in the IRF4 regulatory region, rs62389423 (A allele), showed a putative association with subsets of de novo AML and MDS (OR=1.36; 95% CI,1.21-1.52, Pmeta=1.2x10-7). Although not in linkage disequilibrium with rs12203592, the A allele has also been previously associated with susceptibility to skin cancer and melanoma in multiple GWAS. Analysis of 596 UKbiobank CLL cases and >300,000 controls, shows the A allele correlates with a 50% increased risk of CLL (95% CI, 1.32-1.73, P= 2.4x10-9).Several other GWAS also show an association between additional IRF4 variants and CLL.Analyses in individual subtypes also revealed an intronic variant, rs10098598, in RAD21 to be associated with de novo AML (OR=2.18; 95% CI, 1.60-2.97; Pmeta=8.9x10-7). RAD21, on8q24, a region known to be associated with multiple cancer types, is involved in DNA double-stranded break repair and in chromatid cohesion in mitotic cells. Loss of function would theoretically lead to chromosomal instability and tumorigenesis. Components of the cohesion complex (including RAD21) are somatically mutated in ~12% of de novo AML and MDS patients, where it is frequently an early event.We provide the first GW evidence of association between a common variant and AML susceptibility. This SNP has been shown to be associated with multiple phenotypes, suggesting that there are pleiotropic effects at work. Our RAD21 finding is consistent with the role of cohesion in leukemogenesis and provides some evidence of its role in de novo AML susceptibility. Replication and further subset analyses with genome-wide data in >2000 AML cases are ongoing. [Display omitted] DisclosuresGriffiths:Alexion Inc.: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Celgene, Inc: Honoraria, Research Funding; Novartis, Inc.: Research Funding; Pfizer, Inc.: Research Funding. McCarthy:Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

ARRB1-Induced NOTCH1 Degradation Is Inhibited By Mir-223 in T-ALL

Leukemia is the most common malignant tumor in children under 15 years old, which is divided into several subtypes, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL) and chronic myeloid leukemia (CML), based on the disease phases and effected cells. Each subtype has its specific molecular feature and key regulation factors. In our previous studies, we reported that β-arrestin1 (ARRB1), the pivotal scaffold protein to transduce various cellular signals, could bind with EZH2 to increase Bcr/Abl H4 acetylation level and thus promote CML progression (Brit J Cancer 2014, 111(3): 568-76). ARRB1 could enhance DNMT1 activity and PTEN methylation, decrease PTEN expression and promote self-renew of B-ALL leukemia initiating cells (LICs) (Cancer Lett 2015, 357(1): 170-8.). ARRB1 could increase P300 to bind with SP1 to hTERT promoter, and thus increase hTERT transcription/expression, telomerase activity, telomere length and cell senescence in B-ALL LICs (Cell Death Diff 2017, 8(4): e2756.). However, little is known in the T-ALL, which about 70% have the mutations of NOTCH1 gene. Here, we unveil ARRB1 could curb the progression of T-ALL cells in vitro and in vivo, while the expression of ARRB1 was suppressed by the aberrant increased miR-223. Mechanistically, ARRB1 could recruit DTX1, the E3 ubiquitin ligase, to promote the ubiquitination and degradation of NOTCH1 protein in T-ALL. Furthermore, Overexpression of ARRB1-derived miR-223 sponge BUTR was incompatible with cell proliferation and induces apoptosis in T-ALL cells. Collectively, our results for the first time revealed that ARRB1 acted as a tumor suppressor by promoting NOTCH1 degradation in T-ALL cells where miR-223 effectively antagonized ARRB1 functions. This provides that miR-223 may serve as a valid drug target for developing novel and efficacious T-ALL therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Distribution and Clinical Features of NOTCH1 Signaling Activating Alterations in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

IntroductionNOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations.MethodsA total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS).ResultsIn total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern.Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors.Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations.ConclusionsWe observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

A Novel RAD51 Inhibitor, CYT01B, Shows Anti-Cancer Activity in Preclinical Models of Aid Expressing Lymphomas

Activation Induced Cytidine Deaminase (AID) is a DNA directed cytidine deaminase that is normally only expressed in activated B-cells to promote somatic hypermutations and immunoglobulin class switching. Unlike site-specific recombinases (e.g. RAG1/2), AID lacks target site specificity and can generate DNA damage at widespread locations throughout the genome. In cancer cells, AID expression promotes high levels of DNA replication stress, and results in dependency upon the homologous recombination factor RAD51. We have shown previously that a novel RAD51 inhibitor, CYT01B, functions by disrupting RAD51 focus formation which reduces the nuclear concentration of RAD51 and promotes RAD51 protein degradation. Here we present the in vitro and in vivo pharmacological characterization of CYT01B. We first analyzed the permeability and stability of the compound using Caco-2 and liver microsome assays. CYT01B shows low efflux and is stable with low intrinsic clearance rates (< 30 ml/min/mg protein) in mouse, rat, dog, and human, but not in cynomolgus monkey liver microsomes. In vivo, CYT01B showed oral bioavailability that correlated well with Caco-2 permeability (36.9% in monkeys up to 86.5% in rats). Additionally, CYT01B exhibited a minimum half-life of 4 hours in all species tested (mouse, rat, dog, and monkey). We then examined off target liabilities by performing kinome inhibition and Panlabs safety panel screens. CYT01B showed negligible (<50%) inhibition across 371 kinases tested, while only two targets showed greater than 50% inhibition in the Panlabs panel. CYT01B displayed activity in several human-to-mouse cell line xenograft models. In a systemic model of chronic lymphoblastic leukemia (CLL), the tumor burden in the bone marrow of CYT01B-treated mice was reduced by approximately 30%. In two different subcutaneous engraftment models of either a Burkitt's lymphoma cell line (Daudi) or an acute lymphoblastic leukemia cell line (CCRF-SB), mice treated with CYT01B showed tumor growth inhibition of 49% and 88%, respectively. Lastly, CYT01B was well tolerated in rat 7-day dose range finding studies. Rats were treated with 20, 80, and 240 mg/kg once per day by oral gavage. There were no observed changes in hematology or clinical chemistry, and no observed histopathological toxicities. Taken together, these data demonstrate that CYT01B is a cell permeable, metabolically stable, and orally bioavailable small molecule, with anti-cancer activity in multiple preclinical lymphoid cancer models. Overall, this provides the basis for continued preclinical development of an AID/RAD51 synthetic lethal therapeutic paradigm that may be applicable to various hematologic malignancies. DisclosuresPatrick:Cyteir Therapeutics: Consultancy. Bedard:Cyteir Therapeutics: Consultancy. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Vitamin D and its receptor polymorphisms: New possible prognostic biomarkers in leukemias.

Several factors such as chromosomal translocations, gene mutations, and polymorphisms are involved in the pathogenesis of leukemia/lymphoma. Recently, the role of vitamin D (VD) and vitamin D receptor (VDR) polymorphisms in hematologic malignancies has been considered. In this review, we examine the possible role of VD levels, as well as VDR polymorphisms as prognostic biomarkers in leukemia/lymphoma. Relevant English language literature were searched and retrieved from Google Scholar search engine (1985-2017). The following keywords were used: vitamin D, vitamin D receptor, leukemia, lymphoma, and polymorphism. Increased serum levels of VD in patients with leukemia are associated with a better prognosis. However, low VD levels are associated with a poor prognosis, and VDR polymorphisms in various leukemias can have prognostic value. VD biomarker can be regarded as a potential prognostic factor for a number of leukemias, including acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and diffuse large B-cell lymphoma (DLBCL). There is a significant relationship between different polymorphisms of VDR (including Taq I and Fok I) with several leukemia types such as ALL and AML, which may have prognostic value.

Insights into Chimeric Antigen Receptor Therapy for Chronic Lymphoblastic Leukemia

In a recent study, Fraietta and colleagues identified chimeric antigen receptor (CAR) T cell biomarkers that may predict the success or failure of CAR therapy in patients with refractory chronic lymphoblastic leukemia (CLL). These findings open new prospects for improving T cell product manufacturing and the management of patients with CLL undergoing T cell-based therapies.

A case report of extranodal NK/T-cell lymphoma in patient with chronic lymphocytic leukemia

Rationale:Chronic lymphocytic leukemia often results in secondary tumors, the most common being large B cell lymphoma known as Richter syndrome, followed by extranodal NK/T-cell lymphoma (nasal type) is extremely rare.Patient concerns:A chronic lymphocytic leukemia patient presented with nasal congestion.Diagnoses:Nasal endoscopy identified a left nasal mass, and the pathology suggested extranodal NK/T-cell lymphoma (nasal type).Interventions:The patient received a course of chemotherapy.Outcomes:Pneumonia and coagulopathy occurred after chemotherapy, and the patient died shortly thereafter.Lessons:It is essential to recognize the transformation of disease earlier in chronic lymphoblastic leukemia patient.

The severe cytokine release syndrome in phase I trials of CD19-CAR-T cell therapy: a systematic review.

CD19 chimeric antigen receptor (CAR) T cell therapy has shown impressive results in treating acute lymphoblastic leukemia (B-ALL), chronic lymphoblastic leukemia (B-CLL), and B-cell non-Hodgkin lymphoma (B-NHL) over the past few years. Meanwhile, the cytokine release syndrome (CRS), which could be moderate or even life-threatening, has emerged as the most significant adverse effect in the clinical course of this novel targeting immunotherapy. In this systematic review, we analyzed the incidence of severe CRS in 19 clinical trials selected from studies published between 2010 and 2017. The pooled severe CRS proportion was 29.3% (95% confidence interval [CI] 12.3-49.1%) in B-ALL, 38.8% (95%CI 12.9-67.6%) in B-CLL, and 19.8% (95%CI 4.2-40.8%) in B-NHL. In the univariate meta regression analysis, the proliferation of CD19-CAR-T cell in vivo was correlated with the severe CRS. Specifically, total infusion cell dose contributed to the severe CRS occurring in B-ALL patients but not in B-CLL or B-NHL patients. Tumor burden was strongly associated with the severity of CRS in B-ALL. Besides, post-HSCT CD19 CAR-T cell infusion represented lower severe CRS incidence. Further investigations into the risk factors of CRS in B-CLL and B-NHL are needed.

Developing a quantitative external quality assurance (EQA) module to monitor BCR-ABL1 levels in chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL) using the international scale (IS) to determine a patient's molecular response

Developing a quantitative external quality assurance (EQA) module to monitor BCR-ABL1 levels in chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL) using the international scale (IS) to determine a patient's molecular response

Racial Differences in Four Leukemia Subtypes: Comprehensive Descriptive Epidemiology

Leukemia is a malignant progressive disease and has four major subtypes. Different racial groups differ significantly in multiple aspects. Our goal is to systematically and comprehensively quantify racial differences in leukemia. The SEER database is analyzed, and comprehensive descriptive analysis is provided for the four major subtypes, namely ALL (acute lymphoblastic leukemia), CLL (chronic lymphoblastic leukemia), AML (acute myeloid leukemia), and CML (chronic myeloid leukemia), and for two age groups (≤14 and >14) separately. The racial groups studied include NHW (non-Hispanic White), HW (Hispanic White), BL (Black), and API (Asian and Pacific Islander). Univariate and multivariate analyses are conducted to quantify racial differences in patients’ characteristics, incidence, and survival. For patients’ characteristics, significant racial differences are observed in gender, age at diagnosis, diagnosis era, using radiation for treatment, registry, cancer history, and histology type. For incidence, significant racial differences are observed, and the patterns vary across subtypes, gender, and age groups. For most of the subtypes and gender and age groups, Blacks have the worst five-year survival, and significant racial differences exist. This study provides a comprehensive epidemiologic description of racial differences for the four major leukemia subtypes in the U.S. population.