B-acute Lymphoblastic Leukemia Research Articles

MicroRNA-31 is a potential biomarker for screening B-lymphoblastic leukemia in children.

The present study aimed to investigate the expression and significance of microRNA-31 (miR-31) in children with acute B-lymphoblastic leukemia (B-ALL). Bone marrow specimens and peripheral blood were collected from children with B-ALL (n=38) and healthy controls (n=18). Total RNA was extracted and the expression levels of miR-31 were measured using quantitative PCR. In addition, a receiver operating characteristic curve was generated, and the area under the curve (AUC) was calculated to evaluate the diagnostic value of miR-31 for the development of B-ALL. miR-31 expression was significantly lower in the B-ALL group compared with in the control group (P<0.05). Additionally, the expression levels of miR-31 in the B-ALL group before treatment were markedly lower than in the B-ALL group after treatment, and miR-31 expression was significantly lower after 30 days of treatment compared with after 12 weeks of treatment. Furthermore, miR-31 expression in the group of children ≥10 years of age was higher than that in the group of children <10 years of age. Furthermore, the expression levels of miR-31 were higher in the low-risk group compared with in the medium- and high-risk groups (P<0.05). When the cutoff value was set at 1.8, the AUC of miR-31 for B-ALL diagnosis was 0.915 (95% CI, 0.828-1.000; P<0.0001), with a sensitivity and specificity of 80.8 and 100%, respectively. In conclusion, miR-31 may exert an anticancer role in B-ALL in children and may be a potential marker to assist in diagnosis and prognostic prediction.

Methanolic Extract from Aerial Parts of Artemisia Annua L. Induces Cytotoxicity and Enhances Vincristine-Induced Anticancer Effect in Pre-B Acute Lymphoblastic Leukemia Cells

Background: Nowadays, remarkable attention has been drawn towards the effective therapeutic characteristic of natural products targeting cancerous cells. This study aimed to investigate the anti-cancer effect of Artemisia annua extract (AAE), a Chinese herbal medicine alone and in combination with a microtubule binding agent used in ALL treatment, vincristine (VCR), in B-Acute lymphoblastic leukemia (ALL) Nalm-6 and Reh cells. Materials and Methods: Cytotoxic activity of AAE and VCR was determined using MTT assay in Nalm-6, and Reh cell lines and synergism was evaluated using the CompuSyn software. Caspase 3 activity and Annexin/PI staining were performed for apoptosis assessment. The expression level of apoptosis-related genes, caspase 3, Bax and Bcl-2 were determined using real time-PCR. One-way ANOVA and post hoc Tukey multiple comparisons were used for statistical analysis. Results: Our findings revealed that a single administration of AAE exerted an anti-leukemic effect in both ALL-derived cells in a time- and dose-dependent manner. Interestingly, the growth inhibitory activity of the extract was more potentiated when combined with 0.1 and 1 nM VCR through caspase 3-dependent apoptosis. Moreover, real-time PCR analysis showed that VCR-induced cytotoxicity was augmented by AAE through alteration of Bax, and Bcl-2 mRNA expression. Conclusion: Overall, owing to the nontoxic nature of AAE and its explicit role in enhancing VCR effectiveness, our study provided new insight into the development of a novel combinatorial approach in ALL using natural herbs. The practical implication of the research requires further investigation through clinical trials, opening avenues for forthcoming treatment improvements.

Abstract 542: Anti-CD19 therapy for precursor B-acute lymphoblastic leukemia: Response and resistance mechanisms

Abstract Introduction: CD19 is a transmembrane protein restricted to B-lineage cells and follicular dendritic cells. In conjunction with CD21, CD81, and CD225, CD19 acts as a B cell co-receptor to decrease the threshold for antigen receptor-dependent stimulation. CD19 trafficking and expression are orchestrated by a multitude of molecules. Resistance to blinatumomab (Blina, anti-CD19 therapy) in acute lymphoblastic leukemia (ALL) is thought to be due in part to the emergence of CD19-negative escape variants. Loss of expression of CD81 and/or CD21, which are required for CD19 trafficking, results in the lack of CD19 expression. In addition, alternative CD19 splicing and other non-CD19-mediated resistance mechanisms have been described. Therefore, we aimed to study CD19 trafficking and response to Blina in ALL patients. Methods: Peripheral blood was collected at our Center. Mononuclear cells (PBMCs) were isolated and viably frozen. Cells were then thawed and washed with PBS with 10% FBS, and viable cells were counted using trypan blue dye exclusion. 1x10^6 cells/test were stained for CD19, CD81 (BD Biosciences, San Jose, CA) and CD21 (Biolegend, San Diego, CA). After incubation, cells were washed 2x with FACs buffer, resuspended and acquired on a BD Canto II flow cytometer. Results: Blood was obtained from 10 patients with relapsed or refractory pre-B-ALL with blood blasts prior to Blina treatment. Five patients subsequently achieved complete remission (CR). Cells from four of these patients had CD21 expression on &amp;lt;5% of cells, but CD81 expression on &amp;gt;40% of cells. Of the 5 patients whose disease did not respond to Blina, four also had CD21 expression on &amp;lt;5% of cells and had CD81 expression on an average of 40% of cells. CD19 was expressed on &amp;gt;40% of cells in all samples. Conclusion: Although CD21 and CD81 are required for CD19 trafficking and expression, loss of either of these antigens measured by flow cytometry did not correlate with the response to Blina. PatientAge(years)DiagnosisKaryotypeCD19*CD21*CD81*Response to Blinatumomab176Pre-B-ALL46,XX,t(9;22)(q34.1;q11.2)53241Yes (CR)255Pre-B-ALL46, X,inv(Y)(p11.2q11.23), dic(9;12)(p13;p13), del20(q11.2)42146Yes (CR)347Pre-B-ALL46, XX,t(4;11)(q21;q23)941256Yes (CR)484Pre-B-ALL46,XX,i(9)Iq10)65372Yes (CR)551Pre-B-ALL46, XX, t(9;22)(q34.1;q11.2)77578Yes (CR)633Pre-B-ALL46, XY, +X, der(17;18)(q10;q10)67185No725Pre-B-ALL46, XY, der(20;21)(q10;q10),+2146432No825Pre-B-ALL46, Y, t(X;4)(p11.2;p15), der(9)t(9;14)(p21;q12) del(9)(p21),del(14)q13), +mar[cp14]742429No979Pre-B-ALL46, XY48123No1073Pre-B-ALL46, XY59129No*% cells; CR = complete remission Note: This abstract was not presented at the meeting. Citation Format: Firas El Chaer, Brandon Carter-Cooper, Rena G. Lapidus, Maria R. Baer, Zeba N. Singh, Ashkan Emadi. Anti-CD19 therapy for precursor B-acute lymphoblastic leukemia: Response and resistance mechanisms [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 542.

Успешная терапия резистентного B-острого лимфобластного лейкоза с экстрамедуллярным поражением с применением инотузумаба озогамицина до и после аллогенной трансплантации гемопоэтических стволовых клеток

Успешная терапия резистентного B-острого лимфобластного лейкоза с экстрамедуллярным поражением с применением инотузумаба озогамицина до и после аллогенной трансплантации гемопоэтических стволовых клеток

Identification and characterization of a murine model of BCR‑ABL1+ acute B‑lymphoblastic leukemia with central nervous system metastasis.

Breakpoint cluster region (BCR)-Abelson murine leukemia (ABL)1+ acute B-lymphoblastic leukemia (B-ALL) is a disease associated with a dismal prognosis and a high incidence of central nervous system (CNS) metastasis. However, BCR-ABL1+ B-ALL with CNS infiltration has not been previously characterized, at least to the best of our knowledge. In the present study, a murine model of BCR-ABL1+ B-ALL with CNS metastasis was established using retroviral transduction. The vast majority of BCR-ABL1+ leukemic cells were found to be immature B cells with a variable proportion of pro-B and pre-B populations. The present results indicated that the BCR-ABL1+ B-leukemic cells expressed high levels integrin subunit alpha 6 (Itga6) and L-selectin adhesion molecules, and have an intrinsic ability to disseminate and accumulate in CNS tissues, predominantly in meninges. On the whole, these results provide an approach for addressing the mechanisms of BCR-ABL1+ B-ALL with CNS metastasis and may guide the development of novel therapeutic strategies.

PF168 KINASE AND CYTOKINE RECEPTOR SIGNALING PATHWAY ACTIVATING ALTERATIONS IN UNIFORMLY TREATED B‐ALL

Background:Recent B‐acute lymphoblastic leukemia (B‐ALL) studies revealed a new subgroup of patients with ABL‐class and JAK‐STAT fusions which can be targeted using small‐molecule inhibitors (Roberts et al., 2017). Positivity for ABL‐class or JAK‐STAT fusions was associated with positive post‐induction minimal residual disease (MRD) (Pui et al., 2017) and inferior outcome (Boer et al, 2017) but most studies were conducted in non‐uniformly treated patient populations.Aims:To identify kinase and cytokine receptor pathway activating alterations and determine their clinical significance in uniformly treated B‐ALL.Methods:The study includes 160 BCR‐ABL1‐negative B‐ALL (122 pediatric, 38 adults (≥18 y/o)) patients treated according to MRD‐driven pediatric‐adult NOPHO ALL‐2008 protocol. 101 B‐ALL cases (including high hyperdyploids and low hipodyploids) without canonical B‐ALL aberrations were selected for targeted RNA‐Sequencing (RNA‐Seq). Sequencing was performed using TruSight Pan‐Cancer sequencing panel (Illumina Inc., CA). FISH analysis was used to identify CRLF2‐IGH gene rearrangements.Results:Of 101 B‐ALL patients (75 pediatric, 26 adults), RNR‐Seq and FISH analysis identified three (3%) cases with ABL‐class (ABL1‐ETV6, ABL2‐ZC3HAV1, PDGFRB‐EBF1) fusions and four (4%) cases with JAK‐STAT fusions (JAK2‐BCR, n = 1; CRLF2‐IGH, n = 3). Five of seven ABL‐class and JAK‐STAT mutually exclusive fusions were detected in adults. Remaining ten (9.9%) gene fusions were identified in the pediatric group, of which five cases had PAX5 gene and three had ZNF384 gene rearrangements.Gene mutation analysis detected Ras pathway mutations in a total of 48 (47.5%) cases: NRAS (n = 28), KRAS (n = 17), PTPN11 (n = 7). Other JAK‐STAT and FLT3‐TKD gene mutations were present in 10 (9.9%) and 8 (7.9%) cases, respectively. Thirty‐seven (36.6 %) patients harbored other gene variants (n = 23) or had no recurrent gene variants/fusions (n = 14) detected by RNA‐Seq.ABL‐class and JAK‐STAT fusions were more frequent among adults than pediatric patients (19.2% vs. 2.7%) while ABL‐class fusions were exclusive to adults (p = 0.032). The prognostic analysis was restricted to the adult group. More adult patients with ABL‐class or JAK‐STAT fusions had positive post‐induction day 29 MRD values compared to negative patients (p &lt; 0.001) and were more frequently assigned to NOPHO‐2008 high‐risk groups or had induction failure (80% vs 30.3%, p = 0.05). 75th percentile event‐free (EFS) and overall survival (OS) were 5 vs. 35 (p = 0.008) and 15 vs. 37 (p = 0.07) months in ABL‐class or JAK‐STAT fusion positive vs. negative adult groups, respectively.In multivariate analysis, the positivity for ABL‐class or JAK‐STAT fusions was an independent risk factor for worse EFS (p = 0.032) but not for OS (p = 0.242).Summary/Conclusion:Overall, ABL‐class and JAK‐STAT fusions were infrequent and were more often detectable in adults compared to children. Adult patients with ABL‐class or JAK‐STAT fusions had higher post‐induction MRD values and were more frequently assigned to high‐risk disease groups or had induction failure resulting in lower event‐free survival.

PS965 A NOVEL FAST CD19 CAR‐T CELLS: RAPID AND EFFICIENT TREATMENT FOR REFRACTORY B‐ALL(BP‐CML)

Background:The blastic phase (BP) with B‐acute lymphoblastic leukemia (B‐ALL) of chronic myelogenous leukemia (CML) are generally refractory to treatment with a survival ranging from weeks to months. Although allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is curative, it is important to eliminate the blast of ALL and achieve chronic phase (CP) before HSCT.Aims:To explore the effectiveness and advantages of FasT CD19 CAR‐T therapy, we report a case of B‐ALL (BP‐CML) treated with FasT CD19 CAR‐T.Methods:A 39‐year‐old woman had received irregular treatments for her CP‐CML, which led to B‐ALL (BP‐CML). The classic chemotherapy regimens were ineffective. The patient had T315I/V299L mutation and was resistant to the available TKIs in China. After combination chemotherapy, hematological remission was achieved. However, bone marrow examination showed lymphoblasts and the prolymphocytes was up 4.5%. PCR showed BCR‐ABL p210/ABL 99.1%, and flow cytometry showed abnormal B lymphoblasts was up to 0.5% in total karyocytes. In order to eliminate the minimal residual disease (MRD) of the blasts of B‐ALL prior to HSCT, we employed FasT CD19 CAR‐T therapy. Three days after FC preconditioning (fludarabine 50 mg d1–3, cyclophosphamide 300 mg d1–3), a low dose of FasT CAR‐T cells (5 × 104/kg) was infused. The vein‐to‐vein time of the FasT CAR‐T is 10 days, in which the manufacturing time is only 1 day.Results:Ten days after CAR‐T infusion, the patient's body temperature went up to 39 °C and returned to normal within one day. Flow cytometry showed the number of CAR‐T cells were 9.0 cells /uL blood and 42.37 cells /uL blood on Day 11 and Day 13, respectively. From Day 1–7, few CD19+ B cells detected in peripheral blood by FACS, and the CAR DNA copy could not be detected by Q‐PCR. Between Day 8 and Day 28, CD19+ B cells could not be detected in the blood, and the CAR copy number reached the highest on Day 13 (4670.2 copies/ug DNA) and dropped to 15.4 copies/ug DNA on Day 28. On Day 18, FCM and reexamination of the bone marrow showed CP‐CML, and the patient underwent allo‐HSCT on Day 39.Summary/Conclusion:Low dose FasT CD19 CAR‐T therapy showed an excellent therapeutic effect to eliminate the MRD without serious cytokines release symptom (CRS) and neurotoxicity. The infusion time was 7–10 days earlier than the conventional CD19 CAR‐T cells. The peak CAR‐T cell number and CAR copy number were detected later than the conventional CD19 CAR‐T. The expansion procedure of the novel FasT CD19 CAR‐T cells in vivo instead of in vitro may explain the later CRS and better tumor‐killing activity. Low dose FasT CD19 CAR‐T infusion gives us a new efficient approach for leukemia MRD clearance. There are still many questions that remains answered in clinic. Based on the preclinical experiment, FasT CD19 CAR‐T cells express higher level CARs and cytokines (IL‐2 and IFNγ), which may lead to more efficiency in killing than the conventional CAR‐T cells. The clinical trial (ChiCTR‐OOC‐16008448) is launched to explore new indications, proper dosages and side‐effects. At present, we have successfully treated three r/r B‐ALL patients with FasT CD19 CAR‐T therapy.image

PS943 SAFETY AND EFFICACY OF TISAGENLECLEUCEL (CTL019) IN B ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN AND YOUNG ADULTS: ROBERT DEBRÉ AND SAINT LOUIS HOSPITALS EXPERIENCE

Background:Relapsed/refractory B acute lymphoblastic leukemia (B‐ALL) treatment remains a major challenge. Tisagenlecleucel (CTL019) is a chimeric antigen receptor T cell‐ therapy that reprograms autologous T cells to target CD19+ leukemia cells. This “living drug”, manufactured for each patient, has obtained a marketing authorization in 2018 in the EU for patients under 26 years old in refractory B ALL, in second relapse or more or in first relapse after allogeneic hematopoietic stem cell transplantation (HSCT).Aims:This study reports the feasibility, safety and efficacy of CTL019 in patients treated in 2 centers from the Assistance–Publique‐Hôpitaux de Paris.Methods:Eligible patients were patients included in sponsored‐clinical trials (n = 16) or treated within the French compassionate program (n = 18) with an apheresis performed between mars 1, 2016 and February 15, 2019.Results:As of February 15, 2019, 34 patients referred from 20 french centers were screened and benefited from an apheresis. Twenty‐nine patients were infused with CTL019 at a median age of 13.2 y (range, 4.8–29.2) after fludarabine‐cyclophosphamide based lymphodepletion. Out of 34 patients, 23 (67%) had a prior history of allogeneic SCT. Median number of relapses before apheresis was 2 (range, 1–5). Two patients presented with a primary refractory ALL. Among the 5 patients who did not receive CAR T cells infusion 3 died of progressive disease, one died of septic choc and one was excluded because of age. Out of 29 patients 2 were non‐evaluable for efficacy: one for fatal cytokine release syndrome (CRS) 6 days after CAR T cells infusion, one for insufficient follow up. Among the 27 patients evaluable after one month all were in CR/Cri, 25 (92.6%) being MRD negative. Median relapse free survival (RFS) and overall survival (OS) were not reached with a median follow up of 7.2 months (range, 0.2–30.6). The 18 month‐OS probability is 82.6% (95%CI, 53.2%&gt;94.4%). Eight patients relapsed: 3 CD19+, 4 CD19‐, 1 of undetermined status. Four deaths were reported: 3 due to progressive disease and 1 being associated to a CRS (grade 5). Sixteen patients experienced a CRS (≥ grade 3: n = 12) with a median time of 4 days after CTL019 infusion (range, 0–8): 1/6 patients &lt;10 years and 15/23 patients ≥ 10 years. ICU level care was required for 14 patients (48%). Nine patients received tocilizumab, 4 patients siltuximab and 7 patients corticosteroids. Currently 6 neurological events have been reported, all completely reversible except one (fatality in combination with grade 5 CRS). Two patients received a second CAR T cells infusion at 5 and 7 months of the first one due to B cell recovery at 3 and 4 months after first infusion. No expansion neither B‐cell aplasia was observed in these 2 cases. One patient relapsed 4 months after the second infusion. Of note 2 patients, including the only patient who died of adverse event, were older than 26 years of age, and would note fit the current FDA/EMA indications of tisagenlecleucel. No patient underwent allogeneic HSCT while in remission after CAR T cells infusion.Summary/Conclusion:CTL019 confirms its efficacy in a cohort of patients heavily pretreated for refractory or relapsed B‐ALL without additional therapy after remission. The toxicity profile underlines the need of a strong collaboration between intensivists, neurologists and hematologists to successfully manage these patients.

PS944 LONG-TERM OUTCOME OF ALLOGENEIC-HCT IN ADULT PATIENTS WITH ALL IN FIRST REMISSION; FAVORABLE GVHD-AND-RELAPSE-FREE SURVIVAL OF DOUBLE CORD BLOOD TRANSPLANTATION COMPARED TO CONVENTIONAL DONOR TYPES

Background: Relapsed/refractory B acute lymphoblastic leukemia (B-ALL) treatment remains a major challenge. Tisagenlecleucel (CTL019) is a chimeric antigen receptor T cell- therapy that reprograms autologous T cells to target CD19+ leukemia cells. This “living drug”, manufactured for each patient, has obtained a marketing authorization in 2018 in the EU for patients under 26 years old in refractory B ALL, in second relapse or more or in first relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Aims: This study reports the feasibility, safety and efficacy of CTL019 in patients treated in 2 centers from the Assistance–Publique-Hôpitaux de Paris. Methods: Eligible patients were patients included in sponsored-clinical trials (n = 16) or treated within the French compassionate program (n = 18) with an apheresis performed between mars 1, 2016 and February 15, 2019. Results: As of February 15, 2019, 34 patients referred from 20 french centers were screened and benefited from an apheresis. Twenty-nine patients were infused with CTL019 at a median age of 13.2 y (range, 4.8–29.2) after fludarabine-cyclophosphamide based lymphodepletion. Out of 34 patients, 23 (67%) had a prior history of allogeneic SCT. Median number of relapses before apheresis was 2 (range, 1–5). Two patients presented with a primary refractory ALL. Among the 5 patients who did not receive CAR T cells infusion 3 died of progressive disease, one died of septic choc and one was excluded because of age. Out of 29 patients 2 were non-evaluable for efficacy: one for fatal cytokine release syndrome (CRS) 6 days after CAR T cells infusion, one for insufficient follow up. Among the 27 patients evaluable after one month all were in CR/Cri, 25 (92.6%) being MRD negative. Median relapse free survival (RFS) and overall survival (OS) were not reached with a median follow up of 7.2 months (range, 0.2–30.6). The 18 month-OS probability is 82.6% (95%CI, 53.2%>94.4%). Eight patients relapsed: 3 CD19+, 4 CD19-, 1 of undetermined status. Four deaths were reported: 3 due to progressive disease and 1 being associated to a CRS (grade 5). Sixteen patients experienced a CRS (≥ grade 3: n = 12) with a median time of 4 days after CTL019 infusion (range, 0–8): 1/6 patients <10 years and 15/23 patients ≥ 10 years. ICU level care was required for 14 patients (48%). Nine patients received tocilizumab, 4 patients siltuximab and 7 patients corticosteroids. Currently 6 neurological events have been reported, all completely reversible except one (fatality in combination with grade 5 CRS). Two patients received a second CAR T cells infusion at 5 and 7 months of the first one due to B cell recovery at 3 and 4 months after first infusion. No expansion neither B-cell aplasia was observed in these 2 cases. One patient relapsed 4 months after the second infusion. Of note 2 patients, including the only patient who died of adverse event, were older than 26 years of age, and would note fit the current FDA/EMA indications of tisagenlecleucel. No patient underwent allogeneic HSCT while in remission after CAR T cells infusion. Summary/Conclusion: CTL019 confirms its efficacy in a cohort of patients heavily pretreated for refractory or relapsed B-ALL without additional therapy after remission. The toxicity profile underlines the need of a strong collaboration between intensivists, neurologists and hematologists to successfully manage these patients.

Mechanisms and countermeasures in relapse of B cell acute lymphoblastic leukemia after treatment of CD19 chimeric antigen receptor T cells

Chimeric antigen receptor T cell (CAR-T) therapy is a new type of immunotherapy, which has been developed rapidly in recent years. A number of clinical trials have confirmed that CAR-T therapy can effectively treat a variety of relapsed/refractory (R/R) hematological malignancies. The available results show that 70%-90% of R/R B cell acute lymphoblastic leukemia (B-ALL) patients who receive CD19 CAR-T therapy achieve complete remission (CR). However, ALL can relapse in some patients who achieved CR, and relapse has become one of the obstacles affecting the efficacy of CAR-T. The article aims to summarizes the possible mechanisms and prevention strategies of R/R B-ALL relapse after CD19 CAR-T therapy. Key words: Antigens, CD19; Receptors, antigen; Leukemia, lymphoid; Leukemia, B-cell; Immunotherapy; Recurrence; Chimeric antigen receptor T cell; B-acute lymphoblastic leukemia

Combined Casein Kinase II inhibition and epigenetic modulation in acute B-lymphoblastic leukemia

BackgroundThe tumor suppressor protein phosphatase and tensin homolog (PTEN) is a key regulator of the PI3K/AKT pathway which is frequently altered in a variety of tumors including a subset of acute B-lymphoblastic leukemias (B-ALL). While PTEN mutations and deletions are rare in B-ALL, promoter hypermethylation and posttranslational modifications are the main pathways of PTEN inactivation. Casein Kinase II (CK2) is often upregulated in B-ALL and phosphorylates both PTEN and DNA methyltransferase 3A, resulting in increased PI3K/AKT signaling and offering a potential mechanism for further regulation of tumor-related pathways.MethodsHere, we evaluated the effects of CK2 inhibitor CX-4945 alone and in combination with hypomethylating agent decitabine on B-ALL proliferation and PI3K/AKT pathway activation. We further investigated if CX-4945 intensified decitabine-induced hypomethylation and identified aberrantly methylated biological processes after CK2 inhibition. In vivo tumor cell proliferation in cell line and patient derived xenografts was assessed by longitudinal full body bioluminescence imaging and peripheral blood flow cytometry of NSG mice.ResultsCX-4945 incubation resulted in CK2 inhibition and PI3K pathway downregulation thereby inducing apoptosis and anti-proliferative effects. CX-4945 further affected methylation patterns of tumor-related transcription factors and regulators of cellular metabolism. No overlap with decitabine-affected genes or processes was detected. Decitabine alone revealed only modest anti-proliferative effects on B-ALL cell lines, however, if combined with CX-4945 a synergistic inhibition was observed. In vivo assessment of CX-4945 in B-ALL cell line xenografts resulted in delayed proliferation of B-ALL cells. Combination with DEC further decelerated B-ALL expansion significantly and decreased infiltration in bone marrow and spleen. Effects in patient-derived xenografts all harboring a t(4;11) translocation were heterogeneous.ConclusionsWe herein demonstrate the anti-leukemic potential of CX-4945 in synergy with decitabine in vitro as well as in vivo identifying CK2 as a potentially targetable kinase in B-ALL.

Hemophagocytic Lymphohistiocytosis and Infections: An Update.

Hemophagocytic Lymphohistiocytosis and Infections: An Update.

A rare morphological and immunophenotypic presentation of adult B-cell acute lymphoblastic leukemia with blasts in the monocytic zone on CD45 side scatter

&lt;p class="abstract"&gt;We present a case of B-acute lymphoblastic leukemia in an elderly patient who presented with severe weakness and pancytopenia. The patient was a 75 year old Female whose blasts had an unusual morphology in form of coarse azurophilic granules and cytoplasmic blebs and on flow cytometry the blasts were present in the bright CD45 zone with a high side scatter. Bone marrow aspirate sample was subjected to multicolour flow cytometry using Beckman Coulter Navios® which is an 8 colour flow cytometer. Flow cytometric analysis of the bone marrow aspirate showed blasts in the monocytic zone with a precursor B cell immunophenotype. Complete blood counts showed pancytopenia with peripheral blood film not showing any blasts. Bone marrow aspirate smears showed 20% blasts with coarse azurophilic granules and cytoplasmic blebs. The position of the blasts in this case which were in monocytic zone giving them a bright expression of CD45 and a high side scatter on the CD45 side scatter. This is not the usual position for blasts in B- acute lymphoblastic leukemia as these blasts are less complex. A bright expression of CD45 by blasts in B- acute lymphoblastic leukemia is known to be associated with a poor prognosis but the clinical significance of blasts being bright CD45 with a high side scatter is a very rare occurrence and more number of cases with a similar presentation are required to determine a prognostic significance.&lt;/p&gt;

Chk1 Inhibition Restores Inotuzumab Ozogamicin Citotoxicity in CD22-Positive Cells Expressing Mutant p53.

Inotuzumab ozogamicin (IO) is an anti-CD22 calicheamicin immunoconjugate that has been recently approved for the treatment of relapsed or refractory B-Acute Lymphoblastic Leukemia (r/r B-ALL). We employed both immortalized and primary cells derived from CD22-positive lymphoproliferative disorders to investigate the signaling pathways contributing to IO sensitivity or resistance. We found that the drug reduced the proliferation rate of CD22-positive cell lines expressing wild-type p53, but was remarkably less effective on cells exhibiting mutant p53. In addition, CD22-positive cells surviving IO were mostly blocked in the G2/M phase of the cell cycle because of Chk1 activation that, in the presence of a wild-type p53 background, led to p21 induction. When we combined IO with the Chk1 inhibitor UCN-01, we successfully abrogated IO-induced G2/M arrest regardless of the underlying p53 status, indicating that the DNA damage response triggered by IO is also modulated by p53-independent mechanisms. To establish a predictive value for p53 in determining IO responsiveness, we expressed mutant p53 in cell lines displaying the wild-type gene and observed an increase in IO IC50 values. Likewise, overexpression of an inducible wild-type p53 in cells natively presenting a mutant protein decreased their IC50 for IO. These results were also confirmed in primary CD22-positive cells derived from B-ALL patients at diagnosis and from patients with r/r B-ALL. Furthermore, co-treatment with IO and UCN-01 significantly increased cell death in primary cells expressing mutant p53. In summary, our findings suggest that p53 status may represent a biomarker predictive of IO efficacy in patients diagnosed with CD22-positive malignancies.

Flow cytometric predictive scoring systems for common fusions ETV6/RUNX1, BCR/ABL1, TCF3/PBX1 and rearrangements of the KMT2A gene, proposed for the initial cytogenetic approach in cases of B-acute lymphoblastic leukemia.

In B-acute lymphoblastic leukemia (B-ALL), the identification of cytogenetic prognostic factors is important for stratifying patients into risk groups and tailoring treatment accordingly. The purpose of this study was to propose flow cytometric (FCM) scoring systems (SSs) for predicting t(12;21)(p13;q22), t(9;22)(q34;q11), t(11q23), and t(1;19)(q23;p13.3) translocations. We analyzed retrospectively the FCM immunophenotype of 377 patients with B-ALL with regard to the major cytogenetic findings revealed by interphase fluorescence in situ hybridization (i-FISH). Comparing descriptive data on the expression of each antigen and performing receiver operating characteristic (ROC) analysis, we identified the most reliable predictive markers for each translocation and sought to establish a specific SS for each translocation, based on specific antibody panels. CD27, CD9, CD66c, CD10, CD25, and CD34 were employed for the prediction of t(12;21), CD25, CD38, CD34, and CD66c for t(9;22), NG2, CD10, CD15, CD34, and CD20 for t(11q23), and CD34, cμ, CD123, and CD66c for t(1;19). The sensitivity and specificity, respectively, of each predictive score were 89.29% and 96.15% for t(12;21), 75.00% and 88.19% for t(9;22), 84.21% and 99.04% for t(11q23), and 85.71% and 92.71% for t(1;19). Four highly specific and significantly sensitive FCM-obtained SSs are proposed for the prediction of the four major translocations observed in patients with B-ALL. Prospective evaluation of the proposed SSs could lead to a better targeted cytogenetic investigation and therefore to more cost-effective laboratory practice.

Abstract A152: Investigating metabolic basis of neurotoxicity during CAR-T therapies

Abstract Chimeric antigen receptor T (CAR-T) cell therapies are rapidly becoming first in line therapies for treatment of hematologic malignancies. This is due to the efficacy of the treatment and long-lasting tumor responses. However, CAR-T therapies are associated with unique set of acute toxicities— cytokine release syndrome (CRS) and neurologic toxicities—the cause of which is not clear. As other immune therapies are becoming more widely used, these toxicities are observed in patients undergoing bispecific T-cell engaging antibodies (BiTEs) and checkpoint blockade therapies. Here, we sought to define metabolic changes in patients undergoing chimeric antigen receptor (CAR) T-cell therapy for B-acute lymphoblastic leukemia. We performed metabolic analysis of amino acids in serum samples, and identified significant up-regulation of tryptophan-kynurenine metabolism in patients with high-grade neurotoxicity. In-depth look at tryptophan-kynurenine pathway in cerebrospinal fluid patient samples determined quinolinic acid up-regulation to correlate with low- and high-grade neurotoxicity, indicating local production of this metabolite. We show that quinolinic acid is produced by activated monocytes and microglia in response to Il1beta and TNF. Importantly, tryptophan metabolite—kynurenine— stimulates Il1beta production and further activation of monocytes, possibly exacerbating CSR and neurotoxicity. This study suggests that inhibition of tryptophan-kynurenine pathway may offer some benefit to manage neurotoxicity and CRS associated with CAR-T-cell therapy treatment. Citation Format: Darin Salloum, Bianca Santomasso, Renier J. Brentjens, Hans-Guido Wendel. Investigating metabolic basis of neurotoxicity during CAR-T therapies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A152.

Role of CD71 in acute leukemia- An immunophenotypic marker for erythroid lineage or proliferation?

CD71 or Transferrin receptor is expressed on the surface of erythroid lineage cells. CD71 expression has been found to be significantly increased in rapidly proliferating cells. This cross-sectional study included 37 bone marrow samples of acute leukemia cases diagnosed between October 2016 to April 2018. The samples were analysed on BD FACS Canto II. We evaluated the expression of CD71 on leukemic blasts and compared median fluorescent intensities (MFI) of blasts in different types of acute leukemias. The 37 cases comprised of 21 Acute Myeloid Leukemia (AML), 13 B-Acute Lymphoblastic Leukemia (B-ALL), 2 T- Acute Lymphoblastic Leukemia (T-ALL) and 1 mixed phenotypic acute leukemia (MPAL), T/Myeloid. CD 71 expression was noted in 70.3% (n= 26/37) of acute leukemia cases. CD71 expression was most commonly observed in AML (n= 15/21;71.4%), followed by B-ALL (n= 9/13;69.2%) and T-ALL (n= 1/2;50%). Single case of MPAL revealed blasts positive for CD71. MFI of leukemic blasts of single CD71 positive T-ALL was found to be highest, followed by AML, MPAL (T/Myeloid) and least in B ALL. Of the AML cases, the blasts of AML-M6, acute promyelocytic leukemia and AML-M1 showed higher CD71 expression in terms of MFI. Surface CD71 expression is not only found in erythroid lineage cells, but also in proliferating cells. CD71 MFI is highest in T lymphoblasts followed by leukemic erythroblasts, myeloblasts and least in B lymphoblasts.

Clinical impact of genomic analysis in children with B-acute lymphoblastic leukemia: A pilot study in Slovakia.

Acute lymphoblastic leukemia (ALL) belongs to a genetically heterogeneous disease associated with a wide range of chromosomal and molecular changes. Determining these changes at the time of diagnosis can help the therapeutic decision, and contributes to the prediction of patients' clinical outcomes. A part of B-ALL (B-other) lacks cytogenetic abnormalities with clinical relevance for prognosis. Our first goal was to retrospectively review genetic results of patients from 2013-2017 and identify number of B-other patients in Slovak population. The second goal was to implement single nucleotide polymorphism (SNP) array analysis to improve the diagnosis and risk stratification. In this study we reviewed 133 B-ALL patients. We found that nearly 40% of them (52 cases) belonged to the B-other ALL group. Eighteen B-other ALL patients were subjected to the analysis using SNP-array. Overall, we identified 126 cytogenomic changes and in 4 patients the SNP array revealed clinically relevant markers of adverse prognosis and high relapse risk. Integrating identified genetic changes into clinical practice can bring improvement of prognosis assessment for children with ALL in Slovakia.

Lymphoid enhancer factor 1 gene expression in comparison to other prognostic markers in adult B-acute lymphoblastic leukemia

Background Activation of the canonical Wnt signaling pathway by the key mediator lymphoid enhancer factor 1 (LEF1) plays an important role in the development of several hematological and nonhematological malignancies. Aims The aim was to study LEF1 gene expression in adult B-acute lymphoblastic leukemia (ALL) and its clinical significance in comparison to other prognostic markers to improve risk-adapted treatment stratification for high-risk patients. Patients and methods The study was conducted on 50 adult cases with newly diagnosed B-ALL and 50 adult healthy controls with matched age and sex. The cases were divided into three groups according to risk assessment: low risk (have no risk factor), intermediate risk group (have one or more risk factors), and high risk (BCR-ABL positive). LEF1 gene expression was analyzed using quantitative real-time PCR and its correlation with clinical characteristics and treatment outcome at day 28 after induction chemotherapy and in 6 months follow-up period was assessed. Results LEF1 gene expression showed statistically significant higher expression in ALL cases compared with control (P=0.001). Higher LEF1 gene expression was detected in the high-risk group compared with comparable gene expression in both low- and intermediate- risk groups (P=0.05). Statistically significant positive correlation was detected between LEF1 gene expression and BCR-ABL positive cases. Statistically significant positive correlation was detected between the non-remission group and high LEF1 gene expression (Z=3.026, P=0.002). Conclusion The high LEF1 gene expression at diagnosis may be used in identifying patients with high risk of treatment failure. The high LEF1 gene expression can be a prognostic marker in adult B-ALL patients.

Modeling anti-CD19 CAR T cell therapy in humanized mice with human immunity and autologous leukemia.

BackgroundAdoptive immunotherapy using T cells expressing chimeric antigen receptors (CARs) targeting CD19 has produced remarkable clinical outcomes. However, much of the mechanisms of action, such as the development of memory responses and sources of immune cytokines, remain elusive largely due to the challenge of characterizing human CAR T cell function in vivo. The lack of a suitable in vivo model also hinders the development of new CAR T cell therapies.MethodsWe established a humanized mouse (hu-mouse) model with a functional human immune system and genetically-matched (autologous) primary acute B-lymphoblastic leukemia (B-ALL) that permits modeling of CD19-targeted CAR T cell therapy in immunocompetent hosts without allogeneic or xenogeneic immune responses.FindingsAnti-CD19 CAR T cells were detected in blood of leukemic hu-mice with kinetics and levels similar to those seen in patients receiving CAR T cell therapy. The levels of CAR T cells were correlated inversely with the burden of leukemia cells and positively with the survival times in anti-CD19 CAR T cell-treated leukemic hu-mice. Infusion of anti-CD19 CAR T cells also resulted in rapid production of T cell- and monocyte/macrophage-derived cytokines and an increase in frequency of regulatory T cells as reported in clinical studies.InterpretationThese results provide a proof-of-principle that this novel preclinical model has the potential to be used to model human CAR T cell therapy and facilitate the design of new CARs with improved antitumor activity.