B-acute Lymphoblastic Leukemia Patients Research Articles

Clinical Utility of a Novel Triplex Digital PCR Assay for Clone Monitoring in Sequential and Relapsed Pediatric B-Cell Acute Lymphoblastic Leukemia Patients.

Digital polymerase chain reaction (PCR) studies for clonal disease monitoring in B-acute lymphoblastic leukemia patients are currently limited due to the heterogeneous nature of mutations, which limit cost-effective assay designs. In this study, 70 samples (14 relapse and 56 sequential therapy samples) were tested for 13 recurrent mutations identified on deep sequencing in our published cohort (KRAS, NRAS, NT5C2, PMS2, UHRF1, KMT2D, and TP53 genes) via a novel triplex digital PCR assay. A total of seven major clones of NRAS [five] and NT5C2 [two] were noted in six out of 14 (43%) relapse patients, accounting for 44% of early relapses. In addition, 10 minor clones (PMS2 [two], NRAS [four], NT5C2 [three], and TP53 [one]) were noted in five out of 14 (36%) patients. In the 56 sequential therapy samples, six major clones were noted (NRAS [five], KRAS [one]) in four out of 14 (28.5%) patients, with two increasing in size in maintenance samples, leading to subsequent relapse in both cases. In addition, therapy-acquired minor clones in NT5C2 [four] and PMS2 [one] were seen to emerge in maintenance samples in four out of 14 (28.5%) patients, with concordant detection of such major and minor clones in unpaired relapse samples, indicating the need for their active surveillance during therapy. Overall, digital PCR validated NRAS and NT5C2 major clones in one-third (10 out of 27; 37%) of cases, driving nearly half of early relapses.

Investigates the genetic types of acute lymphoblastic leukemia in full remission Indonesian patients: a cross-sectional analysis

Acute lymphoblastic leukemia (ALL) is most common in children, especially those aged 1–4 years old, and the second most common acute leukemia in adults. The updated International Consensus Classification (ICC) of B-acute lymphoblastic leukemia (B-ALL) and T-acute lymphoblastic leukemia (T-ALL) includes recent clinical, cytogenetic, and molecular data. Transcriptome sequencing (RNA-seq) was performed on 200 bone marrow specimens using TruSeq library preparation and HiSeq 2000/2500 or NovaSeq 6000 sequencers (Illumina). The purpose of this study was to define the frequency of chromosomal abnormalities of ALL patients in adults and children in Indonesian patients after full remission for international collaboration has improved and advanced the diagnosis and treatment of ALL in Indonesia. Our resulting data showed that the most common structural abnormality was t(9;22) in 15% of the patients. The frequency of genetic abnormalities was 69 % and 60% for numerical and/or structural in the B-ALL and T-ALL patients, respectively. The adults had a higher incidence of t(9;22) and a lower incidence of hyperdiploid than children. In conclusion: The results of this study molecular subtypes differ strikingly in their responses to treatment that needs more assessment.

CAR-T Cell Therapy in B-Cell Acute Lymphoblastic Leukemia.

Treatment of refractory and relapsed (R/R) B acute lymphoblastic leukemia (B-ALL) is an unmet medical need in both children and adults. Studies carried out in the last two decades have shown that autologous T cells engineered to express a chimeric antigen receptor (CAR-T) represent an effective technique for treating these patients. Antigens expressed on B-cells, such as CD19, CD20, and CD22, represent targets suitable for treating patients with R/R B-ALL. CD19 CAR-T cells induce a high rate (80-90%) of complete remissions in both pediatric and adult R/R B-ALL patients. However, despite this impressive rate of responses, about half of responding patients relapse within 1-2 years after CAR-T cell therapy. Allo-HSCT after CAR-T cell therapy might consolidate the therapeutic efficacy of CAR-T and increase long-term outcomes; however, not all the studies that have adopted allo-HSCT as a consolidative treatment strategy have shown a benefit deriving from transplantation. For B-ALL patients who relapse early after allo-HSCT or those with insufficient T-cell numbers for an autologous approach, using T cells from the original stem cell donor offers the opportunity for the successful generation of CAR-T cells and for an effective therapeutic approach. Finally, recent studies have introduced allogeneic CAR-T cells generated from healthy donors or unmatched, which are opportunely manipulated with gene editing to reduce the risk of immunological incompatibility, with promising therapeutic effects.

Inotuzumab Use: A Single-Center Experience in B-Cell Acute Lymphoblastic Leukemia

Introduction: Inotuzumab Ozogamicin (InO) is a humanized anti-CD22 monoclonal antibody conjugated to calicheamicin, a cytotoxic antibiotic agent used in B-Acute Lymphoblastic Leukemia (B-ALL). The conjugate is internalized and produces DNA cleavage and subsequent apoptosis. InO has shown better results than conventional chemotherapy. Patients and methods: We present the results of a single-center experience with a small cohort of 13 patients diagnosed with ALL who received InO outside of clinical trials. Results: Of the 13 patients, 69% (9) were male. The median age was 38 years old (range 16-64). 10 patients younger than 55 years 9 patients had relapsed diseases, 3 refractory diseases and one patient was newly diagnosed. Regardless of relapsed disease, 7 patients used InO as a first line of salvage and 2 as a second line. In refractory disease, 1 patient used InO as the second salvage and 2 as third or more. White blood cells range from 1,500 to 285,000 mm3. Peripheral blood blast from 50-80%. Before InO administration, all the patients had bilirubin lower than 2 mg/DL (range 0.15-1.26 mg/dL), an ALT (alanine aminotransferase) medium value of 34 U/dl, and an AST (aspartate aminotransferase) medium value of 33 U/dL. The most common type of leukemia was CALLA in 8 patients: B-ALL Phi positive in 2, Lymphoid blast crisis in1, pre-B ALL 1, and t (8;22) 1 patient. 10 patients had CD-22 positivity in flow cytometry, and 3 had negative results. There was no relation between the intensity of CD 22 and the rate of response. 11 patients received InO as monotherapy, and 2 were associated with Mini-HyperCVAD-InO. 7 patients received 1 cycle and 6 patients received 2 cycles of InO; the average dose received was 2,7 mg/m2. 7 (53%) patients in complete response (CR) plus MDR (Minimal Residual disease) negative after treatment, 4 progressive diseases and 2 missing. Three patients received HCT (hematopoietic cell transplantation) post-InO, and two are waiting in complete remission. One patient relapsed while she was waiting for HCT and died. 4 patients received InO as a salvage after relapse post-HCT. All these patients had progressive diseases and died. In the patients that used InO as a bridge to HCT, the average time to transplant was 94 days. Regardless of Veno-Occlusive Disease (VOD), 1 patient developed it after InO treatment; no patients had VOD associated with HCT. Overall survival: 38 months; progression-free survival: 4.1 months. Mortality rate was 53% (7 patients). Causes were: 4 with progressive disease, 1 hemorrhagic stroke, 1 septic shock, and 1 suicide. Conclusion: We show our small local experience using this new drug in B-ALL patients. Patients who use InO as a bridge to HCT could have a good outcome. We had not found good results when InO was used in relapse post-HCT. There was no relation between the intensity of CD22 at flow cytometry and the rate of responses. The incidence of VOD was low. Rates of response are comparable to other real-world reports. This small experience could be the beginning of a multicenter Real-World study in Latin America.

Extracellular vesicle small RNA cargo discriminates non-cancer donors from pediatric B-lymphoblastic leukemia patients.

Pediatric B-acute lymphoblastic leukemia (B-ALL) is a disease of abnormally growing B lymphoblasts. Here we hypothesized that extracellular vesicles (EVs), which are nanosized particles released by all cells (including cancer cells), could be used to monitor B-ALL severity and progression by sampling plasma instead of bone marrow. EVs are especially attractive as they are present throughout the circulation regardless of the location of the originating cell. First, we used nanoparticle tracking analysis to compare EVs between non-cancer donor (NCD) and B-ALL blood plasma; we found that B-ALL plasma contains more EVs than NCD plasma. We then isolated EVs from NCD and pediatric B-ALL peripheral blood plasma using a synthetic peptide-based isolation technique (Vn96), which is clinically amenable and isolates a broad spectrum of EVs. RNA-seq analysis of small RNAs contained within the isolated EVs revealed a signature of differentially packaged and exclusively packaged RNAs that distinguish NCD from B-ALL. The plasma EVs contain a heterogenous mixture of miRNAs and fragments of long non-coding RNA (lncRNA) and messenger RNA (mRNA). Transcripts packaged in B-ALL EVs include those involved in negative cell cycle regulation, potentially suggesting that B-ALL cells may use EVs to discard gene sequences that control growth. In contrast, NCD EVs carry sequences representative of multiple organs, including brain, muscle, and epithelial cells. This signature could potentially be used to monitor B-ALL disease burden in pediatric B-ALL patients via blood draws instead of invasive bone marrow aspirates.

Risk Factors for CD19-Targeting CAR T Manufacturing Failure and Patient Outcomes: A Report from the UK National CAR T Panel

CAR T manufacturing failure (MF) rates reported in literature range between 1-13%. In some cases the manufacturing process fails to yield a product and in others results in one which does not meet pre-specified criteria for release and labelled an out of specification (OOS) product. We conducted a multicentre retrospective review of factors contributing to MF and its impact on patient outcomes. Patients with relapsed or refractory large B cell lymphoma (LBCL), mantle cell lymphoma (MCL) or B acute lymphoblastic leukemia (B-ALL) approved for CAR T therapy with axicabtagene ciloleucel (axi-cel), brexucabtagene autoleucel (brexu-cel) or tisagenlecleucel (tisa-cel) by the UK national CAR T panel between January 2019 and January 2023 were eligible. Patients with MF were identified from databases of the 11 participating CAR T centres and from the national OOS CAR T panel. In total 1138 patients were approved for CAR T therapy (903 LBCL, 97 MCL and 138 B-ALL). We identified 58 patients who had at least 1 CAR T MF; 38 LBCL (LBCL-MF), 14 MCL and 6 B-ALL with inferred MF frequency of 4.20%, 14.43% and 4.34% respectively. Intended CAR T product was axi-cel in 28 and tisa-cel in 10 LBCL, brexu-cel for all MCL and tisa-cel for all B-ALL patients. Most frequent reasons for MF were poor growth in culture (n=12), low cell viability (n=12) and low T cell purity (n=6). Of the 58 patients with MF, OOS product was available and infused in 19 patients (OOS cohort: 14 LBCL, 2 MCL and 3 B-ALL). Repeat apheresis led to infusion of a product in-specification in 17 patients (MF-delayed-infusion cohort: 10 LBCL, 6 MCL and 1 B-ALL). Another 22 patients did not proceed to CAR T infusion (MF-no-infusion cohort: 14 LBCL, 6 MCL and 2 B-ALL), some despite attempts at remanufacture (n=9) or available OOS product (n=5). We included 25 randomly selected matched (for disease subtype and product) controls (all LBCL: 19 axi-cel and 6 tisa-cel) without MF, 18 of whom received infusion. Univariable analysis of potential baseline clinical factors contributing to risk of MF did not reveal any significant impact of the number of lines of prior therapy, history of prior stem cell transplant or a need for holding therapy prior to apheresis. Bendamustine therapy was significantly associated with risk of MF for all lymphoma patients and the LBCL subset; prior exposure in 4% controls vs 32.7% of all lymphoma MF (p=0.004) and 26.3% of LBCL-MF (p=0.039). This was largely due to therapy within 6 months; 0% of controls vs 19.2% of all lymphoma MF (p=0.026) and 23.7% of LBCL-MF (p=0.009). Factors at apheresis including white cell count, neutrophil count, platelet count, absolute lymphocyte count, CD3 count, CRP, LDH and volume of blood processed had no impact on the risk of MF. With a median follow up of 11.6 months, the 12-month overall survival (OS) from date of approval for CAR T therapy was 45.0% (31.4 - 57.7) for all MF patients (n=58), 49% (24.7 - 69.5) for the OOS cohort (n=19), 64.2% (33.6 - 83.5) for the MF-delayed-infusion cohort (n=17) and 27.3% (11.1 - 46.4) for the MF-no-infusion cohort (n=22). Corresponding 12-month OS was 43.0% (26.7 - 58.3) for all LBCL-MF (n=38), 49.0% (21.6 - 71.7) for LBCL-OOS (n=14), 53.3% (17.3 - 79.8) for the LBCL-delayed-infusion cohort (n=10) and 28.6% (8.8 - 52.4) for the no-infusion cohort (n=14). The 12-month OS for LBCL controls (n=25) was 51.2% (30.3-68.7) for all and 65.5% (38.6 - 82.8) for infused patients (n=18). There was no significant difference between OS for LBCL-MF and LBCL controls (HR 1.38, p=0.31). Though OS was numerically lower for LBCL-OOS compared to infused LBCL controls, it was not statistically different (HR 1.82, p=0.21). For infused patients, the 3 months complete response (CR) rates were similar for the LBCL-OOS (38.5%), LBCL controls (40%) and MF-delayed infusion (53.3%) cohorts. The 3 months overall response rate (OR) was non-significantly lower in the LBCL-OOS (38.5%) compared to LBCL controls (60%) and MF-delayed infusion (53.3%) cohorts. There was no difference between any grade or grade ≥3 CRS or ICANS between the LBCL-OOS, LBCL-control and MF-delayed-infusion cohorts. In summary, outcomes for patients infused with an in-specification or OOS CAR T product following MF are comparable to those of controls. Prior bendamustine therapy within 6 months of apheresis is the only risk factor associated with risk of MF in lymphoma patients. Further analysis will be presented at the meeting.

IGF2BP1 Stabilizes and Inhibits Degradation of Pro-Oncogenic Transcripts in ETV6-RUNX1 Positive B-Cell Acute Lymphoblastic Leukemia

Insulin like growth factor 2 mRNA binding protein 1(IGF2BP1) belongs to a family of oncofetal proteins expressed in embryonic life followed by re-emergence during malignant transformation. It is an RNA binding protein known to be dysregulated and overexpressed in multiple malignancies. We have demonstrated its specific overexpression in ETV6-RUNX1 translocated B-cell Acute Lymphoblastic Leukemia (B-ALL). RNA immunoprecipitation using anti-IGF2BP1 antibody, followed by sequencing (RIP-Seq) in Reh cell line (ETV6-RUNX1 positive) identified multiple oncogenic transcripts belonging to the non-canonical NF-κB and PI3K signaling pathways. Overexpression of genes from these pathways (IL6ST, GADD45A, NFAT5, MDM2, CDK6, FGFR1) was also observed in our B-ALL patient samples specifically in the ETV6-RUNX1 positive patients (Total n=111; ETV6-RUNX1 positive: 39) in comparison to MACS sorted, peripheral blood CD19 positive B-cells from healthy volunteers as well as in comparison to non ETV6-RUNX1 translocated B-Acute lymphoblastic leukemia patients. This was also corroborated by public B-ALL gene expression data from the TARGET database in the cBioportal. However, knockout of IGF2BP1 in Reh cell line using CRISPR-Cas9 technology led to significant downregulation of only some of these targets. This implied that the expression of some of the mRNA targets binding to IGF2BP1 was not influenced by its knockout. To better elucidate the role played by IGF2BP1 in the stability of its binding mRNA targets, we decided to study the rate of degradation of target transcripts. mRNA stability was assessed to quantify the rate of degradation of target mRNA after inhibition of transcription using actinomycin. Since actinomycin causes global transcriptional block, we first quantified the effect of actinomycin on the expression of multiple (n=7) housekeeping genes against each other, and selected 3 ( HGPRT, B2M and PPIA) which showed least change after actinomycin treatment. These 3 housekeeping genes were used for further quantification of target transcripts. Dual specificity phosphatase 1( DUSP1) a transcript known to be rapidly degraded, was used to validate the transcriptional inhibition by actinomycin. We quantified the rate of degradation of target transcripts in Reh cells using BTYNB, a small molecule inhibitor of IGF2BP1. BTYNB binds to IGF2BP1 and prevents binding to target transcripts which leads to their destabilization and reduced half-life. We used a combination of BTYNB and actinomycin to study rate of degradation after IGF2BP1 inhibition. Effect of IGF2BP1 on the stability of three target mRNAs involved in non-canonical NF-κB signalling pathway was quantified by comparing their rate of degradation in BTYNB treated cells against the DMSO (vehicle) treated cells. Rate of degradation was measured by actinomycin treatment of both these groups followed by hourly RNA isolation and qPCR over 12 hours. MYC is a known mRNA binding target of IGF2BP1. BTYNB was also shown to decrease the expression of MYC mRNA by accelerating its degradation. Simultaneously there was an increase in the expression of MYC pre-mRNA indicating a possible for IGF2BP1 in splicing of MYC RNA. In the NF-κB signalling pathway, we had previously identified that NFAT5 expression was not altered significantly after IGF2BP1 knockout even though it binds to IGF2BP1. BTYNB followed by actinomycin treatment demonstrated a dramatic increase in the rate of degradation of NFAT5 implying that IGF2BP1 prevents the degradation of NFAT5 transcript and directly influences its stability. Interestingly, the rates of degradation of other targets like IL6ST and GADD45 were not altered. These findings suggest a heterogeneous influence of IGF2BP1 on the stability of its target transcripts. Some of the transcripts seem to be directly stabilised by IGF2BP1 while others may be influenced indirectly. Taken together, these findings suggest a role of IGF2BP1 in multiple phases of the RNA synthesis and degradation warranting further studies to elucidate role of IGF2BP1 in each of these steps. IGF2BP1 appears to have a critical role in preventing the degradation and stabilizing MYC and NFAT5 transcripts.

Overexpression of CHK1 and CHK2 in pediatric patients of B-acute lymphoblastic leukemia

Background: Despite breakthroughs in the development of chemotherapy drugs to treat pediatric B-acute lymphoblastic leukemia (B-ALL), the relapse rate remains a major therapeutic challenge, requiring more detailed characterization of molecular elements underlying disease development and resistance to treatment. Checkpoint kinase 1 (CHK1) and checkpoint kinase 2 (CHK2) are two critical mediators of the DNA damage response (DDR) mechanism that activate the downstream components responsible for DNA repair, cell cycle regulation, and apoptosis. It has been shown that altered expression of CHK1 and CHK2 in various tumor entities promotes tumorigenesis and disease progression.
 Materials and Methods: In this case-control study, we evaluated the relative expression status of CHK1 and CHK2 genes in pediatric B-ALL patients at diagnosis (n=20), during complete remission (n=23) and relapse phase (n=10), as well as 20 peripheral blood samples from healthy children as a normal control group. The mRNA expression levels of CHK1 and CHK2 were determined by the Real-time PCR method. Data were compared using the Mann–Whitney U test for the relative expression level of target mRNA in different phases of B-ALL. Data were presented as median and statistical significance was described as a P-value less than 0.05.
 Results: Our results revealed that CHK1 expression increased in newly diagnosed patients than in healthy individuals (p ≤ 0.001). Relapsed patients had higher CHK1 expression than the newly diagnosed (p ≤ 0.05) and complete remission (p ≤ 0.001) counterparts. CHK2 was overexpressed in all phases of the diseases (p ≤ 0.001) without any significant alteration among the studied groups.
 Conclusion: Given the CHK1 ability to endow cancer cells with a survival advantage upon chemotherapy, the present study suggests it as a potentially promising target in the fight against B-ALL.

Physical and emotional quality of life in children with B-acute lymphoblastic leukemia randomized to every 4- vs. 12-week vincristine/dexamethasone during maintenance: Children’s Oncology Group AALL0932.

10025 Background: Children with B-acute lymphoblastic leukemia (B-ALL) undergo 2-3 years of treatment that impacts their quality of life. AALL0932 randomized reduction in vincristine and dexamethasone pulses (VCR/DEX every 4 weeks vs. every 12 weeks) during maintenance in the average-risk (AR) subset of NCI standard risk (SR) B-ALL. We longitudinally measured physical and emotional function overall and by treatment. Methods: SR AR B-ALL patients enrolled on AALL0932, aged ≥4 years, with English-speaking parents were evaluated at 5 timepoints (T1-T5: end consolidation, maintenance month 1, maintenance month 10, maintenance month 18 for boys/end of therapy for girls, end of therapy for boys). Parents completed the Pediatric Quality of Life Inventory Generic Core Scales 4.0 and a survey of supportive services received. Age- and sex-matched z scores and proportion impaired ( > 2 SD below normative mean; 2.5% expected in normative population) on the emotional and physical function subscales were measured longitudinally and compared by treatment with the Fisher’s exact test and repeated measures logistic regression. Results: Consent and data were obtained from 555 participants (mean/sd age = 6.0±1.6 years, 46.8% female). Among randomized participants with completed evaluations, 31.3% had impaired physical function at T1 decreasing to 12.5% at T4. Emotional function was impaired in 24.5% of patients at T1 decreasing to 9.8% at T4. Overall 34.9-43.8% received special education/accommodations at T1-T5; there was low use of other services (Table). In adjusted analysis, treatment group was not associated with physical or emotional function. T2 impairment predicted increased risk of impairment at T4 for physical (OR = 4.7, 95% CI 2.3-9.7, p < 0.0001) and emotional (OR = 4.7, CI 2.3-9.5, p < 0.0001) function. Conclusions: Impaired physical and emotional function were prevalent early in SR AR B-ALL therapy, improved by the end of therapy, but remained higher than norms. Outcomes did not vary with VCR/DEX frequency. Patients with early emotional and physical impairment are at elevated risk of later impairment. Clinical trial information: NCT01190930 . [Table: see text]

SMYD2 Expression: Its Relationship to Cytogenetic and Prognosis in a Newly Diagnosed Childhood B-Acute Lymphoblastic Leukemia.

The SMYD2 gene adjusts lysine residues on histones and non-histone proteins such as transcription factors, signaling kinases, and cell cycle regulators affecting the cell fate and other genes' expression. As it acts as an oncogene, SMYD2's expression levels may affect tumor progression. In this prospective study, our main aim was to determine the association of SMYD2 gene expression with the prognostic parameters of childhood B-acute lymphoblastic leukemia (B-ALL) and evaluate its influence on disease prognosis. SMYD2 gene expression was measured in the peripheral blood (PB) samples of 116 newly diagnosed B-ALL patients and 23 controls using real-time polymerase chain reaction (RT-PCR). SMYD2 expression was significantly higher in the childhood B-ALL patients compared with the control group, p-value < 0.001. The patients with unfavorable rather than favorable structural chromosomal abnormalities had significantly higher SMYD2 mRNA levels, p < 0.001. SMYD2 expression levels were positively correlated with total leucocytes count (r = 0.206, p-value = 0.027) and peripheral blood blasts (r = 0.225, p-value = 0.015). There was a statistical difference between the B-ALL cases with high versus low SMYD2 levels regarding translocation (t12; 21), p-value = 0.015. Cox regression analysis identified the increased levels of SMYD2 as an independent prognostic factor affecting both OS and DFS. The SMYD2 gene plays a vital oncogenic role in childhood B-ALL. The association of high SMYD2 levels with unfavorable cytogenetics in childhood B-ALL may be helpful in understanding the relationship between structural chromosomal abnormalities and epigenetic dysregulation. High SMYD2 levels may be useful in the prediction of prognosis in childhood B-ALL.

Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification

PurposeAlthough risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients’ lymphocyte subpopulations.MethodsPeripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of {mathrm{CD}38}^{+}{/mathrm{CD}73}^{+}, and {mathrm{CD}39}^{+}{/mathrm{CD}73}^{+} in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC).ResultsComparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1β, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma.ConclusionAs immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL.

Retrospective Study of B Lymphoblastic Leukemia to Assess the Prevalence of TEL/AML1 in South India: A Study of 214 Cases and Review of Literature

Abstract Introduction Translocation t(12;21)(p13;q22), a recurrent and an invisible chromosomal abnormality, resulting in TEL/AML1 gene fusion, associated with good prognosis, has been described to be a common abnormality, in children with B-acute lymphoblastic leukemia (B-ALL). Objectives The initial observation of very few TEL/AML1 positive patients at this center on testing by fluorescence in situ hybridization (FISH) led to study the prevalence of the abnormality, compare with the global distribution, and evaluate clinical, pathological, molecular, and cytogenetic features in TEL/AML1 positive patients. Materials and Methods A retrospective study of all B-ALL patients tested for TEL/AML1 gene fusion during the period January 2009 to November 2020 was undertaken. Clinicopathological, molecular, cytogenetic, treatment, and follow-up details were collected. All publications dealing with TEL/AML1 gene rearrangement were reviewed post Google and PubMed search. Results TEL/AML1gene rearrangement was assessed by FISH in 178 patients and by reverse transcription polymerase chain reaction in 36 patients and detected as the sole abnormality in 8.4% patients with additional genetic abnormalities noted on FISH evaluation. Normal karyotype was noted in 14/18 (77.7%) of these patients and 2 had complex karyotype. Complete blood count revealed hemoglobin to range from 35 to 116 g/L (median: 74 g/L), white blood count: 1.01–110×109/L (median: 7.8×109/L), platelet counts: 10–115×109/L (median: 42×109/L), blast count in peripheral smear: 0–98% (median: 41%). Immunophenotyping demonstrated 94.4% were CD34 positive, common acute lymphoblastic leukemia associated antigen (CALLA) positive with aberrant expression of CD13, CD33, CD56, singly or in combination in 58.8%. Conclusion TEL/AML1 fusion is rare in Indian patients with B-ALL and appears to be much rarer in our region. The detection of relevant specific abnormalities is of fundamental importance in B-ALL patients and these geographic variations can be used in defining management policies.

Cytogenetic Risk Stratification of B-Acute Lymphoblastic Leukemia and Its Correlation with Other Prognostic Factors.

Purpose of current study was to categorize WHO defined B-Acute Lymphoblastic Leukemia (B-ALL) cases into 3 cytogenetic risk groups (good, intermediate and poor) and to see their correlation with age, NCI risk criteria and treatment response. Clinical and diagnostic details were collected for 78 newly diagnosed B-ALL patients which included bone marrow morphology, flow cytometry immunophenotyping, karyotyping, FISH and RT-PCR. Study cohort comprised 44/78 (56.4%) children including 3 infants and 34/78 (43.6%) adults. Median age for paediatric group was 6 years (3 months-17 years) and for adults was 40.5 years (18 to 75 years). According to NCI risk criteria, excluding infants, 54 (72%) were high risk and 21 (28%) were standard risk. Clonal cytogenetic abnormality was detected in 59/78 cases (75.6%), while 19/78 (24.4%) cases showed normal karyotype. There was significant association of cytogenetic risk groups to age distribution (p value < 0.001) and NCI risk groups (p value < 0.001). There was no significant correlation of CNS involvement with cytogenetic risk groups (p = 0.064). Association of Day 8 steroid response and Day 15 bone marrow status with cytogenetic risk groups was significant (p = 0.006 and p = 0.003 respectively). Post treatment bone marrow status on Day 33 and Day 79 was available for 52 and 42 cases respectively. 9 adults died during induction phase. Day 33 post induction morphological remission was achieved in 51/52 cases (98%) and 1/52 (2.0%) were not in remission. Day 79 post induction morphological remission was achieved in 41/42 cases (98%) and 1/42 (2.0%) were not in remission. Day 33 or End of induction flow MRD (measurable residual disease) was negative in 39/52 (75.0%) patients and positive in 13/52 (25.0%) patients. Day 79 flow MRD was negative in 37/42 (88.1%) and positive in 5/42 (11.9%). Cytogenetic risk groups showed statistically significant Day 33 and Day 79 treatment response (morphologic remission: p = 0.009 and 0.003, flow MRD: p = 0.004 and p = 0.012 respectively). We concluded that cytogenetic risk groups showed statistically significant association with age, NCI risk criteria and treatment response.

Frequency of BCR-ABL Gene Translocation in B-ALL Patients Associated with Clinicopathological Parameters

Background: B-Acute Lymphoblastic Leukemia (B-ALL) accounts for 25% of childhood malignancies. Chromosomal abnormalities like translocations lead to the formation of oncogenes, some of which are strong predictors of prognosis and response to anti-leukemic therapy. This study aimed to find out the frequency of BCR-ABL gene translocation in B-ALL patients by fluorescence in situ hybridization (FISH) and its association with their clinicopathological parameters. Methods: Patients (n=150) aged 1-17 years with a confirmed diagnosis of B-ALL were selected. Peripheral blood and/or bone marrow aspirate samples were obtained and Breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) translocation by FISH was observed. The patient’s demographics, hemoglobin levels, total leucocytes count, platelet count, FISH results, CNS status and risk stratification were recorded. Data was stratified and the Chi-square test was applied, p-value &lt; 0.05 was considered statistically significant. Results: There were 100 (66.67%) males and 50 (33.33%) females. The average age of the patients was 7.03±4.51 years. The frequency of BCR-ABL translocation in B-ALL was 16(10%). A significant association was found between age and BCR-ABL translocation (p-value &lt;0.05), however, an insignificant association was recorded among gender, Hb levels, TLC, platelet count, CNS status, proposed risk stratification system of B-ALL and BCR-ABL translocation (p-value &gt;0.05). Conclusion: The frequency of BCR-ABL translocation in B-ALL was significantly high in the targeted population. FISH improves detection of the BCR-ABL translocation in either metaphase or interphase cells. Therefore, BCR-ABL expression can be considered as a prognostic approach and assist in effective treatment planning and better management of these children. Keyword: Leukemia; In Situ Hybridization; Fluorescence; Pathology.

NT5E gene and CD38 protein as potential prognostic biomarkers for childhood B-acute lymphoblastic leukemia.

The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan-Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.

Identification of diagnosis and prognosis gene markers in B-ALL with ETV6-RUNX1 fusion by integrated bioinformatics analysis

B-acute lymphoblastic leukemia (B-ALL) is characterized by clonal expansion of immature B-lymphocytes in the bone marrow, blood, or other tissues. Chromosomal translocations have often been reported in B-ALL, which are important for its prognosis. B-ALL patients with ETV6-RUNX1 fusion have favorable outcomes, but the mechanisms remain to be clarified. In the present study, we crossed the selected WGCNA module genes and differential expression genes to obtain core genes, and random forest algorithm, a type of supervised learning analysis, was conducted to evaluate the importance of those core genes in distinguishing B-ALL samples with ETV6-RUNX2 fusion with extracting 5 genes as gene markers for ETV6-RUNX2 fusion. Moreover, we calculated the immune infiltration profiles and screened out the ETV6-RUNX2 association immune cells using the CIBERSORT algorithm. In conclusion, combined with various solid informatics methods, we depicted the underlying molecular and immune mechanism of ETV6-RUNX2 fusion and providing potential biological targets for diagnosing and treating B-ALL in the future.

Bone Marrow Lymphoid Infiltrates and Aplasia after CD19-Directed CAR T Cell Therapy in Pediatric B-Lymphoblastic Leukemia

IntroductionChimeric Antigen Receptor (CAR) T cell therapy is used to treated relapsed/refractory B-Acute Lymphoblastic Leukemia (B-ALL) patients. Long term side effects include B cell aplasia and cytopenia that are treated with supportive therapy. Prolonged cytopenia is seen in 16% of CAR T-cell treated B-ALL patients. The etiology of cytopenia is not clear, and could be attributed to the impact of chemotherapy, CAR T cells and disease or patient-specific factors. The impact of CAR T cell infusion on bone marrow cellularity, lymphocyte compartment and its correlation with cytopenia has not been investigated.MethodsWe analyzed pre- and post-CAR bone marrow biopsies in 178 B-ALL patients who received a CD19-directed CAR T-cell product between 2012-2017 and followed for at least 12 months. Responses were categorized into sustained responders, non-responders, CD19-positive relapses and CD19-negative relapses as previously described. Bone Marrow biopsy (BMB) overall cellularity, complete blood counts (CBC) in a 12-month post infusion period were analyzed. BMB were considered mildly, moderately and severely hypocellular based on cellularity of 50, 25 and 5% respectively. Hypocellularity was further stratified by CBC per aplastic anemia (AA) classification guidelines: Non-Severe Aplastic Anemia (NSAA) and Very Severe Aplastic Anemia (VSAA). Immunohistochemistry (IHC) for CD3, CD4, CD8, CD163, granzyme and perforin were performed on pre- and post-CAR BMB. CD3 IHC was digitally scanned and analyzed quantitatively (Leica Aperio ImageScope) and qualitatively. Targeted RNA sequencing-based gene expression profiling (EdgeSeq Immuno-Oncology Panel, HTG Diagnostics) was performed on pre-and post-treatment biopsies, with differential expression assessed by DESeq2 within HTG Reveal software. RNAseq gene expression was deconvoluted to impute relative expression of immune cell subsets.Results31% of patients were mildly hypocellular but none were severely hypocellular at baseline pre-CAR timepoints. The highest proportion of hypocellularity was at the 1-month time point. 81% of patients were mildly hypocellular (≤50%), 42% were markedly hypocellular (≤25%), and 13% were severely hypocellular (≤5%) at the 1-month time point. By month 12, the proportion of mildly hypocellular patients was 74%, markedly hypocellular patients was 26%, and severely hypocellular patients was 7% (Figure 1). The proportion of NSAA and VSAA was 57% and 10% in moderately hypocellular BMB. The proportion of NSAA and VSAA was 64% and 18% in severely hypocellular BMB. Severely hypocellular patients had a higher average day minus 1 disease burden (33% involvement) compared to their moderate (24%) and non-hypocellular patients (16%). VSAA patients had a lower baseline BMB cellularity (47%) compared to NSAA patients (71%) and those without AA (60%)Increased CD3+ T cells were noted in the post-CAR BMB compared to the pre-CAR BMB (Figure 1B). Lymphocyte were singly scattered or formed loose aggregates and tight lymphohistiocytic clusters. IHC and RNAseq analysis showed increased CD8+ granzyme+ cells in the post-CAR BMB compared to pre-CAR BMB. Sustained responders showed higher T cell infiltration compared to other categories of patients (Figure 1C and D). Lymphoid infiltrates did not correlate with hypocellularity or cytopenia.ConclusionWe describe for the first time the changes in bone marrow cellularity and lymphocyte compartment after CD19-directed CAR T-cell infusion in B-ALL. A subset of patients were moderately or severely hypocellular and met criteria for aplastic anemia. However, most of them recovered bone marrow cellularity and CBC. Hypocellularity and AA were correlated with pre-CAR disease burden and baseline cellularity rather than post-CAR lymphocyte infiltration. We also show for the first time that sustained responders to CD19-CAR showed increased CD3+ CD8+ T cell infiltrates compared to CD19-positive relapses and non-responders.Figure 1. A. Bone marrow hypocellularity post-CAR T-cell infusion. Proportion of patients who were mildly hypocellular, markedly hypocellular, and severely hypocellular in the 12month follow up period. B. Increased CD3+ T cells and aggregates after CAR T cell infusion. C and D. Sustained responders show significantly greater CD3+ T cells after CAR T cell infusion compared to other categories. *P<0.05. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Analyses Identify Transcriptional Characterizations of Subsets Associated with Efficacy and Toxicity for CAR-T Immunotherapy in B-ALL Patients

BackgroundThe development of mRNA sequencing has contributed greatly to the mechanism exploration in hematologic malignancies disease. With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize every subset of expression programs and functional states in a comprehensive and unbiased manner. Here, we present a systematic evaluation of engineered chimeric antigen receptor T (CAR-T) products and patient bone marrow profiles in terms of primary resistance and severe cytokine release syndrome (CRS) at the single-cell level.MethodsUsing single-cell mRNA sequencing in conjunction with flow cytometry (FCM), we performed characterization of CD19-targeted CAR-T and mononuclear bone marrow cells from 4 on-trial B acute lymphoblastic leukemia (B-ALL) patients (NCT02965092). Bioinformatics analysis was utilized to explore diversity between patients with different grades of response or CRS. Basing on marker genes, CAR-T products were divided into four groups, which were double-positive T (DPT), CD4 positive T (CD4), CD8 positive T (CD8), and double-negative T (DNT) cells. Meanwhile, both the mononuclear bone marrow cells before and after CAR-T infusion were grouped into six clusters, which were B-ALL, stem, progenitor, B, T, and myeloid cells. The expression and enrichment analyses results were calculated by R (version 3.6.3) and then verified in a 22-sample conventional transcription sequencing cohort of the same clinical trial. Patient efficacy was assessed by the national comprehension cancer network guidelines version 2.2020 for acute lymphoblastic leukemia, and CRS was graded by CTCAE 5.0.ResultsBy FCM detection, the variances of CAR-T infusion products between patients with different clinical outcomes were limited, and nor did mononuclear bone marrow cells. The scRNA sequencing results showed that distinct CAR-T and bone marrow cell subsets indicated differentiated expression in proliferation, cytotoxicity, and intercellular signaling pathways. Expression differentiation variances in CAR-T infusion products were minor than in mononuclear bone marrow cells. CD8+ CAR-T products of complete response (CR) patients were still significantly enriched in pathways such as cell killing (p adjust=0.0012), antigen processing and presentation (p adjust=0.0027), and cell cycle (p adjust=0.0231), exhibiting greater immune function when compared with no response patients. Also, DPT CAR-T products of the non-CRS patients were meaningfully enriched in negative regulation of cytokine production pathway (p adjust=0.0127) when compared with CRS ones. In mononuclear bone marrow cells, B-ALL cells before CAR-T treatment of CR patients presented negatively in cell-cycle (p adjust=0.0019), leading to a low malignant cell proliferation level; and stem-progenitor cells after CAR-T treatment of CR patients showed a stronger ability of neutrophil activation (p adjust<0.0001). As with comparisons between CRS and non-CRS, B-ALL cells before infusion manifested a cell cycle arrest profile (p adjust<0.0006) in non-CRS patients, whereas the immune cells at the same time point were enriched in positive regulation of cell cycle process (p adjust=0.0002).ConclusionsThrough single-cell RNA-seq profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the cell cycle, immune phenotype, and metabolic profiles of subsets during CAR-T therapy, providing a mechanistic basis for ameliorating clinical outcomes and individualized management. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low Rates of Venoocclusive Disease and Non-Relapse Mortality in Pediatric Patients Undergoing Allogeneic Hematopoietic Cell Transplantation Following Inotuzumab Ozogamicin Reinduction for Relapsed or Refractory B Cell Acute Lymphoblastic Leukemia

Background: Inotuzumab ozogamicin (INO) is a humanized, anti-CD22 antibody conjugated to calicheamicin with FDA approval for treatment of adults with relapsed or refractory (r/r) B-acute lymphoblastic leukemia (B-ALL). While it is associated with favorable complete remission rates in r/r B-ALL patients (pts), hepatotoxicity both during treatment and with subsequent hematopoietic cell transplantation (HCT) remains a challenge. In general, venoocclusive disease (VOD) during HCT in pediatric pts is estimated at 10-20%. Following treatment with INO, however, up to 52% of pts undergoing HCT have been reported to develop VOD, of which 18% of cases were fatal (Bhojwani et al. 2019). Here we report our institutional HCT experience in pediatric pts with r/r B-ALL who underwent HCT following INO reinduction and our strategy of using defibrotide prophylaxis to reduce the risk of VOD.Methods: A retrospective chart review was conducted for pts with r/r B-ALL who underwent HCT at our institution from December 2015 to January 2021. Clinical characteristics, laboratory parameters, and radiographic evaluations were reviewed. Patients who received ≥1 course of INO during reinduction were included in the INO group. Development of VOD was defined as a clinical diagnosis of VOD post-HCT based on either the Baltimore, Seattle, or European Society for Blood and Marrow Transplantation (EBMT) diagnostic criteria.Clinical characteristics were summarized. Time to VOD, graft versus host disease (GVHD), non-relapse mortality (NRM), and relapse were estimated under the competing risk setting, in which death without the event of interest was considered to be an absorbing competing risk.Results: Fifty-seven patients with relapsed or refractory B-ALL were transplanted between December 2015 and January 2021. Seventeen (30%) received at least 1 course (3 doses) of INO as part of their pre-HCT reinduction regimen. Fifty-five of 57 pts (97%), including all pts in the INO-group received a myeloablative regimen (51 total body irradiation/cyclophosphamide-based, 4 busulfan-based). Two patients in the non-INO group received a reduced-intensity regimen. Median time from last dose of INO to HCT (Day 0) was 34 days (range 24-71 days). Median number of INO doses received was 6 (range 3-9). Four patients had been transplanted previously, 1 in the INO group and 3 in the non-INO group. Two of these patients developed VOD during their current HCT, both from the non-INO group. Pts receiving INO were more likely to be transplanted in CR3 or higher, and to have required 3 regimens in reinduction to achieve that CR. Median follow-up was 20 months.Rates of VOD were similar between the INO (3 of 17, 18%) and non-INO (4 of 40, 10%) groups (p=0.4, Figure 1). All cases were severe or very severe by EBMT criteria. There was no VOD-related mortality in either group. Defibrotide prophylaxis was used in 82% (14 of 17) of pts in the INO group. Of the 3 patients in the INO group who developed VOD, 2 received defibrotide prophylaxis and 1 did not. Reasons to defer defibrotide prophylaxis included increased risk of CNS bleeding and provider preference. Defibrotide prophylaxis was used in 15% of pts in the non-INO group (6 of 40). In the non-INO group, 1 of 4 patients who developed VOD had received defibrotide prophylaxis. All pts received vitamin E and ursodiol per institutional practice.There were no treatment-related deaths in the INO group and no statistically significant differences were detected in (NRM) (0% vs 10%, p=0.25), median time to engraftment (24 vs 26.5 days, p=0.1), or GVHD (24% vs 55%, p=0.1) in the INO vs non-INO groups.Conclusions: HCT was well tolerated with low rates of VOD and NRM in pediatric pts following reinduction with INO. The results of ongoing prospective studies will provide further insight into the role of INO for reinduction of r/r B-ALL. In our cohort, rates of VOD were similar between pts who had and had not previously received INO, with the majority of pts in the INO group receiving defibrotide prophylaxis during HCT, suggesting that inotuzumab ozogamicin reinduction may safely be used with this approach. [Display omitted] DisclosuresMargossian: Cue Biopharma: Current Employment. Silverman: Takeda, Servier, Syndax, Jazz Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. OffLabel Disclosure:This study evaluates the safety of transplant following inotuzumab ozogamicin re-induction for children with B lymphoblastic leukemia.

A Randomized Phase 3 Trial of Blinatumomab Vs. Chemotherapy As Post-Reinduction Therapy in Low Risk (LR) First Relapse of B-Acute Lymphoblastic Leukemia (B-ALL) in Children and Adolescents/Young Adults (AYAs): A Report from Children's Oncology Group Study AALL1331

A Randomized Phase 3 Trial of Blinatumomab Vs. Chemotherapy As Post-Reinduction Therapy in Low Risk (LR) First Relapse of B-Acute Lymphoblastic Leukemia (B-ALL) in Children and Adolescents/Young Adults (AYAs): A Report from Children's Oncology Group Study AALL1331