Ph-positive ALL Research Articles

Application of Gene Expression Microarray for the Classification of Ph-Like B-Cell Acute Lymphoblastic Leukemia.

Ph-like ALL has gene expression profile similar to Ph-positive ALL but without the BCR::ABL1 fusion. The disease presents higher rates of severe clinical features and is associated with unfavorable outcomes. There is still no standard pipeline for molecular characterization of the disease, and no valid predictor gene panel is available worldwide. We performed expression microarray on 25 B-cell ALL and 6 Ph-positive B-cell ALL to cluster and identify the transcriptional signature of Ph-like ALL. qRT-PCR was used to confirm the expression of candidate genes. Four out of 25 samples (16%) shared gene expression signatures related to and clustered with control Ph-positive samples. Analysis of genes differentially expressed in Ph-like B-cell ALL and evidentially functional in normal blood cell development and leukemogenesis, we selected genes as potential biomarkers for Ph-like B-cell ALL in our dataset: ADGRE2, CD9, EPHA7, FAM129C, TCL1A, and VPREB1. Those genes were filtered by Ph-like gene signatures obtained from distinct reliable data, resulting in five genes, CA6, CHN2, JAK1, JCHAIN, and PON2, selected for validation by qRT-PCR. The Ct values of genes, including CA6 (p = 0.0017), PON2 (p = 0.0210), TCL1A (p = 0.0064), and VPREB1 (p = 0.0338), were significant in Ph-like ALL. GSEA analysis identified VPREB1 as enrichment in the KRAS signaling pathway, and several genes that interact with VPREB1 were reported as critical molecules involved in the leukemogenesis of B-cell ALL. In summary, we demonstrate using a gene expression microarray for classifying Ph-like B-cell ALL and highlight VPREB1 as a potential biomarker for this disease.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

HDAC2-miRNA183 Epigenetic Circuit Contributes to the Growth of Philadelphia-Positive Acute Lymphoblastic Leukemia Via BCR-ABL/PTEN/p-AKT and c-MYC Signaling Pathways

Purpose: To characterize the expression and function of leukemia related/specific microRNAs (miRNAs) in Philadelphia-positive Acute Lymphoblastic Leukemia (Ph-positive ALL), and to identify the mechanistic signaling pathways in Ph-positive ALL. Method: Differentially expressed miRNAs in Ph-positive ALL patient samples and cell line were identified using RNA-seq and qPCR. Cell proliferation and apoptosis were detected with CCK-8 and flow cytometry assays, respectively. Bioinformatics software predicted the upstream and downstream regulatory pathway of miRNA183; the dual luciferase reporter gene analysis verified the regulatory effect of miRNA183 on the target gene BCR-ABL; ChIP experiment detected the recruitment of HDACs in the miRNA183 promoter region, and western blot detected the expression of related protein in the miRNA183 related pathway. Results: We identified thirty-one differentially expressed miRNAs in three Ph-positive ALL samples vs. normal karyotype B-ALL samples. Among these alternately expressed miRNAs, we found miRNA183 was significantly downregulated in Ph-positive ALL. qPCR verified the down-expression of miRNA183 in Ph-positive ALLcasesamples and Ph-positive ALL cell line SUP-B15. Forced over-expression of miRNA183 inhibited proliferation of SUP-B15 cells, but had no effect on NALM-6 cells (normal karyotype B-ALL). Furthermore, either forced over-expression of miRNA183 or silence down of BCR-ABL1 promoted SUP-B15 cell apoptosis.TargetScan software prediction found that miRNA183 and BCR-ABL (e1a2) had potential binding sites, dual luciferase reporter gene experiments further proved that miRNA183 could direct bind to BCR-ABL gene; qPCR and western blot further showed that miRNA183 inhibited the expression of P190. Our results showed that BCR-ABL (e1a2) is the target gene of miRNA183. In addition, forced over-expression of miRNA183 and silence down of BCR-ABL1 regulated the downstream proteins including PTEN, PARP, p-AKT and c-MYC with the same change trend. In complementary loss-of-function studies, inhibition of miRNA183 restored the expression of these proteins.Treatment of SUP-B15 cells with decitabine (DAC) and histone deacetylase inhibitor (HDACi), respectively, resulted in decrease of cell viability in a dose-dependent manner. Surprisingly, miRNA183 levels were significantly increased in HDACi exposure but not in DAC exposure. Furthermore, miRNA183 inhibitor reduced HDACi-induced upregulation of miRNA183 expression and attenuated HDACi-mediated apoptosis in SUP-B15 cells. Biology software prediction showed the presence of a transcription start site (TSS) 5 kb upstream of miRNA183. ChIP assay further demonstrated that HDAC2 but not HDAC1 was specifically recruited to the miRNA183 promoter region. Additionally, after HDACi treatment, HDACi-induced H3K27ac was recruited and accumulated at the miRNA183 promoter region. Western blotassay further proved the changes of the related proteins. Conclusion :Our results indicate that miRNA183 is a potential biomarker for Ph-positive ALL and suggest that a novel “HDAC2-miRNA183 Epigenetic Circuit” may be involved in the growth and progression of Ph-positive ALL by PTEN/p-AKT and c-MYC signaling pathways. Taken together, this study may provide novel approaches to the design of targeted drugs for Ph-positive ALL.

Measurable Residual Disease Results & Survival with Augmented Early Intensification Therapy in Intermediate-Risk & High-Risk Group Pediatric Acute Lymphoblastic Leukemia Patients Treated with ALL IC-BFM 2009 Protocol: A Single-Center Study from India

Introduction Measurable residual disease (MRD) has proven to be one of the most effective disease monitoring responses in ALL. Multiple treatment groups have demonstrated End-of-Induction (day 33) flow MRD of <0.01% has excellent disease-free survival (DFS). Patients with end-of-consolidation MRD of <0.01% has an overall DFS of 75 % as per COG AALL1131 including the very high-risk B-ALL genotypes. We conducted a study to assess the efficacy & toxicity of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 protocol for intermediate-risk & high-risk group pediatric ALL patients. Objectives To evaluate efficacy of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 in terms of achievement of day 78 MRD negative status. To determine the event-free survival in intermediate-risk & high-risk group patients. To appraise the adverse events (AEs) of augmented phase IB Induction. Methodology The study enrolled treatment-naïve ALL patients of 1-25 years' age between April 2021 to February 2023 at AIIMS Rishikesh, India. Patients were risk stratified into Standard (SR), Intermediate (IR) & High risk (HR) groups as per ALL IC BFM 2009 protocol. All patients received Augmented phase IB protocol. The regimen comprised of cyclophosphamide (2 doses), four blocks of cytarabine & 6-mercaptopurine, augmented with vincristine (4 doses), and L-Asparaginase (12 doses). Day 78 bone marrow flowcytometric MRD was performed in all patients irrespective of day 33 marrow morphology & MRD. The study defined complete remission as BM blasts <5% with no extramedullary disease and MRD negativity as <0.01%. The AEs was monitored as per NCI-CTCAE v5. As per the study protocol, patients of B-ALL with day 33 MRD >0.01%, ph-positive ALL, hypodiploidy and T-ALL with day 78 MRD > 0.01% was considered as HR & treated with High-risk blocks consolidation followed by transplant or delayed intensification & maintenance. Results A total of 75 patients enrolled in the study. The M: F ratio was 1.6:1. The median age was 8 years (range 1-25 yrs). B & T-ALL constituted 80% & 20% respectively. ph-positivity accounted 22% of B-ALL. The median Lansky performance was 40 & ECOG was 3 among the participants. The median hb, total white counts, and platelets were 5.9 g/dl, 28 x10 9/L and 30 x10 9/L respectively. At diagnosis, 28% of patients had hyperleukocytosis, 15% with bacterial sepsis, 8% with invasive fungal infections, and 8% with CNS leukemia. At baseline, the distribution of patients in SR, IR & HR was 15%, 68% & 17% respectively. The final risk strata at end-of-induction were 8%, 49% & 43% in SR, IR & HR groups. CNS leukemia accounted for 8% of all patients. Of the total evaluable patients, 86% achieved day 8 good steroid response (absolute blasts count <1000/cu.mm), 73% & 38% achieved day 15 marrow M1 response (<5%blasts) and MRD < 0.1%, 97% & 77% achieved day 33 M1 marrow response and day 33 MRD <0.01% respectively. Among the study participants who were MRD positive on day 15, three-fourths (75%) of them accomplished MRD negativity at day 33. Of the 23% patients with day 33 MRD positivity, 80% achieved day 78 MRD negativity of <0.01% with Augmented phase IB with 3 participants being persistent MRD positive. The median follow-up was 24 months and the overall survival was 74%. The mean survival time was 16.7 months (12.7-20.6) for HR & 21.2 months (18.3-24.1) for IR patients. The subgroup analysis demonstrated an overall survival of 80% for SR, 84% for IR & 64% for HR groups. Nevertheless, the proportion of patients in the SR group (8%) was lower compared to the others. The commonest hematological adverse events were hypofibrinogenemia (22%), followed by febrile neutropenia (19%), anemia (19%), and thrombocytopenia (18%) respectively. The commonest non-hematological AEs were peripheral neuropathy (5%) and typhlitis (5%). Conclusion In our single-center experience, the use of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 protocol in intermediate-risk & high-risk pediatric ALL patients was associated with achievement of day 78 MRD negative status in 80% patients who had day 33 MRD-positivity without significant treatment-related toxicity, and encouraging event-free survival in this patient population.

A pilot study of implication of machine learning for relapse prediction after allogeneic stem cell transplantation in adults with Ph-positive acute lymphoblastic leukemia

The posttransplant relapse in Ph-positive ALL increases the risk of death. There is an unmet need for instruments to predict the risk of relapse and plan prophylaxis. In this study, we analyzed posttransplant data by machine learning algorithms. Seventy-four Ph-positive ALL patients with a median age of 30 (range 18–55) years who previously underwent allo-HSCT, were retrospectively enrolled. Ninety-three percent of patients received prophylactic/preemptive TKIs after allo-HSCT. The values of the BCR::ABL1 level at serial assessments and over variables were collected in specified intervals after allo-HSCT. They were used to model relapse risk with several machine-learning approaches. GBM proved superior to the other algorithms and provided a maximal AUC score of 0.91. BCR::ABL1 level before and after allo-HSCT, prediction moment, and chronic GvHD had the highest value in the model. It was shown that after Day + 100, both error rates do not exceed 22%, while before D + 100, the model fails to make accurate predictions. As a result, we determined BCR::ABL1 levels at which the relapse risk remains low. Thus, the current BCR::ABL1 level less than 0.06% in patients with chronic GvHD predicts low risk of relapse. At the same time, patients without chronic GVHD after allo-HSCT should be classified as high risk with any level of BCR::ABL1. GBM model with posttransplant laboratory values of BCR::ABL1 provides a high prediction of relapse after allo-HSCT in the era of TKIs prophylaxis. Validation of this approach is warranted.

Cost-effectiveness analysis of imatinib versus dasatinib in the treatment of pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia when combined with conventional chemotherapy in China

BackgroundTyrosine kinase inhibitors combined with conventional chemotherapy (CC) in treating Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL) has achieved promising efficacy and safety outcomes. The study was conducted to compare the cost-effectiveness between imatinib (HANSOH Pharma, Jiangsu, China) and dasatinib (CHIATAI TIANQING Pharma, Jiangsu, China) in treating pediatric Ph-positive ALL when combined with CC from the perspective of the health system in China.MethodsA Markov model was established to simulate a hypothetical cohort of pediatric Ph-positive ALL patients receiving imatinib or dasatinib, combined with CC. The model was designed using a 10-year horizon, a 3- month cycle, and a 5% discount rate. Three health states were included: alive with progression-free survival, progressed disease, and death. Patient characteristics and transition probabilities were estimated based on clinical trials. Other relevant data, such as direct treatment costs and health utility data were extracted from published literature and Sichuan Province’s centralized procurement and supervision platform. One-way sensitivity analysis and probabilistic sensitivity analysis were performed to assess the robustness of the results. The willingness-to-pay (WTP) was set as three times China’s GDP per capita in 2021.ResultsIn the base-case analysis, the total medical costs were $89,701 and $101,182, and the quality-adjusted life years (QALYs) gained were 1.99 and 2.70, for imatinib and dasatinib regimens, respectively. The incremental cost-effectiveness ratio for dasatinib versus imatinib was $16,170/QALY. The probabilistic sensitivity analysis indicated that treatment with dasatinib combined with CC achieved a 96.4% probability of cost-effectiveness at a WTP threshold of $37,765/QALY.ConclusionsDasatinib combined with CC is likely to be a cost-effective strategy compared to imatinib combination therapy for pediatric Ph-positive ALL in China at a WTP threshold of $37,765/QALY.

Philadelphia-Like Genetic Rearrangements in Adults With B-Cell ALL: Refractoriness to Chemotherapy and Response to Tyrosine Kinase Inhibitor in ABL Class Rearrangements.

Philadelphia-like (Ph-like) B-cell ALL is a high-risk subtype of B-cell ALL that shares a gene expression profile with Ph-positive ALL, but without a BCR::ABL1 fusion. A subgroup of these patients have fusions or rearrangements involving genes such as ABL1, ABL2, PDGFRβ, JAK2, and EPOR, some of which are potentially sensitive to tyrosine kinase inhibitors (TKIs). Prompt identification of these genetic aberrations are important for prognostication and treatment decisions. We performed a retrospective review of patients with B-cell ALL treated at MD Anderson Cancer Center to identify recurrent genetic fusions commonly seen in Ph-like ALL and focus on patients treated with TKI. We identified 23 patients with recurrent genetic fusions commonly seen in Ph-like ALL; 14 had ABL class fusions (eight ABL1, one ABL2, and five PDGFRβ) and nine had JAK2 class fusions (five JAK2 and four EPOR). Notably, several of these fusions were cryptic by conventional cytogenetics and fluorescent in situ hybridization (FISH) assays and identified only by multiplex fusion assay. Thirteen of these 23 patients received a TKI as part of their treatment; this included ABL1 fusion (n = 8), PDGFRβ fusion (n = 4), and EPOR fusion (n = 1). All four patients with ABL1 fusions who received TKI with induction chemotherapy are alive in first remission. Understanding the genomics of B-cell ALL is important for disease prognostication and for precise treatment planning. Besides conventional cytogenetics and directed FISH testing, multiplex fusion assays can help identify recurrent chromosomal translocations that are seen in patients with Ph-like ALL. Early initiation of TKI appears beneficial; larger studies are required to fully understand the benefit of TKI and to design rational combination therapies for these patients.

Optimal Use of Novel Immunotherapeutics in B-Cell Precursor ALL.

Novel immune therapies are currently being used for patients with R/R ALL based on their ability to induce not only hematologic but also molecular remission. Despite promising results, specific clinical conditions, such as high tumor burden or extra medullary relapse, are still associated with a remarkably poor clinical outcome. Therefore, how to optimize the choice and the timing of such new treatments within different clinical settings remains a matter of debate. In addition, with the aim of increasing the rate and depth of molecular remission, clinical studies are currently evaluating the combination of these immunotherapies with chemotherapy in the contest of frontline treatment. The preliminary data suggest that this approach may increase the cure rate and perhaps reduce the use of allogeneic stem cell transplantation (alloHSCT) in first remission. In Ph-positive ALL, reproducible results are showing that frontline treatment programs, based on the combination of tyrosine kinase inhibitors and immunotherapy, can achieve unprecedented rates of hematologic and molecular remission as well as a long-term cure, even in the absence of chemotherapy and alloHSCT. The results from these studies have led to the development of potentially curative treatment modalities, even for older ALL patients who cannot be treated with conventional intensive chemotherapy. The present review examined the evidence for an appropriate use of the new immunotherapies in ALL patients and provided some appraisal of the current and future possible uses of these drugs for achieving further therapeutic improvement in the treatment of this disease.

126 - Ph-Positive ALL Patients Who Are Treated with Tyrosine Kinase Inhibitors Have Similar Post-Transplant Survival As Ph-Negative Patients

126 - Ph-Positive ALL Patients Who Are Treated with Tyrosine Kinase Inhibitors Have Similar Post-Transplant Survival As Ph-Negative Patients

Dissecting Ph-like ALL: The Role of Genomic Lesion and Minimal Residual Disease in Refining Outcome

Cardinali D and Della Starza I contributed equally Foà R and Chiaretti S contributed equally Background. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a subgroup of B-lineage ALL characterized by a gene expression profile (GEP) that resembles that of Ph-positive ALL but lacks the BCR/ABL1 transcript. Ph-like ALL cases are characterized by CRLF2 overexpression, JAK-STAT pathway mutations, IKZF1 deletions and rearrangements involving cytokine receptors and tyrosine kinases. To identify Ph-like ALL cases, our group developed a predictive tool named "BCR/ABL1-like predictor", which is based on the quantification by quantitative PCR of 10 transcripts, specifically overexpressed by Ph-like ALL cases1. ALL is the first neoplasm where the assessment of early response to therapy by minimal residual disease (MRD) monitoring is pivotal for guiding therapeutic choices. Real-time-quantitative PCR (RQ-PCR) of IG/TR gene rearrangements is the most widely used molecular method for MRD assessment. The GIMEMA LAL2317 is a clinical trial designed for newly diagnosed adult B-lineage Ph-negative ALL that includes two cycles of blinatumomab in the consolidation phase. MRD is evaluated at established time points (TPs), the most important being TP2, i.e., after the first consolidation cycle with high-dose chemotherapy, and TP3, after the first cycle of blinatumomab. Aim of this study. To refine risk relapse categories of Ph-like ALL cases based on their genomic features at presentation in combination with the MRD status. Methods. We performed this sub-analysis on a cohort of Ph-like ALL cases enrolled in the GIMEMA LAL2317 protocol identified according to the BCR/ABL1-like predictor assay and that underwent a centralized comprehensive molecular screening at diagnosis by IG/TR gene rearrangements and targeted DNA/RNA sequencing. MRD status was evaluated at specific TPs, i.e., TP2 and TP3, by RQ-PCR and digital droplet PCR (ddPCR). Results. A Ph-like profile was documented in 31/109 evaluable patients (28.4%); 11 of them (35.5%) experienced a relapse. By targeted RNA sequencing, 9/11 presented a gene fusion at presentation: in 4/9 a CRLF2-P2RY8 gene fusion was identified, whereas the remaining cases had PAX5-ZCCHC7, ETV6-BCL21L4, IKZF1-DDC, JAK2-r and ABL-class fusion in 1 case each (Figure 1A). The MRD status was evaluated at TP2 and TP3 by RQ-PCR in 23/31 patients: 15/23 were negative at both TPs, while 8/23 were positive at TP2 and negative at TP3; in the remaining 8 cases, MRD was not evaluated because of refractoriness (n=3), early death (n=2) or loss to follow-up (n=3). DdPCR analysis was performed in 9 patients and compared to RQ-PCR: 6/9 were concordant by both methods, while 3/9 resulted discordant with RQ-PCR being negative at TP3 while being positive by ddPCR. On the basis of the Ph-like signature and MRD results, we defined 3 subgroups: i) Ph-like ALL cases (n=12, 39%) that did not relapse, being MRD-negative at both TP2 and TP3; 3 cases were positive for fusion genes; ii) relapsed Ph-like ALL cases (n=8, 26%) who were MRD-positive at TP2 but became MRD negative at TP3, with 7 harboring a fusion gene; iii) relapsed Ph-like ALL cases (n=3, 10%), always MRD-negative at both TPs, with 2/3 positive for fusion genes (Figure 1B). Finally, by flow cytometry analysis, available in 7 patients at relapse, 6 maintained CD19 positivity and only 1 proved CD19-negative. Conclusions. Ph-like ALL patients tend to relapse early even after blinatumomab treatment and despite becoming MRD-negative. Relapse is not related to a mechanism of CD19 escape. Based on the gene fusions at presentation and on the MRD status, we could identify 3 subgroups of Ph-like ALL. These findings suggest that Ph-like ALL cases should be followed with different markers in addition to IG/TR, particularly in cases with well-defined fusion genes, who are at a very high risk of relapse. In this sense, a refined and rapid genetic characterization at presentation of Ph-like ALL cases is warranted for a more personalized and targeted patient management. 1. Chiaretti S et al., BJH 2018 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ALL-424 Updated Results from the Phase II Study of Blinatumomab in Combination With Ponatinib in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Blinatumomab and ponatinib both induce high rates of complete molecular remission (CMR) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). Their combination may generate durable responses and decrease the reliance on allogeneic stem cell transplantation (allo-SCT). To evaluate the combination of blinatumomab and ponatinib in patients with Ph-positive ALL. Patients with newly-diagnosed (ND) or relapsed/refractory (R/R) Ph-positive ALL were treated on this phase II trial. Blinatumomab was given for up to 5 cycles at standard doses, and ponatinib was started at 30mg daily during cycle 1, then lowered to 15mg daily upon achieving CMR. Among responders, ponatinib was maintained for a minimum of 5 years. Patients received 12 doses of prophylactic intrathecal chemotherapy. The primary endpoint was the CMR rate in the ND cohort, and the overall response rate (ORR; CR and CRi) in the R/R cohort. Forty-nine patients were treated: 35 ND (median age, 57 years; range, 22-83 years) and 14 R/R (median age, 38 years; range, 24-61 years). Among R/R patients, 43% were in Salvage ≥2. One early death within 4 weeks was observed among ND patients due to intracranial hemorrhage, none among R/R patients. The ORR was 96% in the ND cohort and 92% in the R/R cohort, with respective CMR rates of 85% and 79%. Only one patient with ND disease underwent allo-SCT because of persistently detectable BCR::ABL1 transcript levels; 6 patients (46%) with R/R disease underwent subsequent allo-SCT. After a median follow-up of 11 months (range, 1-46 months), no relapses were observed in the ND cohort with similar 2-year OS and EFS rates of 93%. In the R/R cohort, the estimated 2-year OS and EFS rates were 61% and 42%, respectively. This combination showed a favorable safety profile, with most adverse events of grade 1-2. One patient discontinued ponatinib due to toxicity. The chemotherapy-free combination of blinatumomab and ponatinib demonstrated robust clinical activity in Ph-positive ALL, with high rates of CMR and durable remissions. This strategy will potentially obviate the need for chemotherapy and allo-SCT in many patients, particularly in the frontline setting.

ALL-061 Delineating the Genomic and Proteomic Landscape of BCR-ABL1-like Acute Lymphoblastic Leukemia Subtype: Bench-to-Bedside Translational Research

Baseline detection of Ph/BCR-ABL1-like-ALL cases is extremely beneficial for hematologists/physicians to design personalized medicine and to treat these patients efficiently. Worldwide, researchers use patented TaqMan low-density array (TLDA) gene expression profiling (GEP) to identify these cases. In developing countries, its detection is very challenging and expensive due to the unavailability of patented TLDA and there is a paucity of literature on the identification and characterization of Ph-like ALL cases. Stepwise identification followed by genomic and proteomic characterization of Ph-like ALL cases. This study was conducted at PGIMER, India (2016-2021). Flowcytometric-immunophenotyping and multiplex RT-PCR was performed to detect four recurrent fusion transcripts (n=1021). Transcriptome GEP was performed on 12 Ph-positive ALL cases. Ph-like-ALL cases identified using nCounter Platform (n=96) and RQ-PCR (n=536) were characterized using FISH, DD-PCR, RQ-PCR and MRD-positivity. Nine HKGs' expression was evaluated using 6 algorithms. Furthermore, we built and validated PHi-RACE classifier (n=629 cases) for its rapid detection. Lastly, we delineated the proteomic landscape of Ph-like ALL cases using LC-MS/MS. We studied 1021 treatment-naive and newly diagnosed B-ALL cases in this study cohort. Multiplex assay revealed 18.66% BCR-ABL1, 3.85% TCF3-PBX1, 5.27% ETV6-RUNX1, and 1.21% KMT2A-AFF1. Using transcriptome GEP, we identified 32.05% Ph-like-ALL cases using NanoString and 26.67% using RQ-PCR. These cases were substantially older and represented higher MRD positivity. In CRLF2 subgroup of Ph-like ALLs, we identified chimeric gene fusions including 10% JAK2 rearrangement, 8.33% CRLF2-IGH and CRLF2-P2RY8 translocation with concomitant R683S mutation. We found 8.33% IKZF1 (Δ4-7) deletion using DD-PCR. Furthermore, we developed an online logistic classifier entitled PHi-RACE (sensitivity=95.20%, specificity=83.70%, cut-off >0.28) for its detection using logistic regression. We have validated PHi-RACE classifier (n=93) and identified 56 Ph-like ALL cases. Six algorithms' findings revealed that EEF2, GAPDH and PGK1 represent the best HKGs for its detection. Lastly, from LC-MS/MS findings, we identified 3079 proteins, spliceosomal mediated mRNA splicing pathway and four miRNAs were significantly expressed in Ph-like ALL cases. The authors created and published the PHi-RACE classifier to detect Ph-like ALL cases, which has direct clinical implications. Further, we have delineated the genomic and proteomic landscape of Ph-like-ALL for the first time from developing countries.

PREVENTION AND MANAGEMENT OF RELAPSE OF ACUTE LEUKEMIA AND MYELODYSPLASTIC SYNDROME AFTER ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION IN PEDIATRIC PATIENTS

The best way to manage acute leukemia relapse after HCT is to prevent it, buying time for GVL with immunomodulation and, if no GVHD between days +60 and + 90, prophylactic DLI can be indicate for very high or high risk patients. Short-term low dose of cyclosporine or methotrexate can add safety to pro-DLI, particularly after mismatched or unrelated transplantation. Maintenance with imatinib or dastinib, recommended for Ph-positive ALL, with sorafenib, for FLT3-ITD AML, or azacitidine, for myelodysplastic syndrome patients, can be effective in reducing relapse rates. However, target agent maintenance can add toxicity, depends on patient adherence and demands physician experience to know when is safe to start, how adjust the dose according individual tolerance after transplant and to detect undesirable drug interactions. The second step to avoid hematological relapse is preemptive approach guided by measurable residual disease or mixed chimerism. In patients off immunosuppression, chemotherapy followed by DLI is a useful strategy, and if no response, interferon alpha can be associated to enhance GVL. Target-specific agents can be start at this point either. After relapse, antigen-directed therapy with blinatumumab for CD19 ALL, inotuzumab for CD22 ALL are excellent options to induce MRD negativity and facilitate HCT. Disadvantages of new immunotherapies are: high incidence of VOD with inotuzumab and gemtuzumab; lower response in patients with high leukemia burden or concurrent extramedullary relapse; necessity of consolidation with HCT after a bridging therapy with BiTE and probably with CAR-T cell therapy also. It is important to realize that if remission after chemotherapy is associated with the development of GVHD, then there may be limited benefit (and possibly harm) in consolidating with any kind of cellular therapy. However, for patients who achieved remission without GVHD, either DLI or second transplant can be recommend. Further studies are necessary to determine at which point each strategy might yield the best results.

Is Initial Dasatinib-Blinatumomab Therapy for Ph-Positive ALL in Adults Cost Effective?

Adult, Philadelphia chromosome-positive, Acute Lymphoblastic Leukemia (Ph-positive ALL) had a poor prognosis, but with the use of an initial BCR/ABL tyrosine kinase inhibitor such as Dasatinib in conjunction with chemotherapy such as hyper-CVAD, the projected 24 month disease free survival is 64%. More recently Foa et al. on behalf of the GIMEMA investigators published a phase II trial of Dasatinib and Blinatumomab as initial therapy for adults with ALL. The complete remission rate was 98%, and the projected disease-free survival at 24 months is 88%. We wished to analyze the incremental cost-effectiveness ratio (ICER) of the Dasatinib and Blinatumomab therapy as compared to Dasatinib and hyper-CVAD. The comparison is based on retail costs of chemotherapy for Dasatinib, Blinatumomab and hyperCVAD and excluded hospital and outpatient charges. The cost of hyperCVAD is based on treatment of a person with a BSA I.8m2. The retail cost of therapy is shown below.Drug Dose CostDasatinib 100 mg $505.67Blinatumomab 1 mg $126.81Cyclophosphamide 1,000 mg $231.00Mesna 1,000 mg $42.89Vincristine 2 mg $18.52Dexamethasone 40 mg $4.12Cytarabine 100 mg $22.88Leucovorin 25 mg $7.48The ICER formula is the cost of new therapy minus (-) the cost of standard therapy/quality of adjusted life new therapy - the quality of adjusted life standard (QALY). The yearly cost of Hyper-CVAD and Dasatinib is $147,288.48 per QALY, whereas, the yearly cost of Dasatinib-Blinatumomab is $187,940.46 per QALY. The difference is $42,451.98 which is less than the 50,000-threshold using cost-effective analysis. In conclusion, the combination of Dasatinib-Blinatumomab appears to be cost effective. DisclosuresNo relevant conflicts of interest to declare.

Non-Intensive Continuous Treatment with a Tyrosine Kinase Inhibitor (TKI) Followed By Allo-HSCT Is an Effective Therapeutic Strategy for Adult Ph-Positive Acute Lymphoblastic Leukemia: Outcomes of the Russian Prospective Multicenter RALL Study

Introduction. As Ph-positive (Ph+) ALL in adults remains less favorable in prognosis than other ALL, and by expert opinion needs non-intensive chemotherapy protocols and new generation TKI with the majority of pts undergoing allo-HSCT, the results of treatment based on the different approach: de-escalated but continuous treatment with the change of TKI according to the molecular response and allo-HSCT may be of interest and provide new insights to the treatment of Ph+ ALL.Aim. To evaluate survival and outcomes in different risk groups in pts with Ph+ ALL in the RALL-study (Ph+ALL-2009, Ph+ALL-2012 and Ph+ALL-2012m protocols).Patients and methods. Between January 2010 and June 2021, 74 new Ph+ ALL cases were diagnosed in 6 centers of the RALL-group and 63 of them were evaluable for analysis (median age 37 years (17-73), m/f 32(43%)/42(57%), CNS disease in 13(21%) pts, WBC>30*10 9/l in 27(43%) pts, bcr/abl transcript p190/p210/p190+210 in 31(60%)/12(23%)/9(17%) cases). Standard cytogenetic was performed in all 63 pts, 1 had no mitosis, 6(10%) monosomy 7 and 2 (3%) complex karyotypes were detected.All pts were treated according to RALL protocols with continuous Imatinib. Ph+ALL-2009 protocol included 600 mg Imatinib with prednisone, VNCR, L-asp, Dauno, Cph, followed by 6-MP and MTX. Imatinib had to be changed to Dasatinib (140 mg) after non-achievement of molecular complete response (MolCR) on day 70. MolCR was defined as bcr/abl chimeric transcript <0,01% by PCR with 10 -4 sensitivity. In protocols Ph+ALL-2012 and Ph+ALLm, we de-intensified chemotherapy: reduced Dauno, Cph and L-asp doses, accordingly. All pts were considered as candidates for allogeneic HSCT in CR1 if HLA-identical donor was available. 36 (57%) pts underwent HSCT in the first-line therapy: 2(6%) autologous, 9 (25%) matched related, 20 (56%) matched unrelated and 5 (13%) haplo-HSCT.Results. Hematological complete remission (CR) was achieved in 60 (95%) of 63 pts (1 early death and 2 refractory cases occurred). On day 70, MolCR was achieved in 21(38%) of 56 pts. Death on therapy in CR (within 5 months of induction/consolidation) was registered in 4 (6%) cases. The major causes of the non-relapsed mortality in unrelated allo-HSCT (n=9) were aGVHD and severe infections, at a median +4 months after HSCT.The 5-year overall survival (OS) and disease-free survival (DFS) for all 63 pts were 58% and 45%, respectively. The long-term outcome on different protocols (Ph+ALL-2009, Ph+ALL-2012 and Ph+ALL-2012m) were similar: 3-year OS - 55% vs 51% vs 75% (p=0,27), 3-year DFS - 56% vs 44% vs 50% (p=0,54), respectively.The 5-year OS was 65% vs 61% (p=0,84), and DFS was 57% vs 31% (p=0,24) in transplanted vs non-transplanted patients by landmark analysis with a median 5,3 month of CR. Landmark analysis of 5-year OS for transplanted and non-transplanted pts depending on age showed no significant difference for both groups: >45y 40% vs 80%; and ≤45y 70% vs 49%, respectively (p=0,1625), although data for 5-year OS was still not mature at the time of analysis. DFS was significantly different in transplanted vs. non-transplanted pts: >45y 40% vs 71%; ≤45y 61% vs 0%; respectively (p=0,0439).In a multivariate analysis for Ph+ ALL among common risk factors (age > 45y, WBC>30, LDH>2N, immunophenotype, late MolCR >70d, CNS leukemia) WBC>30, HSCT were significant risk factors for OS and DFS.Conclusions. Our data demonstrate that de-intensification of chemotherapy does not affect the efficacy of Ph+ ALL therapy in the era of TKIs. We confirmed that patients older than 45y old could be treated by chemotherapy with TKI (new generation TKI if needed) only, but all pts younger than 45y should be considered for HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Persistence of the Minimal Residual Disease at the End of Induction Is Not a Factor of Poor Prognosis If Imatinib Was Changed to the Dasatinib By Ph+ RALL Protocol in Ph-Positive Acute Lymphoblastic Leukemia Patients

Introduction. The role of minimal residual disease (MRD) has been confirmed as a clear prognostic factor by some big studies in Ph-positive acute lymphoblastic leukemia (Ph+ ALL) patients. The persistent MRD positivity may be indicative of the emergence of resistant mutations and TKI therapy should therefore be changed in these patients according to the sensitivity. Ph+ RALL protocol suggests that if a patient does not achieve the major molecular complete remission (MolCR) at day +70, Imatinib should be changed to Dasatinib for subsequent therapy. Could the change of Imatinib to Dasatinib in cases with MRD persistence at the end of induction improve the prognosis of the Ph+ ALL patients? It hasn't been described yet.Aim. To evaluate the role MRD persistence at the end of induction (day +70) in Ph+ ALL patients treated by Ph+ RALL protocol.Patients and treatment. From 2010 January to 2017 July, 36 new Ph+ ALL cases were diagnosed in 3 centers of the RALL-group (median age 35 years (17-68), m/f 19(53%)/17(47%), CNS disease=1, WBC> 30*109/l= 16(44%), bcr/abl transcript p190/p210/p190+210 21(59%)/8(22%)/7(19%)). All patients were treated according to RALL protocol with parallel administration of Imatinib. This protocol is based mainly on 600 mg Imatinib with prednisolone, VNCR, L-asp, followed by 6-MP and MTX. Imatinib had to be substituted by Dasatinib (140 mg) after non-achievement of MolCR on day 70 of treatment. MolCR was stated if bcr/abl chimeric transcript was <0,01% by PCR with 10-4 sensitivity. All patients were considered as candidates for allogeneic HSCT if HLA-identical donor was available. 16 pts (44%) underwent HSCT in the first-line therapy: 2 (12,5%) autologous, 4 (25%) matched related and 10 (62,5%) matched unrelated.Results. Hematological complete remission (CR) was achieved in 32 (89%) of 36 pts (except two early deaths and 2 refractory cases). On day 70 MolCR was achieved in 18 (56%) of 32 pts. Death on therapy (within 3 months of induction/consolidation) was registered in 4 (11%) cases, in 2 patients of them after day 70th of the protocol in CR. The major issue is the non-relapsed mortality after unrelated allo-HSCT (n=3, aGVHD and severe infections, at a median +4 months after HSCT).The 5-y overall survival (OS) and disease-free survival (DFS) for all 36 pts was 56% and 47%, respectively.The 5-y OS was 65% vs 51% (p=0,35), and DFS was 49 vs 48% (p=0,9) in MRD-negative and MRD-positive patients on day 70th of the protocol (Picture). In a univariate analysis for Ph+ ALL patients the fulfilled of HSCT in the first-line therapy became statistically significant for DFS. The 5-y OS was 65% vs 48% (p=0,07), and DFS was 60% vs 32% (p=0,01) in transplanted vs non-transplanted patients by Kaplan-Meier analysis.Conclusions. We didn't revealany difference in efficiency of the therapy in Ph+ RALL protocol according to the MRD status at the day 70. We attribute the absence of the effect of MRD status after induction on the results of treatment, to the shift to Dasatinib (140 mg) after non-achievement of MolCR on day 70. The performance of HSCT in the first-line therapy reliably improved the efficacy in Ph-positive ALL patients on RALL protocol. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inotuzumab ozogamicin with bosutinib for relapsed or refractory Philadelphia chromosome positive acute lymphoblastic leukemia or lymphoid blast phase of chronic myeloid leukemia.

Relapsed/refractory (R/R) Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL) and lymphoid blast phase of chronic myeloid leukemia (LBP-CML) have poor outcomes. We designed a phase 1/2 study combining inotuzumab ozogamicin with bosutinib for this patient population. Patients with T315I mutation were excluded. Bosutinib was administered daily at three dose levels (300 mg/d, 400 mg/d, 500 mg/d) in a 3 + 3 design. Inotuzumab ozogamicin was dosed weekly during cycle one, and once every 4 weeks subsequently for a total of six cycles. The primary objective was to determine the safety and the maximum tolerated dose (MTD) of bosutinib in combination with inotuzumab ozogamicin. Eighteen patients were enrolled (Ph-positive ALL, n=16; LBP-CML, n=2). The median age was 62 years (range, 19-74) and the median number of prior therapies was one (range, 1-5). Dose limiting toxicities included grade 3 skin rash and bosutinib 400 mg daily was determined as the MTD. The most frequent grade 3/4 treatment-emergent adverse events were thrombocytopenia (60%) and neutropenia (38%). A complete response (CR) / CR with incomplete count recovery (CRi) was achieved in 15/18 (83%) patients; 11/18 (61%) patients achieved negative measurable residual disease by flow cytometry. Complete molecular response was noted in 10/18 (56%) patients. The 30-day mortality was 0%. After a median follow-up of 44 months, the median duration of response and overall survival were 7.7months and 13.5months, respectively. Six patients had a subsequent allogeneic stem cell transplant. No patient developed veno-occlusive disease. Inotuzumab ozogamicin with bosutinib was well tolerated in R/R Ph-positive ALL and LBP-CML.

Non-inferior long-term outcomes of adults with Philadelphia chromosome-like acute lymphoblastic leukemia

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with inferior outcomes in the chemotherapy setting. We hypothesized that allogeneic hematopoietic cell transplantation (allo-HCT)-based post-remission therapy would improve outcomes of this entity. We examined the frequency and long-term outcomes of adults with Ph-like ALL, particularly focusing on allo-HCT outcomes for Ph-like ALL versus non-Ph-like ALL. Ph-like ALL was determined by anchored multiplex PCR-based targeted next-generation sequencing. Of the 344 patients, 57 (16.6%) had Ph-like ALL, 197 (57.3%) had Ph-positive ALL, and 90 (26.1%) had B-other ALL. To further evaluate the prognosis of Ph-like ALL, outcome analyses were restricted to 147 patients, excluding Ph-positive ALL. The actual allo-HCT rates in complete remission were 87.7% for Ph-like ALL, 71.4% for B-other standard-risk ALL, and 70.4% for B-other poor-risk ALL. Patients with Ph-like ALL had a higher 5-year overall survival (60.6% vs 27.1%; P = 0.008) than B-other poor-risk ALL subgroup, while no difference was observed compared with B-other standard-risk ALL subgroup. Similar results were noted in a separate analysis for patients receiving allo-HCT in complete remission. In multivariate analyses, B-other poor-risk ALL was associated with poorer outcomes. Our data showed that allo-HCT-based post-remission therapy may have contributed to non-inferior outcomes of adult Ph-like ALL.

Minimal residual disease (MRD) positivity at allogeneic hematopoietic cell transplantation, not the quantity of MRD, is a risk factor for relapse of Philadelphia chromosome-positive acute lymphoblastic leukemia

Minimal residual disease (MRD) monitoring by quantitative real-time reverse transcription PCR (qRT-PCR) is the standard of care in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). We evaluated the impact of MRD status at hematopoietic cell transplantation (HCT) on relapse, as measured by a unified protocol at a central laboratory. Only patients with Ph-positive ALL who had minor transcripts (e1a2) and who underwent allogeneic HCT in first complete remission between 2008 and 2017 were included. First, patients with negative-MRD (n = 196) and positive-MRD (n = 61) at HCT were analyzed. As expected, MRD positivity at HCT was significantly associated with an increased risk of hematological relapse (hazard ratio [HR], 2.91; 95% CI 1.67-5.08; P < 0.001) in the multivariate analysis. Next, patients with positive-MRD were divided into low-MRD (n = 39) and high-MRD (n = 22) groups. In the multivariate analysis, high-MRD at HCT was not significantly associated with an increased risk of hematological relapse compared to the low-MRD group (HR 1.10; 95% CI 0.54-2.83; P = 0.620). These results indicate that the therapeutic decisions should be made based on MRD positivity, rather than on the MRD level, at HCT.

Ph-positive and ph-like all: how can we further improve?

Ph-positive and ph-like all: how can we further improve?