Refining measurable residual disease monitoring in Philadelphia positive acute lymphoblastic leukemia adult patients: new insights from methodological and molecular standpoints

Measurable residual disease (MRD) negativity represents the primary endpoint in the management of adult Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. MRD assays must be highly sensitive and specific and the markers must be reliable and representative of the disease. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) can overcome some limitations of standard methodologies. In order to evaluate the best strategy for MRD monitoring, in this study were performed: i) BCR::ABL1-based MRD monitoring between RT-qPCR and ddPCR; ii) MRD evaluation by BCR::ABL1 fusion transcript and IG/TR clonal gene rearrangements ; iii) a comparison of the MRD concordance rate between the two markers; iii) a correlation with biologic features and clinical outcome. Samples derived from 111 adults enrolled in the ongoing phase III GIMEMA ALL2820 clinical trial. At diagnosis, patients underwent a screening for the identification of the predominant IG/TR rearrangement and for the IKZF1plus signature. Firstly, a BCR::ABL1-based comparative study was conducted on 156 MRD samples between the gold standard methodology, i.e. RT-qPCR, and ddPCR showing an optimal correlation degree (R2 = 0.93) and a concordance of 59.6%. Ninety-seven/111 (87.4%) cases were evaluable for IG/TR MRD monitoring. At day +70 (end of induction phase), 97 patients were studied and the concordance rate between BCR::ABL1 and IG/TR MRD monitoring was 46.4%; at day +133 (during consolidation phase), 70 patients were studied and the MRD concordance rate was 41.4%. Five patients experienced a hematologic relapse and a retrospective backtracking was carried out at a previous time-point: 1 was concordantly BCR::ABL1pos and 2 IG/TRpos, 1 was BCR::ABL1pos and IG/TRneg, while the other 2 cases were both BCR::ABL1neg but IG/TRpos at 4.0E-05 and 2.0E-04, respectively, suggesting the presence at the onset of non Ph+ subclone. Overall, the concordance rate between BCR::ABL1 and IG/TR is limited, in line with the literature. Nevertheless, a double-hit strategy may be informative for MRD monitoring and possibly for the distinction between typical/lymphoid Ph+ ALL vs CML-like/multilineage Ph+ ALL and, more important, in some cases for predicting hematological relapse. Finally, a case report of an IKZF1plus patient with an extremely aggressive disease was extensively analyzed

Ponatinib alone or with chemo-immunotherapy in heavily pre-treated Philadelphia-like acute lymphoblastic leukemia: a CAMPUS ALL real-life study

Not available

Optimizing Molecular Minimal Residual Disease Analysis in Adult Acute Lymphoblastic Leukemia

Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients

Optimizing Molecular Minimal Residual Disease Analysis in Adult Acute Lymphoblastic Leukemia

Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.</jats:p

DROPLET DIGITAL PCR DETECTION OF THE T315I BCR::ABL1 KD MUTATION IN ADULT PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Philadelphia chromosome-positive(Ph+) acutelymphoblastic leukemia (ALL) is characterized by thereciprocal translocation t(9;22)(q34;q11).Theadvent of tyrosinekinaseinhibitors (TKIs) has markedly improved the outcome of Ph+ ALL patients and indeed changed the natural history of the disease. At present,TKIs represent the gold standard treatment for patients with Ph+ ALL, with or without chemotherapy 1 . Despitetheefficacy of targeted therapy with TKIs, some patients show drug resistance dueto the onset of BCR::ABL1 kinase domain (KD) point mutations. In particular, the most deleterious oneis represented by theT315I, which causetreatment failure with firstand second generation TKI-based therapies.Sanger sequencing (SS) is the gold standard tool for BCR::ABL1 KD mutational screening.The digital droplet PCR (ddPCR) technology,a third generation PCR, may representa valid alternativeto SS for the detection of BCR::ABL1 KD mutations. Aims: Theaim of this study was to evaluateif ddPCR is at leastas sensitiveas SS for the detection of theT315I mutation, and if the ddPCR can detect the mutation also atvery low levels of minimal residual disease(MRD), ultimately anticipating molecular relapse. Methods: Thestudy comprised samples from patients enrolled in the phaseII GIMEMA LAL2116 chemotherapy-free protocol for newly diagnosed adult Ph+ ALL and based on theadministration of thesecond generation TKI dasatinib followed by the bispecific monoclonal antibody blinatumomab 2 . A BCR::ABL1 KD mutational screening was carried out in all patients with a MRD increase by both SS and ddPCR. In mutation-positive patients, thetimepoint (TP) preceding the MRD increase was evaluated by ddPCR to assess theearlier presence of the mutation. DdPCR was performed as described 3 . Results: Wecarried outa BCR::ABL1 KD mutational screening by SS in 16 patients enrolled in the GIMEMA LAL2116 protocol. Overall, of the 16 patients with a MRD increase(10 during theinduction phase, 6 in theconsolidation phase), 8 resulted wild type(WT) while mutations were detected in 8 patients (7 harboring theT315I mutation and 1 theE255K). In theT315I positive patients, theevaluation by ddPCR of thesameTP samples,confirmed in all cases the presence of the mutation. Moreimportantly, theanalysis ata previous TP, when MRD levels werelower, showed that ddPCR could detect theT315I BCR::ABL1 KD mutation in 6/7 cases, while 1 positive non-quantifiable (PNQ) case proved WT(Table 1). In order to determineif ddPCR could anticipatethe detection of mutations compared to SS, the previous TPs werealso evaluated by SS in 4 cases with available material. In 2 cases the mutation was detected also by SS, whilein 2 it could not befound (Table 1).Likewise, ddPCR for theT315I mutation was also performed in samples that proved negative by SS,and theabsence of mutations was confirmed; this is particularly relevant from a clinical standpointand was corroborated by thefact that only 1/8 experienced a central nervous system relapsein this last cohort,and is currently alivein second complete remission (CR). Summary/Conclusion:The ddPCR proved as reliableand accurateas SS to detect theT315I BCR::ABL1 KD mutation.Furthermore, the ddPCR proved to be moresensitivefor predict molecular relapse beforetheincrease in MRD where, in somecases, theSS failed to detect theT315I mutation.Further efforts are ongoing to expand this screening also to other ABL1 mutations,considering thealways morefrequent use of ponatinib in thefirst-line setting. 1.Foà &amp; Chiaretti, NEJM 2022 2.Foà et al, NEJM 2020 3.Soverini et al,Leukemia 202

CD146 MOLECULE EXPRESSION IN B CELLS ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALLs): A FLOW- CYTOMETRIC MARKER FOR AN ACCURATE DIAGNOSTIC WORK-UP

Background: B-lineage acute lymphoblastic leukemias (B-ALL) harboring the t(9;22)(q34;q11)/BCR::ABL1rearrangement, represent a category with previously dismal prognosis whose management and outcome dramatically changed thanks to the use of tyrosine kinase inhibitors (TKIs) usage and more recently full chemo-free approaches. The prompt identification of these cases represents an important clinical need. Objectives: We sought to identify an optimized cytofluorimetric diagnostic panel to predict the presence of Philadelphia chromosome (Ph) in B-ALLs cases by the introduction of CD146 in our multiparametric flow cytometry (MFC) panels.  Methods: We prospectively evaluated a total of 245 cases of newly diagnosed B-ALLs with a CD146 positivity threshold >10% referred to the Division of Hematology of ‘Sapienza’ University of Rome. We compared results of CD146 expression percentage and its mean fluorescence intensity (MFI) between Ph+ ALLs, Ph-like ALLs and molecularly negative ALLs. Results: Seventy-nine of the 245 B-ALL cases (32%) did not present mutations at molecular testing, with 144/245 (59%) resulted Ph+ ALL and 19/245 (8%) Ph-like ALLs. Comparing the 3 groups we found that: Ph+ B-ALL were characterized by higher expression percentage of myeloid markers such as CD13, CD33 and CD66c and low expression of CD38; Ph+ B-ALL showed a higher CD146 expression percentage  and MFI when compared with both molecular negative B-ALL and Ph-like ALLs; neither the mean percentage of CD146 expression neither CD146 MFI were statically different between molecular negative B-ALL and Ph-like ALLs. Conclusions: Our data demonstrate the association between CD146 expression and Ph+ B-ALLs. CD146 along with myeloid markers may help to identify a distinctive immunophenotypic pattern, useful for rapid identification in diagnostic routine of this subtype of B-ALLs that benefits from a specific therapeutic approach

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

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 201

Measurable Residual Disease Monitoring for Philadelphia Positive Acute Lymphoblastic Leukemia (Ph+ALL) in the Setting of the GIMEMA ALL2820 Trial

Introduction. Measurable residual disease (MRD) negativity represents the primary endpoint in the management of adult Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. MRD assays must be highly sensitive and specific. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) can overcome some limitations of standard methodologies. In order to evaluate the best marker for MRD monitoring, in this study we performed: i) MRD evaluation by BCR::ABL1 fusion transcript and immunoglobulin/T-cell receptor clonal gene rearrangements (IG/TR); ii) a comparison of the MRD concordance rate between the two markers; iii) a correlation with biologic features and clinical outcome. Methods. A total of 167 samples from111 adults enrolled in the ongoing phase III GIMEMA ALL2820 trial were collected. Samples derived from cases from both the experimental and the control arm, based respectively on ponatinib followed by blinatumomab and on a combination of imatinib and conventional chemotherapy. Established time points were day +70 (end of induction) and day +133 (after 2 cycles of blinatumomab or after 6 cycles of chemotherapy, respectively). At diagnosis, patients underwent a screening for the identification of the predominant IG/TR rearrangement by standard PCR and/or by NGS approach [LymphoTrack assay for IGH (FR1/2/3) and IGK] and the IKZF1plus signature by Multiplex Ligation-dependent Probe Amplification (MLPA). MRD monitoring was evaluated by RQ-PCR of BCR::ABL1 and, for translational research purposes, IG/TR monitoring was evaluated by ddPCR. The MRD concordance rate was evaluated comparing the results obtained between the 2 markers. Results. Overall, 97/111 (87.4%) cases were evaluable for IG/TR MRD monitoring. At day +70, the overall concordance rate between BCR::ABL1 and IG/TR was46.4%; 48/97 (49.5%) cases were BCR::ABL1pos; 20 were concordantly IG/TRpos (41.7%), 1 was IG/TRpositive not quantifiable and 27 were IG/TRneg; 34/97 (35%) cases were BCR::ABL1neg, 25 of which were concordantly IG/TRneg (73.5%), 5 were IG/TRpos and 4 were IG/TRpositive not quantifiable; finally, 15/97 (15.5%) cases were BCR::ABL1positive not quantifiable, 5 of which were IG/TRpos and 10 were IG/TRneg. The concordance rate was similar between IKZF1plus vs IKZF1 WT/IKZF1 loss (48.4% vs 44%), and p190 vs p210-p190/p210 (47% vs 42%) cases. At day +133, 70 patients were studied and the overall concordance rate was 41.4%: 28/70 (40%) cases were BCR::ABL1pos, 4 of which were concordantly IG/TRpos (14.3%), 2 were IG/TRpositive not quantifiable and 22 were IG/TRneg; 27/70 (38.6%) cases were BCR::ABL1neg, 25 of which were concordantly IG/TRneg (92.6%) and 2 were IG/TRpos; finally, 15/70 (21.4%) cases were BCR::ABL1positive not quantifiable and IG/TRneg. A lower concordance was observed in the IKZF1plus (24%) vs IKZF1 WT/IKZF1 loss (51%) and in the p210-p190/p210 (27.3%) compared to p190 (48%) cases.Five patients experienced a hematologic relapse. Dual monitoring was feasible in 4/5 cases (in 1 material was lacking) and, in addition to the evaluation at day +70 and +133, a retrospective backtracking was carried out at a previous time-point: 1 was concordantly BCR::ABL1pos (0.26 BCR::ABL1/ABL1 x 100) and IG/TRpos (4.0E-04), 1 was BCR::ABL1pos (7.12 BCR::ABL1/ABL1 x 100) and IG/TRneg, while the other 2 cases were both BCR::ABL1neg but IG/TRpos at 4.0E-05 and 2.0E-04, respectively, suggesting the presence at the onset of non Ph+ subclone. Conclusions. IG/TR MRD monitoring was feasible in 87.4% of Ph+ ALL cases indicating that a fraction of patients is not suitable for this strategy. Overall, the concordance rate between BCR::ABL1 and IG/TR is limited, in line with the literature. While some groups reported a higher predictive prognostic power of IG/TR monitoring, our findings do not confirm these data, also in view of the very low rate of relapses so far observed. Nevertheless, a double-hit strategy may be informative for MRD monitoring and possibly for the distinction between typical/lymphoid Ph+ ALL vs multilineage/CML-like Ph+ ALL