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

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 & Chiaretti, NEJM 2022 2.Foà et al, NEJM 2020 3.Soverini et al,Leukemia 202

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