In Ph+ acute lymphoblastic leukemia, frontline dasatinib plus blinatumomab (dasa+blina) is associated with long-term survival rates of 75-80%. The phase III GIMEMA ALL2820 trial has explored ponatinib with blinatumomab (pona+blina). In the present study, the immune modulation induced by dasa+blina and pona+blina was investigated. Immune cells were analyzed at the end of induction (T0) and after 2, 4 and 5 blinatumomab cycles (T2, T4, T5). Among 153 patients (43 dasa+blina, 110 pona+blina), the dasa+blina combination induced a significantly greater lymphocyte increase at T4 and T5 compared to pona+blina. The Treg counts decreased only in the dasa+blina treated patients. NK and NK-T cells increased significantly in the dasa+blina group, at all timepoints. Complete molecular responders (CMR) after dasatinib induction had significantly higher lymphocytes, T and NK cells compared to non-CMR patients. Bone marrow analyses showed higher activation (CD25, CD69) and lower exhaustion (PD1, TIM3) markers on NK and NK-T cells in dasa+blina treated patients. Dasa+blina patients exhibited a significantly enhanced NK cell capacity compared to ponatinib treated patients. Patients remaining on dasatinib maintained elevated NK cells with a more mature phenotype, suggesting a durable effect. These results highlight the greater dasa+blina immune activation, supporting a potential synergistic effect of the drug combination.

ABSTRACT:Resistance to first-line chemotherapies and crizotinib in anaplastic large cell lymphoma (ALCL) represents a significant challenge, often leading to a dismal outcome. Despite recent advancements, the dissection of the intrinsic and extrinsic molecular alterations underlying crizotinib resistance in ALCL is still poorly understood. Here, we transcriptionally unraveled the bidirectional interplay between anaplastic lymphoma kinase (ALK)-driven ALCL (ALK+ ALCL) and stromal cells in the presence of crizotinib at bulk and single-cell levels and identified that the microenvironment provides prosurvival signals leading to crizotinib persistence in ALK+ ALCL. We detected increased B-cell lymphoma 2 (BCL2) expression and downregulation of pathways related to apoptosis in crizotinib-persister ALK+ ALCL cells. Furthermore, we predicted in silico the ligand-receptor interactions between tumoral and stromal cells, supporting their contribution to ALCL pathogenesis mainly participating in the adhesion/membrane transport, triggering receptors, and promoting activation and microenvironment stimulation in lymphoma cells. Finally, we explored the effect of crizotinib in combination with BH3 mimetics. Pharmacologic and genetic ablation of anti-apoptotic targets displayed a significant synergistic effect with crizotinib, overcoming the stroma-mediated protection of lymphoma cells on drug treatment. Thus, BCL2/B-cell lymphoma-extra large (BCL-XL) targeting is synthetic lethal with crizotinib exposure in ALK+ ALCL and represents an intrinsic- and extrinsic-mediated targetable vulnerability in lymphoma cells challenged with crizotinib. Our data support the evaluation of BCL2 targeting in crizotinib-based regimens in the management of patients with ALK+ ALCL.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the best consolidative treatment for high-risk acute lymphoblastic leukemia (ALL). The Campus ALL study group analyzed the clinical outcomes of patients treated in the real-life with the pediatric-inspired and minimal/measurable residual disease (MRD)-oriented GIMEMA LAL1913 protocol who underwent alloHSCT. Key factors impacting on outcomes were MRD and remission status (1st complete remission vs 2nd complete remission) at transplant. MRD positivity was associated with poorer outcomes, with 3-year overall survival (OS) and disease-free survival (DFS) of 47% and 41% in MRD-positive patients compared to 80% and 70% in MRD-negative patients. Additionally, MRD negativity was associated with improved outcomes also for patients in 2nd complete remission with 3-year OS and DFS rates of 60% and 56%, respectively, compared to only 13% for both outcomes in MRD-positive cases. Patients older than 55 years showed survival rates comparable to younger patients, despite having a slightly higher non-relapse mortality, which remained below 20% at 3 years. These findings underscore the crucial role of alloHSCT in high-risk ALL and emphasize the importance of an early accurate disease risk allocation. The adverse outcome observed with MRD positivity advocates for early pre-transplant intervention with immunotherapy, whenever possible.

The Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) Leucemia Acuta Linfoblastica (LAL) 2317 protocol investigated the frontline chemotherapy-blinatumomab combination in adult Philadelphia chromosome/BCR::ABL1 rearrangement-negative (Ph-) CD19+ B-lineage acute lymphoblastic leukemia (B-ALL) to improve minimal residual disease (MRD) response and clinical outcome. Two cycles of IV blinatumomab were administered after chemotherapy cycles 3 and 6. The primary end point was the rate of molecular MRD negativity after blinatumomab 1. One hundred forty-nine patients were enrolled (median age, 41 years [range, 18-65]); 132 entered remission, 122 received blinatumomab, and 109 had a pre-and post-blinatumomab 1 MRD assessment. MRD negativity increased from 72% to 93% (P < .001) after blinatumomab, with 23 of 30 MRDpositive patients (73%) becoming MRD negative, fulfilling the primary end point. At a median follow-up of 38.1 months (range, 0.5-62.8), the median overall survival (OS) and disease-free survival (DFS) were not reached, and the estimated 3-year OS and DFS were 71% and 65%, respectively, with an excellent outlook for the patients aged 18 to 40 years who achieved an early MRD negativity (DFS, 92%). Pre-blinatumomab MRD predicted a worse outcome, especially in genetically high-risk patients. Notably, the 3-year survival of blinatumomab-treated patients was 82%. Survival and relapse rates were 91% and 15% in patients assigned to standard chemotherapy, 59% and 35% in patients assigned to hematopoietic stem cell transplantation, and 69% and 19% in transplant recipients, respectively. Blinatumomab toxicity was manageable, with only 8 permanent discontinuations. This chemotherapy-blinatumomab risk-oriented program yielded remarkable results that need further improvement in higher-risk patients displaying early MRD persistence. Blinatumomab should be considered as a standard component of induction/consolidation for adult Ph-B-ALL. This trial was registered at www.ClinicalTrials.gov as #NCT03367299.

Early identification of relapsing/refractory diffuse large B-cell lymphoma (DLBCL) represents an unmet clinical need. A real-life cohort of newly diagnosed DLBCL (n = 120) treated with R-CHOP was investigated. Using the standardized uptake value (SUV) threshold of 4.0, PET/CT radiomics variables (SUVmax, tMTV, tTLG and Dmax) were collected. Circulating tumor DNA (ctDNA) analysis by CAPP-seq yielded baseline ctDNA levels and LymphGen molecular clustering. The best cut-off for both PET/CT parameters and ctDNA levels were identified by max-stat statistics. tMTV, tTLG and Dmax retained independent prognostic value when adjusted for ctDNA levels and were grouped together in a variable named high-risk PET. By multivariate analysis, ctDNA-high and high-risk PET independently predicted PFS and were combined into a 2-factor prognostic model (C-indices: 0.712 for PFS and 0.696 for OS). Molecular clustering, by capturing high-risk biological features of DLBCL, further improved outcome prediction. Consistently, BN2/EZB/ST2 clusters maintained an independent association with better PFS when adjusted for the 2-factor model variables and were therefore included in a 3-factor prognostic score (C-indices: 0.745 for PFS and 0.746 for OS), that identified a very high-risk group of patients (n = 22, 40-month PFS 12.1%) which should be prioritized for early response evaluation and for access to novel agents.

The adoption of pediatric-inspired regimens for the treatment of Ph-negative acute lymphoblastic leukemia (ALL) in adults has improved prognosis. However, the feasibility of these intensive regimens in older patients is limited, due to the increased incidence of therapy-related side effects, including those related to asparaginase. In this sub-analysis carried out by the Campus ALL network, 90 ALL patients aged 55 or more (median age 59 years) homogeneously treated in real-life according to the GIMEMA LAL1913 program, were analyzed to evaluate the feasibility and tolerability of pegaspargase (PEG-ASP) treatment. Among the 90 patients analyzed, 86 (96

The introduction of pediatric-inspired regimens in adult Philadelphia-negative acute lymphoblastic leukemia (Ph- ALL) has significantly improved patients' prognosis. Within the Campus ALL network, we analyzed the outcome of adult Ph- ALL patients treated according to the GIMEMA LAL1913 protocol outside the clinical trial to compare the real-life data with the study results. We included 421 consecutive patients; median age 42 years. The complete remission (CR) rate after the first course of chemotherapy was 94%, and measurable residual disease (MRD) negativity after the third course was achieved in 72% of patients. The 3-year overall survival (OS) and disease-free survival (DFS) were 67% and 57%, respectively. In a multivariate analysis, MRD positivity negatively influenced DFS. In a time-dependent analysis including only very high-risk (VHR) and MRD positive cases, transplanted (hematopoietic stem cell transplantation [HSCT]) patients had a significantly better DFS than non-HSCT patients (P=0.0017). During induction, grade ≥2 pegaspargase-related hepato-toxicity was observed in 25% of patients (vs. 12% in the GIMEMA LAL1913 trial, P=0.0003). In this large, real-life cohort of Ph- ALL, we confirmed the very high CR rate and a superimposable OS and DFS compared to the GIMEMA LAL1913 clinical trial (CR rate after C1, 94% vs. 85%, P=0.0004; 3-year OS, 67% vs. 67%, P=0.94; 3-year DFS, 57% vs. 63%, P=0.17). HSCT confirms its important role in VHR and MRD-positive patients. The rate of pegaspargase-related toxicity was significantly higher in the real-life setting, emphasizing the importance of dose adjustment in the presence of risk factors to avoid excessive toxicity.

In the FELIX study,1 the CAR-T product obecabtagene autoleucel (obe-cel) was investigated in relapsed/refractory adult B-cell lymphoblastic leukemia (B-ALL). The high responses associated with the low incidence of grade ≥3 side effects make obe-cel an attractive candidate for a broader use of CAR-T cells in B-ALL. Its impact will have to be weighed with the monoclonal antibody blinatumomab in the frontline treatment of B-ALL.

The greatest improvements in the management of Acute Lymphoblastic Leukemia (ALL) have been witnessed in Ph+ALL patients. The advancements have stemmed from an always more precise genetic characterization at presentation, the use of tailored treatment, the precise monitoring of minima/Measurable Residual Disease (MRD) and, finally, by the inclusion of immunotherapy in the frontline treatment. Prior to the advent of Tyrosine Kinase Inhibitors (TKIs), Ph+ALL was the hematologic malignancy with the worse outcome. The frontline use of TKIs has changed the natural history of the disease. Since year 2000 in Italy all patients enrolled in the GIMEMA multicenter protocols have been treated in induction with a TKI alone (plus steroids) and no systemic chemotherapy. The subsequent advancement has been brought by the addition of the bispecific monoclonal antibody blinatumomab as consolidation, always in the absence of systemic chemotherapy. The results of the GIMEMA LAL2116 (D-ALBA) trial for patients of all ages showed high rates of molecular response following an induction/consolidation treatment with dasatinib and blinatumomab. At 53-months, survival rates of 75%‒80% were recorded, with 50% of patients being managed only with a TKI and blinatumomab, without chemotherapy and transplant. Most MRD+ patients were allografted. IKZF-plus patients have a less favorable outcome and should be identified at diagnosis. When possible, they should undergo an allogeneic transplant. In the subsequent phase 3 GIMEMA ALL2820 trial, patients enrolled in the experimental arm and treated with ponatinib followed by blinatumomab showed even higher rates of molecular response, with estimated OS and DFS of 94.9% and 95.6% at 12-months. Of interest, the combination of dasatinib and ponatinib plus blinatumomab, in the absence of systemic chemotherapy, is associated with a marked host immune activation. The MDACC group also reported the effectiveness of ponatinib combined with blinatumomab, though the combination was associated with greater toxicity. For a review on the treatment of adult Ph+ALL see Chiaretti & Foà. The GIMEMA ALL2820 trial will conclusively show how many patients can be spared systemic chemotherapy and transplant. At the interim analysis, only 10% of patients enrolled in the ponatinib + blinatumomab arm have so far undergone a transplant. I have been asked to cover ‘How I Treat Ph+ALL’, which more appropriately should be ‘How Should I Treat Ph+ LL’ Based on the 25-year experience gathered through the GIMEMA trials, the optimal algorithm should be: i) Identify the presence of the BCR/ABL gene lesion within one week from diagnosis; ii) During this time treat patients with steroids; iii) Start induction with dasatinib or ponatinib plus steroids, with no systemic chemotherapy; iv) CNS prophylaxis should be carried out; v) MRD should be monitored molecularly at given timepoints; vi) After induction, all patients should be consolidated with multiple cycles of blinatumomab (up to 5 in our protocols); vii) TKI should not be stopped. Through this approach the large majority of patients – of all ages – will become molecularly negative. IKZF-plus patients should be identified on the diagnostic material. Transplant should be offered to patients with an unfavorable genetic profile and/or evidence of MRD. All patients should be closely monitored for MRD during the follow-up. The possibility of offering such a personalized frontline management to all patients – including the elderly – strongly relies on adequate and standardized laboratory facilities aimed at a broad diagnostic work-up and at an accurate monitoring of MRD, as well as an optimal and timely access to the different drugs. In the real life, this is often not possible. Patients should then undergo a TKI (plus steroid) induction associated with mild chemotherapy. Many such patients are offered an allogeneic transplant. The future of patients with Ph+ALL of all ages is looking always more favorable if all the pieces of the puzzle are in place. It is likely that with the advent of the subcutaneous formulation of blinatumomab the long-term outcome will look even better.

The Philadelphia (Ph) chromosome is one of the few genetic aberrations in which a casualty has been proven, and as such represents a success in the history of medicine. This is also evident in the setting of Ph+ acute lymphoblastic leukemia (Ph+ ALL), the most frequent genetic subgroup in adult ALL, whose incidence increases with age and whose prognosis, prior to the advent of tyrosine kinase inhibitors (TKIs), was particularly poor. The outcome and management of Ph+ ALL patients have greatly improved since the incorporation of 1st, 2nd and 3rd generations TKIs in the therapeutic backbone, and is further changing with the more recent introduction of immunotherapy. This is allowing long-term survival rates currently ranging between 75 and 80%. The clinical scenario of adult Ph+ ALL has thus changed profoundly, and new challenges are emerging. In this How I treat, illustrative clinical cases are used to discuss the role today of systemic chemotherapy and allogeneic stem cell transplant, the difficulty in treating central nervous system relapses and, more in general, relapses in the current therapeutic era, and the possibility of stopping TKIs. Finally, the challenges related to an optimal management of these patients is discussed.

Infections often complicate pediatric-inspired treatments for adult Philadelphia-negative acute lymphoblastic leukemia (Ph- ALL). Literature data on these complications are difficult to interpret due to the heterogeneity of types of infections analyzed or patients and treatment characteristics. A deeper insight on the infections occurring in the real life in uniformly treated ALL patients is lacking. This study investigated infectious complications in 240 newly diagnosed adult Ph- ALL patients treated in the real life according to the GIMEMA LAL1913 protocol by 18 Italian centers participating in the Campus ALL network. Incidence, etiology of microbiologically documented infections and invasive fungal infections (IFI) and mortality for infection were determined. Potential risk factors and the prophylactic strategies used during the first chemotherapy course (C1) were analyzed. Of 240 patients, 145 (60%) experienced at least one infectious episode, with bacterial infections being the most common (74.3%), followed by viral (13.9%), fungal (10.1%), and Pneumocystis jirovecii (1.7%) infections. The blood stream was the most involved site, pneumonia occurred in 14.6% of cases, half of which being fungal. Infections were prevalent during C1, affecting 40.5% of patients; IFI occurred in 12.5% of patients, most of them in C1. Risk factors for infections included older age (≥ 55 years and particularly > 65 years) and comorbidities only for IFI. The mortality rate for infection was 3.3%. Antibacterial, antiviral, antifungal, and anti-PJ prophylaxis were variably administered and did not associate with a significant reduced infection rate. In conclusion, the rate of infectious complications in the real life of adult Ph- ALL patients treated with a pediatric-inspired intensive regimen is high, mainly during induction and mostly bacterial, particularly in the bloodstream, with a high IFI rate. Older age, mainly over 65 years, is a risk factor for all types of infection. The antimicrobial prophylaxis was not associated to a reduced risk of infection.

Introduction. Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients are commonly treated with tyrosine kinase inhibitors (TKI) in combination with either steroids, chemotherapy and more recently also immunotherapy. In the past, allogeneic transplant (SCT) was considered the only curative option; however, with the introduction of more potent TKIs as well as immunotherapy this milestone is nowadays debated, mostly for patients who achieve a molecular response. Moreover, as in chronic myeloid leukemia (CML), it is becoming relevant to understand if patients may over time discontinue TKI treatment. For this purpose, it is pivotal to report the outcome of Ph+ ALL patients who, for any reason, stopped TKI treatment. Patients and Methods. Forty-seven adult Ph+ ALL patients treated since 1 January 2000, and who did not undergo a SCT, were retrospectively collected from 12 Italian centers participating to the Campus ALL network. Their treatment plan included a TKI +/- chemotherapy according to the center current practice and/or according to the ongoing national trial they were enrolled in. The median age was 63 years (20-84), 20 patients (43%) were males, 30 patients harbored a p190 transcript, 15 a p210 transcript and 2 had both transcripts. Twenty-six patients were treated within a trial, while 21 were off-protocol. For 32 patients, induction was based only on a TKI plus steroids, whereas the remaining 15 received a combination of a TKI plus chemotherapy (in 3 cases high intensity protocols such as the NILG (Northern Italian Leukemia Group) and HCVAD regimens, in 12 low dose chemotherapy). The reasons not to transplant patients were: donor unavailability (5), clinical decision, mainly due to age/unfitness (30), patient's refusal (4), and achievement of a molecular response (i.e. at least BCR-ABL/ABL ≤0.01%) (8). The median follow-up was 60 months, with a median overall survival (OS) not reached at the last follow-up and a median disease-free survival (DFS) of 12 years. At the last follow-up, 12/47 patients had experienced a relapse. At the end of the induction (i.e. after 3 months, at a median of +82 days, from treatment initiation), 27 patients had achieved a molecular response. Overall. a molecular response was obtained in 40/46 pts (data not available for 1 case) after a median of 5 month, and was associated with a significantly lower hazard of disease recurrence (HR=0.057, 95% CI: 0.006-0.51, p=0.011) after adjusting for gender, fusion protein, association with chemotherapy, CMR at 3 months. Results. TKI treatment was discontinued in 14 of the 47 patients evaluated. The reasons for discontinuation were either toxicity (6) or clinical/patient decision (8). Eight patients harbored a p190 transcript, 5 a p210 transcript and 1 carried both. At the time of discontinuation, the median age was 75 years (23-87) and patients had been on a TKI for a median of 57.5 months (8-160) prior to stopping treatment. For these cases, the median WBC at diagnosis were 8.6 x 10 9/l (2.1-306 x 10 9/l). In 7 patients the first TKI used was imatinib, in 3 dasatinib, in 3 ponatinib and in 1 nilotinib. All patients but 1 had achieved a molecular response before stopping treatment; the only patient who never achieved a molecular response prior to discontinuing the TKI had a rapid relapse (1 month). At the last follow-up, 9 cases were still in treatment-free remission (TFR), whereas 5 eventually relapsed after 1, 1, 4, 14 and 30 months, respectively. Re-challenge with a TKI led to a hematological and molecular response in 3 of the 5 patients. The remaining 2 patients, who had been previously heavily treated and whose TFR was of only 1 month, received salvage standard chemotherapy but eventually died from ALL progression. The median time of TFR for the entire cohort of 14 patients was 20.5 months (1-82). When considering only the 9 patients who remain off treatment, the median time of TFR was 29.5 months (7-82) (Table 1, Figure 1). Conclusions. We report the outcome of 14 Ph+ ALL patients who discontinued TKI treatment due to various reasons. Nine of them are still on TFR after more than 2 years at the last follow-up, reinforcing the concept that a TFR is feasible in selected adult Ph+ ALL patients. Of further relevance, TKI re-challenge was successful in 3/5 patients who relapsed, similarly to what observed in CML.

Experts from the European Leukemia Net (ELN) working group for adult acute lymphoblastic leukemia have identified an unmet need for guidance regarding management of adult ALL from diagnosis to aftercare. The group has previously summarized their recommendations regarding diagnostic approaches, prognostic factors and assessment of ALL (cross-reference). The current recommendation summarizes clinical management. It covers treatment approaches including the use of new immunotherapies, application of MRD for treatment decisions, management of specific subgroups and challenging treatment situations as well as late effects and supportive care. The recommendation provides guidance for physicians caring for adult ALL patients which has to be complemented by regional expertise preferably provided by national academic study groups.

Introduction. The outcome of adult patients with newly diagnosed Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL) has markedly improved in recent years, first due to the introduction of tyrosine kinase inhibitors (TKI) and more recently with the combination with immunotherapy (blinatumomab). In the previous GIMEMA LAL2116 trial, we showed the effectiveness of a chemo-free approach based on dasatinib followed by blinatumomab (Foà et al, NEJM 2020), with long-term (median follow-up 53 months) overall survival (OS) and disease-free survival (DFS) of 80.7% and 75.8%, respectively (Foà et al, JCO 2023). Nine relapses were recorded. To improve the results obtained and reduce relapses, in the experimental arm of the current phase III trial ALL2820 trial, ponatinib was used in place of dasatinib. The trial is expected to complete enrolment in 2024. Here we report the updated results in terms of complete hematologic remission (CHR), measurable residual disease (MRD) and OS, as well as toxicity profile of the experimental arm. Methods. Between September 2021 and July 2024, 200 newly diagnosed adult Ph+ ALL patients were enrolled. One hundred thirty three patients were randomized to the experimental arm that contemplated a steroid pre-phase followed by a 70-day induction with ponatinib at 45 mg or 30 mg according to age (less or more than 65 years) followed by at least 2 cycles (maximum 5) of blinatumomab. Median age was 57 years (20-87), 27% of patients being >65 years; 52% were female, the white blood count was 11x109/l (1-244); the p190 protein was detected in 70% of cases and 30% carried the p210 or 190/p210 protein. Results. By the end of the induction, 110 patients (95%) achieved a CHR; 4 (3%) died in induction (pneumonia in 2, ileus paralyticus in 1 and death of unknown causes in 1) and 2 (1.5%) went off-study due to cardiac toxicity, while being in CHR. The remaining 17 cases are still receiving induction. A MRD response (complete molecular response plus positive non quantifiable) was recorded in 45% of cases. By the end of the second cycle of blinatumomab, 93 of the 95 evaluable patients (98%) were in CHR, 1 withdraw consent due to non-compliance and 1 experienced a hematologic relapse. MRD response raised to 73%. Overall, 52 grade >3 adverse events due to ponatinib were recorded, the most frequent being hepatic (20), hematologic (12) and pancreatic (8); cardiac toxicities were relatively rare, with only 5 events; finally, 5 infectious episodes (2 sepsis) and 2 cutaneous events were also reported. The combination of ponatinib with blinatumomab led to 18 grade >3 adverse events: 8 were hematologic (5 in a single case), 4 were infections, 2 cytokine release syndromes and 2 neurologic related events were documented, as well as 2 gastrointestinal events. Forty percent of patients required a dose reduction and/or a transient interruption: the most frequent toxicities were represented by grade 3 transaminitis in 10 patients and grade 2 in 6; an increase in pancreatic enzymes was observed in 6 cases (4 grade 3 and 2 grade 2); a cardiac toxicity was recorded in 6 patients, leading to a permanent discontinuation only in 2 cases. Other adverse events requiring a dose reduction were cutaneous rush, pharyngeal abscess, COVID-19 infection, and hematologic toxicity, documented in 1 case each. There was not a significant enrichment of toxicity leading to dose reduction/transient interruption in elderly. So far, only 3 relapses (3%) have been recorded: 1 was BCR::ABL1-negative, suggesting the presence at diagnosis of a non Ph+ subclone, and 1 was recorded in a case who discontinued treatment for cardiovascular toxicity. Transplant allocation was based on an IKZF1plus profile at presentation and MRD persistence; so far, only 12% of patients have been allografted. At a median follow-up of 6.4 months (range 0.1-32.3), the estimated 18-month OS is 91.6%. Conclusions. The intermediate analysis of the GIMEMA ALL2820 trial shows the feasibility and efficacy of a frontline chemo-free induction/consolidation approach with ponatinib and blinatumomab for Ph+ ALL adults of all ages. The combination was overall well tolerated, with very few treatment discontinuations, also in the elderly, suggesting that a ponatinib dose adjustment according to age may prevent severe toxicities. Lastly, with the biology-driven transplant allocation only 12% of patients have been transplanted so far.

Introduction. Expression of the CD49d integrin chain has prognostic impact on ibrutinib (IB)-treated CLL patients (Tissino et al, J Exp Med, 2018; Tissino et al, Blood 2020). In addition, a 4-factor (4-f) score, based on TP53 aberration, β2-microglobulin (β2M), lactate dehydrogenase (LDH) and previous therapy lines was proposed to identify patients at a higher risk of treatment failure or death during IB therapy (Ahn et al, J Clin Oncol, 2020; Morabito et al, Am J Hematol, 2021). The clinical impact of TP53 disruption was recently refined by demonstrating that only the co-presence of TP53 deletion and mutations, and not the single aberrations, has prognostic value in the IB setting (Bomben et al, Leukemia 2022). The aim here is to integrate these observations in a comprehensive scoring for the management of IB-treated patients. Methods. This study is a retrospective/multicenter analysis of 410 CLL patients treated with IB in the current clinical practice (December/2013-March/2021). Outcome data were updated as of March/2023. Median follow-up from IB treatment was 29.5 months (95% CI 26.7-33.1 months); 57 patients were treatment naïve (0 previous lines), while 353 had ≥1 previous lines. CLL patients were characterized for CD49d expression and for the 4-f variables, TP53 disruption was evaluated by FISH and TP53 mutational status by NGS (low-VAF mutations included). OS or PFS were measured from the date of IB treatment to the date of death (OS), or progression/death (PFS) or last follow-up. Results. 1) The canonical 4-f was applied as previously described, identifying 111 low-, 163 intermediate (int)-, and 136 high-risk patients. Patients in the low-risk group had a longer OS than patients in the int-, and high-risk groups ( P=0.0115 and P=0.0011), while no difference was found comparing patients in the int- vs. high-risk groups (P=0.3036). 2) We generated a modified version of the 4-f score by considering: i) as TP53 disrupted only patients presenting a concomitant TP53 deletion and mutation (n=93); ii) since no difference in terms of OS was found comparing previously untreated patients (n=57) and patients treated with 1 previous line (n=157; P=0.78), these two groups were combined (n=214) and kept separated from patients who had received >1 previous lines of therapy (n=196). Accordingly, the 4-f-modified identified 218 low-, 109 int-, and 83 high-risk patients, the 3 groups presented different OS, the latter model outperforming the canonical 4-f score (C-indices 0.616 vs 0.639; P<0.0001). 3) CD49d-high CLL cases (i.e. CLL with CD49d expression ≥30% and/or with CD49d-bimodal expression; n=268) had shorter OS and PFS intervals ( P=0.0005 and 0.0194, respectively) than CD49d-low cases (n=142). By combining these observations, CD49d remained an independent prognostic factor for OS ( P=0.004) in a multivariable model, which included the 4-f-modified int-risk ( P=0.0585) and high-risk ( P=0.0011) groups. Driven by these findings, we re-analyzed the single parameters of the 4-f score along with CD49d expression. According to a new multivariable analysis (Fig. A), LDH was not included in the final model, which in turn included CD49d expression as independent OS predictor ( P=0.0011). 4) We then redesigned a novel 4-f model excluding LDH data in favor of CD49d expression data (4-factor-CD49d). Each of the four factors contributed one point and stratified patients (n=407/410) into three groups with a significantly different risk for OS probability, merged on a one-to-one risk group comparison basis: score 0-1, low-risk (n=185); score 2-3, int-risk (n=211); score 4, high-risk (n=11; Fig. B). The inclusion of CD49d into this novel 4-f model improved its prediction capability (C-index=0.663; P<0.0001 versus both the original 4-f and the 4-f-modified). Similar results were obtained when considering PFS as clinical readout. Conclusions. Evidence is provided that in the context of CLL patients treated with IB in a real-world setting, variables like B2M, the number of previous lines of therapy and assessment of TP53 disruption remain important tools for identifying patients at a higher risk of relapse/death. Here we emphasize the need of a comprehensive assessment of TP53 aberrations considering TP53 deletions and mutations simultaneously, and propose to add the assessment CD49d expression, integrating these four variables into a novel 4-f-CD49d scoring system. Further validation in independent cohorts is needed.

Introduction . Digital twins are defined as virtual representations of real patients, generated from multi-layer longitudinal data. In the medical setting, this approach can be leveraged to enhance diagnosis, prognosis, and prediction of optimal treatment within a specific patient population, enabling the simulation of virtual scenarios to improve the decision-making process. In B-lineage acute lymphoblastic leukemia (B-ALL), the prognosis of Philadelphia-negative (Ph-neg) patients is still suboptimal if compared with the results achieved in Ph-positive ALL (Foà et al, NEJM 2020 and JCO 2024). The frontline LAL1913 GIMEMA trial for adult Ph-neg ALL (Bassan et al, Blood Adv 2023) based on a pediatric-inspired and minimal residual disease (MRD)-oriented strategy provided promising results on 183 patients: complete remission (CR) was achieved in 91% of cases, a post-consolidation MRD negativity in 75.5%, and the 3-year overall survival (OS) was 67%. The protocol was later used in the real-life Campus ALL setting in 421 patients (Lazzarotto et al, P396 EHA 2024; Haematologica, in press) and the LAL1913-like scheme is currently considered in Italy as standard of care (SOC). In the present study we aimed at generating digital twins of Ph-neg ALL to be used as a control group in in silico trials, as reported for acute myeloid leukemia (Piciocchi et al, EJHaem 2024). Methods. Data from the GIMEMA LAL1913 study and real-life data of patients treated with a LAL1913-like therapeutic scheme were merged to form a unique cohort named realPh-neg cohort of 604 patients. To create the synthetic cohort, named synthPh-neg, a machine learning generative model was constructed from the real data of the realPh-neg cohort, capturing its patterns and statistical properties, without identifiable data from the original dataset. To develop the synthetic cohort, the R package “synthpop” (Nowok et al, J Stat Soft 2016) and different parametric methods were used to generate variables according to their types. Survival variables were simulated using classification and regression trees (CART). Next, we verified the adherence of the virtual cohort to the original one in terms of age, WBC count, gender, risk category, cytogenetic class, CR. Virtual and real cohorts were also compared in terms of survival outcomes and by stratifying according to lineage, age, risk category. Results. Using the real-patient dataset, the synthPh-neg cohort of 3020 patients, corresponding to 5 times the realPh-neg cohort, was generated. The comparison between the original and synthetic cohorts showed that the clinico-biological characteristics and CR of the 2 cohorts did not differ. In detail, the CR rate was 92% in the realPh-neg cohort and 93% in the synthLAL1913 cohort (p=0.75). Moreover, OS and disease-free survival (DFS) were superimposable (p=0.69 and p=0.82, respectively). At 3 years the OS was 67.6% (63.4%-72.1%) in the original cohort and 68.7% (66.7%-70.6%) in the synthPh-neg cohort. DFS at 3 years was 59.5% (55.0%-64.3%) in the original and 58.7% (56.7%-60.8%) in the synthetic cohort. The similarities between the original and the synthetic dataset were maintained also when stratifying patients according to the biologic features at diagnosis. Conclusions. We generated a large cohort of digital Ph-neg ALL patients which faithfully reproduces the features and outcome of the original dataset. Since the LAL1913 scheme is the current SOC in Italy, synthPh-neg may qualify as the optimal control group in in-silico trials when testing innovative treatments. In this case, enrolled patients would receive only the novel - potentially more active - treatment, thus allowing a quicker evaluation of its safety and efficacy. Indeed, though randomized trials represent the gold-standard, the rapid development of novel drugs requires the evolution of clinical trial design, especially in rare diseases/subsets for which the conduct of a randomized trial would be challenging. In Ph-neg ALL, this is particularly relevant, given the biologic and clinical heterogeneity of ALL. To this end, GIMEMA has recently opened 2 phase II trials for the treatment of Ph-like ALL and very high-risk T-ALL respectively with ponatinib and daratumumab incorporated in the LAL1913 backbone (Eudra CT Number 2022-000633-17 and2024-511627-34-00) . In the future, a subset of digital twins belonging to synthPh-neg ALL could be used as a control group in phase III virtual studies.