ABSTRACT Background The level of measurable residual disease (MRD) is one of the most important features correlating depths of response and long‐term outcomes in multiple myeloma (MM) and MRD evaluation is currently the gold standard tool for assessing treatment response. Nevertheless, reproducibility across laboratories is a major concern, as discrepancies among results make comparability impractical. Aims: herein, we report preliminary results from the “Italian MM‐MRD network” project. Patients & Methods MRD in bone marrow (BM) samples have been measured from newly diagnosed MM patients using next‐generation flow‐cytometry (NGF) or next‐generation sequencing (NGS) approaches in different laboratories. Results The NGF workgroup (7 laboratories) implemented the Euro‐Flow Standard‐Operating‐Protocol to reach minimum 1 × 10−5 sensitivity. The inter‐operator retrospective study (Stage 1) showed high inter‐center concordance in monoclonal plasma cells detection (ICC = 0.90, p < 0.001), whereas moderate concordance was observed in the inter‐laboratory correlation (Stage 2) in in‐vivo samples (ICC = 0.63, p < 0.001), reaching a median limit‐of‐detection (LOD) and limit‐of‐quantification (LOQ) of 8 × 10−6 and 2 × 10−5, respectively. Greater variability was also observed in the analysis of other BM cell populations. The NGS workgroup (4 laboratories) employed a targeted amplicon‐based approach to detect clonotypic IGH/IGK gene rearrangements in diagnostic samples, subsequently used to track MRD in mock samples. The experimental design was divided into three quality‐control (QC) rounds, focused on finding a shared strategy for clonotype identification (QC1: 100% concordance among centers), or quantifying MRD in mock samples (concordance: 81% [QC2]; 91% [QC3]). The 10−5‐sensitivity level was successfully reached in most of tested dilutions (QC2: 19/20 = 95%; QC3: 19/23 = 83%). Conclusion Overall, this pilot study provided preliminary data for MRD harmonization across Italian centers, paving the way for an expanded network, aiming at reducing variability, improving comparability, and enabling broader use of MRD‐monitoring in clinical practice.

In the original publication [...].

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder caused by the BCR::ABL1 fusion gene, resulting from a reciprocal translocation between chromosomes 22 and 9. Quantification of BCR::ABL1 transcript levels in peripheral blood by RT-qPCR represents the gold standard for molecular response (MR) monitoring, providing essential clinical information on treatment efficacy. Xpert® BCR-ABL Ultra is a fully automated in vitro diagnostic test that quantitatively detects e13a2 and e14a2 BCR::ABL1 transcripts using a single-use cartridge that integrates RNA extraction, cDNA synthesis, nested real-time PCR, and signal detection within a rapid, closed, and user-friendly system. In this study, we evaluated Xpert® BCR-ABL Ultra as an alternative to validated systems currently used by four highly specialized Italian laboratories affiliated with the Italian national laboratory network for CML. A total of 129 peripheral blood samples from CML patients at various disease stages, along with two external quality control materials, were analyzed. We assessed the test’s repeatability, specificity, and stability. Concordance of BCR::ABL1%IS values generated by the different methods was evaluated using EUTOS criteria and Bland–Altman analysis. Finally, MR value concordance was analyzed based on European LeukemiaNet recommendations or calculated using the formula 2 − log10(BCR::ABL1%IS). Xpert® BCR-ABL Ultra demonstrated high repeatability and stability. The BCR::ABL1%IS values obtained with this assay showed strong concordance with those generated by local reference methods, and MR classifications were consistent across platforms. These findings confirm the robustness, accuracy, and efficiency of the Xpert® BCR-ABL Ultra assay, supporting its use as a reliable alternative to currently validated systems for the routine clinical monitoring of CML patients.

INTRODUCTION:Epigenetics has been shown to be relevant in oncology: BMI1 overexpression has been reported in leukemias, EZH2 mutations have been found in follicular lymphoma, and USP22 seems to stabilize BMI1 protein. In this study, we measured the expression of BMI1, EZH2, and USP22 in lymph nodes from 56 diffuse large B-cell lymphoma (DLBCL) patients. METHODS:A new multiplex digital droplet PCR (ddPCR) has been set up to measure the expression of 4 genes (BMI1, EZH2, USP22, and GAPDH) in the same reaction on RNA extracted from paraffin-embedded tissues. RESULTS:The specificity of ddPCR was confirmed by a 100% alignment on the BLAST platform and its repeatability demonstrated by duplicates. A strict correlation between expression of BMI1 and EZH2 and BMI1 and USP22 has been found, and high expression of these genes was correlated with extra-nodal lymphomas. Progression-free survival (PFS) and overall survival (OS) were conditioned by IPI, bone marrow infiltration, and the complete response achievement. High levels of BMI1 and USP22 did not condition the response to therapy, but impaired the PFS, especially for patients defined at "high risk" based on the cell of origin (no germinal center [GCB]), high BCL2 expression, and IPI 3-5. In this subgroup, the probability of relapse/progression was twice higher than that of patients carrying low BMI1 and USP22 levels. CONCLUSION:High expression of BMI1 and of USP22 might be a poor prognostic factor in DLBCL, and might represent the target for novel inhibitors.

The recent introduction of targeted therapies for Systemic Mastocytosis (SM) has raised the awareness about the importance of timely and accurate diagnosis- that, outside reference centers, is hampered by the rarity of this malignancy, by the heterogeneity of clinical symptoms and presentation and by the need to integrate the specific expertise of different healthcare professionals. Molecular testing plays a key role in the diagnosis, classification and management of SM: detection of KIT D816V activating mutation, underlying approx 95% of SM cases, is among the diagnostic criteria and quantitation of the allele burden (AB) helps in subtype classification, provides prognostic information and might serve, in future, for minimal residual disease monitoring. Screening for KIT D816V requires sensitive PCR-based methods, since disease burden, especially in the indolent forms, may be very low. The importance of reliable and accurate molecular testing in SM prompted the Rete Italiana MAstocitosi (RIMA) to undertake, with the sponsorship of the GIMEMA Working Party on Chronic Myeloproliferative Neoplasms, an initiative aimed to i) map the status of KIT D816V mutation testing in Italy; ii) foster the creation of a network of specialized, reference laboratories available to support clinicians in the diagnosis of SM. A survey was conducted among 35 molecular biology labs at major hematological centers all across Italy to assess whether and with which methodology KIT D816V mutation testing was offered. Twenty-nine labs (83%) declared they were routinely performing KIT mutation testing, either by Sanger sequencing (n=4), NGS (n=4), digital PCR (with either home-brew assays or commercial kits; n=17), semi-quantitative real time PCR (either home-brew or commercial; n=2), quantitative ARMS-PCR (home-brew; n=1), or qualitative ASO-PCR (home-brew; n=1). After a pilot experience that involved 7 labs in 2023, a second control round of proficiency testing was conducted among 10 labs (Milan, Monza, Bergamo, Vicenza, Pisa, Pescara, Rome, Bari, Palermo, Nuoro) selected among those who reported to be using a quantitative assay on a digital PCR platform (BioRad QX200 or QX600, n=8; Thermo Fisher QuantStudio Absolute Q, n=1; Qiagen QIAcuity One, n=1). KIT D816V-mutated and wild-type DNAs isolated from the HMC-1.2 and HL-60 cell lines, respectively, were mixed in variable proportions to mimic different ABs (5%; 0.5%; 0.3%; 0.1%; 0.05%; 0.02%; 0%) and used to prepare 10 identical batches of 7 blinded vials. Dilutions were first externally assessed and validated by the UK Wessex Genomics Laboratory Service (WGLS) using an accredited droplet dPCR assay with an LoD=0.01% and then shipped to the participating labs, where they were analyzed according to local procedures. Labs were asked to score each sample as positive or negative for KIT D816V and to provide a quantitative estimate of the AB. Agreement between measurements was assessed using Weighted Deming Linear Regression and Bland-Altman bias analyses. The vial containing no KIT D816V DNA was scored negative by 9/10 labs. The 0.02% vial was scored positive for the D816V by 5 labs, borderline by one lab and negative by 4 labs (in accordance with their reported LoDs of 0.1%, 0.1%, 0.06% and 0.04%). The remaining dilutions were correctly scored positive by all labs. In these samples, quantitation of AB showed excellent correlation and concordance among labs and between labs and the WGLS, with Pearson R correlation coefficients ranging from 0.9997 to 0.9999 and Bland-Altman plots yielding a mean bias ranging from -0.17 to 0.02. Overall, our experience shows that the rarity of the disease and the lack of awareness about the diagnostic challenges in detecting KIT D816V result into routine use of an array of heterogeneous and sometimes inadequately sensitive methods (like Sanger sequencing and NGS, that may yield false-negative results leading to missed/delayed SM diagnosis in a not negligible proportion of patients). Our control round demonstrates that digital PCR, regardless of the platform, provides a reliable and reproducible option for KIT D816V mutation detection and quantitation, although further efforts should be made to enhance LoD in some cases. Our experience also shows that lab networking and exchange of positive controls and expertise plays a critical role in guaranteeing high standards of molecular testing in rare diseases like SM. Supported by Istituto Gentili.

Introduction. Minimal residual disease (MRD) analysis has been largely used in follicular lymphoma (FL) for outcome prediction. However, despite the broad employment of rituximab maintenance for long-term disease control, there are few published data describing the prognostic impact of MRD monitoring during anti-CD20 maintenance or pre-emptive rituximab strategies. Therefore, we performed a wide and comprehensive MRD analysis at several predefined time points in the phase III, “FOLL12” trial [Luminari JCO 2022], sponsored by the Fondazione Italiana Linfomi (FIL). This is the largest MRD-driven clinical trial ever conducted in FL comparing conventional rituximab maintenance (after R-CHOP or BR) vs a combined PET/MRD response-adapted post-induction approach. We here present for the first time the MRD results stratified by post-induction treatment arm. Methods. Peripheral blood(PB) and bone marrow (BM) samples were centralized at the four Italian Euro-MRD certified laboratories of the FIL MRD Network. MRD was assessed with consensus primers on IGH::BCL2 rearrangements by RQ-PCR at end of induction (EOI) and every six months thereafter during either two years rituximab maintenance (reference arm, REF) or a response-adapted approach, offering up to three cycles of four, weekly pre-emptive rituximab infusions only in case of MRD positivity (experimental arm, EXP), till month 24. Results. High MRD negativity rates were obtained at EOI in both treatment arms (REF vs EXP: 92% vs 88% p=0.255), but the MRD kinetics changed overtime during the 24 months after EOI: if 60% of patients steadily maintained MRD negativity, 27% experienced at least one MRD recurrence (being MRD negative at EOI) while 13% converted at least once from MRD positive to MRD negative; importantly, only 1% of patients steadily maintained MRD positivity overtime without clinical relapse. Actually, focusing on the time points after EOI MRD negativity rates decreased in patients of the EXP arm vs patients receiving standard rituximab maintenance, with an overall higher incidence of MRD positivity in EXP vs REF during the 2 years after EOI (relative risk of MRD positivity: 1.78, 95% CI 1.30-2.45, p<0.001). In particular, patients in REF were characterized by lower rates of MRD recurrence vs EXP (18% vs 36%, p=0.001), accounting for an overall doubled risk ratio of MRD recurrence in EXP vs REF (RR 2.03, 95%CI 1.31-3.16, p=0.001). Consequently, sustained MRD negativity at 12 months after EOI was more frequent in REF than in EXP: 90% (111/124) vs 78% (103/132), p=0.017. On the other hand, the effect of either therapeutic strategy in inducing MRD negativization in MRD positive cases was limited and similar: 14% of patients in REF vs 12% in EXP, respectively (RR 1.15, 95% CI 0.61-2.17, p=0.671). Interestingly, among MRD positive patients, no difference in MRD quantitative burden was noted between the two study arms. MRD positivity at EOI was predictive of a worse PFS in EXP arm (5-yr PFS 26% vs 61%, HR 3.05, 1.78-5.22, p<0.001), while MRD positive patients at EOI receiving maintenance rituximab were not strongly associated with adverse prognosis at present clinical update, if compared to MRD negative ones (5-yr PFS 82% vs 76% p=0.542). However, the persistence or reappearance of an MRD positive signal in PB during the second year after EOI (time points +12, +18 and +24 months) predicted a worse PFS, adjusted by treatment arms (HR 2.58, p=0.003). Finally, it is interesting to note that MRD negative patients in REF experienced a statistically significant better outcome than MRD negative patients in EXP (5-yr PFS 76% vs 61% p=0.002), highlighting that rituximab maintenance benefits also patients with sustained MRD negativity, by preserving this favorable status overtime and delaying the occurrence of clinical relapse. Conclusions. These data suggest that a regular MRD monitoring in PB is needed to better understand the complex MRD kinetics in an indolent disease such as FL, in order to early identify patients at risk of relapse. Notably, rituximab maintenance in FL improved patients' outcome independently from their MRD status at EOI, mainly by halving the risk of MRD recurrence overtime, thus favoring a sustained MRD negativity and eventually delaying the occurrence of clinical relapse.

Topic: 8. Chronic myeloid leukemia - Clinical Background: Chronic myeloid leukemia is characterized by rearrangement of the BCR::ABL1 oncogene resulting in the production of p210 and p190 fusion proteins with deregulated tyrosine kinase activity. Monitoring BCR::ABL1 transcript levels in PB patients on TKI therapy using RT-qPCR is the gold standard for management of CML. Xpert BCR-ABL Ultra (named Xpert® BCR-ABL Ultra p210) is a cartridge-based assay that automates all quantitative process integrating all steps directly from clinical samples in less than 3 hours. We need to understand the differences in measurement to ensure that variability between the two methods is controlled for treatment response assessments. Aims: The aim of the study was to compare two different methodological approaches. This was done by measuring the agreement between the values obtained from the analysis of PB samples from CML patients. These analysis were performed in parallel with the use of the Cepheid cartridge and with the individual assay used in 4 reference laboratories of the Italian Labnet Network. These assays have been validated and described in the Italian Laboratory Recommendations (RIL). Furthermore, the methodological comparison was done through the use of a reference material such as a well-known ACROMETRIX BCR::ABL1 Reference Panel and the analysis of two samples received from the UK NEQAS Control Panel. Methods: 25-30 CML peripheral blood samples were evaluated using the Xpert BCR-ABL Ultra test and each of the 4 comparator assays of 4 study sites. The ACROMETRIX material is a panel of 5 vials with a known of concentration of BCR::ABL1 aligned with the International scale (I.S). The two samples supplied by the UK NEQAS panel consist of lyophilized samples with BCR-ABL1 IS levels ranging from 10% to 0.0032. Results: The analysis included a total of 193 measurements, which were quantified by both the Cepheid and the Standard in-house method. The measurements were divided by level of disease, with 31 for MR1, 36 for MR2, 54 for MR3, 26 for MR4, 28 for MR4.5, and 18 for MR5. For each method and level of disease, the median, interquartile range, minimum, and maximum were reported in the table. Bias and 95% Limit of Agreements from Bland-Altman analysis were also computed. The agreement on MR assignment was evaluated using Cohen’s Kappa (squared weights) and more conservative equal weights, which yielded values of 0.88 (p<0.001) and 0.75 (p<0.001), respectively. These results indicate substantial to almost perfect agreement (McHugh ML. Interrater reliability: the kappa statistic. Biochem Med (Zagreb). 2012;22(3):276-82. PMID: 23092060; PMCID: PMC3900052.) TABLEFinally, we evaluated the agreement between the standard and Cepheid results according to Branford et al. (Blood 2008; 112: 3330–3338) by applying three criteria. The criteria take into account the ratio of the standard to the Cepheid result. Overall, 193 observations gave the following results: N=109 (56.5%) between 0.5 and 2.0 fold difference N=155 (80.3%) between 0.33 and 3.0 fold difference N=182(94.3%) between 0.2 and 5.0 fold difference The data obtained fall within the required ranges and confirm the level of agreement between the two analysis systems. Summary/Conclusion: Statistical analysis of the data obtained with the two systems and on the three different types of material demonstrated a good correlation at all different levels of disease. The CEPHEID cartridge system could therefore be considered as an alternative to the analysis systems currently validated within the Italian network CML LabNet. Acknowledgements Thanks to Elena Bergatto and Marco Vigliano for the support of the project Keywords: Chronic myeloid leukemia, Peripheral blood

Supplemental Figure Legend from Minimal Residual Disease after Conventional Treatment Significantly Impacts on Progression-Free Survival of Patients with Follicular Lymphoma: The FIL FOLL05 Trial

Cox proportional hazard Models for PFS: role of the molecular tumor burden at diagnosis.

CONSORT diagram of the study.

Digital droplet PCR (ddPCR) is a recent version of quantitative PCR (QT-PCR), useful for measuring gene expression, doing clonality assays and detecting hot spot mutations. In respect of QT-PCR, ddPCR is more sensitive, does not need any reference curve and can quantify one quarter of samples already defined as "positive but not quantifiable". In the IgH and TCR clonality assessment, ddPCR recapitulates the allele-specific oligonucleotide PCR (ASO-PCR), being not adapt for detecting clonal evolution, that, on the contrary, does not represent a pitfall for the next generation sequencing (NGS) technique. Differently from NGS, ddPCR is not able to sequence the whole gene, but it is useful, cheaper, and less time-consuming when hot spot mutations are the targets, such as occurs with IDH1, IDH2, NPM1 in acute leukemias or T315I mutation in Philadelphia-positive leukemias or JAK2 in chronic myeloproliferative neoplasms. Further versions of ddPCR, that combine different primers/probes fluorescences and concentrations, allow measuring up to four targets in the same PCR reaction, sparing material, time, and money. ddPCR is also useful for quantitating BCR-ABL1 fusion gene, WT1 expression, donor chimerism, and minimal residual disease, so helping physicians to realize that "patient-tailored therapy" that is the aim of the modern hematology.

Background. Immunochemotherapy is effective in follicular lymphoma (FL), but most patients (pts) eventually relapse. MRD analysis, based on the detection of Bcl-2/IGH rearrangement by highly sensitive PCR-based tools, is effective in identifying pts at risk of relapse [Ladetto Blood 2012; Pott EHA23]. However, several issues are still unresolved, including: i) which is the best tissue source and the most reliable technique; ii) which are the most predictive time points; iii) which is the role of disease kinetics during the long natural history of FL. The FIL FOLL12 prospective, phase III randomized clinical trial (EudraCT: 2012-003170-60) included a systematic MRD analysis on both peripheral blood (PB) and bone marrow (BM) taken at eight different pre-planned time points, by both nested and real time quantitative (RQ)-PCR. Therefore, it allows addressing these unresolved issues.

Hypereosinophilia (HE) is defined as a persistent increase in absolute eosinophil counts (AEC) to levels >1.5 × 109/L. HE can be ‘primary’ or ‘secondary’, more often accompanying infections, allergic disorders, autoimmune diseases or myeloproliferative/lymphoproliferative neoplasms [1,2]. In 2016, the WHO revised the classification of eosinophilic disorders, distinguishing myeloid/lymphoid neoplasms with eosinophilia and recurrent genetic lesions (such as rearrangement of PDGFRA, PDGFRB, FGFR1 and PCM1-JAK2), chronic eosinophilic leukemia (CEL) not otherwise specified (NOS), and idiopathic HE [3,4].

SARS-CoV-2 is the viral agent responsible for the pandemic that in the first months of 2020 caused about 400,000 deaths. Among compounds proposed to fight the SARS-CoV-2-related disease (COVID-19), tyrosine kinase inhibitors (TKIs), already effective in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (CML), have been proposed on the basis of their antiviral action already demonstrated against SARS-CoV-1. Very few cases of COVID-19 have been reported in Ph+ ALL and in CML Italian cohorts; authors suggested that this low rate of infections might depend on the use of TKIs, but the biological causes of this phenomenon remain unknown. In this study, the CML model was used to test if TKIs would sustain or not the viral replication and if they could damage patient immunity. Firstly, the infection and replication rate of torquetenovirus (TTV), whose load is inversely proportional to the host immunological control, have been measured in CML patients receiving nilotinib. A very low percentage of subjects were infected at baseline, and TTV did not replicate or at least showed a low replication rate during the follow-up, with a mean load comparable to the measured one in healthy subjects. Then, after gene expression profiling experiments, we found that several “antiviral” genes, such as CD28 and IFN gamma, were upregulated, while genes with “proviral” action, such as ARG-1, CEACAM1, and FUT4, were less expressed during treatment with imatinib, thus demonstrating that TKIs are not detrimental from the immunological point of view. To sum up, our data could offer some biological explanations to the low COVID-19 occurrence in Ph+ ALL and CML patients and sustain the use of TKIs in COVID-19, as already proposed by several international ongoing studies.

The most of patients affected by chronic myeloid leukemia is cured with Tyrosine Kinase Inhibitors (TKI). However, about 30% must change therapies for intolerance or poor efficacy. The failure of therapy is associated in the majority of cases to the appearance of ABL1 mutations. Ponatinib (ICLUSIG, INCYTE®) is able to overcome most of the known mutations, including the T315I. But when compound mutations appear, the TKIs alone are not enough and a combination therapy is required to overcome this condition. We describe here the evolution of the mutational status of 3 patients with compound mutations, treated with the association of ponatinib plus alfa interferon. This combination resulted safe and effective for all. In the first patient, the T315I plus the V289A and the M244V mutations were discovered; the second patient showed the T315I plus the A288T and the G390R and the third one, without T315I, the K419E plus the G254R were found. After one year of the combination therapy, all cases showed the disappearanced of the compound mutations and the T315I. In addition, the use of next generation sequencing resulted fundamental for detecting and monitoring resistant patients.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) interfere with cellular metabolism contributing to oncogenesis. Mutations of IDH2 at R140 and R172 residues are observed in 20% of acute myeloid leukemias (AML), and the availability of the IDH2 inhibitor Enasidenib made IDH2 mutational screening a clinical need. The aim of this study was to set a new quantitative polymerase chain reaction (PCR) technique, the drop-off digital droplet PCR (drop-off ddPCR), as a sensitive and accurate tool for detecting IDH2 mutations. With this technique we tested 60 AML patients. Sanger sequencing identified 8/60 (13.5%) mutated cases, while ddPCR and the amplification refractory mutation system (ARMS) PCR, used as a reference technique, identified mutations in 13/60 (21.6%) cases. When the outcome of IDH2-mutated was compared to that of wild-type patients, no significant difference in terms of quality of response, overall survival, or progression-free survival was observed. Finally, we monitored IDH2 mutations during follow-up in nine cases, finding that IDH2 can be considered a valid marker of minimal residual disease (MRD) in 2/3 of our patients. In conclusion, a rapid screening of IDH2 mutations is now a clinical need well satisfied by ddPCR, but the role of IDH2 as a marker for MRD still remains a matter of debate.

Minimal residual disease (MRD) in non-Hodgkin's lymphomas (NHLs) still represents matter of interest and debate: indeed, the new available treatments offer higher rates of complete responses and MRD negativity than in the past, with a positive impact on the long-term survival. Furthermore, the introduction of more sensitive and accurate molecular techniques, such as digital PCR (ddPCR) and the next generation sequencing techniques (NGS), increased the possibility of identifying molecular targets to be followed after therapy (such as rearrangement of immunoglobulins, fusion genes, or mutations). This review focused on how molecular biology can help to detect MRD in different types of NHLs and how MRD can change the clinical practice in 2019. In follicular lymphoma (FL), contamination of the grafts and molecular disease persistence after transplantation represent a negative prognostic factors. The combination of Rituximab or Obinutuzumab with Bendamustine seems to be the most effective way to clear MRD in FL patients receiving chemo-immunotherapy (further studies are in progress), and also (90)Yttrium-Ibritumomab-Tiuxetan offers a deep clearance of molecular disease. Finally, molecular MRD can further stratify PET-negative cases, with subjects both PET- and MRD-negative presenting the best outcome. In aggressive lymphomas, MRD has a relevant prognostic power and can represent the platform for immunotherapy (such as CAR-T). In diffuse large B-cell lymphoma (DLBCL), the assessment of MRD in the plasma (where cell-free DNA and exosomes circulate) seems to be more predictive than the bone marrow analysis or peripheral blood mononuclear cells. Finally, NGS technologies could be more useful than the classical "patient allele-specific PCR" because they can identify any possible clone emerging during the treatment or follow-up, even if different from that identified at diagnosis, thus predicting relapse. After all, the present available molecular approaches can move MRD from the bench side to the clinical practice.

Background In addition to morphological and cytogenetic features, acute myeloid leukemias are characterized by mutations that can be used for target-therapy; also the minimal/measurable residual disease (MRD) could be an important prognostic factor. The purpose of this retrospective study was to investigate if somatic mutations could represent an additional prognostic value in respect of MRD alone. Method At baseline, 98 patients were tested for NPM1 , FLT3 , and for WT1 expression; 31 for ASXL1 , TET2 , IDH1 , IDH2 , N - RAS , WT1 , c - KIT , RUNX1 , and DNMT3A . The same genes have been also tested after induction and consolidation. Results Overall, 60.2% of our patients resulted mutated: 24.5% carried mutations of FLT3 - ITD , 38.7% of NPM1 , 48.4% of c - KIT , 25.8% of N - RAS and 19.3% of IDH2 . The probability of achieving a complete response (CR) was higher for younger patients, with low ELN risk score, NPM1 -mutated, with low WT1 levels, and without FLT3 . The presence of additional mutations represented a poor predictive factor: only 19% of these cases achieved CR in comparison to 43% of subjects without any of it. Concerning survival, it was conditioned by a lower ELN risk score, younger age, reduction > 1 log of the NPM1 mutational burden, disappearance of FLT3 mutations and lower WT1 expression. Regarding the role of the additional mutations, they impaired the outcome of 20% of the already MRD-negative patients. Concerning the possibility of predicting relapse, we observed an increase of the NPM1 mutational burden at the time-point immediately preceding the relapse (about 2 months earlier) in 50% of subjects. Similarly concerning WT1 , an increase of its expression anticipated disease recurrence in 64% of cases. Conclusions We demonstrated that additional somatic mutations are able to impair outcome of the already MRD-negative subjects. About MRD, we suggest a prognostic role also for the WT1 expression. Finally, we considered as relevant the assessment of NPM1 quantity clearance instead of the presence/absence of mutations alone. Still remains in doubt the utility in terms of long-term prognosis of a baseline more complex mutational screening; we could hypothesize that it would be useful for those patients where other markers are not available or who reached the MRD negativity.

The Polycomb gene BMI1 expression exerts a negative predictive impact on several hematological malignancies, such as acute and chronic myeloid leukemia (CML), myelofibrosis, and follicular lymphoma. As already demonstrated in CML, BMI1 is responsible for the resistance to the tyrosine kinase inhibitors (TKIs) in a BCR-ABL1-independent way. Even if, it is unknown where BMI1 in CML is expressed (in progenitors or more mature cells). We decided, therefore, to evaluate if and where the BMI1 protein is located, focusing mainly on the CD34+/CD38-/CD26+ CML progenitors. To begin we measured, by flow cytometry, the proportion of CD34+/CD26+ cells in 31 bone marrow samples from 20 CML patients, at diagnosis and during treatment with imatinib. After that the bone marrow blood smears were stained with antibodies anti-CD26, BCR-ABL1, and BMI1. These smears were observed by a confocal laser microscope and a 3D reconstruction was then performed. At diagnosis, CD34+/CD26+ cells median value/μL was 0.48; this number increased from diagnosis to the third month of therapy and then reduced during treatment with imatinib. The number and behavior of the CD26+ progenitors were independent from the BCR-ABL1 expression, but they summed up what previously observed about the BMI1 expression modulation. In this work we demonstrate for the first time that in CML the BMI1 protein is co-expressed with BCR-ABL1 only in the cytoplasm of the CD26+ precursors; on the contrary, in other hematological malignancies where BMI1 is commonly expressed (follicular lymphoma, essential thrombocytemia, acute myeloid leukemia), it was not co-localized with CD26 or, obviously, with BCR-ABL1. Once translated into the clinical context, if BMI1 is a marker of stemness, our results would suggest the combination of the BMI1 inhibitors with TKIs as an interesting object of research, and, probably, as a promising way to overcome resistance in CML patients.

IntroductionZinc plays an important role in thymic function and immune homeostasis. We performed a prospective clinical trial using a high-dose zinc oral supplementation to improve the immune reconstitution after hematopoietic stem cell transplant (HSCT).Patients and methodsWe enrolled 18 patients undergoing autologous HSCT for multiple myeloma. Nine patients were randomized to receive only a standard antimicrobial prophylaxis; whereas, nine patients received in addition 150 mg/day of zinc from day +5 to day +100 after transplant.ResultsCD4+ naïve lymphocytes and TRECs showed a significant increase from day +30 until day +100 only in the zinc-treated group. Moreover, the load of Torquetenovirus, a harmless virus that replicates in course of immunedepression, increased at day +100 only in the control group. No severe adverse events were reported during the zinc consumption.ConclusionFirst data from the ZENITH trial suggest that high-dose zinc supplementation is safe and may enhance the thymic reconstitution after HSCT. Registered: http://Clinicaltrials.gov (NCT03159845); and EUDRACT: 2014-28 004499-47.