Prognostic Role of Minimal Residual Disease before and after Haematopoietic Stem Cell Transplantation in pediatric ALL patients and evaluation of droplet digital PCR applicability in pre-HSCT MRD monitoring

Acute Lymphoblastic Leukemia (ALL) represents the most frequent cancer in childhood. Currently, more than 80% of children with ALL can be cured through intensive and risk-adapted chemotherapy protocols, but unfortunately, the remaining 20% ultimately relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered beneficial for approximately 10% of patients who are at high risk (HR) at frontline therapy according to the AIEOP-BFM protocol criteria, and for the majority of patients after ALL relapse. However, also after HSCT, relapse remains the leading cause of treatment failure in pediatric ALL. The strongest prognostic factor in childhood ALL is the monitoring of Minimal Residual Disease (MRD). MRD is defined as the persistence, in bone marrow (BM), of leukemic cells not identifiable through cyto-morphological methods. MRD diagnostics has been implemented into major frontline treatment protocols for pediatric ALL, in which it is routinely used to stratify patients into different risk classes: standard risk (SR), medium risk (MR) or high risk (HR) of relapse. The aim of MRD-based stratification is to refine therapy based on risk-class, maximizing cure and minimizing toxicities. Also for relapsed ALL patients and in patients undergoing HSCT, MRD assessment has been identified as one of the most relevant predictors of prognosis, useful to identify good and poor responders to the therapy. Nevertheless, the clinical significance of MRD in pediatric ALL patients given allogeneic HSCT has not yet been fully validated. The most widely used approach to detect MRD is represented by real-time quantitative PCR (RQ-PCR), a very sensitive and specific molecular assay. RQ-PCR is based on the patient-specific junctional regions of Immunoglobulin (Ig) and T-cell Receptor (TCR) genes rearrangements, detected on BM aspirates collected at diagnosis (or relapse) of ALL patient. In the first project of my PhD training (described in Chapter 1) we quantify MRD by RQ-PCR immediately before HSCT, in order to assess its clinical significance and impact on transplant outcome in a large cohort (119) of pediatric ALL patients in first, second or subsequent complete remission (respectively 1CR, 2CR or others CR). In addition, we consecutively analyzed MRD by RQ-PCR in 98/119 and 59/119 ALL patients, respectively during the first (post-HSCT1) and third (post-HSCT3) trimester after HSCT. The aim of these analyses was to address the question of whether MRD evaluation could provide further information to predict the risk of post-transplant leukemia recurrence. The overall 10-year event-free survival probability (EFSp) for patients with any level of positive MRD pre-HSCT was lower (39% for MRD < 1x10-3 and 18% for MRD ≥ 1x10-3) as compared with negative MRD patients (EFSp = 73%). When patients were analyzed according to the number of CR at HSCT, we observed that different levels of positivity had a different impact on EFSp: low-level MRD positivity had a negative impact only in patients transplanted in second or higher CR; while in first CR, only a high MRD positivity increased the risk of relapse. So pre-transplant MRD assessment confirmed to be a strong predictor of outcome and its effect was consistent throughout the different disease remissions. We also evaluated the EFSp according to the MRD assessment at post-HSCT1 and post-HSCT3. MRD negativity at early post-transplant was associated with a good EFSp (63%), that was even better when negativity was confirmed also at 3th trimester post-HSCT (pEFS = 84%). Also the variations of MRD levels over time were important. In particular the change between 1st and 3th trimester allowed to identify 2 categories of patients, with a dramatically different outcome: a group of patients with very poor prognosis (patients with an MRD increasing from post-HSCT1 to post-HSCT3) with an EFSp of only 8%, and a group of patients with very good prognosis (patients with unchanged negative MRD or decreasing to negative MRD and those with unchanged low-positive MRD) with an EFSp ≥ 80%. Overall, these results confirm that MRD assessment is important both before and after transplant, for early identification of patients with the highest risk of ALL recurrence and with a strong indication to a prompt immunological intervention and to adoption of new drugs. The second project (described in Chapter 2) was a preliminary study. We focused on a third generation PCR, the droplet digital PCR (ddPCR), that allows for an absolute quantification, with accurate concentration of target DNA. Instead, RQ-PCR allows for a relative quantification, since is based on the comparison with a calibration standard curve made with the diagnostic DNA of patient, for MRD level quantification in follow-up sample. Thus, availability of diagnostic sample can limit RQ-PCR assay. A broad spectrum of molecular markers has been yet interrogated using ddPCR for diagnostic purposes in various malignancies. Recently, the absolute method was evaluated for MRD quantification in lymphoproliferative disorders of adult, such as lymphomas and ALL; these reports showed a good correlation between quantitative PCR and ddPCR. However, there are still no studies in pediatric ALLs. In the light of this, we performed ddPCR analyses on BM samples of 65 pediatric ALL transplanted patients with the same primers and probes used for RQ-PCR and in the same reaction conditions. Comparing head-to-head the MRD results obtained with the two molecular approaches, we aimed to investigate the applicability of ddPCR for MRD assessment also in this context. First, we evaluated if positive but not-quantifiable (PNQ) MRD performed by RQ-PCR can be quantified by ddPCR; then we also evaluated the prognostic impact of pre-HSCT MRD levels assessed by ddPCR. A good level of concordance was found in results of both analyses (Pearson r = 0.98, P < 0.0001) and ddPCR was also able to quantify a various number of sample not-quantifiable by conventional RQ-PCR. Our results suggest that ddPCR has sensitivity, accuracy and reproducibility at least comparable with RQ-PCR. Statistical analyses have shown no significant differences in prognostic impact on outcome, if patients were stratified according to MRD levels detected by RQ-PCR and ddPCR, since EFSp of PNQ patients was very similar to that of MRD NEG by ddPCR (71% vs 68%, respectively). Despite this, the digital method was able to measure a positive and quantifiable value for 12 ALL patients who relapsed after HSCT, while RQ-PCR technique failed to identify relapse in advance. These preliminary data confirm that ddPCR may be an accurate and applicable tool for MRD evaluation also in the context of pediatric ALL clinical trials, but highlight the importance of extending the analysis on other retrospectively collected cases, to better define the role of ddPCR for prospective MRD evaluation in pediatric ALLs

Prognostic role of minimal residual disease before and after hematopoietic stem cell transplantation in childhood acute lymphoblastic leukemia

More than 80% of children with acute lymphoblastic leukemia (ALL) can be cured through intensive and risk-oriented chemotherapy protocols. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered bene\ufb01cial for approximately 10% of the patients who are at veryhigh risk at frontline therapy and for the majority of patients after relapse. Consequently, it is critically important to identify prognostic factors in this group of patients in order to tailor risk-adapted therapy. In this retrospective study, we aimed to assess the prognostic role of minimal residual disease (MRD) before HSCT and at di\ufb00erent time points after transplantation in children with ALL

Applicazione Della Droplet Digital Pcr Per La Quantificazione Della Malattia Residua Minima Nella Leucemia Linfoblastica Acuta Pediatrica

La real-time PCR quantitativa (RQ-PCR) viene attualmente utilizzata per la quantificazione relativa della Malattia Residua Minima (MRM) nei la quantificazione relativa della Malattia Residua Minima (MRM) nei pazienti pediatrici affetti da LeucemiaLinfoblastica Acuta (LLA). Il recente sviluppo della Droplet Digital PCR (ddPCR) per la quantificazione assoluta potrebbe consentire di caratterizzare meglio i pazienti attualmentedefiniti positivi non quantificabili (NQ) in RQ-PCR

Distribution of HbS Allele and Haplotypes in a Multi-Ethnic Population of Guinea Bissau, West Africa: Implications for Public Health Screening

BACKGROUND: Sickle Cell Disease (SCD) is an inherited condition that is widespread globally and especially in malaria-endemic West African countries. Limited epidemiological data on SCD are available for Guinea Bissau, where newborn screening is not yet implemented, routine diagnosis is not available, and care is case directed. METHODS: Dried blood spots were collected from children accessing two hospitals managed by Italian Non-Governmental Organizations in the capital city of Bissau and sent to Padova for Hemoglobin (Hb) quantification through HPLC and molecular analysis. Beta globin gene analysis was performed in all; and Hb haplotype of the HbSS and HbSA patients was performed in South Africa. One hundred samples belonging to the most frequent ethnic groups were randomly selected for detection of G6PD mutations. RESULTS: Samples from 848 consecutive children (498 males and 350 females, mean age 6.8 years) accessing the two hospitals were analyzed: 6.95% AS (4.42% allelic frequency), 0.94% SS, and 0.23% AC. 376G G6PD allelic frequency was 24%; 14.8% in AS individuals. The Senegal haplotype was the most prevalent (31%), and the proposition of chromosomes with the atypical haplotype was surprisingly high (56%). CONCLUSION: Our study demonstrates a significant frequency of the HbS allele in the population of Guinea Bissau supporting the implementation of screening strategies. The differences among ethnic groups can help guide targeted interventions for SCD awareness campaigns and determine priority areas for public health interventions. The pilot analysis on haplotypes reveals a large proportion of the atypical haplotype, which may be indicative of a genetically heterogeneous population

Comparison of MRD detection by RQ-PCR and droplet digital PCRin pediatric Acute Lymphoblastic Leukemia before Hematopoietic Stem Cell Transplantation

Real-time quantitative PCR (RQ-PCR) is a standardized tool for Minimal Residual Disease (MRD) monitoring in pediatric patients with Acute Lymphoblastic Leukemia (ALL), showing a prognostic impact also in the field of Hematopoietic Stem Cell Transplantation (HSCT). This approach can be limited by the availability of diagnostic material. Recently, droplet digital PCR (ddPCR) was introduced and has already led to several research breakthroughs in various fields. The main advantage of this method is that there is no need for external references, since it allows for absolute quantification of target DNA. We evaluated the applicability of ddPCR for MRD analysis in pediatric ALL transplanted patients and we compared the prognostic impact of MRD assessed by RQ-PCR and ddPCR before HSCT

Pre- and post-transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre-HSCT and during the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). The overall event-free survival (EFS) was 50%. The cumulative incidence of relapse and non-relapse mortality was 41% and 9%. Any degree of detectable pre-HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity <1 x 10-3 or 651 x 10-3, respectively, versus 73% in MRD-negative patients (P < 0001). This effect was maintained in different disease remissions, but low-level MRD had a very strong negative impact only in patients transplanted in second or further CR. Also, MRD after HSCT enabled patients to be stratified, with increasing MRD between post-HSCT1 and post- HSCT3 clearly defining cohorts with a different outcome. MRD is an important prognostic factor both before and after transplantation. Given that MRD persistence after HSCT is associated with dismal outcome, these patients could benefit from early discontinuation of immunosuppression, or pre-emptive immuno-therapy

Droplet Digital PCR Improves IG-/TR-based MRD Risk Definition in Childhood B-cell Precursor Acute Lymphoblastic Leukemia

: Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real-time quantitative polymerase chain reaction (RQ-PCR) represents the gold standard for molecular MRD assessment and risk-based stratification of front-line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin-Frankfurth-Munschen (BFM) group AIEOP-BFM ALL2009 and ALL2017, B-lineage ALL patients with high RQ-PCR-MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP-BFM ALL2000 study, these patients are treated as high-risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ-PCR ("positive not-quantifiable," POS-NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients' risk definition, we analyzed MRD in 209 pediatric B-lineage ALL cases classified by RQ-PCR as POS-NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP-BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ-PCR MRD ≥ 5.0 × 10-4 at day+33 and POS-NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients' subgroup, while ddPCR NEG or ddPCR-POS-NQ patients had a significantly better outcome (P &lt; 0.001). Overall, in 112 MR cases and 52 standard-risk patients, MRD negativity and POS-NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ-PCR in the measurement of MRD, particularly in late follow-up time points, and may thus allow improving patients' stratification in ALL protocols