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
