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Letters to the Editor
Supplementary Methods). Using this model, we generated receiver operating characteristic (ROC) curves comparing PB and BM in relapse prediction (Figure 1E), although it should be pointed out that a ROC curve is a suboptimal tool in this setting, due to the use of multiple longitudinal determinations for each patient. The area under the curve was 0.9201 (95% CI: 0.8438-0.9964) for PB and 0.7045 (95% CI: 0.4292-0.9799) for BM, with superior perform- ance for PB at all possible thresholds. Based on the results of the ROC analysis and on the maximal reproducible sensitivity of the method, we further refined our model considering positive only values above 0.13% for PB and 0.24% for BM.
Based on these criteria, we documented that PB chimerism predicted four of seven relapses (57%) (Figure 1F) and gave false positive results in three of 13 patients from the study group who did not relapse (23%) (Figure 1G) and in one of seven patients from the control group (14%) (Figure 1H), all in the first 4 months after allogene- ic HSCT. BM chimerism analysis predicted relapse in two of seven patients (29%) (Figure 1I) and provided false positive results in six of 13 non-relapsing patients from the study group (46%) (Figure 1J) and in one of the seven patients in the control group (14%) (Figure 1K). Of note, BM false positive results spanned over the entire follow- up, including some at very late time-points. Overall, PB chimerism showed a sensitivity of 57.14% and a speci- ficity of 76.92% in relapse prediction, while BM analyses showed a sensitivity of 28.57% and a specificity of 45.45%, without statistically significant differences. Finally, we compared the median time from detection of increasing mixed chimerism to relapse for the two sites of sampling: the median time was 17 days for PB (range, 8- 44) and 33 days for BM (range, 30-59), indicating that in most cases relapse was anticipated by a single episode of over-threshold increasing mixed chimerism. Two patients from the study cohort relapsed after the first year of follow-up (on days 603 and 661). In line with the short temporal window of prediction of chimerism by quanti- tative PCR, none of these patients had scored positive during the study period.
This study is the first, to our knowledge, to prospec- tively address the clinical utility of quantitative PCR- based chimerism monitoring in AML patients with a high or very high disease risk index undergoing allogeneic HSCT. These patients represent a significant clinical chal- lenge since, due to the aggressiveness and chemoresis- tance of their disease, even if they achieve remission, this is often short-lived. This has prompted the search for approaches to anticipate the detection of leukemia recur- rence and to allocate pre-emptive therapies rationally.
Although a number of recent studies have documented encouraging results obtained by quantitative PCR chimerism monitoring,6,7,9 semiquantitative analysis of short tandem repeat polymorphisms8,10 remains the gold standard, probably because there are approved guide- lines.11 The higher sensitivity of quantitative PCR could be perfect for PB monitoring, allowing a tighter follow-up of high-risk patients. Indeed, our results suggest clear, although not statistically significant, superiority of PB monitoring over BM. This was not only due to the more frequent sampling, allowing increasing mixed chimerism to be captured before overt relapse, but also to the higher specificity of positive signals, since BM analysis detected significant "background noise" at all time-points, possibly explained by aspiration of host BM stromal cells. Despite being superior, results obtained from PB monitoring are far from perfect, and evidently inferior to those obtained in similar studies in which specific mutations are
tracked.12 In our study cohort, only four of 20 (20%) patients carried nucleophosmin-1 mutations and for all of them, relapse was predicted by both mutation monitor- ing and PB quantitative PCR chimerism.
One of the main issues evidenced by our study is that the median time to relapse in high-risk patients is excep- tionally short. This makes the value of early determina- tions crucial, especially in order to understand whether early positive chimerism is the sign of still active clear- ance of the patient’s hematopoiesis or the first sign of dis- ease reappearance. Tighter monitoring and the use of techniques with higher accuracy than quantitative PCR, such as droplet digital PCR,13-15 may be key to address this crucial hurdle. It should be noted in fact that in several true positive cases from our study, multiple serial deter- minations confirmed the initial positivity, whereas in false positive cases there was often a decrease of chimerism at the netx time-point. The short experimental turn-around time of quantitative PCR and its relatively affordable cost could easily allow weekly PB determina- tions in very high-risk patients or in case of a first warn- ing.
The very narrow time window of prediction also raises the relevant issue of whether and how the information acquired could be translated into a therapeutic benefit: whereas immune interventions are known to take 2 to 4 weeks to show any effect, new drugs, such as FLT3 inhibitors, have extremely rapid activity. This might allow physicians to implement them in a pre-emptive fashion, based on an increase in host chimerism, sparing the toxicities and economic burden that accompany pro- phylactic administration16 and possibly also the cytope- nias that are commonly observed upon use in overt relapse.17
In conclusion, even considering its limited sensitivity and specificity in relapse prediction, the low invasive- ness, rapid analytical turnaround and nearly universal applicability of PB quantitative PCR-based monitoring might make it a useful complement to the still too limited armamentarium that is available for monitoring high-risk AML. We do, however, have to note that the cohort ana- lyzed in our study is rather small, due to its single-center nature and to the intrinsic hurdles of prospective trials; therefore, further and larger multicenter studies should be undertaken to confirm these results, and to address the crucial issue of standardization of this method across different laboratories.
Valentina Gambacorta,1 Riccardo Parolini,1
Elisabetta Xue,1,2 Raffaella Greco,2 Evelien E. Bouwmans,3 Cristina Toffalori,1 Fabio Giglio,2 Andrea Assanelli,2 Maria Teresa Lupo Stanghellini,2 Alessandro Ambrosi,4 Benedetta Mazzi,5 Wietse Mulder,3 Consuelo Corti,2 Jacopo Peccatori,2 Fabio Ciceri2,4# and Luca Vago1,2#
1Unit of Immunogenetics, Leukemia Genomics and Immunobiology, IRCCS San Raffaele Scientific Institute, Milano, Italy; 2Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milano, Italy; 3GenDx, Utrecht, the Netherlands; 4Vita-Salute San Raffaele University, Milano, Italy and 5HLA and Chimerism Laboratory, IRCCS San Raffaele Scientific Institute, Milano, Italy
#FC and LV contributed equally as co-senior authors. Correspondence:
LUCA VAGO - vago.luca@hsr.it doi:10.3324/haematol.2019.238543
Received: October 15, 2019. Accepted: September 3, 2020.
haematologica | 2021; 106(5)