General limitation of the study
The main limitation is the small sample size. However, if a significant finding was found in a small sample, like our pilot study, it encourages further and larger study. The lower relapse rate 4 (27%) versus 7 (47%) and later TTR 12.9 versus 1.8 months in pegIFN arm versus observation arm, respectively, might provoke argument that the finding merely because, as a form of CML treatment, pegIFN delays the relapse. Thus, the duration of follow-up must be long enough to overcome this potential confounder. We reported our study after the last recruited patient completed a year of follow-up and by chance, the pegIFN arm had longer follow-up 38.1 months (range 15.9–49.4) compared to observation arm 23.8 months (1.5–51.0). Hence, this could likely eliminate the confounder. Of course, a longer follow-up of the study is needed.
Special note is needed during reading data on the first documented and duration of MMR, MR4 and MR4.5 in Table 1 and Fig. 2 because of the timing starting qPCRIS in the two centers where our patients had their qPCRIS monitoring prior to enrollment (Ampang Hospital in March 2010, while Singapore General Hospital from 15th May 2012 (personal communication with Dr. Charles Chuah)). Ampang Hospital issued the qPCRIS reports according to a new guideline(2) starting from 15th January 2016. All enrolled patients’ reports were carefully checked and primary data on the RNA replicates was checked if needed to ensure inclusion criteria was fulfilled.
Three important questions on pegIFN during the design of the study are which pegIFN to be used, the starting dose, and the duration. There is no comparison study of pegIFN-α-2a and 2b in CML. We picked pegIFN-α-2a because the stock flow was steadier in Malaysia. The starting dose of 180 µg was selected based on the available literature, as single agent at 180 µg(31), 270 µg(34), and 450 µg(35), or combined with TKI as first line treatment in CML at 45 to 90 µg(36–38) and 180 µg(31). We do not know the appropriate duration of pegIFN-α-2a. Previous history of IFN usage, not reported it was either conventional IFN or pegIFN, for > 1.5 years was associated with higher TFR.(39) Usuki et al. used conventional IFN 3 MU 2–5 times a week for two years as consolidation.(32) Hardan et al. used pegIFN-α-2a for a year as consolidation.(31) Looking at these studies, we decided 1-year duration of pegIFN- α-2a in this pilot study, but the question of an adequate duration of pegIFN to increase TFR would continue until further study is available.
In this study, pegIFN was given for a year, regardless pegIFN was withhold in between. Only 8 out of 15 (53%) patients completed 52 doses of pegIFN with mean dose of 43 out of 52 doses (range 20 to 52). Seven patients did not complete 52 doses because of transaminitis (n = 5), missed dose (n = 1) and relapse (n = 1). Two of the four patients with transaminitis ≥ 5 times ULN refused to restart pegIFN when transaminitis resolved. This factor of incomplete pegIFN course should be considered during interpretation of the study’s finding, too. We would suggest at least 52 completed tolerable doses of pegIFN instead of the 1-year bound in future similar study.
From this study, we found the likely tolerable pegIFN-α-2a dose is correlated with age (see Supplementary Fig. 1). Among 13 patients who required pegIFN dose reduction, 9 (69%) patients could have avoided/reduced the need of dose reduction if the starting dose was lower. This would certainly help future similar study to achieve the target accumulative dose of pegIFN-α-2a, reduce unnecessary side effects, and improve compliance. We would suggest the starting pegIFN-α-2a dose as 180 µg weekly in patients age below 40 years old, 135 µg weekly in patients age 40 to 60 years old and 90 µg weekly in patients age more than 60 years old in future similar study.
Fluctuation of qPCRIS level
Fluctuation of qPCR level below MMR (0.1%IS) is a known phenomenon after stopping TKI.(40) To our knowledge, there was no report on fluctuation that exceeding MMR, which is probably the reason it is recommended as a criterion of relapse(41) and used in most of the stop TKI trials(42). We would like to report two cases of fluctuation exceeding MMR here.
Two patients in the observation arm, both from the same study site, relapsed at different time point (one at 12 months (Dec 2017) and one at 14 months (Feb 2018) after stopping TKI) according to the relapse criteria no.1, i.e. loss of MMR. TKI was reinitiated as per protocol. However, a repeated qPCRIS, which was not prohibited in the study protocol, was done prior to the initiation of TKI, which showed DMR. Investigations showed no evidence of wrong sampling or laboratory error. After discussion, investigators decided to stop their TKI after two months of TKI intake. These two patients remained in TFR up to the time of writing. Their qPCRIS trend and blood count changes compared to the other two relapsed patients at the same study site and other study sites are shown in Supplementary Fig. 2 and Supplementary Fig. 3, respectively.
These two “relapse” cases challenge MMR as a relapse criterion and raise doubt on the four relapse cases prior to the incidence. We re-examined these four cases and could only truly confirm relapse in one case (observation arm), in which the previous two readings already showed 1-log increment from 0.0059%IS to 0.0896%IS, fulfilled relapse criterion no.2, before loss MMR with reading of 0.2594%IS. We could not rule out the possibility of fluctuation in other three cases confidently because no repeat qPCRIS was done before restarting TKI. One patient (observation arm) had two prior readings of 0.0117%IS and 0.0774%IS before loss of MMR 0.1931%IS. One patient (pegIFN arm) had two prior readings of 0.0225%IS and 0.0680%IS before loss of MMR 0.1085%IS. One patient (pegIFN arm) had two prior readings of 0.0203%IS and 0.0664%IS prior to loss of MMR 0.1653%IS. Following the incidence, study protocol was amended to include a repeated qPCRIS on the time of restarting TKI after loss of MMR, which means relapse criteria no. 1 – loss of MMR – must be confirmed by two successive readings, and re-stopping TKI if the repeated qPCRIS does not confirm the loss of MMR. In fact, at the time of writing (Apr 2020), there was another patient (pegIFN arm), who had such fluctuation, with two prior reading of 0.0108%IS and 0.0337%IS before loss of MMR 0.1046%IS and the repeated reading of 0.0232%IS. He was restarted and planned on TKI for two months, just like the previous two cases of fluctuation, but had not came back for review due to Malaysia and Singapore lock-down during COVID-19.
Gender effect on the efficacy of pegIFN?
The limitation of this pilot study is small sample size, which caused the significant difference in the gender ratio between the two arms. PegIFN arm had more male patients whereas observation arm had more female patients. Earlier single arm observation study (13) suggested female is less likely to maintain TFR, but the finding was disputed after a longer follow-up(43). Other single arm observation studies also did not find gender as a predictor of TFR.(39, 41, 44–46) Thus, it is unlikely gender distribution affects the outcome in the observation arm, but we cannot extrapolate the same assumption to pegIFN arm. In fact, there are data to suggest pegIFN is less effective in female compared to male in the treatment of Hepatitis C using pegIFN and ribavirin.(47–49) A significant more female in relapse pegIFN arm compared to non-relapse pegIFN arm (p = 0.03) in our small sample size warrants careful interpretation of the outcomes.
TKI and DMR duration
Relapse patients in pegIFN arm had shorter duration of TKI and shorter duration of DMR compared to non-relapse patients in pegIFN arm and either relapse or non-relapse patients in observation arm (see Fig. 2). The clinical predictors of TFR found in many single arm observation studies are longer duration of TKI and/or longer duration of DMR.(39, 43, 45, 46) This factor should be considered during interpretation of the outcomes.
First, the increment of the blood counts during the first two visits and plateau off after that period in the observation arm (see Supplementary Fig. 4) provides further evidence for the myelosuppression effect of TKI. It also explains partly the improvement in QoL (see Table 2) and justifies the need of TFR. Second, there was a groove in the trend of the blood counts in the pegIFN arm during the six months stopping TKI corresponding to the adjustment of pegIFN dosage during that period (see Supplementary Fig. 4). However, the blood count trend and the use of pegIFN did not affect the QoL (see Table 2). Third, absolute basophil count (ABC), but not total white cell count (TWC), was shown to correlate with percentage of Philadelphia chromosome in marrow metaphases of pre-clinical CML.(50) However, it seems that ABC also correlate with qPCRIS (see Supplementary Fig. 3 (c)) at lower disease burden as in pre-clinical CML. TWC seems to correlate with qPCRIS, too. These findings were unexpected. Thus, we examined whether these correlations could be used as surrogate marker of the fluctuation. The data is immature, but TWC and ABC might be the surrogate marker of the fluctuation (see Supplementary Fig. 3 (a)).