This study was the first human clinical trial designed to investigate the protective role of DFO against DOX-induced liver injury in pediatric patients with cancer. We could show that DOX didn’t have significant toxicity to the liver tissue at least in the short-term, though transient transaminases were seen in a subgroup of patients. There was no intergroup difference in the LFT parameters of patients treated with DFO and the group. Within each group, all parameters remained comparable except a small rise in ALT level in groups 2 & 3 and a minor drop in total protein in group 3, though within the normal range (Table 3). Due to lack of similar clinical trials in the past, we couldn’t compare our results with them. However, in a few animal studies like Saad’s study [20], they reported that DFO with the dosage of 15–20 times of DOX dose may decrease serum AST levels in rat models. This was not observed in our study.
We also checked some indirect serum markers of fibrosis, and found that APRI and FIB-4 score were significantly decreased in patients who were pre-treated with higher dose DFO (group 3, 50 mg/kg) (one-sided P values 0.015 and 0.02 respectively). However, statistical adjustment for age, sex and baseline APRI revealed that only treatment in group 2 is independently accompanied with 83% decrease in the risk of DOX-related liver fibrosis. The implication of these circulating factors to predict liver fibrosis is becoming more popular because they are available in many laboratories, they are almost non expensive, and can be repeated several times. Moreover, they may be representative of the fibrosis in the whole liver, thus avoiding small sampling error which is a technical defect in percutaneous liver biopsy [21].
The predictive value of indirect serum markers of liver fibrosis was previously mentioned in conditions such as viral hepatitis and fatty liver disease [1, 22]. Unalp-Ardia similarly reported that APRI can be used to predict liver fibrosis with higher scores indicative of advanced liver disease [23]. Moreover, it has been claimed that the combination of APRI with FibroMeter may show an accurate lower cost alternative to liver biopsy to evaluate fibrosis [24]. A meta- analysis showed that APRI > 1 had a sensitivity of 76% and a specificity of 72% to predict cirrhosis in patients infected with hepatitis C virus (HCV). For significant fibrosis, an APRI threshold of 0.7 was 77% sensitive and 72% specific. They concluded that APRI may obviate the need for staging liver biopsy in a subset of patients with HCV infection [14].
Moreover, FIB-4 index as another non-invasive serum marker to delineate liver fibrosis has been implicated in a variety of illnesses including hepatitis B virus (HBV) infection. Mallet investigated the accuracy of FIB-4 index in a group of chronic HBV-infected patients and concluded that a cut-off value ≤ 1.45 can differentiate moderate fibrosis from severe fibrosis with a negative predictive value of 86%, a sensitivity of 71.1% and a specificity of 73.1%. He asserted that it is even more precise than APRI to exclude significant fibrosis [1]. Shah et al. reported that in patients with NAFLD, the FIB-4 index is superior to other non-invasive markers of fibrosis [22]. In addition, a systematic review and meta-analysis showed APRI and FIB-4 can identify hepatitis B-related fibrosis with a moderate sensitivity and accuracy. They suggested that an APRI threshold of 0.5 and 1.5 and an FIB4 threshold of 1.45 and 3.25 had acceptable sensitivity and specificity to delineate mild from significant fibrosis [17]. Though we showed that treatment with higher dose DOX (50 mg/kg) may be associated with significant decrease in FIB-4 score (Table 3), we were not able to show its independent association with FIB-4 score.
We also checked FibroTest which is a commercially available algorithm combining different elements, and has been proved to have high predictive value in advanced fibrosis. A cut-off value of 0.58 has been reported to associate with severe fibrosis (F ≥ 3) [19, 25]. Only one patient who was treated with high-dose DFO (group 3) exceeded this cut-off point, and the results were comparable in the 3 groups.
The same finding was confirmed with TE that none of our patients experienced significant fibrosis following DOX treatment. They all had a METAVIR score in the F0-F1 zones, compatible with no or mild fibrosis. Meanwhile, we could show that DFO even further decreased serum markers of hepatic fibrosis when administered at a dose of 10 times DOX dose. It may be promising that DFO may play a role in decreasing the chance of liver fibrosis in the long-term. Despite normal LFT, METAVIR score, APRI, FIB-4 index, and FibroTest, nobody can guarantee that these patients are protected from liver fibrosis and cirrhosis when they reach their adulthood. Therefore, there is always a concern that survivors of pediatric malignancy may suffer from multi-organ damage in the future particularly if they are treated with toxic agents such as anthracyclines with proven cardiac and possibly hepatic and renal complications. It is also important to consider that non-invasive predictors of liver fibrosis such as serum markers and FibroScan are more reliable for detection of advanced fibrosis, and are less sensitive in the early stages of liver fibrosis (F ≤ 2) [21]. Although none of our patients suffered from severe fibrosis as a result of chemotherapy effect, it is highly recommended to follow these children for at least a decade to investigate the long-term toxicity of chemotherapy agents on the liver and other organs.
Our study had some strengths and limitations. Regarding the strengths, it was the first randomized clinical trial in patients with cancer and especially in the pediatric age group which assessed the role of DFO as a rescue therapy to prevent liver damage induced by DOX. Up to now, only limited animal studies were conducted and this issue makes out our study as the first study of its own. Secondly, we tried to assess liver fibrosis with different modalities including FibroScan, LFT and non-invasive markers of liver fibrosis such as FibroTest, APRI and FIB-4 index. This probably helped us to increase the accuracy of our assessment.
On the other hand, the study faced some limitations. First of all, the small number of our cases and the short period of follow up hinder the generalizability of the results. A larger multi-center study with longer follow up is required to assess the reproducibility of our results. Secondly, the heterogeneity of the study population in terms of their primary diagnosis is another issue that may have confounded the results as the chemotherapy protocols were not the same in all participants. Therefore, the interaction of other chemotherapy agents with DOX and their hepatic side effects cannot be overlooked. Lastly, we didn’t have tissue biopsy to assess liver fibrosis in our patients because of ethical issues and the potential hazards of liver biopsy in cancer patients. Though FibroScan, and non-invasive serum markers of fibrosis are good alternative to liver biopsy, they are more informative in advanced fibrosis rather than early stages of liver fibrosis [26].
At the end, considering all these pros and cons of the study, our results may shed light for future researches to solve the dark sides of this puzzle. It is highly advised to run a large multi-center trial in a cohort of patients with similar malignancy such as leukemia to know whether DFO may prove beneficial in long-term protection of liver injury related to anthracyclines.