We looked at our CML patients with regards to imatinib therapy, a front-line treatment for CML in our government hospitals in view of treatment discontinuation for optimal CML responders as suggested by the European LeukemiaNet 2013. Data showed that half of our responsive patients received imatinib therapy for more than 5 years with mean age of 48 years old, average of 5.5 years for patients with MMR and 6 years for DMR. These indicated on average, MMR achieved by 18 months extended survival for at least 4 years and DMR is achievable with longer treatment in our CML patients. DMR was observed in almost half of the responsive patients and if consistent these patients can be considered for imatinib discontinuation studies, as minimum requirements are at least 24 months of DMR with MR4 or deeper [26]. DMR was reported suitable for imatinib discontinuation as it has survival benefits and lesser events [7]. However, determining the right time for imatinib discontinuation is critical. Long term TFR is still low, with relapse after discontinuation is rather high in remission patients. Weighted mean molecular relapse rate of CML was 42% with mean molecular remission was more than 37 months before discontinuation of imatinib. Shorter duration on imatinib and shorter duration with undetectable level of BCR-ABL transcripts were pointed out as reasons for relapse [27]. Therefore sufficient duration on imatinib and consistency in DMR is crucial before imatinib discontinuation.
Management of CML has remarkably improved over the years with high success rate for treatment using TKIs in chronic patients. This is assisted by new analysis techniques complementing existing ones for a comprehensive detection and monitoring of CML. RT-qPCR, a sensitive technique has become a gold standard at molecular response and is used to analyse treatment response especially from TKIs as first line. Since RT-qPCR has limitations at very low-levels thus new biomarkers are needed not only to assist analysis but also to understand better molecular and cellular interactions and associations in order to prolong TFR.
Numerous novel findings were reported from CML studies using NGS, a new technology with relatively high sensitivity and wide coverage. In CML patients treated with imatinib, NGS has enabled identification of novel BCR-ABL1 fusions gene comprising BCR intron 14 and ABL1 intron 2 breakpoints, giving partial deletion of SH3 domain [28]. Meanwhile with whole genome sequencing, NGS has enabled identification of e13a2-like BCR-ABL1 fusion comprising broken BCR exon 13 and 9 ABL1 intron 1 nucleotides forming a novel chimeric exon [29]. In our study, NGS has assisted in identifying miRNAs at molecular response in local CML patients responsive and non-responsive to imatinib therapy. Validation by custom array real-time RT-qPCR has identified hsa-miR 181-5p, hsa-miR-182-5p and hsa-miR-26a-5p as potential new therapeutic biomarkers in response to treatment using TKIs in CML. Hsa-miR 181-5p could be a tumour suppressor miRNA and could be involved in major or partial suppression of CML. Meanwhile hsa-miR-182-5p and hsa-miR-26a-5p could be oncomiRs in which their expressions may indicate non-responsiveness to TKIs treatment or relapse.
Hsa-miR-181a-5p was significantly down-regulated in both the responsive and non-responsive to imatinib groups in comparison with the control group. The down-regulation indicated significant interactions between hsa-miR-181a-5p and TKI therapies. These showed that imatinib, nilotinib, bosutinib and dasatinib taken by CML patients over the time had caused inhibition to CML cell proliferation, led to apoptosis thus reduced the amount of BCR-ABL1 in CML patients. Fold down-regulation of 2.14 observed in the responsive to imatinib group represented 3 to 4.5 log reduction of BCR-ABL1 levels from IRIS standardized baseline with average 5.7 years of intake. Similarly 2.3 fold down-regulation observed in the non-responsive to imatinib group represented less than 1 to 5 log reductions except in 6 (21%) patients in which were either on TKI break or imatinib. The log reduction to MMR and DMR in the imatinib responsive group was consistent with down-regulation of hsa-miR-181a-5p. Various log reduction seen in the non-responsive to imatinib group was also consistent with down-regulation of hsa-miR-181a-5p. Thus indicated significant down-regulation of hsa-miR-181a-5p has positive correlation to log reduction or reduced levels of BCR-ABL1 in CML patients treated with TKIs. Therefore hsa-miR-181-5p has potential to be used as a therapeutic biomarker for positive performance of TKIs shown by reduced levels of BCR-ABL1 indicating patients were responding to TKIs treatment in CML. Hsa-miR-181-5p could be a tumor-suppressive miRNA which prevents mRNA from coding specific protein directly or associated to CML in patients treated with TKIs.
On the other hand, hsa-miR-182-5p and hsa-miR-26a-5p were observed significantly up-regulated and only in the non-responsive to imatinib group with 2.08 and 2.39 fold up-regulation respectively in comparison with healthy donors. These were patients whereby their latest available BCR-ABL1IS ratios were mostly more than 0.1 % that were neither in MMR nor DMR, with substantial average and only 7% (2) were treated with imatinib. Fold regulation of these miRNAs in the imatinib responsive group, whereby BCR-ABL1 in these patients were minimal or absent (MMR or DMR), were not significant in comparison with healthy donors. Thus indicated imatinib had reduced the amount of BCR-ABL1 in these responsive patients to the extent not significant in comparison with healthy donors. Thus the significant up-regulation could indicate that hsa-miR-182-5p and hsa-miR-26a-5p have positive correlation to substantial presence of BCR-ABL1 in CML patients treated with TKIs. Therefore, these miRNAs could be used as new therapeutic biomarkers for substantial presence of BCR-ABL1 in CML patients treated with TKIs. Hsa-miR-182-5p and hsa-miR-26a-5p could be oncogenic miRNAs (oncomiRNAs) and could be associated to CML in patients treated with TKIs.
Studies have shown hsa-miR-181-5p as a therapeutic biomarker, has potential use for clinical improvement in cancers. This is demonstrated in gastric cancer cells whereby hsa-miR-181-5p has been extensively studied and was reported as a potential regulator of MEG2, a tumor suppressor gene [30], regulated RASSF6 and in combination predicts poor prognosis in gastric cancer [31]. It also inhibited MTMR3 expression in AGS gastric cancer cells and was identified as a novel autophagy [32]. In non-small-cell-lung cancer tissues and cell lines, hsa-miR-181a-5p was reported significantly reduced and has potential role in tumor suppression by partially targeting Kras [33], a protein that regulates cell growth. In breast (BC) and colon cancers (CC), hsa-miR-181a-5p was reportedly down-regulated and inversely related to matrix metalloprotinase-14, which is elevated in tumors, in which, in order to prevent cancer metastasis in BC and CC, is by elevating hsa-miR-181a-5p [34]. In hepatocellular carcinoma, miR-181 was reported significantly turned on the MAPK/JNK pathway, the regulator of cell proliferation and by limiting it, would suppress the pathway [35]. Hsa-miR-181a-5p, a tumor suppressor is considered as prognostic marker in Acute Myeloid Leukaemia patients treated with intensive induction chemotherapy and autologous stem cell transplant [36].
Higher expression of hsa-miR-182-5p as observed in our study was also seen in other studies as in prostate cancer (PCa), lung squamous cell carcinoma tissues analysed from The Cancer Genome Atlas database, the Gene Expression Omnibus database, and real-time qPCR [37] and also in colorectal cancer cells using tumorigenic variant cell line MICOL-14tum compared to MICOL-14h-tert cells [38]. In PCa, biphasic role of hsa-miR-182-5p was observed which was higher expression in localized PCa and contrarily lower expression in aggressive tumor [39]. Lower expression was also observed in renal cancer whereby hsa-miR-182-5p was downregulated in tumor tissue compared with adjacent normal tissues and overexpression decreased tumor growth in mice, demonstrating antitumor effect [40].
Hsa-miR-26a-5p expression in our study is consistent with a larger study on CML patients indicating miR-26a expressions are influenced by response to TKIs used [41,42]. The study showed miR-26a was differentially expressed in HL-60.BCR-ABL cells treated with tyrosine kinase inhibitors when compared to HL-60 cells. miR-26a expression levels increased gradually with both dasatinib and nilotinib treatments. The increased expression levels with dasatinib and nolitinib were consistent with our findings of hsa-miR-26a expression was upregulated in CML patients molecularly non responsive to imatinib in comparison with controls whereby most patients were treated with nilotinib or dasatinib. Thus with dasatinib and nilotinib treatments, upregulation of hsa-miR-26a was observed in CML patients non-responsiveness to imatinib. However, HL-60.BCR-ABL cells treated with imatinib mesylate showed increased miR-26a expression at 4 hour but decreased expression at 8 hour. This correlates with miR-26a expression was lower in chronic phase patients (62.5% Complete Cytogenetic Response (CCyR) and 37.5% imatinib resistant) in comparison to healthy individuals. Moreover, miR-26a expression was observed lower in imatinib resistant patients compared to patients who achieved CCyR (<1% BCR-ABL1). In our study hsa-miR-26a-5p expression was insignificant in CML patients molecularly responsive to imatinib when compared to controls as these were patients either in MMR or DMR (≤ 0.1% to undetectable BCR-ABL1). These showed that with imatinib, decreasing expressions of hsa-miR-26a were observed from responsiveness towards resistance. Thus these indicated imatinib, nilotinib and dasatinib have altered BCR-ABL kinase activity differently.
These studies showed hsa-miR-181-5p, hsa-miR-26a-5p and hsa-miR-26a-5p are promising therapeutic biomarkers and can be manipulated to improve cancer treatment. In this study, only 18 miRNAs from NGS profiles were validated using real time RT-qPCR thus the significant miRNAs observed could be solely or partially involved in the decreasing and/or increasing of BCR-ABL1 in CML patients. Therefore further studies are needed to determine their targets and effect of involvements in CML. Knowing their interactions and targets in CML will give their net effects that allow manipulation by inhibit oncomiRs or stimulate tumor suppressor miRNAs. These will give new values for TKIs responses in which could assist in determining accurate time for TKIs discontinuation and better prediction of relapse in CML. Timely discontinuation of TKIs in CML remission patients is crucial for long-term TFR, and prolong TFR would subsequently cure CML.