In our pilot study, we showed that the expression of microRNAs, which can act as an epigenetic regulator of PD-L1 expression, could be a valuable factor for non-invasive diagnosis of NSCLC. Each of the microRNAs showed lower expression in NSCLC patients when compared to healthy subjects. However, there are no statistical differences in expression of these molecules in patients with early and advanced NSCLC. We evaluated microRNAs expression in plasma because it is an easily obtainable material compared to tumor tissue. It is also advantageous due to the potential use of microRNAs expression assessment in treatment monitoring.
Initially, our assumption was to assess the expression of PD-L1 and TP-53 mRNA as well as microRNAs. Our interest was aroused by an article of Tojyo et al. who found the positive correlation between PD-L1 and TP-53 protein expression, examined with immunohistochemistry method in tissue of oral squamous cell carcinoma . However, it turned out that we cannot fully achieve our goals due to the fact that PD-L1 expressions was detected in only 6 of NSCLC patients and in none of the healthy persons. This may be important because predictive factors for immunotherapy are constantly searched and the value of the liquid biopsy used to evaluate PD-L1 mRNA expression is not fully understood. We analyzed PD-L1 mRNA expression with the use of probes which flank the end of the third and fourth exons of NM_001314029.1 (variant 4), NM_014143.3 (variant 1) and NR_052005.1 (variant 3) transcripts (product length 77 nt). Exons 3 and 4 encode the intracellular domain of the PD-L1 protein . The fact that we did not detect PD-L1 mRNA expression in plasma from all patients may indicate the need to use other probes, e.g. complementary to the exons encoding the extracellular domain of PD-L1. However, it seems that the expression of PD-L1 mRNA in serum or plasma is very low. This is definitely an issue that should be developed in further studies on a larger group of patients.
Despite the lack of success in examination of PD-L1 mRNA expression, we have made some remarkably interesting observations regarding TP-53 mRNA and microRNAs expression which seem to be good diagnostic factors in the non-invasive diagnosis of NSCLC. Most of the studies on TP-53 gene concern the identification of mutations in this gene in tumor tissue or in free-circulating DNA (fc-DNA) [19, 20, 21]. To our best knowledge, there are no reports on TP-53 mRNA expression in liquid biopsy and its diagnostic and clinical significance. Our research showed that TP-53 mRNA expression could differentiate NSCLC patients from healthy subjects. The ROC analysis confirmed that a diagnostic test based on examination of TP-53 mRNA expression is significantly sensitive and specific in distinguishing between these two groups. However, our study has some limitations related to low numbers of study groups and further studies should be conducted using the liquid biopsy methods on an enlarged group of NSCLC patients. The expansion of the studied group to include patients with other types of cancers also seems to be an important aspect, because the reduction of TP-53 protein expression resulting from mutations or chromosomal aberrations may be observed in many types of cancers, including hematological malignancies.
An interesting observation in our study is the occurrence of negative correlation between TP-53 mRNA and microRNA-34a-5p expression only in advanced NSCLC patients. This dependence can be related to neoplastic transformation and to increase in tumor aggressiveness. Li et al. examined 60 samples of NSCLC tumors and corresponding para-carcinoma tissues. Authors showed that the miR-34a expression in tumor samples was lower than in corresponding non-tumor tissues . On the other hand, there are reports about elevated expression of miR-34a in liquid biopsy (serum) in breast cancer patients. The expression of miR-34a was similar in patients and healthy controls .
We investigated the expression of two microRNAs from miR-34a family: miR-34a-5a and miR-34a-3p. During the formation of microRNAs, mature molecules temporarily form into a double-stranded form, in which one of the strands is called the leading strand and the other is the passenger strand. For some time, only the leader strand was considered to be capable of post-transcriptional silencing, however studies have shown that the passenger strand (usually at the 3' end) is also active. Then, miR-34a-3p is sometimes described as microRNA from the passenger strand. Expression of miR-34a-3p was significantly higher in tumors of non-muscle-invasive bladder cancer patients without recurrence after surgery and decreased expression of miR-34a-3p was associated with significantly shorter recurrence-free survival . Fawzy et al. during functional enrichment pathway analysis of miR-34a target genes indicated that both miR-34a were involved in fatty acid biosynthesis and fatty acid metabolism. However, miR-34a-5p was also involved in tumor suppressor or oncogenes function, and may play a role in development of colorectal cancer, thyroid cancer, NSCLC, chronic myeloid leukemia, bladder cancer, pancreatic cancer, glioma, and melanoma. Fawzy et al. conducted their study in tissue of colorectal cancer .
Furthermore, we demonstrated reduced expression of miR-17 in plasma from NSCLC patients compared to healthy donors. Jurkovicova et al. showed that the expression of miR-17 in liquid biopsy is similar in non-invasive breast cancer patients and in healthy subjects. Whereas, expression of this molecule is significantly reduced in invasive breast cancer patients compared to healthy volunteers. Moreover, expression of miR-17 is gradually decreased with the decrease in the grade of tumor differentiation . Zeng and colleagues showed that the expression of this microRNA is significantly reduced in patients with benign gastric diseases or with gastric cancer (GC) compared to healthy people. They found that miR-17 can distinguish healthy person and GC patients with AUC of 0.879 and with 80.6% of sensitivity as well as 87.5% of specificity . Hetta et al. showed results different from ours in a group of 40 NSCLC patients and 20 healthy controls. They indicated that miR-17 was significantly upregulated in NSCLC patients . It should be noted that our study group was twice as large as the group analyzed by Hetta et al. It also indicates the need to continue studies with liquid biopsy in a much larger population of patients.
Results of our study showed that the level of every analyzed microRNA was decreased in plasma of cancer patients compared to healthy donors. It could be assumed that all these molecules play the role of tumor suppressors. However, at least Jurkovicowa indicated that traditional oncomiR with high expression tumor tissue could have low expression in cancer patients’ plasma (this may concern e.g. miR-17 expression) . The concentration of microRNAs in serum or plasma may depend on the stage of the disease, the grade of tumor differentiation, the increase in cancer cell apoptosis or the efficiency of the mechanisms of microRNAs secretion into the bloodstream. Thus, the expression of the microRNAs in the tumor tissue will not always be faithfully reflected in the serum or plasma microRNAs expression profile. Nevertheless, the profile of microRNAs expression in liquid biopsy may accurately detect early neoplastic lesions undetectable in imaging diagnostics or serve as a marker of response to treatment, or prognosis of the disease course.
miR-93 control the EMT (epithelial to mesenchymal transition) and is overexpressed in several cancers. On the other hand, it has been found that miR-93 inhibited cell proliferation and cell cycle, which suppressed tumor growth and enhanced the therapeutic effect of paclitaxel in cell cultures . There is evidence that miR-93 can act as a tumor suppressor or oncomiR by enhancing tumor growth, angiogenesis and metastasis [30, 31, 32, 33]. Wang et al. found significantly reduced expression of miRNA-93 in plasma from 55 colorectal cancer patients compared to healthy subjects (57 cases) with AUC = 0.739 for microarray test and AUC = 0.828 for qRT-PCR method of microRNAs assessment . In our study, AUC was 0.833 for qRT-PCR. The test evaluated this microRNA and it was similar to observation of Wang et al. Therefore, the microRNAs expression and hence the ROC analysis may vary depending on the research method. Ono et al. noted that the results of microRNAs expression analysis and its possible usefulness in diagnostic tests were influenced by many factors, including the testing method . They analysed different types of cancers, including lung, head and neck, liver, stomach, kidney, breast, melanoma and myeloma. They considered the following factors affecting the expression of microRNAs: type of anticoagulant used in blood collection, quantity and quality of RNA, type of microRNA isolation method, and finally the research methods, including qRT-PCR, Affymetrix GeneChip miRNA Arrays 4.0, Agilent Oligonucleotides Microarrays, Exiqon miRCURY LNA microRNA arrays, µParidiclo® Technology, 3D-Gene® and Next-Generation Sequencing (NGS). They found that relatively easy and simple qRT-PCR method is the best method for microRNAs testing with high sensitivity, specificity and accuracy as well as highly reproducible results. One disadvantage of this method was the inability to detect new and unknown microRNAs, which was possible with NGS. However, NGS is still more expensive, complicated and less available compared to the qRT-PCR technique .
Kanaan et al. indicated that plasma microRNAs are reliable, non-invasive, and inexpensive markers for colorectal adenomas. Panel of eight microRNAs in liquid biopsy, including miR-142, distinguished polyps from controls with high accuracy with AUC of 0.868. They studied 380 plasma-miRNAs expression using microfluidic array technology in 12 healthy people, 9 patients with colorectal adenomas and 20 patients with colorectal cancer (CRC) . The studied groups were not very numerous and the microarray analysis is expensive. Moreover, the analysis of 380 microRNAs was a great achievement, especially that there were molecules with diagnostic potential, including miR-142. Gao et al. also drew attention to this microRNA. They tested serum from 363 CRC patients and 156 healthy controls . They found that expression of miR-142 was significantly lower in CRC patients than in healthy volunteers witch AUC of 0.74. Moreover, they proved that low miR-142 expression was associated with worse prognosis for colorectal cancer patients qualified for surgery . Liu observed that in lung cancer tissue miR-142 expression is reduced. Authors suggested that this molecule acts as a tumor suppressor and promotes NSCLC progression via MALAT1/β-catenin (Metastasis Associated Lung Adenocarcinoma Transcript 1) signalling pathway .
There is no evidence in literature, that circulated miR-519 and miR-526 are the markers of non-invasive cancer detection. Nevertheless, expression of miR-519 was low in early breast cancer tissue compared to normal adjacent tissue . Nong et al. indicated that miR-519 directly regulates PD-L1 expression. Application of miR-519 inhibited invasiveness and tumor growth as well as induced apoptosis of pancreatic cancer in a mouse model .
In cancer diagnostic panels, not one but many microRNAs should be used. Disturbances in the expression of various microRNAs are observed in various types of cancer. Therefore, the selection of microRNAs for diagnostic panels is difficult, requires in-depth and extensive studies and must be very precise. We focused on those that could be related to PD-L1 gene expression and our results showed that it was the right choice. Our study indicated that seven microRNAs could be used for non-invasive lung cancer diagnosis. Kanaan et al. showed that 8 microRNAs out of 380 molecules may have a diagnostic value in neoplastic diseases . However, their choice became possible after analysis of broad microRNAs panel. Our selection of microRNAs, that potentially regulate PD-L1 expression, is related to the role of PD-L1 in tumor progression. The tumor cells with PD-L1 expression have the ability to escape immune surveillance. This property of PD-L1-positive tumor cells is observed not only in lung cancer but also in other immunogenic cancers such as colon, kidney, stomach, pancreatic cancer. Unfortunately, our study failed to determine an expression of PD-L1 mRNA in liquid biopsy and the existence of a correlation between PD-L1 mRNA and microRNAs expression has not been confirmed. The detection method of mRNA expression analysis should be enhanced, e.g. by PCR pre-amplification method. Further research in this area seems very important.