Persistent and novel changes in plasma microRNA profiles in patients with non-small cell lung cancer following tumour resection

Abstract

Background: Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancers, the leading cause of cancer mortality. microRNAs (miRNA, miR) have emerged as important components of carcinogenesis and promising biomarkers. We aimed to analyse global plasma miRs in NSCLC patients before and at least one year after tumour resection.

Methods: Plasma was collected from the peripheral blood of 24 donors without cancer and of NSCLC patients before surgery (n=36) and at least 1 year after surgery (n=12). Next-generation sequencing (NGS)-based miR profiling was performed. Patients were followed-up for 4 to 12 years after surgery to assess disease recurrence.

Results: Untreated NSCLC patients exhibited significant changes in plasma miR levels compared to cancer-free donors (48 up- and 17 down-regulated miRs). miR profiles in patients with adenocarcinoma (ADC) (n=18) and squamous cell carcinoma (SCC) significantly differed (16 and 86 miRs up-, and 15 and 16 miRs down-regulated, respectively). A subset of pre-surgery deregulated miRs was found to be associated with recurrence (49 miRs). Six miRs were shown to have independent prognostic value. After tumour resection, some pre-surgery miR alterations returned to control levels (18 miRs), some others persisted (27 miRs), while also novel plasma miR changes emerged (75 miRs) in patients with no clinical evidence of recurrence.

Conclusions: Untreated NSCLC patients present deregulated plasma miRs, some of which may have a potential of prognostic markers. After tumour excision plasma miR profiles change, some miR levels normalise, some changes persist and novel miR changes are observed despite no clinical symptoms of recurrence. Plasma miR profiles in NSCLC patients may suggest systemic abnormalities predisposing to lung cancer and/or reflect a systemic response to pre-cancer/dormant cancer cells.

Keywords: Non-small cell lung cancer (NSCLC); circulating microRNA; systemic; tumour resection.

Figure 1

A diagram showing plasma samples of NSCLC divided into analysed groups. ADC, adenocarcinoma; AS, after surgery; BS, before surgery; C, control group; Rec, recurrence; Non-rec, without recurrence; NSCLC, non-small cell lung cancer; SCC, squamous cell carcinoma.

Figure 2

Volcano plot of miRs significantly differentiating all NSCLC patients before surgery (BS_All; n=36) from controls (C; n=24). The most important miRs, according to P value and log₂FC analysis, are marked with a name. FC, fold change; miRs, microRNAs; NSCLC, non-small cell lung cancer; NS, not significant.

Figure 3

The ROC curves representing the sensitivity and specificity of plasma miRs levels in differentiating NSCLC patients from the control group, along with plots of individual miR levels: 122b-3p (A), 185-5p (B), 20a-5p (C), 20b-5p (D), 144-3p (E), 423-5p (F), 451a (G), and 590-5p (H), generated using a linear regression model adjusted for sex, smoking status, and age. Values are accompanied by 95% CI shown in square brackets. AUC, area under the curve; CI, confidence interval; miRs, microRNAs; NSCLC, non-small cell lung cancer; ROC, receiver operating characteristic.

Figure 4

Plasma miRs in patients BS, differentiating those with non-small cell lung adenocarcinoma (BS_ADC; n=18) and squamous cell carcinoma (BS_SCC; n=18) from controls (C; n=24). The green circle delineates miRs with significantly altered levels in both histopathological subtypes (BS_All; n=36). The gray and yellow circles delineate miRs with significantly altered plasma levels in BS_ADC and BS_SCC, respectively. ADC, adenocarcinoma; BS, before surgery; miRs, microRNAs; SCC, squamous cell carcinoma.

Figure 5

Plasma miRs differentiating non-small cell lung cancer patients before surgery with (BS_Rec; n=14) and without (BS_Non-rec; n=22) later recurrence, and the control group (C; n=24). In a subgroup of twelve non-small cell lung cancer patients (without recurrence) with paired plasma samples before surgery (BS_Paired) and after surgery (AS_Paired), the purple color indicates plasma miRs, the levels of which are also altered after tumour removal (AS_Paired). The green color indicates miRs with the altered levels before surgery (BS_Paired) that normalise after tumour removal (AS_Paired). The black colour indicates plasma miRs with normal levels either before (BS_Paired) or after surgery (AS_Paired). AS, after surgery; BS, before surgery; Rec, recurrence; miRs, microRNAs.

Figure 6

Differences in plasma miR levels in a subgroup of 12 NSCLC patients BS and C. (A) The PCA exhibiting differences between NSCLC patients with SCC (n=5) and ADC (n=7) and the C (n=24). (B) Heatmap showing sample grouping according to the statistically significant differences in miR levels identified by NGS (patient and control samples are marked as BS and C, respectively). ADC, adenocarcinoma; BS, before surgery; C, controls; miR, microRNA; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; PCA, principal component analysis; SCC, squamous cell carcinoma.

Figure 7

Differences in plasma miR levels in a subgroup of 12 NSCLC patients AS. (A) The PCA exhibiting differences between NSCLC patients AS (n=12) and the C (n=24). (B) Heatmap showing sample grouping according to the statistically significant differences in miR levels identified by NGS (patient and control samples are marked as AS and C, respectively). AS, after surgery; C, controls; miR, microRNA; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; PCA, principal component analysis.

Figure 8

Changes in plasma miR levels in NSCLC patients with paired plasma samples before surgery (BS_Paired; n=12) and after surgery (AS_Paired; n=12), in relation to the controls (C; n=24). The red font marks plasma miRs up-regulated in NSCLC patients with recurrence. The white font marks plasma miRs down-regulated in NSCLC patients with recurrence. The asterisk marks post-surgery up-regulated miRs with down-regulated pre-surgery level. The yellow, blue and green boxes mark up-regulated, down-regulated and normalised miRs, respectively. miR, microRNA; NSCLC, non-small cell lung cancer.