MicroRNA expression profiling of sputum for the detection of early and locally advanced non-small-cell lung cancer: a prospective case–control study

Abstract

Background

Non-small-cell lung cancer (nsclc) is associated with very poor overall survival because 70% of patients present with locally advanced or metastatic disease at the time of diagnosis. Micrornas (mirnas) are a class of short, noncoding rna molecules whose presence in samples of biologic fluids such as sputum has demonstrated promise as a potential means of detecting nsclc. We investigated the stage-specific nsclc detection potential of an efficient panel of 3 mirnas (mir-21, mir-210, mir-372) using a single sputum sample.

Methods

A single spontaneously expectorated sputum sample was prospectively collected from 21 early nsclc (≤stage ii) patients, 22 advanced nsclc (≥stage iii) patients, and 10 control subjects. Mirna expression profiles were determined by quantitative real-time polymerase chain reaction and were analyzed by unsupervised hierarchical cluster analysis.

Results

Mean tumour size (±95% confidence interval) in the early and advanced nsclc patients was 3.3 cm ± 0.9 cm and 4.8 cm ± 0.7 cm respectively. Adenocarcinoma constituted 61.9% of the early and 45.5% of the advanced nsclc cases respectively. In comparing the early nsclc group with the control group, the mirna panel yielded a diagnostic sensitivity of 67% and a specificity of 90.0%. For the advanced nsclc group, the mirna panel detected nsclc with a sensitivity and specificity of 64% and 100% respectively.

Conclusions

A sputum mir-21, mir-210, and mir-372 expression profile might provide a sensitive and highly specific means for detecting nsclc. Sputum mirna analysis demonstrates promise as a potential complementary screening tool.

INTRODUCTION

Non-small-cell lung cancer (nsclc) represents the 2nd most commonly diagnosed malignancy in Canada¹. It is the leading cause of cancer-related mortality in both men and women, accounting for 27% of all predicted cancer-related deaths in 2015¹. Because more than half of all patients present with metastatic disease at the time of diagnosis, the overall current 5-year survival is an abysmal 17.4%². Diagnosis at an early stage has a significant effect on the long-term survival rate. The 10-year survival of individuals with stage i nsclc can approach 90%³.

The efficacy of chest radiography and sputum cytology for screening purposes has not been supported in the literature⁴. The U.S. National Lung Screening Trial demonstrated that a reduction in lung cancer mortality can be achieved through the use of low-dose computed tomography (ct) imaging in high-risk populations⁵. The long-term sequelae of repeated exposure to ct radiation and the morbidity related to the invasive investigations of incidentally discovered nodules leaves room for the development of a complementary screening modality.

Since their discovery in 1993, small noncoding endogenous single-stranded rnas (approximately 22 nucleotides), called “micrornas” (mirnas), have received significant attention⁶. Currently, posttranslational regulation of mirnas is implicated in multiple biologic processes such as proliferation, apoptosis, cell differentiation, oncogenesis, and metastasis^7–13. The role of mirnas in lung cancer initiation, progression, and treatment response prediction is the subject of ongoing evaluation^14,15. In the setting of lung cancer detection, sputum provides a potential source of mirnas because of their presence in cancer cells exfoliated into the airway and ultimately expectorated as sputum. However, the extracellular milieu also contains detectable mirnas because of a variety of transport mechanisms such as exosomes, microvesicles, rna binding protein complexes, or apoptotic bodies^16,17. Even in extremely small quantities, mirnas can be detected by sensitive techniques such as real-time quantitative polymerase chain reaction (rt-qpcr). Furthermore, mirnas display remarkable stability, even under adverse experimental conditions^18–20. Those features favourably support the future utilization of mirnas as a practical screening biomarker.

Our previous work used single sputum samples from 24 prospectively accrued patients of all nsclc stages and from 6 healthy control subjects to evaluate a 5-mirna panel (mir-21, mir-143, mir-155, mir-210, mir-372) for the detection of nsclc, demonstrating 83.3% sensitivity and 100% specificity²¹. A post-hoc analysis revealed that 3 mirnas (mir-21, mir-210, mir-372) were primarily responsible for the diagnostic power of the panel. Individually, those 3 mirnas have been used in nsclc blood and sputum evaluations because of their overexpression, as documented in our own experience and in the literature^21,22. This specific 3-mirna panel has not previously been reported in the literature. The first aim of the present study was therefore to determine whether this small and cost-effective 3-mirna panel could accurately diagnose the presence or absence of nsclc. Second, we investigated whether nsclc stage influenced the ability of the mirna panel to detect the presence of malignancy. To those ends, we prospectively recruited two cohorts:

• ■ An early-stage group consisting of individuals with stages i and ii nsclc

• ■ An advanced-stage group consisting of patients with stages iii and iv nsclc

Both groups had been staged according to the 7th edition of the American Joint Committee on Cancer staging system²³. To our knowledge, the screening potential of an mirna panel has not previously been investigated in a prospective stage-dependent manner.

METHODS

Patient Selection and Data Collection

Adults diagnosed with nsclc of any histologic type, which had been confirmed either by bronchial wash cytology or by transbronchial or percutaneous fine-needle aspiration, were recruited. Participants were categorized as having either advanced or early nsclc. The early nsclc cohort comprised individuals staged either i or ii. Reported staging was based on the final postoperative pathology review. The advanced nsclc cohort comprised individuals staged either iii or iv. Advanced nsclc was staged using standard clinical practices including imaging modalities such as ct and positron-emission tomography, with nodal evaluation conducted either by endobronchial ultrasound-guided biopsy or cervical mediastinoscopy. Tumour size was based on the reported maximum tumour size.

Participants 18–75 years of age, with a minimum life expectancy of 3 months or more, who were medically stable, with an Eastern Cooperative Oncology Group score of 2 or less, were eligible²⁴. All nsclc-negative controls had undergone either radiography or ct imaging of the chest within 12 months of sputum collection, confirming the absence of intrathoracic pathology. The presence of pulmonary medical disorders such as asthma or chronic obstructive pulmonary disease were not an indication for exclusion, given their prevalence in the nsclc population. Individuals with a history of malignancy except for nonmelanoma skin cancer were excluded from study recruitment.

All study participants completed a detailed questionnaire compiling information about their past medical history, smoking history, potential lifetime occupational hazard exposure, functional status, and demographic information. Imaging, pathology reports, and past medical and surgical histories were also retrieved for each study participant from our health region’s electronic medical records system. Control subjects and early-stage patients were prospectively recruited from a preoperative clinical evaluation by a thoracic surgeon; advanced-stage patients were recruited from our local medical oncology treatment facility (Cross Cancer Institute, Edmonton, AB) before initiation of any treatment.

The study was approved by the Human Research Ethics Board of the University of Alberta (Edmonton, AB) and the Alberta Cancer Research Ethics Committee (Alberta Health Services, Edmonton, AB). All study participants provided informed consent.

Collection and Processing of Sputum

Sputum samples were collected from a single spontaneous expectoration obtained before any nsclc-directed treatment (surgery, chemotherapy, radiation). Participants were instructed to rinse their mouth with water after waking in the morning, to inspire, to hold that breath, and subsequently, to produce a deep cough. The sputum was directly collected into sterile containers and stored at 4°C until further processing was performed within 24 hours of collection. Code identifiers were applied to each sample container, blinding the individual performing the mirna analysis, and thus preventing subsequent statistical bias.

Based on our previous experience, we did not evaluate sputum cytology as a surrogate marker for sputum adequacy²¹. Sputum cytology is associated with a high rate of false positives for the detection of nsclc²⁵. Furthermore, cytology does not evaluate the extracellular mirna component present in sputum^16,17. Xie et al.¹⁶ demonstrated a significant difference in sensitivity when they compared sputum cytology with mirna expression. Total rna isolation, rna reverse transcription, and rt-qpcr, which were conducted according to the manufacturer’s recommendations, are described in our previous study²¹. The procedure is briefly summarized in the subsections that follow.

RNA Isolation and Reverse Transcription

Sputolysin solution (Sigma–Aldrich, St. Louis, MO, U.S.A.) was added to the sputum to fully homogenize the sample. Total rna was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.). Total rna was quantified using a ultraviolet spectrophotometer (DU 7000: Beckman Coulter, Brea, CA, U.S.A.).

The TaqMan MicroRNA Reverse Transcription Kit for individual mirnas (Applied Biosystems, Foster City, CA, U.S.A.) was used for reverse transcription. Reverse transcription was performed using a StepOnePlus rt-pcr instrument (Applied Biosystems). The reverse transcription products were stored at –80°C until subsequent rt-qpcr analysis.

RT-qPCR

The assays for individual mirnas were performed in duplicate using the reverse transcription reaction product already described. The cycle threshold (cyth) was defined as the number of cycles required for the fluorescent signal to cross the threshold in pcr. The SDS software application (Applied Biosystems) was used to identify cyth values. Expression values of mir-21, mir-210, and mir-372 were normalized to the commonly used endogenous control U6 and to a reference sample derived from normal lung fibroblast cells (MRC-5)^26,27. The comparative method (ΔΔCτ method) was used to quantify the rt-qpcr mirna expression data.

The relative mirna expression of a given sample was determined using

$$\Delta \Delta \text{Rn}=2^{-\Delta \Delta \text{C}\tau },$$

where Rn represents the required quantity of mirna to be tested, and ΔΔCτ was calculated as

$$\Delta \Delta \text{C}\tau ={\left({\text{CyTh}}_{\text{m}}={\text{CyTh}}_{\text{ec}}\right)}_{\text{sample}}-{\left({\text{CyTh}}_{\text{m}}={\text{CyTh}}_{\text{ec}}\right)}_{\text{references}},$$

with cyth_m being the cyth for the measured mirna and cyth_ec being the cyth for the endogenous control mirna (U6) for the sputum samples to be tested (sample) and for the reference sample [reference (from the MRC-5 cell line)].

Statistical Analysis

The normalized sputum expression profiles for each individual (the 3-mirna panel) were evaluated using hierarchical cluster analysis as validated in our previous work²¹. Cluster analysis is a statistical method that groups similar (or related) objects within a group (“cluster”), thereby placing dissimilar objects in other groups²⁸; it is useful for analyzing complex biologic data. The “nearest neighbour cluster” method and the squared Euclidean distance were used to measure the intervals. Two separate cluster analyses were used to compare the control samples with the samples from the early- and advanced-stage nsclc samples. Hierarchical cluster analyses were performed using the SPSS software application (version 13: SPSS, Chicago, IL, U.S.A.).

Study population data are expressed as means or medians (± 95% confidence intervals) for continuous variables and as frequencies with proportions for categorical data. Cohort population comparisons were made using the Student t-test when the data were normally distributed and the Mann–Whitney U-test when the data were non-normally distributed continuous variables.

RESULTS

The study enrolled 10 control subjects, 21 patients with early nsclc, and 22 patients with advanced nsclc. Table i presents cohort-specific characteristics. Table ii shows characteristics, lung disease status, and smoking history for the individual control participants. Tables iii and iv show the patient and tumour characteristics for the individual patients with early and advanced nsclc respectively.

TABLE I.

Characteristics of study participants overall

Characteristic	Study group
	Control	Non-small-cell lung cancer
		Early	Advanced
Participants (n)	10	21	22
Sex ratio (men:women)	4:1	4.3:1	2.1:1
Age (years)
Median	58	70	68
Range	30–77	46–84	49–82
Mean smoking history (pack–years)	41.7±17.0	46.1±13.6	33.4±10.2
Chronic obstructive pulmonary disease (%)	50.0	42.9	40.9
Mean maximum tumour diameter (cm)	NA	3.3±0.9	4.8±0.7
NSCLC stagea [n (%)]
IA	NA	10 (45.5)
IB		4 (18.2)
IIA		4 (18.2)
IIB		3 (13.6)
IIIA			10 (45.5)
IIIB			4 (18.2)
IV			8 (36.4)
Histologic subtype (%)
Adenocarcinoma	NA	61.9	45.5
Squamous cell carcinoma		23.8	22.7
Other		14.3	31.8

^aBased on the 7th edition of the American Joint Committee on Cancer staging system²³.

NA = not applicable; NSCLC = non-small-cell lung cancer.

TABLE II.

Characteristics of control participants

Specimen ID	Sex	Age	Lung disease	Smoking history
				Status	Pack–years
CTR01	Male	51	—	Never	0
CTR02	Male	41	—	Current	10
CTR03	Female	30	—	Never	0
CTR04	Male	30	—	Never	0
CTR05	Male	55	—	Never	0
CTR06	Male	62	COPD	Current	44
CTR07	Female	66	COPD	Current	55
CTR08	Male	63	COPD	Previous	59.5
CTR09	Male	67	COPD	Previous	40
CTR10	Male	77	COPD	Never	0

COPD = chronic obstructive pulmonary disease.

TABLE III.

Characteristics of participants with early-stage non-small-cell lung cancer (NSCLC)

Specimen ID	Age	Sex	Cancer characteristics			Lung disease	Smoking history
			Histology	Stagea	Maximum diameter (cm)		Status	Pack–years
E01	64	M	Adenocarcinoma	IA	1.1	COPD	Current	50
E02	63	M	Adenocarcinoma	IA	1.8	COPD	Current	102
E03	71	F	Adenocarcinoma	1B	3.5	—	Previous	27
E04	72	M	Adenocarcinoma	IIB	8.0	COPD	Previous	26
E05	66	M	Squamous cell carcinoma	IA	2.0	COPD	Current	108
E06	80	M	Squamous cell carcinoma	IA	2.4	—	Current	30
E07	51	M	Adenocarcinoma	IIA	4.2	—	Previous	16.5
E08	70	M	Adenocarcinoma	IIA	3.2	—	Previous	22.75
E09	73	M	NSCLCb	IIB	7.0	COPD	Previous	59
E10	84	F	Adenocarcinoma	IA	0.9	COPD	Previous	28
E11	74	M	Adenocarcinoma	IA	4.1	—	Previous	48
E12	62	M	Large-cell	IB	3.5	—	Current	44
E13	59	F	Adenocarcinoma	IIB	2.1	—	Previous	46
E14	46	M	Adenocarcinoma	IA	2.4	—	Never	0
E15	71	M	Adenocarcinoma	IIA	2.0	COPD	Previous	20
E16c	61	F	Squamous cell carcinoma, neuroendocrine	IB	2.7 2.8	COPD	Previous	50
E17	82	M	Adenocarcinoma	IA	1.4	—	Previous	102
E18	82	M	Squamous cell carcinoma	IA	0.9	—	Current	70
E19	64	M	Large-cell	IA	3.0	—	Current	82.25
E20	81	M	Squamous cell carcinoma	IIA	5.5	COPD	Previous	11
E21	69	M	Adenocarcinoma	IB	8.0	—	Current	27

^aBased on the 7th edition of the American Joint Committee on Cancer staging system²³.

^bPoorly differentiated tumour reported as NSCLC not otherwise specified.

^cDiagnosed with 2 synchronous tumours.

COPD = chronic obstructive pulmonary disease.

TABLE IV.

Characteristics of participants with advanced-stage non-small-cell lung cancer (NSCLC)

Specimen ID	Age	Sex	Cancer characteristics			Lung disease	Smoking history
			Histology	Stagea	Maximum diameter (cm)		Status	Pack–years
A01	62	F	Adenocarcinoma	IIIB	4.1	—	Current	42
A02	68	F	NSCLCb	IV	NAc	—	Previous	25
A03	70	F	Adenocarcinoma	IIIA	3.1	COPD	Previous	20
A04	72	M	NSCLCb	IIIA	2.7	COPD	Current	48
A05	68	M	Adenocarcinoma	IIIA	4.5	—	Current	90
A06	54	F	Squamous cell carcinoma	IIIA	6.5	—	Current	18
A07	73	M	Adenocarcinoma	IIIA	7.3	—	Previous	2.8
A08	49	M	Adenocarcinoma	IIIA	2.6	Asthma, COPD	Current	17.5
A09	60	F	Squamous cell carcinoma	IV	7.6	—	Previous	17.5
A10	62	M	NSCLCb	IV	6.3	COPD	Previous	52.5
A11	69	M	NSCLCb	IIIA	5.3	COPD	Current	54
A12	73	M	NSCLCb	IV	2.1	—	Previous	11.75
A13	72	M	Adenocarcinoma	IIIA	5.6	—	Previous	50
A14	75	F	Adenocarcinoma	IV	4.7	—	Previous	0
A15	69	F	Squamous cell carcinoma	IV	5.2	—	Current	50
A16	64	M	Adenocarcinoma	IIIA	4.8	—	Never	0
A17	63	M	Adenocarcinoma	IV	3.3	COPD	Previous	9
A18	74	M	Adenocarcinoma	IIIA	8	COPD	Previous	6
A19	59	M	NSCLCb	IIIB	5.2	COPD	Current	30
A20	61	M	NSCLCb	IV	2.5	—	Current	30
A21	59	M	Squamous cell carcinoma	IIIB	5.5	COPD	Current	60
A22	82	M	Adenocarcinoma	IIIB	4.5	—	Never	0

^aBased on the 7th edition of the American Joint Committee on Cancer staging system²³.

^bPoorly differentiated tumour reported as NSCLC not otherwise specified.

^cCould not be accurately measured on computed tomography imaging because of lobar consolidation.

NA = not available; COPD = chronic obstructive pulmonary disease.

The sex ratios (male:female) for the participants in the early nsclc group, the advanced nsclc group, and the control group were 4.3:1, 2.1:1, and 4:1 respectively. Median age in the groups was 70 years (range: 46–84 years), 68 years (range: 49–82 years), and 58.5 years (range: 30–77 years), and concurrent chronic obstructive pulmonary disease was diagnosed in 42.9%, 40.9%, and 50.0% of the participants.

Most of the patients with early and advanced nsclc (≥86%) had a current or previous history of smoking; 50% of the control cohort had a smoking history. Mean pack–years for the individuals with smoking histories in the control, early nsclc, and advanced nsclc groups were 41.7 ± 17.0, 46.1 ± 13.6, and 33.4 ± 10.2 respectively.

For the patients with early and advanced nsclc, mean tumour size in the largest measured diameter was 3.3 ± 0.9 cm and 4.8 ± 0.7 cm respectively. Adenocarcinoma and squamous cell carcinoma constituted 62.0% and 23.8% of the tumours in the early nsclc group, and 45.5% and 22.7% of the tumours in the advanced nsclc group. No study participant had a prior history of malignancy. Every participant in the early-stage nsclc group underwent curative-intent resection.

Figures 1 and 2 present dendrograms comparing the control subjects with, respectively, the early and the advanced nsclc patients, based on their individual normalized mirna expression results (Table v). In the dendrograms, a horizontal line segregates the study participants into 2 large clusters, differentiating those with and without nsclc. Individuals clustered closer to 0 demonstrate a greater degree of relatedness according to their 3-mirna expression profiles.

FIGURE 1.

Dendrogram resulting from the hierarchical cluster analysis comparing the microRNA expression profiles of sputum samples from control subjects (Cnn) and from patients (Enn) with early-stage non-small-cell lung cancer (NSCLC). The full-width horizontal line segregates the presence from the absence of NSCLC.

FIGURE 2.

Dendrogram resulting from the hierarchical cluster analysis comparing the microRNA expression profiles of sputum samples from control subjects (Cnn) and from patients (Ann) with advanced-stage non-small-cell lung cancer (NSCLC). The full-width horizontal line segregates the presence from the absence of NSCLC.

TABLE V.

Proportional expression of the three microRNAs, normalized to the U6 endogenous control

Specimen ID	MIR-21	MIR-210	MIR-372
CTR01	0.68	0.13	4.09
CTR02	11.86	0.02	18.05
CTR03	20.82	1.30	153.42
CTR04	92.26	24.68	1488.08
CTR05	24.12	1.15	781.75
CTR06	114.98	2.21	1001.33
CTR07	80.41	12.50	438.19
CTR08	2.26	2.65	5.51
CTR09	1.53	0.14	47.53
CTR10	31.76	5.37	413.17
E01	16.02	0.02	0.57
E02	103.42	0.63	78.69
E03	461.21	0.64	211.39
E04	7.55	0.29	0.74
E05	4.92	0.00	2.22
E06	66.38	0.32	1.01
E07	0.22	0.19	26.02
E08	2.76	0.35	0.45
E09	46.17	1.22	23.43
E10	14.55	2.01	0.74
E11	125.22	16.08	266.88
E12	30.54	0.05	0.00
E13	1067.99	3.23	182.95
E14	41.69	0.38	0.00
E15	16.67	1.61	493.21
E16	5280.68	25.25	4337.94
E17	1180.01	13.19	1427.50
E18	180.54	34.64	2696.89
E19	47.19	4.69	137.38
E20	72.44	7.92	1597.47
E21	13.75	2.69	259.27
A01	1331.10	47.00	0.00
A02	133.58	1.62	279.67
A03	12.97	8.95	3103.17
A04	109.46	75.65	798.36
A05	34.91	22.78	0.00
A06	17.45	44.34	0.00
A07	34.44	207.81	0.00
A08	225.37	128.25	0.00
A09	30.65	111.07	0.00
A10	9.01	6.81	0.00
A11	0.41	1.20	36.25
A12	6.63	19.18	307.71
A13	19.00	0.92	0.00
A14	1362.23	24.10	2202.74
A15	3096.91	6.19	3121.79
A16	10664.25	65.54	1195.18
A17	829.72	2.53	199.99
A18	397.63	37.19	305.01
A19	1.78	0.00	0.03
A20	47.27	0.14	5.12
A21	50.97	1.29	15.53
A22	515.34	130.80	198.26

In the early nsclc group, the mir-21, mir-210, and mir-372 expression profile yielded a diagnostic sensitivity of 67% and a specificity of 90%. False negatives were generated for participants E07, E13, E14, E17, E19, E20, and E21. Only 1 participant with squamous cell carcinoma generated a false-negative result; of the remaining 6 participants, 5 had adenocarcinoma, and 1 had large-cell nsclc. Just 1 control subject (C02) generated a false-positive result. The positive and negative predictive values of the panel in the early nsclc group were 93% and 56% respectively.

Comparing the control subjects with the patients who had advanced nsclc, a similar sensitivity and specificity (64% and 100%) was observed. False negatives were generated for participants A02, A03, A04, A11, A12, A14, A15, and A18. As in the comparison involving the early-stage group, only 1 participant with squamous cell carcinoma generated a false-negative result; of the remaining 7 participants, 4 had nsclc not otherwise specified, and 3 had adenocarcinoma. No false-positive results were observed, leading to positive and negative predictive values of 100% and 56% respectively.

DISCUSSION AND CONCLUSIONS

The burden that lung cancer places on society leads to a great demand for the development and validation of biomarkers that can easily be measured in accessible biologic fluids to accurately detect nsclc. Sputum is one such source that has been investigated to determine if it contains elements that reflect the presence or absence of malignancy. Cytology alone—or even in combination with chest radiography—is not recommended for screening purposes⁴. Aberrant over- or underexpression of mirna in lung cancer tissue has clearly been shown¹⁵. The mirnas play an important role in controlling cell growth, the cell cycle, and apoptosis. An understanding of specific mirnas and their roles in various malignancies has grown tremendously in recent years. The promising results from the present study, which used a small 3-mirna panel to detect nsclc, have, interestingly, also been implicated in a variety of solid malignancies.

Overexpression of mir-21 has been associated with malignancies such as glioma and colorectal and prostate cancers^29–31. A recent meta-analysis that evaluated 598 specimens of lung cancer and 528 normal lung tissue controls found that mir-21 and mir-210 upregulation were statistically significant in the tumour tissue group³², and mir-21 is known to target multiple negative regulators of the Ras/mek/erk pathway, thereby promoting proliferation³³. Furthermore, mir-21 inhibition of pro-apoptotic gene products further contributes to tumourigenesis and tumour progression³².

Overexpression of mir-210 has also been demonstrated in lung, esophageal, and pancreatic cancer^34,35, and this mirna is often referred to as the “master” hypoxia mirna³⁶. The response to hypoxia has been well demonstrated to result in upregulation of mir-210^37,38. Using the nsclc cell line A549, Grosso et al.³⁸ demonstrated that mir-210 confers radioresistance through hypoxia inducible factor 1.

The oncogenic role of mir-372 has been demonstrated by its downregulation of the tumour suppressor gene large tumour suppressor homolog 2³⁹. Using the lung adenocarcinoma cell line CL 1-0, Lai et al.⁴⁰ demonstrated that the overexpression of mir-372 corresponded with increased invasiveness.

We initially hypothesized that advanced nsclc would result in an increase of mirna within the sputum milieu, correlating with an enhanced rate of sensitivity in the advanced nsclc group. Our results demonstrate that nsclc stage does not appear to play a significant role in the results of our diagnostic test, such that reasonable sensitivity and very high specificity were achieved in patients with an early-stage nsclc diagnosis.

Our future direction with this test will focus on the interest in low-dose ct screening for high-risk individuals. The U.S. National Lung Screening Trial highlights a tremendous challenge facing widespread ct screening. The authors observed that 4.2% of participants required at least 1 diagnostic surgical procedure³. The cost to the health care system of subsequent investigations and their associated morbidity leaves room for the development of complementary diagnostic tests for future integration within low-dose ct screening programs. We have demonstrated that sensitivity is enhanced with mirna analysis of bronchoalveolar lavage compared with sputum (86%)⁴¹. The feasibility of routine bronchoscopy inhibits its usefulness in a screening setting⁴¹. Noninvasive biomarker-based techniques such as mirna analysis of sputum could further aid in cancer-risk stratification for pulmonary nodules, thereby minimizing unnecessary invasive testing. Further prospective evaluation in individuals with pulmonary nodules will have to be undertaken if sputum mirna analysis is to be validated for clinical application.

Interestingly, mir-372 expression demonstrated the greatest variability in our advanced-stage cohort. The exact reason for that variability is unclear, because mir-372 overexpression is correlated with lung cancer invasiveness. Squamous cell carcinoma constituted only 1 of the observed false negatives in each group. That observation could be related to the greater sensitivity for the detection of squamous cell carcinoma also seen in sputum cytology, because squamous cell carcinomas are frequently more centrally located⁴².

Similar results with a small, focused mirna panel have been demonstrated in the literature. Xing et al.⁴³ used a 3-mirna panel (mir-21, mir-31, mir-210) to evaluate sputum, yielding a sensitivity of 80.52% and a specificity of 86.08%. The mounting body of literature highlights the exciting potential of a noninvasive tool of this kind in nsclc diagnosis. Because of sample size limitations and variability in expression values, we were unable to demonstrate meaningful associations between individual mirnas and tumour subtype or patient factors. Large population-based studies will be required to answer such questions.

To summarize, hierarchical cluster analysis of sputum mirna expression yields a highly specific test for the detection of early and advanced nsclc. Future studies using internal and external validation to reconfirm the results of the present study are warranted. We propose that a test such as the one described here could have future clinical applicability as a complementary diagnostic investigation in low-dose ct screening programs.

CONFLICT OF INTEREST DISCLOSURES

We have read and understood Current Oncology’s policy on disclosing conflicts of interest, and we declare that we have none.