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. 2017 Mar 24;13(5):3586–3598. doi: 10.3892/ol.2017.5909

Evaluation of microRNA-205 expression as a potential triage marker for patients with low-grade squamous intraepithelial lesions

Hong Xie 1,2, Ingrid Norman 3, Anders Hjerpe 4, Tomislav Vladic 5, Catharina Larsson 2, Weng-Onn Lui 2, Ellinor Östensson 5,6, Sonia Andersson 5,✉
PMCID: PMC5431461  PMID: 28529583

Abstract

High-risk human papillomavirus (HPV) testing is a recommended triage approach for females with atypical squamous cells of undetermined significance (ASCUS), but due to its poor specificity this approach is not recommended for patients with low-grade squamous intraepithelial lesions (LSIL). The objective of the current study was to determine microRNA (miR)-205 expression levels in liquid-based cytology (LBC) samples, and evaluate their ability to predict cervical intraepithelial neoplasia grade 2/3 or worse (CIN2/3+) in females with minor cytological abnormalities. LBC samples were obtained from patients attending the Swedish Cervical Cancer Screening Program. The Mann-Whitney U test, one-way analysis of variance, Kruskal-Wallis test, Spearman rank order correlation analysis, and Pearson's χ2 test were used to assess the results. Accuracy analyses indicated that high miR-205 expression had a significantly higher specificity to high-risk HPV testing, and a sensitivity similar to that of high-risk HPV testing to predict CIN2+ and CIN3+ in women with LSIL, but not those with high-grade squamous intraepithelial lesions. Although further research is required for females with LSIL, miR-205 expression in LBC samples may be a novel triage marker for, or a beneficial supplement to high-risk-HPV testing in these patients.

Keywords: liquid-based cytology, microRNA-205, specificity, human papillomavirus, cervical intraepithelial lesions

Introduction

Cervical cancer is a leading cause of cancer-associated mortality among females worldwide. It accounts for 13% of all female cancer cases, with >500,000 new cases and ~275,000 mortalities occurring annually (1). In Sweden, 450 new cases and 150 mortalities occur each year (2). According to reports from the organized Swedish Cervical Cancer Screening Program, ~30,000 women exhibit some form of cellular abnormality and require follow-up with colposcopy and biopsy (3).

Persistent infection with human papillomavirus (HPV) is the causative agent in cervical cancer (4). HPV depends on differentiated keratinocytes; the infection of the squamous epithelia alone is not sufficient for the infection to progress to neoplasia (5). The expression of the HPV oncoproteins E6 and E7 is able to inactivate p53 and retinoblastoma proteins, leading to methylation and mutation of the host genome DNA and resulting in the initiation of and progression towards cancer (6,7). The use of high-risk HPV (8) testing in primary screening for cervical disease has exhibited a high sensitivity (9), but the specificity of this method is low, and thus a follow-up test must be administered prior to treatment (10).

The implementation of organized cervical cancer screening programs has reduced the incidence of cervical cancer considerably (11). However, several previous studies have demonstrated that conventional cytology has a limited sensitivity (only 50–70%) to detect cervical intraepithelial neoplasia (CIN) (12,13). Liquid-based cytology (LBC) was developed to improve diagnostic reliability (14), as it offers the possibility to use the same sample for HPV testing and triage. Such triage is recommended for women with atypical squamous cells of undetermined significance (ASCUS) due to its high sensitivity, but it is not recommended for women with low-grade squamous intraepithelial lesions (LSIL) due to the high prevalence of high-risk HPV in this population, which generally leads to poor specificity (15). The low predictive value of HPV testing among females with minor cytological abnormalities may create unnecessary concern among healthy patients and contribute to a significant risk of over-diagnosis and over-treatment. The use of predictive biomarkers is a novel approach to improving the diagnosis and management of patients with LSIL.

MicroRNA (miRNA) is a small, non-coding RNA that is ~22 nucleotides in length. miRNA has an important role in pathological processes, including viral infection and cancer development (4). Generally, miRNA negatively regulates gene expression at the post-transcriptional level via transcription inhibition and/or translation suppression (16). Previous studies have identified altered miRNA expression profiles in human cervical cancer tissues and cell lines, and several of them, including miRNA (miR)-145, miR-21 and miR-205, are consistently dysregulated in cervical cancer tissue compared with normal cervical tissue (17–19). In our previous study, it was revealed that miR-205 expression was significantly increased in cervical cancer tissue compared with matched normal cervical tissue, and that miR-205 has an oncogenic role in cervical cancer through the promotion of cell proliferation and migration (20). This prompted the further investigation of the potential value and clinical applications of miR-205 in the present study.

Recently, miRNAs were suggested as potential biomarkers for the diagnosis or prognosis of different cancer types, including cervical cancer (21–24). Due to the requirement for non-invasive detection methods, the majority of the applications focused on serum or plasma samples. For example, serum miR-203 expression was an independent predictive marker for lymph node, peritoneal and distant metastases, and a poor prognosis marker in patients with gastric cancer (8). In patients with colorectal cancer, circulating miR-103, miR-720 and miR-372 were potential novel biomarkers: High serum miR-103 expression levels were significantly associated with histological differentiation grade and lymphatic invasion; high serum miR-720 levels were significantly associated with lymph node metastasis; and high miR-372 levels were significantly associated with tumor size, tumor-node-metastasis stage and poorer overall survival (25,26). Downregulation of miR-205 expression in colorectal cancer predicts the risk of lymph node metastasis (27). Circulating miR-205 and let-7f together were reported to be diagnostic biomarkers for ovarian cancer (28). Serum miR-205 expression was revealed to be significantly downregulated in patients with glioma compared with healthy controls and was a novel and valuable biomarker for the diagnosis of glioma, and a prognostic factor for those with advanced-grade tumors (29). Ma et al (30) reported that upregulated serum miR-205 is a predictive marker for the prognosis of cervical cancer, and Zhao et al (31) reported that high circulating miR-20a expression levels represent a potential marker for detecting lymph node metastasis in early-stage cervical cancer. However, only a limited number of studies have performed miRNA detection in cervical exfoliated cells (32,33).

The aim of the present study was to investigate whether miR-205 expression may be used as a novel triage approach to predict high-grade CIN in LBC samples from patients attending the population-based Swedish Cervical Cancer Screening Program.

Materials and methods

Study population

Between 2008 and 2012, LBC samples were collected from 140 women with squamous intraepithelial lesions or squamous cell carcinoma detected within the framework of the Swedish Cervical Cancer Screening Program in Stockholm, Sweden (34). Cervical cells for LBC were obtained from the ectocervix and endocervix of the uterus, preserved in PreservCyt medium (ThinPrep®, Hologic, Boxborough, MA, USA) at −20°C, and evaluated at the Department of Clinical Pathology and Cytology, Karolinska University Hospital (Solna-Stockholm, Sweden). Cytological results were categorized according to the Bethesda classification (35), with modifications based on Swedish recommendations: Samples with coilocytosis, but without cellular atypia, were classified as ‘within normal limits’ (WNL), and LSIL included mild dysplasia only. The diagnosis and staging of CIN was based on colposcopy and histology, and grouped into normal histology (WNL), CIN grade 1 (CIN1), CIN grade 2 (CIN2) and CIN2 or worse (CIN2+). Histological information and high-risk-HPV test results were retrieved from the medical and laboratory records at the Karolinska University Hospital.

This study was approved by the Ethical Review Board at Karolinska Institutet (Stockholm, Sweden) and written informed consent was obtained from all participants prior to sample collection.

RNA extraction

Cervical cells were collected by centrifugation and washed with cold PBS twice, followed by total RNA extraction using the mirVana™ miRNA isolation kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), all according to the manufacturer's protocol. RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and stored at −80°C for further use.

TaqMan RT-qPCR

miR-205 expression was quantified by TaqMan reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the StepOne Plus real-time PCR system (Thermo Fisher Scientific, Inc.). cDNA was synthesized from 100 ng of RNA using the TaqMan miRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The pre-designed TaqMan assays for miR-205 (ID 000509) and the reference material RNU6B (ID 001093) were purchased from Thermo Fisher Scientific, Inc. (20). All reactions were performed in triplicate, according to the manufacturer's protocol. The relative expression of miR-205 was normalized to RNU6B and reported as 2−∆∆Cq (36).

HPV DNA detection

HPV testing was performed at Karolinska University Hospital. Briefly, DNA was extracted from the LBC suspensions using the MagNA Pure LC Robot (Roche Diagnostics, Basel, Switzerland). HPV DNA detection and genotyping were carried out using the Linear Array HPV Genotyping test (Roche Diagnostics, Mannheim, Germany) and Cobas 4800 (Roche Diagnostics, Basel, Switzerland), which detects 37 HPV types: High-risk-HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59/68/73, and 82); probable high-risk-HPV types (HPV26, 53, and 66); and low-risk or undetermined-risk HPV types (HPV6, 11, 40, 42, 43, 44, 54, 55, 61, 62, 64, 67, 69, 70, 71, 72, 81, 83, 84, IS39, and CP6108).

Statistical analysis

Data were entered into Statistica 7.0 (Statsoft, Inc., Tulsa, OK, USA). The difference in miR-205 expression between all HPV-positive and all HPV-negative samples was analyzed using the Mann-Whitney U test. The associations between miR-205 expression levels and diagnoses (including cytology, histology and the final histopathological diagnosis) were analyzed by the Kruskal-Wallis one-way analysis of variance (ANOVA) test. The correlation of miR-205 expression with age was analyzed with the Spearman Rank Order correlation and Pearson's χ2 test. Sensitivity and specificity calculations were performed using VassarStats online software (http://vassarstats.net/). P<0.05 was considered to indicate a statistically significant difference.

Results

Cytology, histology, final diagnosis and HPV status

The median age of the 140 females in the study sample was 32.5 years (range, 23–59 years). Of these patients, 123 (123/140, 87.9%) had histological information available, and 115 (115/140, 82.1%) had HPV test results available in the medical and laboratory records at the Karolinska University Hospital. Among the patients with HPV results, 93 were HPV-positive (93/115, 80.9%) and 22 were HPV-negative (22/115, 19.1%) (Table I).

Table I.

Summary of clinical features of the study sample (N=140).

Characteristic (N with results available) N %
Cytology (N=140)
  WNL 18 12.86
  LSIL 45 32.14
  HSIL 74 52.86
  Cancer 3 2.40
Histology (N=123)
  WNL 9 7.32
  CIN1 35 28.46
  CIN2 28 22.76
  CIN3 47 38.21
  Cancer 4 3.25
Final histopathological diagnosis (N=140)
  WNL 16 11.43
  CIN1 29 20.71
  CIN2 44 31.43
  CIN3 47 33.57
  Cancer 4 2.86
HPV testing (N=115)
  Positive 93 80.87
  Negative 22 19.13
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N, number; WNL, within normal limits (normal cytology); LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; CIN1, cervical intra-epithelial neoplasia grade 1; CIN2, cervical intra-epithelial neoplasia grade 2; CIN3, cervical intra-epithelial neoplasia grade 3; HPV, human papillomavirus.

Of the 93 HPV-positive women, only one (no. 43) was infected with a low-risk HPV type (HPV54). Eighty-seven patients were infected with at least one high-risk HPV type, and 43 (43/93, 46.2%) were infected with either HPV16 or 18, the two most common high-risk HPV types (Table II).

Table II.

Detailed clinical information and miR-205 expression in 140 patients.

HPV
Sample ID Age miR-205 (2−∆ΔCq) Cytology diagnosis Histology diagnosis Final diagnosis Status Subtype HR/LR-HPV
2 37 61.0439 LSIL CIN1 CIN1 Positive 31 HR-HPV
3 30 6.7449 WNL n.a. WNL n.a.
4 29 34.5873 HSIL CIN3 CIN3 n.a.
5 35 2.2973 HSIL CIN3 CIN3 n.a.
6 34 19.0510 LSIL CIN3 CIN3 Positive 16 HR-HPV
7 32 23.3276 HSIL CIN3 CIN3 Positive 16 HR-HPV
8 39 25.2251 HSIL CIN2 CIN2 n.a.
9 41 3.8929 LSIL CIN1 CIN1 Negative
10 30 3.3291 HSIL CIN2 CIN2 Positive 18 HR-HPV
11 37 20.8132 HSIL CIN2 CIN2 Positive
12 26 13.9071 HSIL CIN2 CIN2 Positive 58 HR-HPV
13 34 2.4690 HSIL CIN3 CIN3 Positive 58 HR-HPV
14 33 4.7576 HSIL CIN1 CIN2 n.a.
15 30 29.0087 LSIL CIN2 CIN2 Positive 39 HR-HPV
19 28 1.2505 LSIL CIN1 CIN1 Positive
20 28 20.1998 HSIL CIN3 CIN3 Positive 16,31 HR-HPV
22 43 7.4901 WNL n.a. WNL Negative
23 42 2.2771 LSIL CIN1 CIN1 Positive 16,52,82 HR-HPV
24A 59 2.0112 WNL n.a. WNL Negative
24B 25 7.2827 LSIL CIN2 CIN2 Positive 31,51,73 HR-HPV
25 27 39.7380 LSIL CIN2 CIN2 Positive 31,59 HR-HPV
28 59 17.1738 HSIL CIN2 CIN2 n.a.
29 43 0.6120 WNL CIN1 CIN1 Positive 51 HR-HPV
30 43 53.3738 HSIL CIN3 CIN3 n.a.
31 31 12.1099 HSIL CIN1 CIN2 Negative
32 44 42.0013 HSIL CIN3 CIN3 Positive 18 HR-HPV
33 43 7.4764 HSIL CIN1 CIN2 Positive 45 HR-HPV
34 31 59.1805 HSIL CIN2 CIN2 Positive
35 26 3.8242 HSIL CIN1 CIN2 Negative
36 28 19.4811 HSIL CIN3 CIN3 n.a. 16,51
37 27 2.4561 LSIL CIN1 CIN1 Positive 53,73 HR-HPV
38 32 1.4752 LSIL WNL CIN1 Positive 82 HR-HPV
39 26 14.5685 HSIL WNL CIN2 Positive 31,56 HR-HPV
40 30 14.2268 HSIL CIN3 CIN3 n.a.
41 28 6.1169 HSIL WNL CIN2 Negative
42 33 12.0710 HSIL CIN3 CIN3 n.a.
43 39 3.5259 HSIL n.a. CIN2 Positive 54 LR-HPV
44 35 10.6758 HSIL CIN2 CIN2 Positive 16 HR-HPV
45 43 7.5600 HSIL CIN3 CIN3 Positive 56, HR-HPV
46 26 72.3169 LSIL CIN2 CIN2 Positive 39,51,58,73 HR-HPV
47 26 41.7024 HSIL CIN3 CIN3 Positive 16 HR-HPV
48 43 22.4166 WNL n.a. WNL Positive
49 45 16.2120 WNL n.a. WNL Positive
50 41 7.5508 WNL n.a. WNL Negative
51 35 7.5375 WNL n.a. WNL Negative
52 26 32.8494 HSIL CIN3 CIN3 Positive 18,31,51,52,66,68 HR-HPV
53 39 14.3435 LSIL CIN1 CIN1 Positive 18,51 HR-HPV
54 39 8.1765 HSIL CIN2 CIN2 Negative
55 29 54.1454 HSIL CIN1 CIN2 Positive 16,33,59 HR-HPV
56 43 33.0009 HSIL CIN1 CIN2 Positive 59 HR-HPV
57 33 6.3544 HSIL CIN3 CIN3 n.a.
58 34 3.6957 WNL n.a. WNL Positive 18 HR-HPV
59 43 2.4636 HSIL CIN3 CIN3 Positive 52 HR-HPV
60 54 28.7410 WNL n.a. WNL Negative
61 46 18.0521 WNL n.a. WNL Negative
62 27 7.9717 HSIL CIN2 CIN2 Positive 16 HR-HPV
64 51 6.7104 WNL n.a. WNL Negative
65 41 0.7032 HSIL CIN3 CIN3 Positive 52 HR-HPV
66 29 12.8313 HSIL CIN2 CIN2 n.a.
67 42 6.9052 HSIL CIN2 CIN2 Positive 16 HR-HPV
68 32 1.3904 LSIL WNL CIN1 Negative
69 28 0.3772 HSIL CIN2 CIN2 n.a.
70 47 6.7330 Cancer Cancer Cancer n.a
71 29 7.7228 WNL CIN1 CIN1 Positive 16 HR-HPV
72 28 9.6434 HSIL CIN2 CIN2 Positive 16 HR-HPV
73 26 6.9220 HSIL CIN2 CIN2 Positive 16,33 HR-HPV
74 41 7.0546 HSIL CIN3 CIN3 n.a.
75 32 4.2851 LSIL CIN2 CIN2 Positive 33,73 HR-HPV
76 44 11.3330 HSIL WNL CIN2 n.a.
78 51 2.6267 WNL n.a. WNL Negative
79 32 8.7506 LSIL CIN1 CIN1 Positive 73 HR-HPV
80 28 1.6829 HSIL CIN2 CIN2 n.a.
81 30 3.4284 WNL n.a. WNL Negative
82 48 17.9020 LSIL WNL CIN1 Positive 16 HR-HPV
84 30 13.8683 LSIL CIN1 CIN1 Positive 33 HR-HPV
85 48 6.9998 WNL n.a. WNL Negative
86 31 6.0450 HSIL CIN3 CIN3 n.a.
87 30 3.0692 HSIL CIN2 CIN2 n.a.
88 56 12.7754 WNL n.a. WNL Negative
89 28 16.8543 WNL WNL WNL Negative
90 33 2.0479 HSIL WNL CIN2 Positive 51 HR-HPV
91 27 40.2667 LSIL CIN1 CIN1 Negative
93 31 28.9839 HSIL CIN3 CIN3 Positive HR-HPV not 16,18 HR-HPV
94 29 45.6632 HSIL CIN2 CIN2 n.a.
95 37 6.2884 LSIL CIN1 CIN1 Positive 31,39,56,53 HR-HPV
97 28 38.5117 HSIL CIN1 CIN2 Positive 18 HR-HPV
98 29 2.4868 HSIL CIN2 CIN2 Positive 45,51 HR-HPV
99 28 8.1134 HSIL CIN1 CIN2 Positive 51 HR-HPV
100 31 1.6449 HSIL CIN3 CIN3 n.a.
101 29 21.7971 HSIL CIN1 CIN2 Positive 16 HR-HPV
111 31 8.9870 HSIL CIN3 CIN3 n.a.
113 51 7.1208 Cancer CIN3 CIN3 Positive 16 HR-HPV
115 26 5.0528 HSIL CIN3 CIN3 n.a.
116 45 2.5974 HSIL n.a. CIN2 Positive 51,52 HR-HPV
117 30 8.9810 HSIL CIN3 CIN3 Positive 16 HR-HPV
119 58 8.5443 LSIL Cancer Cancer Positive 18 HR-HPV
121 36 0.6870 HSIL CIN3 CIN3 Positive 51 HR-HPV
124 29 3.5774 HSIL CIN3 CIN3 Positive 16 HR-HPV
126 29 10.9771 HSIL CIN3 CIN3 Positive 31 HR-HPV
127 38 0.4523 HSIL CIN3 CIN3 Negative
129 30 2.6331 HSIL CIN3 CIN3 Positive 16 HR-HPV
130 37 0.1385 HSIL CIN3 CIN3 Positive 58 HR-HPV
132 29 9.6822 HSIL CIN3 CIN3 Positive 16,45 HR-HPV
133 30 3.2601 HSIL CIN3 CIN3 Positive 16 HR-HPV
135 28 7.0403 HSIL CIN3 CIN3 Positive 16,66 HR-HPV
136 28 16.9954 HSIL CIN3 CIN3 Positive 18 HR-HPV
137 44 0.2899 HSIL Cancer Cancer n.a
138 51 37.3282 HSIL WNL CIN2 Positive 16 HR-HPV
139 26 42.2245 HSIL CIN3 CIN3 Positive 16,68 HR-HPV
140 36 7.2177 HSIL CIN3 CIN3 Positive 16 HR-HPV
141 42 13.0473 HSIL CIN3 CIN3 Positive 16 HR-HPV
142 30 5.8978 HSIL CIN3 CIN3 Positive 16 HR-HPV
143 34 3.6293 LSIL CIN2 CIN2 Positive 16,52 HR-HPV
144 31 10.5154 LSIL CIN2 CIN2 Positive 18,31,58 HR-HPV
145 27 2.4491 HSIL CIN3 CIN3 Positive 16 HR-HPV
146 44 4.5693 Cancer CIN3 CIN3 Positive 16 HR-HPV
147 30 2.1592 LSIL CIN2 CIN2 Positive 16,31,33,39 HR-HPV
148 55 4.4372 HSIL Cancer Cancer n.a
150 23 2.2068 LSIL CIN1 CIN1 Positive 51 HR-HPV
151 34 0.1408 LSIL CIN1 CIN1 Positive 51 HR-HPV
152 40 22.2206 LSIL CIN1 CIN1 Positive 51,52,82,83 HR-HPV
153 39 3.6158 LSIL CIN3 CIN3 Positive 31 HR-HPV
155 39 2.4600 LSIL CIN2 CIN2 Positive 16 HR-HPV
156 23 4.3218 LSIL CIN1 CIN1 Positive 53 HR-HPV
157 49 2.9542 LSIL CIN1 CIN1 Positive 52,73 HR-HPV
158 47 19.4705 LSIL CIN1 CIN1 Positive 52 HR-HPV
159 47 1.5333 LSIL CIN3 CIN3 Positive 52 HR-HPV
160 23 5.2855 LSIL CIN1 CIN1 Positive 31,73 HR-HPV
161 24 1.7701 LSIL CIN1 CIN1 Positive 59 HR-HPV
162 37 8.7852 LSIL CIN1 CIN1 Positive 68 HR-HPV
163 23 0.7902 LSIL CIN3 CIN3 Positive 16,39,58,73 HR-HPV
164 33 5.0789 LSIL CIN1 CIN1 Positive 31 HR-HPV
165 39 4.6425 LSIL CIN1 CIN1 Positive 31 HR-HPV
166 23 7.9566 LSIL CIN3 CIN3 Positive 31 HR-HPV
167 23 29.6942 LSIL CIN1 CIN1 Positive 31,33,53 HR-HPV
168 44 2.9572 LSIL CIN1 CIN1 Positive 56 HR-HPV
169 25 4.1910 LSIL CIN1 CIN1 Positive 51 HR-HPV
170 34 85.2947 LSIL CIN3 CIN3 Positive 35 HR-HPV
171 23 42.8047 LSIL CIN2 CIN2 Positive 51 HR-HPV
172 38 0.4877 LSIL CIN3 CIN3 Positive 16 HR-HPV
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HPV, human papillomavirus; LR, low-risk; HR, high-risk; LSIL, low-grade squamous intraepithelial lesion; CIN1, cervical intra-epithelial neoplasia grade 1; WNL, within normal limits; n.a., not applicable; HSIL, high-grade squamous intraepithelial lesion; CIN3, cervical intra-epithelial neoplasia grade 3; CIN2, cervical intra-epithelial neoplasia grade 2; miR, microRNA.

Sensitivity and specificity of high miR-205 expression levels to predict CIN2+ and CIN3+ in LSIL and HSIL

Sensitivity and specificity analyses were performed among patients with LSIL and high-grade squamous intraepithelial lesions (HSIL), based on high miR-205 expression levels and HPV positivity. The specificity of HPV testing to predict the absence of CIN2+ and cervical intraepithelial neoplasia grade 3 or worse (CIN3+) was 0.11 [95% confidence interval (CI), 0.03–0.30] and 0.08 (95% CI, 0.02–0.23), respectively, in women with LSIL. The specificity of high miR-205 expression levels was 0.63 (95% CI, 0.42–0.80) and 0.57 (95% CI, 0.40–0.72), which was significantly higher than that of HPV testing. Although positivity for HPV16, HPV18, or HPV16/18 exhibited a higher sensitivity (0.88, 0.96, and 0.85, respectively, to predict CIN2+; 0.83, 0.94, and 0.73, respectively, to predict CIN3+) than high miR-205 expression levels, these values were not statistically significant (Table III).

Table III.

Overview of the sensitivity and specificity, PPV, NPV and risk of disease in the LSIL group.

Triage group Outcome Test TP FP FN TN N Prevalence (95% CI) Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) PLR (95% CI) NLR (95% CI)
LSIL CIN2+ high 10 10   8 17 45 0.40 0.56 0.63 0.50 0.68 1.50 0.71
miR-205 (0.26–0.55) (0.31–0.78) (0.42–0.80) (0.28–0.72) (0.46–0.84) (0.79–2.85) (0.40–1.23)
LSIL CIN2+ HPV+ 18 24   0   3 45 0.40 1.00 0.11 0.43 1.00 1.12 0
(0.26–0.55) (0.78–1.00) (0.03–0.30) (0.28–0.59) (0.31–1.00) (0.98–1.29)
LSIL CIN2+ HPV16+   6   3 12 23 44 0.41 0.33 0.88 0.67 0.66 2.89 0.75
(0.27–0.57) (0.14–0.59) (0.69–0.97) (0.31–0.91) (0.48–0.80) (0.83–10.07) (0.54–1.06)
LSIL CIN2+ HPV18+   2   1 16 25 44 0.41 0.11 0.96 0.67 0.61 2.89 0.92
(0.27–0.57) (0.02–0.36) (0.78–1.00) (0.13–0.98) (0.45–0.75) (0.28–29.51) (0.78–1.09)
LSIL CIN2+ HPV16+18+   8   4 10 22 44 0.41 0.44 0.85 0.67 0.69 2.89 0.66
(0.27–0.57) (0.22–0.69) (0.64–0.95) (0.35–0.89) (0.50–0.83) (1.02–8.16) (0.43–1.01)
LSIL CIN3+ high   4 16   4 21 45 0.18 0.50 0.57 0.20 0.84 1.16 0.88
miR-205 (0.09–0.33) (0.17–0.83) (0.40–0.72) (0.07–0.44) (0.63–0.95) (0.53–2.54) (0.42–1.83)
LSIL CIN3+ HPV+   8 34   0   3 45 0.18 1.00 0.08 0.19 1.00 1.09 0
(0.09–0.33) (0.60–1.00) (0.02–0.23) (0.09–0.35) (0.31–1.00) (0.99–1.20)
LSIL CIN3+ HPV16+   3   6   5 30 44 0.18 0.38 0.83 0.33 0.86 2.25 0.75
(0.09–0.33) (0.10–0.74) (0.66–0.93) (0.09–0.69) (0.69–0.95) (0.71–7.14) (0.43–1.30)
LSIL CIN3+ HPV18+   1   2   7 34 44 0.18 0.12 0.94 0.33 0.83 2.25 0.93
(0.09–0.33) (0.01–0.53) (0.80–0.99) (0.02–0.87) (0.67–0.92) (0.23–21.89) (0.71–1.21)
LSIL CIN3+ HPV16+18+   4   8   4 28 44 0.18 0.50 0.78 0.33 0.88 2.25 0.64
(0.09–0.33) (0.17–0.83) (0.60–0.89) (0.11–0.65) (0.70–0.96) (0.89–5.67) (0.32–1.31)
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PPV, positive predictive value; NPV, negative predictive value; LSIL, low-grade squamous intraepithelial lesions; TP, true positive; FP, false positive; FN, false negative; TN, true negative; CI, confidence interval; PLR, positive likelihood ratio; NLR, negative likelihood ratio; N, number; WNL, within normal limits (normal cytology); CIN2+, cervical intra-epithelial neoplasia grade 2 or worse; CIN3+, cervical intra-epithelial neoplasia grade 3 or worse; HPV, human papillomavirus.

Although the specificity of HPV testing to predict CIN3+ in patients with HSIL was lower than that of high miR-205 expression levels (0.16, 95% CI: 0.05–0.37; 0.38, 95% CI, 0.23–0.56, respectively), this trend was also not statistically significant (Table IV).

Table IV.

Overview of the sensitivity and specificity, PPV, NPV and risk of disease in the HSIL group.

Triage group Outcome Test TP FP FN TN N Prevalence (95% CI) Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) PLR (95% CI) NLR (95% CI)
HSIL CIN2+ high 41   0 33   0 74 1 0.55 n.a. 1 0 n.a. n.a.
miR-205 (0.94–1.00) (0.43–0.67) (0.89–1.00) (0–0.13)
HSIL CIN2+ HPV+ 46   0   7   0 53 1 0.87 n.a. 1 0 n.a. n.a.
(0.92–1.00) (0.74–0.94) (0.90–1.00) (0–0.44)
HSIL CIN2+ HPV16+ 22   0 29   0 51 1 0.43 n.a. 1 0 n.a. n.a.
(0.91–1.00) (0.30–0.58) (0.82–1.00) (0–0.15)
HSIL CIN2+ HPV18+   5   0 46   0 51 1 0.10 n.a. 1 0 n.a. n.a.
(0.91–1.00) (0.04–0.22) (0.46–1.00) (0–0.10)
HSIL CIN2+ HPV16+18+ 27   0 24   0 51 1 0.53 n.a. 1 0 n.a. n.a.
(0.91–1.00) (0.39–0.67) (0.84–1.00) (0–0.17)
HSIL CIN3+ high 20 21 20 13 74 0.54 0.50 0.38 0.49 0.39 0.81 1.31
miR-205 (0.42–0.66) (0.34–0.66) (0.23–0.56) (0.33–0.65) (0.23–0.58) (0.54–1.22) (0.87–1.96)
HSIL CIN3+ HPV+ 25 21   3   4 53 0.53 0.89 0.16 0.54 0.57 1.06 0.67
(0.39–0.66) (0.71–0.97) (0.05–0.37) (0.39–0.69) (0.20–0.88) (0.86–1.32) (0.15–2.94)
HSIL CIN3+ HPV16+ 14   8 14 15 51 0.55 0.50 0.65 0.64 0.52 1.44 0.77
(0.40–0.69) (0.31–0.69) (0.43–0.83) (0.41–0.82) (0.33–0.70) (0.73–2.81) (0.51–1.16)
HSIL CIN3+ HPV18+   3   2 25 21 51 0.55 0.11 0.91 0.60 0.46 1.23 0.98
(0.40–0.69) (0.03–0.29) (0.70–0.98) (0.17–0.93) (0.31–0.61) (0.22–6.76) (0.85–1.12)
HSIL CIN3+ HPV16+18+ 17 10 11 13 51 0.55 0.61 0.57 0.63 0.54 1.40 0.70
(0.40–0.69) (0.41–0.78) (0.35–0.76) (0.42–0.80) (0.33–0.74) (0.80–2.43) (0.41–1.18)
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PPV, positive predictive value; NPV, negative predictive value; HSIL, high-grade squamous intraepithelial lesions; TP, true positive; FP, false positive; FN, false negative; TN, true negative; CI, confidence interval; PLR, positive likelihood ratio; NLR, negative likelihood ratio; CIN2+, cervical intraepithelial neoplasia grade 2 or worse; CIN3+, cervical intraepithelial neoplasia grade 3 or worse; HPV, human papillomavirus; n.a., not available.

The sensitivity of high miR-205 expression to predict CIN2+ and CIN3+ was 0.56 (95% CI, 0.31–0.78) and 0.50 (95% CI, 0.17–0.83), respectively, among patients with LSIL, whereas HPV testing had a corresponding sensitivity of 1.0 (95% CI, 0.78–1) and 1.0 (95% CI, 0.60–1), respectively. Furthermore, when divided by HPV type, the individual sensitivity values (0.33, 0.11 and 0.44 for CIN2+; 0.38, 0.12 and 0.50 for CIN3+) were not higher than those for high miR-205 expression levels; the ANOVA test revealed that the differences between HPV testing and high miR-205 expression levels were not statistically significant (Table III). Similar results were obtained in the HSIL group, in which the sensitivity of HPV testing to predict CIN2+ and CIN3+ was 0.87 (95% CI, 0.74–0.94) and 0.89 (95% CI, 0.71–0.97), respectively, which was higher than that of high miR-205 expression levels (0.55, 95% CI, 0.43–0.67 for CIN2+ and 0.50, 95% CI, 0.34–0.66 for CIN3+; Table IV).

miR-205 expression is not associated with HPV status, but may differ by HPV type

Using the relative quantification method (2−∆ΔCq), as normalized to RNU6B, the relative miR-205 expression in all 140 LBC samples was calculated, and the associations between miR-205 expression and HPV positivity in the 115 samples that had this information available were analyzed using the Mann-Whitney U test. No statistically significant difference in miR-205 expression was observed between HPV-positive (n=93) and HPV-negative (n=22) samples (P=0.97; Z-score=0.039; two-tailed), indicating that miR-205 expression was not associated with HPV positivity. Similar results were obtained using the χ2 test (Table V). A univariate test for miR-205 expression in all 140 samples revealed significant differences (P=1×10−6), indicating the role of an unknown variable. Therefore, the association between miR-205 expression and HPV type, particularly HPV16 and 18, was investigated using the ANOVA Kruskal-Wallis test. Although the mean miR-205 expression levels in HPV18-positive samples (mean value, 18.98; n=9) were higher than those in HPV16-positive samples (mean value, 12.27; n=34), due to small sample size and large variation between samples, they were not statistically significant (P=0.279).

Table V.

Correlation of clinical features of LBC samples with miR-205 expression levels.

Characteristics All cases High miR-205 (>median) Low miR-205 (<median) P-valuea
Age (n=140)
  <32.5 70 39 31 0.1763
  >32.5 70 31 39
HPV (n=115)
  Positive 93 47 46 0.7352
  Negative 22 12 10
HPV subtypes (n=90)
  HPV16, HPV18 43 23 20 0.5267
  Non HPV16, non HPV18 47 22 25
Cytology (n=140)
  LSIL 45 20 25 0.3093
  HSIL 74 40 34
Histology (n=123)
  CIN1 35 17 18 0.8391
  CIN2+ 79 40 39
Final diagnosis (n=140)
  CIN1 29 11 18 0.1657
  CIN2+ 95 50 45
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a

Two-tailed χ2 test (without Yates correlation). High or low miR-205 expression based on the median expression level. LBC, liquid-based cytology; HPV, human papillomavirus; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; CIN1, cervical intra-epithelial neoplasia grade 1; CIN2+, cervical intra-epithelial neoplasia grade 2 or worse.

miR-205 expression and age

Spearman Rank Order correlation analyses did not reveal any significant correlations between miR-205 expression and age (R=−0.0836; P=0.324); similar results were obtained using χ2 tests (Table V).

miR-205 expression and cervical cancer progression

No significant difference between the LSIL and the HSIL group was observed based on cytology diagnosis, histology diagnosis or final histopathological diagnosis (P=0.64, 0.70 and 0.32, respectively), indicating that miR-205 expression alone was not able to distinguish the progression of cervical cancer in LBC samples. Based on the median expression levels of miR-205 in the 140 LBC samples, the correlations between miR-205 expression and different characteristics, including age, HPV positivity, HPV type, and final histopathological diagnosis were evaluated using a two-tailed χ2 test; however, no significant differences were observed (Table V).

Discussion

Cervical cancer develops from well-recognized, pre-malignant forms. The detection of these forms through population-based screening programs is able to reduce the number of cases of cervical cancer dramatically (37). However, more robust and reliable molecular markers are required in current screening programs in order to distinguish between lesions with invasive potential and lesions that will spontaneously regress.

miRNAs are well described non-coding RNAs involved in human cancer, which typically negatively regulate gene expression by transcription repression or translation inhibition (38). Dysregulated miRNA profiles have been identified in various human cancer types, including cervical cancer (17,39). However, the majority of previous studies were based on tissue samples or serum samples; there is a lack of knowledge concerning miRNA expression in LBC samples. miR-205 is frequently dysregulated in many cancer types and functions as a either a tumor suppressor or an oncogene, depending on the cellular context (20). miR-205 expression in tumor tissue or serum is associated with the development and progression of tumors (40). Our previous studies revealed that miR-205 is highly expressed in cervical tumor tissue compared with matched normal cervical tissue, and further demonstrated that miR-205 has an oncogenic role by promoting cell proliferation and migration in cervical cancer cells (17,20). In the present study, miR-205 was selected as an example to evaluate the possibility of miRNA detection by RT-qPCR in LBC samples and to assess the potential value of miR-205 in clinical applications.

The preliminary results revealed that high miR-205 expression levels had a significantly higher specificity than HPV testing to predict the absence of CIN2+ or CIN3+ in women with LSIL, whereas the corresponding sensitivities were not significantly different. This demonstrates that there may be promising clinical applications for miR-205 expression. HPV testing is not recommended to triage women with LSIL due to its poor specificity, but this may be improved by the addition of the evaluation of miR-205 expression in these patients.

Certain miRNAs have been associated with HPV infection in cervical cancer. For example, miR-218 was specifically underexpressed in HPV16-positive cervical cancer cell lines, cervical lesions and cancer tissues when compared with HPV-negative C33A cells and normal cervical cells (41). Wang et al (42) revealed that HPV16 E6 expression is regulated via the histone acetyltransferase p300 and reported that increases in the expression of miR-16, miR-25, miR-92a and miR-378, and decreased expression of miR-22, miR-27a, miR-29a and miR-100 may be attributed to the HPV oncoproteins E6 and E7. In the present study, the association between high miR-205 expression and the presence of HPV was also analyzed, but no significant differences were observed, indicating that miR-205 expression is not associated with HPV infection.

In addition, no significant association between miR-205 expression and cancer stage was detected based on cytology, histology or final histopathological diagnosis. This may indicate that miR-205 expression levels do not increase at specific stages, but may increase continually during cancer progression. To better address this question, analyses are required to be performed on more than one sample from the same patient, on specially paired samples or on series of samples.

The present study cohort was taken from patients attending the population-based organized cervical cancer screening program in Sweden, and the majority of the samples were pre-malignant. However, the majority of the cells in the samples were normal, and thus it was difficult to distinguish if the miR-205 molecules extracted were from abnormal or normal cells. Theoretically, other single-cell-based detection methods, such as in situ hybridization (43,44) or microfluidic flow cytometry (45,46) are practical and ideal methods for LBC.

In conclusion, the findings from this screening-based population study revealed that high miR-205 expression levels in patients with LSIL provided statistically higher specificity than HPV testing to predict the absence of CIN2+ and CIN3+. Therefore, the data suggest that miRNA detection in LBC samples may have a potential application as an adjunct to HPV testing in the triage of women with LSIL. Further studies in larger cohorts or testing for a panel of miRNAs is required before recommendations may be suggested.

Acknowledgements

The present study was supported by the Swedish Research Council (grant nos. 523-2009-3517 and 521-2010-3518), the Swedish Cancer Foundation (grant no. CAN2011/471), the King Gustaf V Jubilee Fund and the Cancer Research Funds of Radiumhemmet (grant no. 154022). The authors would like to thank Ms. Trudy Perdrix-Thoma from Professional Standards Editing, Inc. (222 Lovejoy Avenue, Waterloo, IA 50,701, USA), for the English language revision.

Glossary

Abbreviations

ASCUS

atypical squamous cells of undetermined significance

CI

confidence interval

CIN

cervical intraepithelial neoplasia

CIN1

cervical intraepithelial neoplasia grade 1

CIN2

cervical intraepithelial neoplasia grade 2

CIN2+

cervical intraepithelial neoplasia grade 2 or worse

CIN3+

cervical intraepithelial neoplasia grade 3 or worse

HPV

human papillomavirus

HSIL

high-grade squamous intraepithelial lesion

LBC

liquid-based cytology

LSIL

low-grade squamous intraepithelial lesions

miRNA

microRNA

RT-qPCR

reverse transcription-quantitative polymerase chain reaction

WNL

within normal limits (normal cytology)

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