Open Access

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

  • Authors:
    • Hong Xie
    • Ingrid Norman
    • Anders Hjerpe
    • Tomislav Vladic
    • Catharina Larsson
    • Weng‑Onn Lui
    • Ellinor Östensson
    • Sonia Andersson
  • View Affiliations

  • Published online on: March 24, 2017     https://doi.org/10.3892/ol.2017.5909
  • Pages: 3586-3598
  • Copyright: © Xie et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


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.

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 (1719). 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 (2124). 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).

Table I.

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

Characteristic (N with results available)N%
Cytology (N=140)
  WNL1812.86
  LSIL4532.14
  HSIL7452.86
  Cancer32.40
Histology (N=123)
  WNL97.32
  CIN13528.46
  CIN22822.76
  CIN34738.21
  Cancer43.25
Final histopathological diagnosis (N=140)
  WNL1611.43
  CIN12920.71
  CIN24431.43
  CIN34733.57
  Cancer42.86
HPV testing (N=115)
  Positive9380.87
  Negative2219.13

[i] 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.

Table II.

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

HPV

Sample IDAgemiR-205 (2−∆ΔCq)Cytology diagnosisHistology diagnosisFinal diagnosisStatusSubtypeHR/LR-HPV
23761.0439LSILCIN1CIN1Positive31HR-HPV
3306.7449WNLn.a.WNLn.a.
42934.5873HSILCIN3CIN3n.a.
5352.2973HSILCIN3CIN3n.a.
63419.0510LSILCIN3CIN3Positive16HR-HPV
73223.3276HSILCIN3CIN3Positive16HR-HPV
83925.2251HSILCIN2CIN2n.a.
9413.8929LSILCIN1CIN1Negative
10303.3291HSILCIN2CIN2Positive18HR-HPV
113720.8132HSILCIN2CIN2Positive
122613.9071HSILCIN2CIN2Positive58HR-HPV
13342.4690HSILCIN3CIN3Positive58HR-HPV
14334.7576HSILCIN1CIN2n.a.
153029.0087LSILCIN2CIN2Positive39HR-HPV
19281.2505LSILCIN1CIN1Positive
202820.1998HSILCIN3CIN3Positive16,31HR-HPV
22437.4901WNLn.a.WNLNegative
23422.2771LSILCIN1CIN1Positive16,52,82HR-HPV
24A592.0112WNLn.a.WNLNegative
24B257.2827LSILCIN2CIN2Positive31,51,73HR-HPV
252739.7380LSILCIN2CIN2Positive31,59HR-HPV
285917.1738HSILCIN2CIN2n.a.
29430.6120WNLCIN1CIN1Positive51HR-HPV
304353.3738HSILCIN3CIN3n.a.
313112.1099HSILCIN1CIN2Negative
324442.0013HSILCIN3CIN3Positive18HR-HPV
33437.4764HSILCIN1CIN2Positive45HR-HPV
343159.1805HSILCIN2CIN2Positive
35263.8242HSILCIN1CIN2Negative
362819.4811HSILCIN3CIN3n.a.16,51
37272.4561LSILCIN1CIN1Positive53,73HR-HPV
38321.4752LSILWNLCIN1Positive82HR-HPV
392614.5685HSILWNLCIN2Positive31,56HR-HPV
403014.2268HSILCIN3CIN3n.a.
41286.1169HSILWNLCIN2Negative
423312.0710HSILCIN3CIN3n.a.
43393.5259HSILn.a.CIN2Positive54LR-HPV
443510.6758HSILCIN2CIN2Positive16HR-HPV
45437.5600HSILCIN3CIN3Positive56,HR-HPV
462672.3169LSILCIN2CIN2Positive39,51,58,73HR-HPV
472641.7024HSILCIN3CIN3Positive16HR-HPV
484322.4166WNLn.a.WNLPositive
494516.2120WNLn.a.WNLPositive
50417.5508WNLn.a.WNLNegative
51357.5375WNLn.a.WNLNegative
522632.8494HSILCIN3CIN3Positive 18,31,51,52,66,68HR-HPV
533914.3435LSILCIN1CIN1Positive18,51HR-HPV
54398.1765HSILCIN2CIN2Negative
552954.1454HSILCIN1CIN2Positive16,33,59HR-HPV
564333.0009HSILCIN1CIN2Positive59HR-HPV
57336.3544HSILCIN3CIN3n.a.
58343.6957WNLn.a.WNLPositive18HR-HPV
59432.4636HSILCIN3CIN3Positive52HR-HPV
605428.7410WNLn.a.WNLNegative
614618.0521WNLn.a.WNLNegative
62277.9717HSILCIN2CIN2Positive16HR-HPV
64516.7104WNLn.a.WNLNegative
65410.7032HSILCIN3CIN3Positive52HR-HPV
662912.8313HSILCIN2CIN2n.a.
67426.9052HSILCIN2CIN2Positive16HR-HPV
68321.3904LSILWNLCIN1Negative
69280.3772HSILCIN2CIN2n.a.
70476.7330CancerCancerCancern.a
71297.7228WNLCIN1CIN1Positive16HR-HPV
72289.6434HSILCIN2CIN2Positive16HR-HPV
73266.9220HSILCIN2CIN2Positive16,33HR-HPV
74417.0546HSILCIN3CIN3n.a.
75324.2851LSILCIN2CIN2Positive33,73HR-HPV
764411.3330HSILWNLCIN2n.a.
78512.6267WNLn.a.WNLNegative
79328.7506LSILCIN1CIN1Positive73HR-HPV
80281.6829HSILCIN2CIN2n.a.
81303.4284WNLn.a.WNLNegative
824817.9020LSILWNLCIN1Positive16HR-HPV
843013.8683LSILCIN1CIN1Positive33HR-HPV
85486.9998WNLn.a.WNLNegative
86316.0450HSILCIN3CIN3n.a.
87303.0692HSILCIN2CIN2n.a.
885612.7754WNLn.a.WNLNegative
892816.8543WNLWNLWNLNegative
90332.0479HSILWNLCIN2Positive51HR-HPV
912740.2667LSILCIN1CIN1Negative
933128.9839HSILCIN3CIN3PositiveHR-HPV not 16,18HR-HPV
942945.6632HSILCIN2CIN2n.a.
95376.2884LSILCIN1CIN1Positive31,39,56,53HR-HPV
972838.5117HSILCIN1CIN2Positive18HR-HPV
98292.4868HSILCIN2CIN2Positive45,51HR-HPV
99288.1134HSILCIN1CIN2Positive51HR-HPV
100311.6449HSILCIN3CIN3n.a.
1012921.7971HSILCIN1CIN2Positive16HR-HPV
111318.9870HSILCIN3CIN3n.a.
113517.1208CancerCIN3CIN3Positive16HR-HPV
115265.0528HSILCIN3CIN3n.a.
116452.5974HSILn.a.CIN2Positive51,52HR-HPV
117308.9810HSILCIN3CIN3Positive16HR-HPV
119588.5443LSILCancerCancerPositive18HR-HPV
121360.6870HSILCIN3CIN3Positive51HR-HPV
124293.5774HSILCIN3CIN3Positive16HR-HPV
1262910.9771HSILCIN3CIN3Positive31HR-HPV
127380.4523HSILCIN3CIN3Negative
129302.6331HSILCIN3CIN3Positive16HR-HPV
130370.1385HSILCIN3CIN3Positive58HR-HPV
132299.6822HSILCIN3CIN3Positive16,45HR-HPV
133303.2601HSILCIN3CIN3Positive16HR-HPV
135287.0403HSILCIN3CIN3Positive16,66HR-HPV
1362816.9954HSILCIN3CIN3Positive18HR-HPV
137440.2899HSILCancerCancern.a
1385137.3282HSILWNLCIN2Positive16HR-HPV
1392642.2245HSILCIN3CIN3Positive16,68HR-HPV
140367.2177HSILCIN3CIN3Positive16HR-HPV
1414213.0473HSILCIN3CIN3Positive16HR-HPV
142305.8978HSILCIN3CIN3Positive16HR-HPV
143343.6293LSILCIN2CIN2Positive16,52HR-HPV
1443110.5154LSILCIN2CIN2Positive18,31,58HR-HPV
145272.4491HSILCIN3CIN3Positive16HR-HPV
146444.5693CancerCIN3CIN3Positive16HR-HPV
147302.1592LSILCIN2CIN2Positive16,31,33,39HR-HPV
148554.4372HSILCancerCancern.a
150232.2068LSILCIN1CIN1Positive51HR-HPV
151340.1408LSILCIN1CIN1Positive51HR-HPV
1524022.2206LSILCIN1CIN1Positive51,52,82,83HR-HPV
153393.6158LSILCIN3CIN3Positive31HR-HPV
155392.4600LSILCIN2CIN2Positive16HR-HPV
156234.3218LSILCIN1CIN1Positive53HR-HPV
157492.9542LSILCIN1CIN1Positive52,73HR-HPV
1584719.4705LSILCIN1CIN1Positive52HR-HPV
159471.5333LSILCIN3CIN3Positive52HR-HPV
160235.2855LSILCIN1CIN1Positive31,73HR-HPV
161241.7701LSILCIN1CIN1Positive59HR-HPV
162378.7852LSILCIN1CIN1Positive68HR-HPV
163230.7902LSILCIN3CIN3Positive16,39,58,73HR-HPV
164335.0789LSILCIN1CIN1Positive31HR-HPV
165394.6425LSILCIN1CIN1Positive31HR-HPV
166237.9566LSILCIN3CIN3Positive31HR-HPV
1672329.6942LSILCIN1CIN1Positive31,33,53HR-HPV
168442.9572LSILCIN1CIN1Positive56HR-HPV
169254.1910LSILCIN1CIN1Positive51HR-HPV
1703485.2947LSILCIN3CIN3Positive35HR-HPV
1712342.8047LSILCIN2CIN2Positive51HR-HPV
172380.4877LSILCIN3CIN3Positive16HR-HPV

[i] 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.

Table III.

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

Triage groupOutcomeTestTPFPFNTNNPrevalence (95% CI)Sensitivity (95% CI)Specificity (95% CI)PPV (95% CI)NPV (95% CI)PLR (95% CI)NLR (95% CI)
LSILCIN2+high1010  817450.400.560.630.500.681.500.71
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)
LSILCIN2+HPV+1824  0  3450.401.000.110.431.001.120
(0.26–0.55)(0.78–1.00)(0.03–0.30)(0.28–0.59)(0.31–1.00)(0.98–1.29)
LSILCIN2+HPV16+  6  31223440.410.330.880.670.662.890.75
(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)
LSILCIN2+HPV18+  2  11625440.410.110.960.670.612.890.92
(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)
LSILCIN2+HPV16+18+  8  41022440.410.440.850.670.692.890.66
(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)
LSILCIN3+high  416  421450.180.500.570.200.841.160.88
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)
LSILCIN3+HPV+  834  0  3450.181.000.080.191.001.090
(0.09–0.33)(0.60–1.00)(0.02–0.23)(0.09–0.35)(0.31–1.00)(0.99–1.20)
LSILCIN3+HPV16+  3  6  530440.180.380.830.330.862.250.75
(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)
LSILCIN3+HPV18+  1  2  734440.180.120.940.330.832.250.93
(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)
LSILCIN3+HPV16+18+  4  8  428440.180.500.780.330.882.250.64
(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)

[i] 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.

Table IV.

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

Triage groupOutcomeTestTPFPFNTNNPrevalence (95% CI)Sensitivity (95% CI)Specificity (95% CI)PPV (95% CI)NPV (95% CI)PLR (95% CI)NLR (95% CI)
HSILCIN2+high41  033  07410.55n.a.10n.a.n.a.
miR-205 (0.94–1.00)(0.43–0.67) (0.89–1.00)(0–0.13)
HSILCIN2+HPV+46  0  7  05310.87n.a.10n.a.n.a.
(0.92–1.00)(0.74–0.94) (0.90–1.00)(0–0.44)
HSILCIN2+HPV16+22  029  05110.43n.a.10n.a.n.a.
(0.91–1.00)(0.30–0.58) (0.82–1.00)(0–0.15)
HSILCIN2+HPV18+  5  046  05110.10n.a.10n.a.n.a.
(0.91–1.00)(0.04–0.22) (0.46–1.00)(0–0.10)
HSILCIN2+HPV16+18+27  024  05110.53n.a.10n.a.n.a.
(0.91–1.00)(0.39–0.67) (0.84–1.00)(0–0.17)
HSILCIN3+high20212013740.540.500.380.490.390.811.31
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)
HSILCIN3+HPV+2521  3  4530.530.890.160.540.571.060.67
(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)
HSILCIN3+HPV16+14  81415510.550.500.650.640.521.440.77
(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)
HSILCIN3+HPV18+  3  22521510.550.110.910.600.461.230.98
(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)
HSILCIN3+HPV16+18+17101113510.550.610.570.630.541.400.70
(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)

[i] 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.

Table V.

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

CharacteristicsAll casesHigh miR-205 (>median)Low miR-205 (<median) P-valuea
Age (n=140)
  <32.57039310.1763
  >32.5703139
HPV (n=115)
  Positive9347460.7352
  Negative221210
HPV subtypes (n=90)
  HPV16, HPV184323200.5267
  Non HPV16, non HPV18472225
Cytology (n=140)
  LSIL4520250.3093
  HSIL744034
Histology (n=123)
  CIN13517180.8391
  CIN2+794039
Final diagnosis (n=140)
  CIN12911180.1657
  CIN2+955045

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

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)

References

1 

Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 127:2893–2917. 2010. View Article : Google Scholar : PubMed/NCBI

2 

The National Board of Health and Welfare: Cancer incidence in Sweden 2008. The National Board of Health and Welfare. Stockholm: 2009.

3 

National Quality Register for Cervical Screening: Prevention of cervical cancer in Sweden. Annual report 2014 with data to 2013. National Quality Register for Cervical Screening. (Stockholm, Swedish). 2014.

4 

Bosch FX and Muñoz N: The viral etiology of cervical cancer. Virus Res. 89:183–190. 2002. View Article : Google Scholar : PubMed/NCBI

5 

Woodman CB, Collins SI and Young LS: The natural history of cervical HPV infection: Unresolved issues. Nat Rev Cancer. 7:11–22. 2007. View Article : Google Scholar : PubMed/NCBI

6 

Münger K: Disruption of oncogene/tumor suppressor networks during human carcinogenesis. Cancer Invest. 20:71–81. 2002. View Article : Google Scholar : PubMed/NCBI

7 

Pett M and Coleman N: Integration of high-risk human papillomavirus: A key event in cervical carcinogenesis? J Pathol. 212:356–367. 2007. View Article : Google Scholar : PubMed/NCBI

8 

Imaoka H, Toiyama Y, Okigami M, Yasuda H, Saigusa S, Ohi M, Tanaka K, Inoue Y, Mohri Y and Kusunoki M: Circulating microRNA-203 predicts metastases, early recurrence and poor prognosis in human gastric cancer. Gastric Cancer. 19:744–753. 2016. View Article : Google Scholar : PubMed/NCBI

9 

Naucler P, Ryd W, Törnberg S, Strand A, Wadell G, Elfgren K, Rådberg T, Strander B, Johansson B, Forslund O, et al: Human papillomavirus and Papanicolaou tests to screen for cervical cancer. N Engl J Med. 357:1589–1597. 2007. View Article : Google Scholar : PubMed/NCBI

10 

Giorgi-Rossi P, Franceschi S and Ronco G: HPV prevalence and accuracy of HPV testing to detect high-grade cervical intraepithelial neoplasia. Int J Cancer. 130:1387–1394. 2012. View Article : Google Scholar : PubMed/NCBI

11 

Bergström R, Sparén P and Adami HO: Trends in cancer of the cervix uteri in Sweden following cytological screening. Br J Cancer. 81:159–166. 1999. View Article : Google Scholar : PubMed/NCBI

12 

Cuzick J, Clavel C, Petry KU, Meijer CJ, Hoyer H, Ratnam S, Szarewski A, Birembaut P, Kulasingam S, Sasieni P and Iftner T: Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer. 119:1095–1101. 2006. View Article : Google Scholar : PubMed/NCBI

13 

Nanda K, McCrory DC, Myers ER, Bastian LA, Hasselblad V, Hickey JD and Matchar DB: Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: A systematic review. Ann Intern Med. 132:810–819. 2000. View Article : Google Scholar : PubMed/NCBI

14 

Strander B, Andersson-Ellström A, Milsom I, Rådberg T and Ryd W: Liquid-based cytology versus conventional Papanicolaou smear in an organized screening program: A prospective randomized study. Cancer. 111:285–291. 2007. View Article : Google Scholar : PubMed/NCBI

15 

Persson M, Elfström KM, Brismar Wendel S, Weiderpass E and Andersson S: Triage of HR-HPV positive women with minor cytological abnormalities: A comparison of mRNA testing, HPV DNA testing and repeat cytology using a 4-year follow-up of a population-based study. PLoS One. 9:e900232014. View Article : Google Scholar : PubMed/NCBI

16 

Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI

17 

Lui WO, Pourmand N, Patterson BK and Fire A: Patterns of known and novel small RNAs in human cervical cancer. Cancer Res. 67:6031–6043. 2007. View Article : Google Scholar : PubMed/NCBI

18 

Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C and Zheng ZM: Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One. 3:e25572008. View Article : Google Scholar : PubMed/NCBI

19 

Witten D, Tibshirani R, Gu SG, Fire A and Lui WO: Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls. BMC Biol. 8:582010. View Article : Google Scholar : PubMed/NCBI

20 

Xie H, Zhao Y, Caramuta S, Larsson C and Lui WO: MiR-205 expression promotes cell proliferation and migration of human cervical cancer cells. PLoS One. 7:e469902012. View Article : Google Scholar : PubMed/NCBI

21 

Hagrass HA, Sharaf S, Pasha HF, Tantawy EA, Mohamed RH and Kassem R: Circulating microRNAs-a new horizon in molecular diagnosis of breast cancer. Genes Cancer. 6:281–287. 2015.PubMed/NCBI

22 

He Y, Lin J, Kong D, Huang M, Xu C, Kim TK, Etheridge A, Luo Y, Ding Y and Wang K: Current state of circulating microRNAs as cancer biomarkers. Clin Chem. 61:1138–1155. 2015. View Article : Google Scholar : PubMed/NCBI

23 

Lin XJ, Chong Y, Guo ZW, Xie C, Yang XJ, Zhang Q, Li SP, Xiong Y, Yuan Y, Min J, et al: A serum microRNA classifier for early detection of hepatocellular carcinoma: A multicentre, retrospective, longitudinal biomarker identification study with a nested case-control study. Lancet Oncol. 16:804–815. 2015. View Article : Google Scholar : PubMed/NCBI

24 

Mu YP, Sun WJ, Lu CW and Su XL: MicroRNAs may serve as emerging molecular biomarkers for diagnosis and prognostic assessment or as targets for therapy in gastric cancer. Asian Pac J Cancer Prev. 16:4813–4820. 2015. View Article : Google Scholar : PubMed/NCBI

25 

Nonaka R, Miyake Y, Hata T, Kagawa Y, Kato T, Osawa H, Nishimura J, Ikenaga M, Murata K, Uemura M, et al: Circulating miR-103 and miR-720 as novel serum biomarkers for patients with colorectal cancer. Int J Oncol. 47:1097–1102. 2015.PubMed/NCBI

26 

Yu J, Jin L, Jiang L, Gao L, Zhou J, Hu Y, Li W, Zhi Q and Zhu X: Serum miR-372 is a diagnostic and prognostic biomarker in patients with early colorectal cancer. Anticancer Agents Med Chem. 16:424–431. 2016. View Article : Google Scholar : PubMed/NCBI

27 

Orang AV, Safaralizadeh R, Hosseinpour Feizi MA and Somi MH: Diagnostic and prognostic value of miR-205 in colorectal cancer. Asian Pac J Cancer Prev. 15:4033–4037. 2014. View Article : Google Scholar : PubMed/NCBI

28 

Zheng H, Zhang L, Zhao Y, Yang D, Song F, Wen Y, Hao Q, Hu Z, Zhang W and Chen K: Plasma miRNAs as diagnostic and prognostic biomarkers for ovarian cancer. PLoS One. 8:e778532013. View Article : Google Scholar : PubMed/NCBI

29 

Yue X, Lan F, Hu M, Pan Q, Wang Q and Wang J: Downregulation of serum microRNA-205 as a potential diagnostic and prognostic biomarker for human glioma. J Neurosurg. 124:122–128. 2016. View Article : Google Scholar : PubMed/NCBI

30 

Ma Q, Wan G, Wang S, Yang W, Zhang J and Yao X: Serum microRNA-205 as a novel biomarker for cervical cancer patients. Cancer Cell Int. 14:812014. View Article : Google Scholar : PubMed/NCBI

31 

Zhao S, Yao D, Chen J and Ding N: Circulating miRNA-20a and miRNA-203 for screening lymph node metastasis in early stage cervical cancer. Genet Test Mol Biomarkers. 17:631–636. 2013. View Article : Google Scholar : PubMed/NCBI

32 

Malta M, Ribeiro J, Monteiro P, Loureiro J, Medeiros R and Sousa H: Let-7c is a candidate biomarker for cervical intraepithelial lesions: A pilot study. Mol Diagn Ther. 19:191–196. 2015. View Article : Google Scholar : PubMed/NCBI

33 

Tian Q, Li Y, Wang F, Li Y, Xu J, Shen Y, Ye F, Wang X, Cheng X, Chen Y, et al: MicroRNA detection in cervical exfoliated cells as a triage for human papillomavirus-positive women. J Natl Cancer Inst. 106:pii: dju241. 2014. View Article : Google Scholar : PubMed/NCBI

34 

Fröberg M, Johansson B, Hjerpe A and Andersson S: Human papillomavirus ‘reflex’ testing as a screening method in cases of minor cytological abnormalities. Br J Cancer. 99:563–568. 2008. View Article : Google Scholar : PubMed/NCBI

35 

Solomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M, Raab S, Sherman M, Wilbur D, Wright T Jr, et al: The 2001 bethesda system: Terminology for reporting results of cervical cytology. JAMA. 287:2114–2119. 2002. View Article : Google Scholar : PubMed/NCBI

36 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

37 

Canavan TP and Doshi NR: Cervical cancer. Am Fam Physician. 61:1369–1376. 2000.PubMed/NCBI

38 

Esquela-Kerscher A and Slack FJ: Oncomirs-microRNAs with a role in cancer. Nat Rev Cancer. 6:259–269. 2006. View Article : Google Scholar : PubMed/NCBI

39 

Lee YS and Dutta A: MicroRNAs in cancer. Annu Rev Pathol. 4:199–227. 2009. View Article : Google Scholar : PubMed/NCBI

40 

Qin AY, Zhang XW, Liu L, Yu JP, Li H, Wang SZ, Ren XB and Cao S: MiR-205 in cancer: An angel or a devil? Eur J Cell Biol. 92:54–60. 2013. View Article : Google Scholar : PubMed/NCBI

41 

Martinez I, Gardiner AS, Board KF, Monzon FA, Edwards RP and Khan SA: Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene. 27:2575–2582. 2008. View Article : Google Scholar : PubMed/NCBI

42 

Wang X, Wang HK, Li Y, Hafner M, Banerjee NS, Tang S, Briskin D, Meyers C, Chow LT, Xie X, et al: MicroRNAs are biomarkers of oncogenic human papillomavirus infections. Proc Natl Acad Sci USA. 111:4262–4267. 2014. View Article : Google Scholar : PubMed/NCBI

43 

Nielsen BS: MicroRNA in situ hybridization. Methods Mol Biol. 822:67–84. 2012. View Article : Google Scholar : PubMed/NCBI

44 

Ge J, Zhang LL, Liu SJ, Yu RQ and Chu X: A highly sensitive target-primed rolling circle amplification (TPRCA) method for fluorescent in situ hybridization detection of microRNA in tumor cells. Anal Chem. 86:1808–1815. 2014. View Article : Google Scholar : PubMed/NCBI

45 

Wu M, Piccini M, Koh CY, Lam KS and Singh AK: Single cell microRNA analysis using microfluidic flow cytometry. PLoS One. 8:e550442013. View Article : Google Scholar : PubMed/NCBI

46 

Wu M, Piccini ME and Singh AK: MiRNA detection at single-cell resolution using microfluidic LNA flow-FISH. Methods Mol Biol. 1211:245–260. 2014. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

May-2017
Volume 13 Issue 5

Print ISSN: 1792-1074
Online ISSN:1792-1082

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Xie H, Norman I, Hjerpe A, Vladic T, Larsson C, Lui WO, Östensson E and Andersson S: Evaluation of microRNA‑205 expression as a potential triage marker for patients with low‑grade squamous intraepithelial lesions. Oncol Lett 13: 3586-3598, 2017.
APA
Xie, H., Norman, I., Hjerpe, A., Vladic, T., Larsson, C., Lui, W. ... Andersson, S. (2017). Evaluation of microRNA‑205 expression as a potential triage marker for patients with low‑grade squamous intraepithelial lesions. Oncology Letters, 13, 3586-3598. https://doi.org/10.3892/ol.2017.5909
MLA
Xie, H., Norman, I., Hjerpe, A., Vladic, T., Larsson, C., Lui, W., Östensson, E., Andersson, S."Evaluation of microRNA‑205 expression as a potential triage marker for patients with low‑grade squamous intraepithelial lesions". Oncology Letters 13.5 (2017): 3586-3598.
Chicago
Xie, H., Norman, I., Hjerpe, A., Vladic, T., Larsson, C., Lui, W., Östensson, E., Andersson, S."Evaluation of microRNA‑205 expression as a potential triage marker for patients with low‑grade squamous intraepithelial lesions". Oncology Letters 13, no. 5 (2017): 3586-3598. https://doi.org/10.3892/ol.2017.5909