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Abstract

BACKGROUND:

MicroRNAs constitute promising biomarkers.

OBJECTIVE:

The aim was to investigate diagnostic and prognostic implications of miR-182-5p and miR-205-5p in p16-positive and p16-negative oropharyngeal squamous cell carcinomas (OPSCCs).

METHODS:

Expression of miR-182-5p, miR-205-5p were determined via quantitative real-time-PCR in fresh frozen tissues of 26 p16-positive, 19 p16-negative OPSCCs and 18 HPV-negative oropharyngeal controls. Associations between miRNA-expression, clinicopathological characteristics and prognosis were analyzed.

RESULTS:

Higher miR-182-5p expression was associated with significant inferior disease-specific survival for p16-positive OPSCCs (HR $=$ 1.98E $+$ 09, 95% CI 0–Inf; $P=$ 0.028) and a similar trend was observed for p16-negative OPSCCs (HR $=$ 1.56E $+$ 09, 95% CI 0–Inf; $P=$ 0.051).

Higher miR-205-5p expression was associated with an inferior progression-free survival (HR $=$ 4.62, 95% CI 0.98–21.83; $P=$ 0.034) and local control rate (HR $=$ 2.18E $+$ 09, 95% CI 0–Inf; $P=$ 0.048) for p16-positive OPSCCs.

CONCLUSIONS:

Results indicate that miR-182-5p and miR-205-5p can further stratify patients with p16-positive OPSCC into prognostic groups.

Keywords

Oropharyngeal squamous cell carcinomas p16 microRNA miR-182-5p miR-205-5p

1. Introduction

An increasing subset of oropharyngeal squamous cell carcinomas (OPSCC) is associated with human papillomavirus (HPV) infections and/or overexpression of the protein p16 [1, 2]. The latter is considered as a surrogate marker to detect high-risk-HPV-infections [3, 4, 5].

Distinction between HPV/p16-positive and HPV/p16-negative OPSCCs was recently introduced to the 4th edition of the World Health Organization classification for malignant tumors [5] and the 8th edition of the Union International Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC) cancer staging manual [1, 3]. This is consistent with demographic, molecular, morphological, clinical and prognostic diversities of HPV/p16-positive or -negative OPSCCs [3, 5]. Nevertheless, trials having investigated modified therapeutic strategies for HPV/p16-positive OPSCCs [6, 7] have so far not resulted in altered standard treatment recommendations [8].

Additional biomarkers could provide new insights into the pathobiological basis underlying HPV/p16-positive and HPV/p16-negative OPSCCs, and therefore might also help to establish a more precise and individualized oncological management of patients with OPSCCs [8, 9, 10].

MicroRNAs (miRNAs, miRs) are approximately 22-nucleotide-long, single stranded RNAs [11]. Hybridizing with complementary sequences of messenger RNAs, they can epigenetically influence the expression of multiple genes [9, 11]. Dysregulation of miRNAs was related to pathogenesis, progression, immune and treatment response of tumors [9, 10, 11], but also to viral infections like HPV [12, 13]. Combined with their stable tumor-type specific expression, stability, and reliable detectability in clinical specimens, they were considered as potential biomarkers, as well as therapeutic targets for various cancers [9, 11, 14].

To date, only a few studies have been investigating the association of miRNAs with clinicopathological characteristics and prognosis within a uniform OPSCC cohort, and in addition, considering the tumor’s p16-/HPV-status [15, 16, 17, 18, 19, 20, 21, 22]. None of them focused on miR-182 or miR-205. Both were shown to modulate important carcinogenic genes and pathways in HNSCCs (miR-182-5p [23, 24, 25, 26, 27, 28, 29]; miR-205-5p [30, 31, 32, 33, 34, 35]). In particular both miRNAs were discussed in context with TP53 mutation and dysregulation (miR-182 [28, 36, 37]; miR-205-5p [36, 38]), which is common in p16/HPV-negative OPSCCs, but untypical for p16/HPV-positive OPSCCs [39]. Additionally, both were discussed in context with HPV-infections of cells (miR-182-5p [12, 40]; miR-205-5p [13]). Furthermore, both miRNAs were suggested as miRNAs of potential prognostic relevance in HNSCCs (miR-182-5p [16]; miR-205-5p [36, 41]) and a variety of other cancers (miR-182-5p [42, 43, 44, 45, 46, 47]; miR-205-5p [48, 49, 50, 51, 52, 53]). Even though to the best of our knowledge the potential prognostic implications of both as single markers remained so far unexplored in a selective OPSCC dataset. Therefore, the aim of the present study was to investigate the diagnostic and prognostic implications of miR-182-5p and miR-205-5p in a well characterized cohort of patients with p16-positive and p16-negative OPSCCs treated with curative intent.

2. Patients and methods

2.1 Patients and specimens

Patients with OPSCC, who received treatment with curative intent at an academic tertiary referral center were included. Time span of inclusion was from 07/2011 to 04/2016, with subsequent systematic follow-up. Cases were identified, and data obtained from the hospital’s cancer database and original medical records. It was analyzed anonymously. The study was conducted according to the criteria of the Declaration of Helsinki and was approved by the institutional review board (Ethics Committee of the University Medical Center Göttingen) according to the national regulations (application number: 9/12/10). Written informed consent for anonymized analysis of their data and tissues was obtained from all patients prior to treatment at the University Medical Center Göttingen.

Due to the aim of assessing oncologic outcomes in the setting of curative treatment the following patients were excluded from analysis: one patient was excluded due to distant metastasis, two patients, who were treated with palliative intent, one patient due to complete rejection of any therapy, and further two patients due to lack of follow-up data of at least twelve months after primary diagnosis. Furthermore, of 34 cases no sufficient material for p16-immunohistochemistry and micro-RNA quantification was available. Subsequently, 45 patients with histopathological proven OPSCC were included to the study. Tumors’ main sublocalizations were assigned according to the predominantly infiltrated region by the surgeon. Control specimens were obtained from 18 non-cancer patients, who underwent tonsillectomy.

2.2 Staging, treatment and follow-up

Regarding staging, the 7th edition of classification of the UICC [54] and the AJCC [55] was applied.

The therapeutic strategy was primary radiotherapy in 8.9% ( $n=$ 4) of cases and transoral resection of the primary tumor by CO ${}_{2}$ -laser microsurgery (TLM) in 91.1% ( $n=$ 41) cases. Furthermore, the surgical treatment concept also included neck dissection in all cases and was finally completed with postoperative (chemo-)radiotherapy in 68.3% ( $n=$ 28 of 41). In all cases decision on therapeutic approach was not based upon HPV- or p16-status.

Patients received a systematic follow-up after treatment for at least five years. After 60 months without recurrence a patient was considered as cured, but follow-up examinations continued on a regular basis.

2.3 Tissue analyses

All included malignant tissue was harvested during initial panendoscopy and biopsy or surgery of the primary tumor. Fresh frozen (FF) tissues were snap-frozen in liquid nitrogen and stored at $-$ 80 ${}^{\circ}$ C until further processing. FF specimens were used for miRNA quantification and formalin-fixed paraffin embedded (FFPE) tissues for p16-immunhistochemistry (study group) and HPV genotyping (control group).

2.3.1 p16-immunohistochemistry and HPV-typing

For tumor cases without an assessment for p16-status at time of initial diagnosis, p16-immunohistochemistry was performed using the FFPE tissue of the oropharyngeal primaries. Monoclonal p16 antibodies (p16 (JC8): sc-56330, Santa Cruz Biotechnology, Inc., Dallas, TX) (1:50; pH 9) combined with diaminobenzidine and the EnVision Flex $+$ System (both Dako, Agilent Technologies Inc., Santa Clara, CA) were used. Exclusively strong and diffuse nuclear and cytoplasmic p16-immunostaining was classified as p16-positive. Lack of staining, weak or moderate staining was considered as p16-negative [3, 4, 5].

In the non-cancerous control group HPV-genotyping was performed in order to rule out HPV-infections. Therefore, DNA was isolated from FFPE tissues using innuPREP FFPE DNA Kit – IPC16 combined with the InnuPure ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ C16 touch (both Analytik Jena AG, Jena, Germany). The subsequent procedure to test for HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 was performed as described previously [56].

2.3.2 RNA extraction, cDNA synthesis, microRNA quantification

Expression levels of hsa-miR-182-5p (UUUGGCAAUGGUAGAACUCACACU), hsa-miR-205-5p (UCCUUCAUUCCACCGGAGUCUG), and the housekeeping miRNA miR-103a-3p (AGCAGCAUUGUACAGGGCUAUGA) were determined using quantitative real-time-PCR (qRT-PCR). The middle section of 21 slices of the FF tissues was stained with hematoxylin and eosin and was considered as representative to estimate the amount of tumor cells ( $\geqslant$ 60% within the study group), as well as to reconfirm diagnosis by a pathologist. Total RNA from the remaining 20 slices (5 $\mu$ m each) was isolated using TRIzol™ Reagent (Thermo Fisher Scientific Inc., Waltham, MA) combined with ethanol and chloroform. Sufficient RNA quality and quantity were then assessed with Agilent 2100 Bioanalyzer combined with the RNA 600 Nano Kit (both Agilent Technologies Inc., Santa Clara, CA).

MiRNA cDNA was synthesized using the Applied Biosystems™ TaqMan™ Advanced miRNA cDNA Synthesis Kit. Finally, the expression of miRNAs was assessed by qRT-PCR with TaqMan™ Fast Advanced Master Mix technology combined with specific miRNA Assays (all Thermo Fischer Scientific Inc., Waltham, MA; Assay-ID: 478253_mir (hsa-miR-103a-3p), 477935_mir (hsa-miR-182-5p), 477967_mir (hsa-miR-205-5p)). All qRT-PCRs were conducted in duplicates. Exclusively, if the discrepancy between the duplicates was less than one cycle, the mean of the threshold cycle (CT) values was included in the further analysis. For other cases, the qRT-PCR was repeated. Among all samples the maximum detected CT value was 35 of 40 total cycles. miRNA expression was normalized to the housekeeping miRNA miR-103a-3p. This miRNA was recommended for normalization by Thermo Fisher Scientific.

2.4 Statistics

For descriptive analysis, the mean value with corresponding standard deviation, median, minimum and maximum or frequencies with corresponding proportions in percentages were calculated for each variable. Clinicopathological parameters were tested for differences between p16-positive and p16-negative patients using Pearson’s Chi Squared Test.

The Kaplan-Meier method [57] was applied for calculation of overall survival (OS), disease-specific survival (DSS), progression-free survival (PFS), recurrence-free survival (RFS) and local control rate (LCR). The date of initial histopathologic diagnosis was defined as the starting point for interval calculations. Recurrence was defined as the same tumor entity occurring not earlier than 3 months after initial diagnosis, otherwise it was considered as residual disease with subsequent completion of treatment. With regard to OS, death from any possible cause was counted as an event and all other observations were considered as censored. Regarding DSS, exclusively death related to the oropharyngeal primary tumor was considered as an event, while all other observations were censored. Calculating the PFS, events were defined as local, regional, or locoregional recurrence, as well as distant metastasis and death from all causes. Loss of follow-up was defined as censored observations. Regarding RFS, local, regional, or locoregional recurrence, distant metastasis and death related to the oropharyngeal primary tumor were counted as events. Correspondingly, death from other causes, as well as loss of follow-up were considered as censored. Referring to the LCR, local recurrence was considered as an event, whereas alive patients without local recurrence or death regardless the reason were accounted as censored. The association of variables with the relative risk of having an event was assessed by univariate Cox regression proportional hazard models. Calculated hazard ratios (HR) are shown with the respective 95% confidence interval (CI). Log-rank test was applied in order to test for differences of risks between comparison groups.

Relative differences of miRNA expression levels between tumors and benign controls were calculated applying the 2 ${}^{-\Delta\Delta\text{CT}}$ method [58] and significance was assessed by the Mann-Whitney-U-test. Associations of miRNA expression and clinicopathological characteristics within the tumor dataset were further analyzed by the Mann-Whitney-U-test and Kruskal-Wallis-test for categorical variables and Spearman’s rank correlation test for continuous variables. For analysis of the prognostic impact of miRNAs the median miRNA expression level was used as a cutoff value for the dichotomization into high- (greater/equal than cutoff) and low-expressors within respective groups (study cohort: complete, p16-stratified subgroups).

Statistica 13.3 (Dell Inc., Round Rock, Texas) was used to calculate Spearman’s rank correlation, Mann-Whitney-U test, Kruskal-Wallis test, Pearson’s Chi-squared test and Kaplan-Meier estimates. Survival analysis and visualization was conducted using the R statistical platform (R Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, version 3.5.1 [July 2, 2018]) in combination with the survival and Survminer R packages. Box plots were generated using GraphPad Prism Version 7.0e (Graph Pad Software Inc., San Diego).

miRNA $\Delta$ CT values were log transformed and mean normalized. For all tests the level of significance was defined as 5% ( $P<$ 0.05).

3. Results

3.1 Patients and therapy

This study included 45 patients diagnosed with OPSCC. Oropharyngeal tissue of 18 patients with a non-malignant diagnosis served as a control group.

Primary tumors originated from tonsillar regions (51.1%, $n=$ 23) followed by base of tongue (24.4%, $n=$ 11), posterior wall (11.1%, $n=$ 5), lateral wall (6.7%, $n=$ 3), and palatal arch (2.2%, $n=$ 1). For two tumors (4.4%) no assignment to one specific oropharyngeal subsite was made by the surgeon. A positive p16-overexpression was detected in 57.8% ( $n=$ 26) of the OPSCCs, whereas 42.2% ( $n=$ 19) were p16-negative tumors.

The distribution of patient, disease, and treatment characteristics within the complete cohort and p16-stratified subgroups is depicted in Table 1. As shown, patients with p16-positive vs. p16-negative OPSCC shared the same baseline characteristics ( $P>$ 0.05; Table 1).

Table 1
Patient and disease characteristics and follow-up data of the complete cohort and the p16-stratified subgroups

Characteristic	Complete cohort ( $n=$ 45)		p16-positive subgroup ( $n=$ 26)		p16-negative subgroup ( $n=$ 19)		$P^{\text{a}}$
	[ $n$ ]	[%]	[ $n$ ]	[%]	[ $n$ ]	[%]
All patients	45	100	26	57.8	19	42.2
Sex
Female	9	20	6	23.1	3	15.8	0.546
Male	36	80	20	76.9	16	84.2
Age [years]
Mean $\pm$ SD	64.2 $\pm$ 11.6		64.2 $\pm$ 9.7		64.3 $\pm$ 6.4
Median (min-max)	63.7 (47–91.4)		63.1 (47–91.4)		63.8 (54.7–77.4)
T categorization ${}^{\text{b}}$
pT1	2	4.4	0	0	2	10.5	0.077
pT2	11	24.4	5	19.2	6	31.6
pT3	22	48.9	17	65.4	5	26.3
pT4a	6	13.3	2	7.7	4	21.1
pT4b	4	8.9	2	7.7	2	10.5
Local tumor extension ${}^{\text{b}}$
Circumscribed (c/pT1-pT2)	13	28.9	5	19.2	8	42.1	0.095
Advanced (c/pT3-pT4a/b)	32	71.1	21	80.8	11	57.9
N categorization ${}^{\text{b}}$
c/pN0	9	20	3	11.5	6	31.6	0.333
pN1	10	22.2	6	23.1	4	21.1
pN2a	2	4.4	2	7.7	0	0
pN2b	16	35.6	10	38.5	6	31.6
pN2c	7	15.6	5	19.2	2	10.5
N3	1	2.2	0	0	1	5.3
Lymph node metastases
N0	9	20	3	11.5	6	31.6	0.097
N $+$	36	80	23	88.5	13	68.4
Staging ${}^{\text{b}}$
I	1	2.2	0	0	1	5.3	0.443
II	1	2.2	0	0	1	5.3
III	15	33.3	8	30.8	7	36.8
IVa	24	53.3	16	61.5	8	42.1
IVb	4	8.9	2	7.7	2	10.5
Prognostic groups ${}^{\text{b}}$
Local (I–II)	2	4.4	0	0	2	10.5	0.091
Advanced (III–IVa/IVb)	43	95.6	26	100	17	89.5
Extracapsular spread
No data	5	11.1	2	7.7	3	15.8	0.068 ${}^{\text{c}}$
Positive	17	37.8	13	50	4	21.1
Negative or c/pN0	23	51.1	11	42.3	12	63.2
Lymphatic invasion
No data	9	20	6	23.1	3	15.8	0.296 ${}^{\text{c}}$
Positive	17	37.8	11	42.3	6	31.6
Negative	19	42.2	9	34.6	10	52.6
Vascular invasion
No data	9	20	5	19.2	4	21.1	0.355 ${}^{\text{c}}$
Positive	7	15.6	3	11.5	4	21.1
Negative	29	64.4	18	69.2	11	57.9
Histopathologic differentiation
High	1	2.2	0	0	1	5.3	0.365 ${}^{\text{d}}$
Moderate	35	77.8	22	84.6	13	68.4
Poor	9	20	4	15.4	5	26.3
Keratinization
Positive	27	60	16	61.5	11	57.9	0.805
Negative	18	40	10	38.5	8	42.1

Table 1, continued
Characteristic	Complete cohort ( $n=$ 45)		p16-positive subgroup ( $n=$ 26)		p16-negative subgroup ( $n=$ 19)		$P^{\text{a}}$
	[ $n$ ]	[%]	[ $n$ ]	[%]	[ $n$ ]	[%]
Treatment
Primary CRT	4	8.9	2	7.7	2	10.5	0.741 ${}^{\text{e}}$
Primary surgery $+$ ND $\pm$ (C)RT	41	91.1	24	92.3	17	89.5
TLM $+$ ND	13	31.7	7	29.2	6	29.4
TLM $+$ ND $+$ RT	7	17.1	3	12.5	4	23.5
TLM $+$ ND $+$ CRT	21	51.2	14	58.3	7	41.2
Follow-up [months]
Mean $\pm$ SD	28.4 $\pm$ 16.7		29.8 $\pm$ 17.1		26.5 $\pm$ 16.4
Median (min–max)	26.2 (0–61.1)		26 (0–60.3)		26.2 (0.3–61.1)
Alcohol consumption
Yes	24	53.3	13	50	11	57.9	0.600
No	21	46.7	13	50	8	42.1
Tobacco consumption
No data	1	2.2	1	3.8	0	0	0.066 ${}^{\text{c}}$
Former/current	28	62.2	13	50	15	78.9
Never	16	35.6	12	46.2	4	21.1
Tobacco and alcohol consumption
Incomplete data	1	2.2	1	3.8	0	0	0.625 ${}^{\text{c}}$
Yes	19	42.2	10	38.5	9	47.4
No	25	55.6	15	57.7	10	52.6

Abbreviations: SD, standard deviation; min, minimum; max, maximum; c, clinical; p, pathological; (C)RT, (chemo) radiotherapy; ND, neck dissection; TLM, transoral laser microsurgery; OPSCC, oropharyngeal squamous cell carcinoma; $P$ , $P$ -value. Notes: ${}^{\text{a}}$ Pearson’s Chi-Squared test. ${}^{\text{b}}$ TNM-classification and prognostic stages defined according to the 7 ${}^{\text{th}}$ edition of classification of the Union International Contre le Cancer (UICC) [53] and the American Joint Committee on Cancer (AJCC) [54]. ${}^{\text{c}}P$ -value of the Pearson’s Chi-Squared test with exclusion of patients with no data. ${}^{\text{d}}P$ -value of the Pearson’s Chi-Squared test between OPSCCs with high/moderate vs. poor differentiation. ${}^{\text{e}}P$ -value of the Pearson’s Chi-Squared test between patients that were treated with primary surgery vs. primary (chemo)radiotherapy.

3.2 Oncologic results

Median follow up was 26.2 months $\pm$ 16.7 months with a maximum of 61.1 months. With regard to the complete cohort the five-year estimates of OS, DSS, PFS, RFS, and LCR were 64.5%, 79.6%, 43.2%, 49.2% and 83.7%, respectively. Supplementary Fig. S1 illustrates the survival curves for p16-positive vs. p16-negative OPSCCs. As shown, patients with p16-positive tumors demonstrated a trend towards superior PFS and RFS (PFS: HR 0.46, 95% CI 0.2–1.03, $P=$ 0.052; RFS: HR 0.45, 95% CI 0.18–1.12, $P=$ 0.079). The results for OS, DSS and LCR were as followed: OS: HR 0.6, 95% CI 0.22–1.66, $P=$ 0.324; DSS: HR 0.68, 95% CI 0.17–2.72, $P=$ 0.584; LCR: HR 0.7, 95% CI 0.14–3.46, $P=$ 0.658.

3.3 miR-182-5p and miR-205-5p expression in tumor versus normal tissues

Based on a literature research [23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 59, 60, 61, 62], miR-182-5p and miR-205-5p were selected for this study. To analyze the potential dysregulation of miR-182-5p and miR-205-5p in malignant tissue, expressions were examined in FF samples of 26 p16-positive and 19 p16-negative primary OPSCCs, as well as 18 HPV-negative control specimens.

Compared to benign controls, expression of miR-182-5p showed no significant differences within either the complete cohort of tumors, or the p16-stratified subgroups (complete cohort: relative fold change (RFC) $=$ 0.92, $P=$ 0.78; p16-positive: RFC $=$ 1.08, $P=$ 0.58; p16-negative: RFC $=$ 0.58, $P=$ 0.82).

In contrast, miR-205-5p was significantly overexpressed in tumor tissues compared to benign controls in both, the complete cohort as well as the p16-negative subgroup (complete cohort: RFC $=$ 1.93, $P=$ 0.02; p16-negative: RFC $=$ 2.07, $P=$ 0.03). A statistical trend towards higher miR-205-5p expression was also observed in p16-positive tumors compared to benign controls (p16-positive: RFC $=$ 1.84, $P=$ 0.06).

Figure 1 illustrates the expression of miR-182-5p and miR-205-5p in the complete cohort as well as the p16-stratified subgroups compared to benign controls, respectively.

3.4 miRNAs and clinicopathological characteristics

In order to examine associations between miR-182 -5p and miR-205-5p and clinicopathological characteristics within the complete cohort as well as the p16-stratified subgroups the Mann-Whitney-U-Test, Kruskal-Wallis-Test and Spearman’s rank correlation test were applied.

Table 2
Associations of miR-182-5p and miR-205-5p with clinicopathological characteristics in the complete cohort as well as in the p16-stratified subgroups

	Complete cohort ( $n=$ 45)		p16-positive subgroup ( $n=$ 26)		p16-negative subgroup ( $n=$ 19)
Characteristic	miR-182-5p	miR-205-5p	miR-182-5p	miR-205-5p	miR-182-5p	miR-205-5p
	$P$	$P$	$P$	$P$	$P$	$P$
Sex ${}^{\text{a}}$	0.419	0.744	0.378	0.523	0.955	0.065
Age ${}^{\text{b}}$	0.790	0.984	0.653	0.694	0.694	0.792
T categorization ${}^{\text{c}}$	0.923	0.453	0.499	0.516	0.393	0.919
Local tumor extension ${}^{\text{a}}$	0.831	0.754	0.558	0.745	0.836	0.967
N categorization ${}^{\text{d}}$	0.897	0.482	0.907	0.613	0.844	0.419
Lymph node metastases ${}^{\text{a}}$	0.523	0.435	0.688	0.748	0.759	0.273
Extracapsular spread ${}^{\text{e,g}}$	0.722	0.722	0.908	0.772	0.671	0.585
Prognostic groups ${}^{\text{a}}$	0.526	0.215	1.000	1.000	0.740	0.207
Histopathologic differentiation ${}^{\text{f}}$	0.989	0.989	0.915	0.500	0.817	0.487
Keratinization ${}^{\text{a}}$	0.107	0.048	0.133	0.280	0.650	0.063
Lymphatic invasion ${}^{\text{a,g}}$	0.824	0.612	0.879	0.224	0.551	0.704
Vascular invasion ${}^{\text{a,g}}$	0.576	0.497	0.175	0.880	0.648	0.215
Alcohol consumption ${}^{\text{a}}$	0.027	0.334	0.101	0.473	0.127	0.773
Tobacco consumption ${}^{\text{a,g}}$	0.335	0.168	0.314	0.265	0.803	0.726
Tobacco and alcohol consumption ${}^{\text{a,g}}$	0.142	0.093	0.677	0.127	0.079	0.540

Abbreviations: miR, microRNA; $P$ , $P$ -value. Notes: Significant $P$ -values ( $P<$ 0.5) in bold. ${}^{\text{a}}$ Mann-Whitney-U-test. ${}^{\text{b}}$ Spearman’s rank correlation test. ${}^{\text{c}}$ Kruskal-Wallis test between tumors of the four TNM main categories (T1 vs. T2 vs. T3 vs. T4a/b). ${}^{\text{d}}$ Kruskal-Wallis test between N0 vs. N1 vs. N2 vs. N3. ${}^{\text{e}}$ Mann-Whitney-U-test between patients with extracapsular spread vs. patients without extracapsular spread negative or N0. ${}^{\text{f}}$ Mann-Whitney-U-test between tumors with high/moderate versus poor differentiation. ${}^{\text{g}}$ Patients without available data excluded.

Figure 1.

Expression of miR-182-5p in oropharyngeal squamous cell carcinoma (OPSCC) tissue compared to benign control tissue in the complete cohort (A), p16-positive subgroup (B), and p16-negative subgroup (C), respectively. Expression of miR-205-5p in OPSCC tissues compared to benign control tissues in the complete cohort (D), p16-positive subgroup (E), p16-negative subgroup (F), respectively. The relative fold change (RFC) was computed with the 2 ${}^{-\Delta\Delta\text{CT}}$ method with normalization to miR-103a-3p. $P$ -values were calculated by Mann-Whitney-U-test. NS, $P>$ 0.05; ${}^{*}P<$ 0.05. CT, threshold cycle; qRT-PCR, quantitative real-time polymerase chain reaction.

As shown in Table 2, among each of the three subgroups no associations were found between the expression levels of miR-182-5p and miR-205-5p with respect to sex, age, T-categorization (T1 vs. T2 vs. T3 vs. T4a/b), local tumor extent (circumscribed T1-2 vs. advanced T3-4), N-categorization (N0 vs. N1 vs. N2 vs. N3), lymph node metastases (N0 vs. N $+$ ), extracapsular spread (ECS neg. and c/pN0 vs. ECS pos.), prognostic groups (early stage I-II vs. advanced stage III-IVa/b disease), histopathologic differentiation (high/moderate vs. poor differentiation), lymphatic invasion, vascular invasion, tobacco consumption, as well as tobacco and/or alcohol consumption, respectively. This indicated that the expression of miR-182-5p and miR-205-5p was independent from these clinicopathological characteristics.

Patients with a positive history of alcohol consumption exhibited significant higher miR-182-5p levels compared to those patients, who had negated any drinking history ( $P=$ 0.03) within the complete cohort. This difference was not observed in the p16-stratified subgroups (p16-positive: $P=$ 0.13; p16-negative: $P=$ 0.65). Furthermore, considering the whole study population, miR-205-5p levels were significantly lower in keratinized OPSCCs compared to tumors without keratinization ( $P=$ 0.05). Again, this difference was not observed in the p16-stratified subgroups (p16-positive: $P=$ 0.28; p16-negative: $P=$ 0.06).

3.5 miRNAs for prognostic stratification

After dichotomization according to high or low levels of miR-182-5p and miR-205-5p expression based on a median separation, the survival differences between the resulting groups were analyzed within the complete cohort as well as the p16-stratified subgroups, respectively. The corresponding Kaplan-Meier curves are displayed in Fig. 2 (miR-182-5p) and Fig. 3 (miR-205-5p).

Figure 2.

Association between high ( $\geqslant$ median) and low ( $<$ median) expression of miR-182-5p with overall survival (A–C), disease-specific survival (D–F), progression-free survival (G–I), recurrence free survival (J–L), and local control rate (M–O) of the complete cohort (A, D, G, J, M), p16-positive subgroup (B, E, H, K, N), p16-negative subgroup (C, F, I, L, O) with median separation. Patients at risk are shown below the diagrams. $P$ -values were calculated by log-rank test. HR, hazard ratio; 95% CI, 95% confidence interval.

Figure 3.

Association between high ( $\geqslant$ median) and low ( $<$ median) expression of miR-205-5p with overall survival (A–C), disease-specific survival (D–F), progression-free survival (G–I), recurrence free survival (J–L), and local control rate (M–O) of the complete cohort (A, D, G, J, M), p16-positive subgroup (B, E, H, K, N), p16-negative subgroup (C, F, I, L, O) with median separation. Patients at risk are shown below the diagrams. $P$ -values were calculated by log-rank test. HR, hazard ratio; 95% CI, 95% confidence interval.

High expression levels of miR-182-5p were associated with a significant inferior DSS and RFS within the complete cohort (DSS: HR $=$ 1.79E $+$ 09, 95% CI 0–Inf, $P=$ 0.003; RFS: HR $=$ 2.62, 95% CI 0.99–6.92; $P=$ 0.043). With regard to the p16-positive subgroup patients with higher miR-182-5p levels also demonstrated significant inferior DSS estimates (HR $=$ 1.98E $+$ 09, 95% CI 0–Inf; $P=$ 0.028). Within the p16-negative group a higher level of miR-182-5p was also associated with a trend towards an inferior DSS (HR $=$ 1.56E $+$ 09, 95% CI 0–Inf; $P=$ 0.051). Since there were no events in the low-expressor groups, it was not possible to calculate the 95% CI for these comparisons, nor to determine exact hazard ratios.

With regard to miR-205-5p, patients with higher expression levels demonstrated a significant inferior PFS, RFS, as well as LCR within the complete cohort (PFS: HR $=$ 2.58, 95% CI 1.1–6.07; $P=$ 0.024; RFS: HR $=$ 2.94, 95% CI 1.11–7.78; $P=$ 0.023; LCR: HR $=$ 6.64, 95% CI 0.77–57.23; $P=$ 0.047). The associations between high miR-205-5p expression levels and a reduced PFS, as well as LCR were also significant with regard to the p16-positive subgroup (PFS: HR $=$ 4.62, 95% CI 0.98–21.83; $P=$ 0.034; LCR: HR $=$ 2.18E $+$ 09, 95% CI 0–Inf; $P=$ 0.048). Among patients with p16-negative OPSCCs no prognostic differences were observed between patients with relatively low and high expression of miR-205-5p (Table 3).

Table 3

Association between high ( $\geqslant$ median) and low ( $<$ median) expression of miR-182-5p and miR-205-5p with the prognosis of the complete cohort as well as the p16-stratified subgroups

Endpoint	Complete cohort ( $n=$ 45)		p16-positive subgroup ( $n=$ 26)		p16-negative subgroup ( $n=$ 19)
	miR-182-5p	miR-205-5p	miR-182-5p	miR-205-5p	miR-182-5p	miR-205-5p
OS
HR (95% CI)	2.17 (0.74–6.35)	1.6 (0.57–4.50)	3.14 (0.61–16.24)	2.85 (0.55–14.76)	1.52 (0.36–6.4)	1.06 (0.26–4.28)
$P^{\text{a}}$	0.148	0.369	0.150	0.192	0.562	0.934
DSS
HR (95% CI)	1.79E $+$ 09 (0–Inf)	1.84 (0.44–7.7)	1.98E $+$ 09 (0–Inf)	3.31 (0.34–31.88)	1.56E $+$ 09 (0–Inf)	0.42 (0.04–4.1)
$P^{\text{a}}$	0.003	0.399	0.028	0.273	0.051	0.445
PFS
HR (95% CI)	1.97 (0.86–4.51)	2.58 (1.1–6.07)	1.87 (0.53–6.65)	4.62 (0.98–21.83)	1.38 (0.48–4)	1.96 (0.67–5.75)
$P^{\text{a}}$	0.102	0.024	0.323	0.034	0.551	0.211
RFS
HR (95% CI)	2.62 (0.99–6.92)	2.94 (1.11–7.78)	2.12 (0.51–8.89)	3.41 (0.69–16.92)	1.85 (0.54–6.38)	1.97 (0.59–6.55)
$P^{\text{a}}$	0.043	0.023	0.293	0.111	0.321	0.261
LCR
HR (95% CI)	1.11 (0.22–5.49)	6.64 (0.77–57.23)	0.73 (0.07–8.11)	2.18E $+$ 09 (0–Inf)	1.72 (0.16–18.96)	3.23 (0.28–37.48)
$P^{\text{a}}$	0.9	0.047	0.800	0.048	0.654	0.326

Abbreviations: 95% CI $=$ 95% confidence interval; DSS, disease-specific survival; HR, Hazard Ratio; inf, infinite; LCR, local control rate; OS, overall survival; miR, microRNA; PFS, progression-free survival; $P$ , $P$ -value; RFS, recurrence-free survival. Notes: Significant $P$ -values ( $P<$ 0.5) in bold. ${}^{\text{a}}P$ -value calculated with log-rank test.

Table 3 summarizes the analysis of miR-182-5p and miR-205-5p for prognostic stratification of the complete cohort as well as the p16-stratified subgroups.

3.6 Analysis of potential cofounders

In order to identify potential prognostic relevant cofounders, the association of clinicopathological characteristics and survival endpoints were analyzed. Therefore, patients were stratified according to clinicopathological characteristics, that were also analyzed for difference of miRNA expression and their association with prognosis. Results are illustrated in Supplementary Table S1. Referring to the complete cohort, lower histopathological differentiation (G1-2 vs. G3) was significantly associated with an inferior OS (HR $=$ 2.84, 95% CI 0.96–8.41, $P=$ 0.049). There was no further significant association between any other tested characteristics and one of the other endpoints in this group.

In the p16-positive subgroup, nodal involvement (N $+$ vs. N0) was significantly associated with a superior OS (HR $=$ 0.21, 95% CI 0.04–1.1, $P=$ 0.042), but all of the patients ( $n=$ 3) without lymph node metastases had an advanced local primary tumor (pT3-4a), of which two of them died early due to postoperative complication and intercurrently 13.5 months after initial diagnosis. Regarding the other endpoints and characteristics, no further statistical trend or significance towards a survival discrepancy was observed within the p16-positive subgroup.

In the p16-negative subgroup an advanced T-category (c/pT1-2 vs. c/pT3-4a/b) was significantly associated with worse PFS and RFS (PFS: HR $=$ 5.00, 95% CI 1.34–18.67, $P=$ 0.009; RFS: HR $=$ 4.00, 95% CI 1.01–15.87, $P=$ 0.034). Comparing patients without lymph node metastases and those with lymph node metastases, but without extracapsular spread (N0 and N $+$ ECS neg.) to those with extracapsular spread (N $+$ ECS pos.), the latter showed an inferior prognosis for OS and PFS (OS: HR $=$ 5.81, 95% CI 1.12–30.05, $P=$ 0.019; PFS: HR $=$ 4.97, 95% CI 1.3–18.96, $P=$ 0.010).

Finally, comparing all these results (association of clinicopathological characteristics with prognosis; Supplementary Table S1) with the univariate analyses of the miRNAs (association of miRNA expression with prognosis; Table 3), there was no overlap between a significant association of any of the clinicopathological characteristics, or one of the miRNAs with one of the prognostic endpoints in the same group, respectively. Therefore, the significant associations observed in the univariate analysis between miR-182-5p or miR-205-5p and prognosis were considered as independent of any of the tested clinicopathological characteristics and thus, no further multivariate testing was performed.

4. Discussion

An increasing subset of OPSCC is associated with HPV-infections [1, 63, 64, 65]. Corresponding to the observed prognostic superiority of patients with p16-/HPV-positive disease, trials investigated altered therapeutic treatment strategies for “HPV associated” OPSCCs [6, 7]. Nevertheless, to which extent the HPV/p16-status can be applied for appropriate patient selection is still widely debated [8, 66, 67].

Additional biomarkers could help in order to refine the prognostic value of HPV/p16 and provide more detailed information about the molecular [15, 16, 20] and clinical [68] diversity between HPV/p16-positive and HPV/p16-negative OPSCCs [8, 9, 10].

MiRNAs were described as potential important players in carcinogenesis, immune, and treatment response [9, 10, 11] that are also dysregulated in the course of HPV-infections [12, 13]. Combined with their relatively stable tumor-type specific expression, as well as stability and reliable detectability in clinical specimens, they were suggested as potential biomarkers and therapeutic targets for various cancers [9, 11].

A limited number of studies investigated miRNAs’ associations with clinicopathological characteristics and prognosis within a uniform OPSCC cohort with in addition considering the tumor’s p16- or HPV-status [15, 16, 17, 18, 19, 20, 21, 22]. The current study analyzed the diagnostic and prognostic implications of miR-182-5p and miR-205-5p in a well characterized, p16-stratified cohort of 45 patients with OPSCCs treated with curative intent.

4.1 miR-182

miR-182 was considered to act oncogenic but also tumor-suppressive in HNSCC. For example higher expression of this miRNA was indicated to enhance cell-cycle progression, colony formation, invasiveness and proliferative capacity e.g. by directly suppressing genes like RASA1 and SPRED1, leading to an activation of the Ras-MEK-ERK pathway [27]. The proliferation of tumor cells could also be affected by miR-182-5p through targeting genes like CAMK2N1 [25]. Another way miR-182 could mediate cell migration and the evasion of invasion control and thereby influence the evolution and progression of HNSCCs is by suppress the gene of the Metastasis Suppressor Protein 1 [24]. Nevertheless, enhanced proliferative, migratory, or invasive ability of HNSCC cells, which was associated with miR-182 expression could be reverted by an induced expression of the genes $\beta$ -TrCP2 [28] and Numb [23]. This points to the regulation of miR-182 as a new approach for future therapies. However, there is evidence that restoration of homeostasis, rather than reduced expression of miR-182, should then likely be directed. For instance, inhibition of the repressive function of miR-182 on genes such as PDK4 could also mediate dysregulation of cellular metabolism, facilitating carcinogenesis and proliferation [29]. Furthermore, pro-apoptotic and therefore tumor-suppressive effects of miR-182 possibly through repression of the gene CRR9 have been described, for example, with respect to LSCCs [26].

In the present study miR-182-5p was not significantly dysregulated between OPSCCs and benign tissues, irrespective of the p16-status. Unaltered tissue levels of miR-182-5p are in accordance with another study focusing on OPSCCs [16]. In contrast, miR-182 was described to be downregulated in tissue and cell lines of laryngeal cancer (LSCC) [26]. Whereas, an upregulation was observed in cell lines [24, 25, 27] and specimens deriving from oral SCC (OSCC) [24, 25, 27, 69], as well as pooled HNSCC datasets [23, 28, 36, 70]. Interestingly, Ganci et al. observed a significant upregulation of miR-182-5p in TP53-mutated only, and not in the TP53 wild-type HNSCC [36]. Consistent with this, Wang et al. described an upregulation of miR-182 predominantly in p53-overexpressing HNSCC tumor tissue [28]. Therefore, this potential dependency of miR-182-5p expression and tumor’s TP53/p53-status, suggests a promising approach for further investigations of the differences of miR-182-5p dysregulation and its role in cancer of the head and neck region.

Furthermore, in vitro and in vivo experiments indicated, that the oncoprotein E7 of high-risk HPV is able to cause an upregulation of miR-182 in tumor, as well as adjacent normal cells via activating the TGF- $\beta$ /Smad4 signaling pathway [12]. Additionally, miR-182 itself was reported to have a predicted binding-site for the mRNA of the HPV16 oncoprotein E6 [19]. Therefore, dysregulation of miR-182 could not only be dependent on tumor’s TP53/p53-, but also its HPV/p16-status. Nevertheless, in the current study no significant differential tissue levels of miR-182-5p were observed between p16-positive vs. p16-negative OPSCCs or within p16-stratified subgroups sharing certain clinicopathological characteristics. This indicated an independent expression of miR-182-5p of these variables. In accordance with this, miR-182 was not among the HPV/p16-associated miRNAs reported for OPSCCs [16, 71, 72].

In contrast to the current study, associations between miR-182-5p with poor differentiation, advanced T-category, and lymph node metastases were observed within an OSCC dataset [24], indicating that miR-182-5p might serve as a marker for a more aggressive diseases. These aspects are consistent with the above-mentioned oncogenic potential of miR-182 [23, 24, 25, 27, 28], its association with an enhanced proliferation, cell-cycle progression, invasiveness, colony formation, migration, and reduced apoptosis in vitro [25, 27, 28], as well as an extended tumor volume, and growth rate in vivo [25, 28]. Moreover, it is in accordance with the herein observed association between higher levels of miR-182-5p with an inferior DSS and RFS in the complete cohort, an unfavorable DSS within the p16-positive subgroup, and a similar trend in the p16-negative subgroup. This is in line with a meta-analysis focusing on different types of cancer [46] and the inverse association between miR-182 with prognosis, e.g. within patients with glioma [44], prostate [43], hepatocellular [42], and colorectal [45, 47] cancer. Nevertheless, it contradicts the positive prognostic association found in, e.g. patients with NSCLC [59].

In summary, the results of the present study indicate, that although miR-182-5p was not dysregulated in OPSCCs compared to benign tissues, higher expression levels in OPSCC at initial diagnosis may help to identify patients with an inferior prognosis. Consequently, they could be excluded from, e.g. de-escalation trials for p16-positive OPSCCs.

4.2 miR-205

Within HNSCC miR-205 could act tumor-suppressive via targeting genes like Tissue Inhibitor of Metallopro- teinases-2 leading to a reduced activation of Pro-matrix Metalloproteinase-2 and finally in a reduced migration and invasion of cancer cells [30]. Furthermore miR-205 was negatively associated with the expression of proliferative markers like the dihydrofolate reductase and the proliferating cell nuclear antigen as well as the apoptotic regulator Bcl-2. In vivo, the induced expression of miR-205 was further associated with an increase in apoptotic cells in xenograft tumors [31]. Nevertheless, miR-205 could also positively influence the progression of HNSCCs, e.g. through inducing genomic instability by targeting the DNA repair proteins of BRCA1 and RAD17 [35] or leading to stemmness associated characteristics and an enhanced tumor growth via targeting COMMD1 [32]. Also through the repression of CDK2AP1 and the induction of proliferation and invasion, miR-205 could potentially contribute the progression of HNSCCs [33]. Last miR-205 was also discussed to negatively affect the response to radiotherapy by regulating the well-known tumor suppressor PTEN, leading to an activation of AKT in HNSCC [34].

In the current study, both, p16-positive and p16-negative OPSCCs exhibited at least a trend towards an upregulation of miR-205-5p compared to controls, indicating miR-205-5p elicits oncogenic functions in OPSCCs. These results are consistent with the previously described upregulation of miR-205 in tissue of laryngeal [33, 73, 74], oral [41, 69], nasopharyngeal carcinomas [75], as well as in tumor samples [62, 76, 77], sera [78], fine needle aspirations of lymph node metastases [79], and diverse cell lines [80, 81] of HNSCC. Interestingly, Ganci et al. identified an upregulation of miR-205-5p, as already described above for miR-182-5p, exclusively in TP53-mutated, but not TP53-wild-type HNSCC [36]. This corresponds to the observed upregulation of miR-205-5p in HNSCC cell lines, that overexpressed mutant p53 [38]. However, miR-205 was also considered as a marker for squamous epithelium irrespective of the tissue’s dignity [82], consistent with the unaltered levels of miR-205 observed, e.g. in OSCC [82, 83, 84]. Furthermore also downregulation of miR-205 was reported, e.g. in tissues of LSCCs [31], OSCCs [30], pooled HNSCC datasets [85]. Summarized, these results indicate a dysregulation of miR-205-5p possibly occuring in diverse HNSCCs, including p16-positive and p16-negative OPSCCs at time of initial diagnosis. However, in context with the reported ability of miR-205 to influence oncogenic, as well as tumour-suppressive genes and pathways, its potential role and therefore its up- or downregulation appears target- and tumor-specific.

Moreover, in vitro studies demonstrated an association between expression of HPV16’s oncoproteins E6 and E7 with depletion of miR-205 [13]. Although, in line with the present study, no altered tissue levels of miR-205 were reported between HPV/p16-positive vs. negative HNSCCs by others [15, 16, 62, 71, 72].

However, within the present study miR-205-5p was not significantly associated with any of other clinicopathological features within the p16-stratified subgroups and therefore was (as miR-182-5p) considered as independent from them. Nevertheless, for instance, in pooled HNSCC and LSCC datasets, high miR-205 expression levels were previously associated with absence of features that were considered indicative for more aggressive and moreover metastatic disease [31, 62]. In contrast, in a cohort including OSCCs, as well as OPSCCs an upregulation of miR-205 was significantly associated with lymph node metastases and a trend towards association with higher pathological stage [60], supporting the consideration of miR-205 as a marker for more advanced and aggressive tumors.

This is also consistent with the above described oncogenic potential of miR-205 [32, 34, 35], its association with a malign cellular phenotype [32, 34, 35], its upregulation in tumor tissues, and its association with an inferior prognosis within the current study’s complete cohort (PFS, RFS, LCR), as well as p16-positive subgroup (PFS, LCR). Fittingly, miR-205-5p expression alone, as well as in a pattern with other miRNAs, was identified as a predictor for lower RFS of HNSCC patients independent from other clinicopathological characteristics [36]. Furthermore, an inverse association between miR-205 and prognosis was reported for patients with OSCC [41], as well as outside the HN-region, e.g. for patients with endometrial [53], ovarian [51], non-small cell lung [48], esophageal SC [52] carcinomas. In contrast, higher miR-205 levels were associated with a beneficial prognosis of patients with, e.g. colon [49], prostate [50], breast [53] cancer.

Summarized, the comparison of miR-205-5p expression in tumor vs. control tissues supports that higher miR-205 levels could help to detect metastatic HNSCC cells within lymph nodes [86], or residual tumor cells at the resection margin [77]. Furthermore, results indicate that higher miR-205-5p levels could help in identifying patients with p16-positive OPSCCs and an inferior prognosis.

4.3 Strengths and limitations

In accordance with the 8th edition of the AJCC/UICC cancer staging manual [1, 3] and the 2017 (4th) edition of World Health Organization classification of malignant tumors [5], the need for distinction between HPV/p16-positive and HPV/p16-negative OPSCCs was addressed in the current study. Therefore, the strength of the study lies in the p16-stratified analyses of miRNAs based on FF tissue of the local primaries, strict clinical and laboratory inclusion criteria, and detailed clinical data, which was added by results of further pathological assessment when necessary. This way we were able to provide information to both, potential diagnostic and prognostic implications of miR-182-5p and miR-205-5p.

Limitations of this study are the small sample size, the paucity of comparable analyses in literature, and the absence of an adequate validation cohort. Potential causes for variability and sometimes conflicting results of miRNA analyses include for instance distributions of ethnicity, lifestyle factors like tobacco or alcohol consumption, and other clinicopathological characteristics like p16-/HPV-status within the study populations. Also, basic methodical differences like detection methods (microarray vs. qPCR vs. ISH etc.), type (FFPE vs. FF vs. cell lines vs. saliva etc.) and origin of the investigated biomaterials (pooled datasets vs. specific sites), as well as control groups (non-cancer patients’ specimen vs. adjacent normal tissue from cancer patients) may have contributed to partly diverging results.

Prospective validation in extended cohorts, the examination of miR-182-5p and miR-205 in the context of recurrent or metastatic disease, as well as analysis in different biomaterial (e.g. non-invasive acquirable saliva samples) could further emphasize the translational relevance of the current study’s results. Our findings could serve as a basis for further research on miRNA-based biomarkers in p16-positive and p16-negative OPSCCs.

4.4 Conclusion

In conclusion, miR-182-5p and miR-205-5p further stratified patients with p16-positive OPSCC into prognostic groups. These findings may have implications for the development of a more precise and individualized oncological management.

Author contributions

Conception: Bernhard G. Weiss, Mahalia Zoe Anczykowski, Julia Kitz, Mark Jakob.

Interpretation or analysis of data: Bernhard G. Weiss, Mahalia Zoe Anczykowski, Friedrich Ihler, Mattis Bertlich, Jennifer L. Spiegel, Frank Haubner, Martin Canis, Stefan Küffer, Julia Hess, Kristian Unger, Julia Kitz, Mark Jakob.

Preparation of the manuscript: Bernhard G. Weiss, Mahalia Zoe Anczykowski, Jennifer L. Spiegel.

Revision for important intellectual content: Bernhard G. Weiss, Mahalia Zoe Anczykowski, Friedrich Ihler, Mattis Bertlich, Jennifer L. Spiegel, Frank Haubner, Martin Canis, Stefan Küffer, Julia Hess, Kristian Unger, Julia Kitz, Mark Jakob.

Supervision: Bernhard G. Weiss, Mahalia Zoe Anczykowski, Julia Kitz, Mark Jakob.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-203149.

sj-docx-1-cbm-10.3233_CBM-203149.docx - Supplemental material

Supplemental material, sj-docx-1-cbm-10.3233_CBM-203149.docx

sj-pdf-1-cbm-10.3233_CBM-203149.pdf - Supplemental material

Supplemental material, sj-pdf-1-cbm-10.3233_CBM-203149.pdf

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MicroRNA-182-5p and microRNA-205-5p as potential biomarkers for prognostic stratification of p16-positive oropharyngeal squamous cell carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Patients and methods

2.1 Patients and specimens

2.2 Staging, treatment and follow-up

2.3 Tissue analyses

2.3.1 p16-immunohistochemistry and HPV-typing

2.3.2 RNA extraction, cDNA synthesis, microRNA quantification

2.4 Statistics

3. Results

3.1 Patients and therapy

Table 1 Patient and disease characteristics and follow-up data of the complete cohort and the p16-stratified subgroups

3.3 miR-182-5p and miR-205-5p expression in tumor versus normal tissues

3.4 miRNAs and clinicopathological characteristics

Table 2 Associations of miR-182-5p and miR-205-5p with clinicopathological characteristics in the complete cohort as well as in the p16-stratified subgroups

4. Discussion

4.1 miR-182

4.2 miR-205

4.3 Strengths and limitations

4.4 Conclusion

Author contributions

Supplementary data

sj-docx-1-cbm-10.3233_CBM-203149.docx - Supplemental material

sj-pdf-1-cbm-10.3233_CBM-203149.pdf - Supplemental material

References

Table 1
Patient and disease characteristics and follow-up data of the complete cohort and the p16-stratified subgroups

Table 2
Associations of miR-182-5p and miR-205-5p with clinicopathological characteristics in the complete cohort as well as in the p16-stratified subgroups