Open Access

Absence of high-risk HPV 16 and 18 in Chinese patients with oral squamous cell carcinoma and oral potentially malignant disorders

Virology Journal201613:81

https://doi.org/10.1186/s12985-016-0526-2

Received: 14 February 2016

Accepted: 11 April 2016

Published: 20 May 2016

Abstract

Background

The critical role of human papillomavirus (HPV) in cancer has been recognized, but the involvement of HPV in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD) is still controversial. The aim of this study was to identify and verify the prevalence of high-risk HPV infection (HPV16 and 18) in Chinese patients with OSCC or OPMD using real-time PCR and DNA sequencing.

Methods

Paired tissue and serum DNA samples were extracted from 40 Chinese patients with OSCC and 59 with OPMD. A SYBR Green-based real-time PCR assay was developed to detect the E6 gene of HPV16 and HPV18. Suspicious positive samples were then sequenced to eliminate false positives.

Results

We found that none of the tissue and serum samples of OSCCs and OPMDs were positive for HPV16 E6 or 18 E6, using both real-time PCR and DNA sequencing. Overall, 3 of 198 (1.52 %) and 7 of 198 (3.54 %) samples were false-positive for HPV16 E6 and HPV18 E6, respectively, using real-time PCR.

Conclusion

The lack of HPV16 and HPV18 detected in this study indicates that high-risk HPV 16 and 18 infections are uncommon in Chinese patients with OSCC and OPMD. Real-time PCR followed by DNA sequencing for HPV DNA detection is an effective strategy to rule out false positives.

Keywords

HPV16HPV18Oral squamous cell carcinomaOral potentially malignant disorders

Background

Head and neck squamous cell carcinoma (HNSCC), which includes squamous cell carcinomas of the oral cavity, oropharynx, larynx, and hypopharynx, is the sixth most common cancer worldwide [1, 2]. Two main risk factors related to HNSCC are tobacco use and alcohol consumption [3]. Recently, investigators have suggested that human papillomavirus (HPV) is a potential etiological factor of HNSCC in patients who do not smoke or drink alcohol, particularly in oropharynx squamous cell carcinoma (OPSCC) [4, 5]. The oncogenic proteins E6 and E7 of high-risk HPVs, such as HPV16 and HPV18, are considered to be associated with the carcinogenic process of OPSCC by inactivating the tumor suppressor genes p53 and Rb [6, 7]. However, the rate of detecting HPV in OSCC varies widely (0–100 %), and the role of HPV in oral carcinogenesis has long been controversial [8].

HPV has been detected in not only cervical cancer but in cervical premalignant lesions as well, and the detection rate is known to increase with the severity of disease abnormality [9]. Oral lesions and conditions associated with a risk of malignant transformation have been referred to as oral potentially malignant disorders (OPMD) and include oral leukoplakia (OLK), lichen planus, and erythroplakia [10]. Recent studies have revealed a varying rate of detected HPV in OPMD [8]. A better understanding of the true presence of HPV in OSCC and OPMD may thus contribute to further studies of these diseases.

Different techniques have been used to detect HPV, including in situ hybridization (ISH), Southern blot hybridization, dot blot hybridization, hybrid Capture 2 (hc2), conventional PCR, and real-time PCR [11]. ISH, Southern blot and dot blot hybridization are time-consuming procedures that require relatively large amounts of purified DNA [11]. Hc2 assay cannot genotype single HPV subtypes [11]. Of these methods, studies using PCR techniques have reported a higher sensitivity for HPV detection [12]. However, conventional PCR assays may have a lower sensitivity and specificity [11]. Real-time PCR has a sensitivity of 92 % and a specificity of 97 % in detecting HPV and is able to genotype and quantitate HPV viral load [13].

The aim of our study was to identify the detection rate of high-risk HPV types 16 and 18 in Chinese patients with OSCC and OPMD using real-time PCR and DNA sequencing.

Methods

Subjects

A total of 99 patients including 40 OSCC and 59 OPMD patients were enrolled from the Department of Oral Mucosal Diseases and the Department of Oral Maxillofacial Surgery at the Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine. Paired tissue and serum samples were collected from each patient. Tissue samples were immediately frozen at −80 °C after surgery. Serum was obtained from the supernatant of the collected whole blood and stored at −80 °C until processing. Histological diagnoses were made by one pathologist who was on duty and confirmed by a superior pathologist according to the World Health Organization criteria [14, 15]. This study was approved by an Independent Ethics Committee of Shanghai Ninth People's Hospital affiliated to Shanghai Jiao Tong University, School of Medicine (#200703), and signed informed consent was obtained from each patient. The baseline characteristics of the patients are presented in Table 1.
Table 1

The baseline characteristic of patients

ID

Diagnosis

Age

Gender

Smoking

Alcohol

Stage TNMa

Notes

Tumor Site

Type

CXJ 1

OSCC

77

M

Past

Never

 

real-time PCR

Tongue

 

CXJ 2

OLK

48

F

Never

Sometimes

 

real-time PCR

Gingiva

 

CXJ 3

OLK

36

M

Current

Current

 

real-time PCR

Buccal

 

CXJ 4

OSCC

63

M

Never

Past

 

real-time PCR

Tongue

 

CXJ 5

OSCC

54

M

Current

Past

 

real-time PCR

Buccal

Papillary

CXJ 6

OSCC

60

M

Current

Current

 

real-time PCR

Buccal

 

CXJ 7

OSCC

41

M

Current

Current

 

real-time PCR

Tongue

 

CXJ 8

OSCC

53

M

Past

Current

 

real-time PCR

Buccal

 

CXJ 9

OSCC

41

M

Current

Current

 

real-time PCR

Floor of mouth

 

CXJ 10

OSCC

69

F

Never

Never

T1M0N0

real-time PCR & DNA sequencing (18 ZDNA)

Gingiva

 

CXJ 11

OSCC

56

M

Current

Never

 

real-time PCR & DNA sequencing (16 SDNA)

Buccal

 

CXJ 12

OSCC

60

F

Never

Never

 

real-time PCR

Gingiva

 

CXJ 13

OSCC

58

M

Current

Sometimes

 

real-time PCR

Gingiva

 

CXJ 14

OSCC

57

F

Never

Never

T2N0M0

real-time PCR

Tongue

 

CXJ 15

OSCC

55

M

Sometimes

Sometimes

 

real-time PCR

Hard palate

Papillary

CXJ 16

OSCC

75

M

Never

Sometimes

 

real-time PCR

Buccal

 

CXJ 17

OSCC

66

F

Never

Never

T4N1M0

real-time PCR

Buccal

 

CXJ 18

OSCC

63

M

Never

Never

T4N0M0

real-time PCR

Buccal

 

CXJ 19

OSCC

43

M

Current

Current

T4N0M0

real-time PCR

Gingiva

 

CXJ 20

OLK

65

M

Never

Sometimes

 

real-time PCR

Hard palate

 

CXJ 21

OLK

56

M

Current

Sometimes

 

real-time PCR

Buccal

 

CXJ 22

OLK

78

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 24

OSCC

59

M

Current

Current

 

real-time PCR

Gingiva

 

CXJ 25

OSCC

54

F

Never

Never

T1N0M0

real-time PCR

Tongue

 

CXJ 26

OSCC

72

M

Never

Never

T3N0M0

real-time PCR

Tongue

 

CXJ 27

OSCC

75

F

Never

Never

T1N0MO

real-time PCR

Tongue

 

CXJ 28

OSCC

40

M

Sometimes

Never

 

real-time PCR

Gingiva

 

CXJ 30

OLK

56

F

Never

Never

 

real-time PCR & DNA sequencing (16 SDNA)

Gingiva

 

CXJ 31

OLK

60

M

Current

Never

 

real-time PCR

Gingiva

 

CXJ 32

OSCC

44

M

Current

Current

 

real-time PCR

Floor of mouth

 

CXJ 33

OLK

65

M

Never

Never

 

real-time PCR

Buccal

 

CXJ 34

OSCC

81

M

Never

Never

 

real-time PCR

Lip

 

CXJ 35

OLK

63

M

Never

Never

 

real-time PCR

Gingiva

 

CXJ 36

OSCC

58

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 37

OLK

72

M

Never

Never

 

real-time PCR

Gingiva

 

CXJ 38

OLK

75

M

Never

Never

 

real-time PCR

Buccal

 

CXJ 39

OLK

73

M

Past

Never

 

real-time PCR

Tongue

 

CXJ 40

OSCC

60

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 41

OLK

36

M

Never

Sometimes

 

real-time PCR

Gingiva

 

CXJ 42

OLK

57

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 43

OLK

51

M

Past

Past

 

real-time PCR

Tongue

Verrucous

CXJ 44

OLK

54

M

Past

Never

 

real-time PCR

Gingiva

 

CXJ 45

OLK

56

M

Never

Sometimes

 

real-time PCR

Tongue

 

CXJ 46

OLK

66

F

Current

Never

 

real-time PCR

Gingiva

 

CXJ 47

OLK

62

M

Never

Past

 

real-time PCR

Tongue

 

CXJ 48

OLK

50

F

Never

Never

 

real-time PCR

Gingiva

 

CXJ 49

OSCC

63

M

Current

Sometimes

 

real-time PCR

Buccal

 

CXJ 50

OLK

53

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 51

OLK

54

M

Current

Past

 

real-time PCR

Soft palate

Verrucous

CXJ 52

OLK

30

M

Current

Sometimes

 

real-time PCR

Tongue

 

CXJ 53

OLK

62

M

Current

Sometimes

 

real-time PCR

Soft palate

 

CXJ 54

OLK

64

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 55

OSCC

70

M

Past

Current

 

real-time PCR

Buccal

 

CXJ 56

OLK

50

F

Never

Never

 

real-time PCR & DNA sequencing (16 SDNA, 18 SDNA)

Tongue

 

CXJ 57

OSCC

73

F

Never

Never

 

real-time PCR & DNA sequencing (18 SDNA)

Buccal

 

CXJ 58

OLK

59

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 59

OLK

62

F

Never

Never

 

real-time PCR

Gingiva

 

CXJ 60

OLK

57

F

Current

Never

 

real-time PCR

Tongue

 

CXJ 61

OLK

51

M

Current

Never

 

real-time PCR

Tongue

 

CXJ 62

OLK

50

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 63

OSCC

67

M

Current

Never

 

real-time PCR

Buccal

 

CXJ 64

OLK

64

M

Never

Never

 

real-time PCR

Tongue

 

CXJ 65

OLK

45

F

Never

Never

 

real-time PCR

Gingiva

 

CXJ 66

OLK

60

M

Never

Never

 

real-time PCR

Buccal

 

CXJ 67

OLK

66

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 68

OSCC

38

M

Current

Sometimes

 

real-time PCR

Tongue

 

CXJ 69

OSCC

61

M

Past

Past

 

real-time PCR & DNA sequencing (18 ZDNA)

Buccal

 

CXJ 70

OLK

52

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 71

OLK

35

M

Past

Sometimes

 

real-time PCR

Buccal

 

CXJ 72

OLK

58

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 73

OLK + EK

37

M

Past

Past

 

real-time PCR

Tongue

 

CXJ 74

OSCC

34

M

Current

Current

 

real-time PCR

Tongue

 

CXJ 75

OSCC

53

M

Current

Current

 

real-time PCR

Tongue

 

CXJ 76

OLK

71

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 77

OSCC

58

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 78

OLK

58

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 79

OLK + EK

37

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 80

OLK

53

M

Past

Current

 

real-time PCR

Tongue

 

CXJ 81

OSCC

58

M

Past

Sometimes

 

real-time PCR

Tongue

 

CXJ 82

OLK

55

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 83

OLK

53

M

Current

Current

 

real-time PCR

Tongue

 

CXJ 84

OLK

53

M

Current

Current

 

real-time PCR

Hard palate

Verrucous

CXJ 85

OLK

54

F

NA

NA

 

real-time PCR

Tongue

 

CXJ 86

OLK

54

F

Never

Never

 

real-time PCR

Tongue

 

CXJ 87

OLK

63

M

Sometimes

Current

 

real-time PCR

Tongue

 

CXJ 88

OLK

72

M

Never

Current

 

real-time PCR

Gingiva

 

CXJ 89

OLK

79

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 90

OLK

55

M

Past

Sometimes

 

real-time PCR

Tongue

 

CXJ 91

EK

45

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 94

OLP

54

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 95

OLP

54

F

Never

Never

 

real-time PCR & DNA sequencing (18 SDNA)

Buccal

 

CXJ 96

OLP

29

M

Current

Sometimes

 

real-time PCR

Buccal

 

CXJ 97

OLP

40

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 98

OLP

58

F

Never

Never

 

real-time PCR

Buccal

 

CXJ 99

OLP

28

M

Current

Never

 

real-time PCR

Buccal

 

CXJ 100

OSCC

28

M

Current

Current

 

real-time PCR & DNA sequencing (18 ZDNA)

Buccal

 

CXJ 101

OSCC

62

F

Never

Never

 

real-time PCR

Gingiva

 

CXJ 102

OSCC

68

M

Never

Never

 

real-time PCR

Tongue

 

CXJ 103

OSCC

59

M

Never

Never

 

real-time PCR & DNA sequencing (18 ZDNA)

Buccal

 

OSCC oral squamous cell carcinoma, OLK oral leukoplakia, OLP oral lichen planus, EK oral erythroplakia, ZDNA tissue DNA, SDNA serum DNA, NA data not available

aUnion for International Cancer Control; T, tumor size; N, lymph node; M, Metastasis

Cell culture

The CAL27 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MA, USA) and was grown in Dulbecco’s Modified Eagle Medium (HyClone, Logan, UT, USA) containing 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin solution at 37 °C in 5 % CO2.

DNA extraction

Twenty 20-μm sections were cut from the frozen tissue samples, and DNA was extracted using the QIAamp DNA Micro Kit (Qiagen, Düsseldorf, Germany). Serum DNA extraction was performed using the QIAamp DNA Blood Mini Kit (Qiagen, Düsseldorf, Germany). CAL27 cells were detached by trypsinization and extracted DNA with QIAamp DNA Mini Kit (Qiagen, Düsseldorf, Germany). The plasmid pB-actin 16 E6 and pB-actin 18 E6 were bought from Addgene (Cambridge, MA, USA). Plasmid DNA was extracted using the QIAfilter MidiKit (Qiagen, Düsseldorf, Germany). Purified plasmid DNA were sequenced and blasted with HPV16 E6 (NC_001526.2) and HPV18 E6 (NC_001357.1) NCBI reference sequence. The extracted DNA was stored at −80 °C until further use.

Real-time PCR and sequencing

Real-time PCR was performed by LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) together with 0.5 μmol/L of each primer and 50 ng DNA in a 10 μl reaction were utilized. Positive controls were performed, which including HPV plasmid DNA, HPV containing cell line DNA and small amount of plasmids added to clinical sample DNA (Fig. 1). Negative controls were also performed, which including pure water, pure water instead of 2 × master mixture, pure water instead of positive control DNA (Fig. 1). A standard curve was developed for both HPV16 E6 (Fig. 2a) and HPV18 E6 (Fig. 2b) using a series of 10-fold diluted plasmid DNA 1 ng to 0.1 pg. The quantitated data was normalized by beta-actin (ACTB) using CAL27 genomic DNA. The reaction was performed by initiation at 95 °C for 5 min followed by 35 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s. Each sample was performed in triplicate. A sample was considered positive for HPV infection if two or three wells of the triplicate showed an amplifying curve. It was under suspicion if the amplifying curve was detected later than the 30th cycle of the reaction or had a deformed shape. The suspicious samples of HPV16 E6 or HPV18 E6 were then sequenced to rule out false positives. All primers are shown in Table 2.
Fig. 1

Positive and negative controls for HPV16 and HPV 18 with real-time PCR. a Positive and negative controls for HPV16; b Positive and negative controls for HPV18. Standard curve 1–5, 10-fold diluted HPV16 E6 or HPV18 E6 plasmid DNA ranging from 1 ng/well to 0.1 pg/well. Positive control 1, cilnical DNA sample added with 0.1 pg HPV16 E6 or HPV18 E6 DNA. Positive control 2, 50 ng Hela cell DNA. Negative control 1, pure water. Negative control 2, pure water instead of 2 × master mixture. Negative control 3, pure water instead of positive control DNA

Fig. 2

Standard curves for HPV16 and HPV 18 E6. a Standard curves for 10-fold diluted HPV16 E6 plasmid DNA ranging from 0.1 ng/well to 0.1 pg/well; b Standard curves for 10-fold diluted HPV18 E6 plasmid DNA ranging from 1 ng/well to 0.1 pg/well

Table 2

Sequence of HPV16 and HPV18 E6 primers and ACTB primers used for real-time PCR

Name

Sequence

HPV16 E6-F

GTCATATACCTCACGTCGCAG

HPV16 E6-R

AGCGACCCAGAAAGTTACCAC

HPV18 E6-F

GTTTCTCTGCGTCGTTGGAG

HPV18 E6-R

GGTGCCAGAAACCGTTGAAT

ACTB-F

TCCCTCTCAGGCATGGAGTC

ACTB-R

AATGCCAGGGTACATGGTGG

Results

Real-time PCR was conducted to detect HPV16 E6 and HPV18 E6 DNA. We found that zero of the 99 tissue samples (0 %) showed a standard amplifying curve for HPV 16 E6, but a few samples showed late or deformed amplifying curves in one of the triplicates, which were clearly not considered to be positive (Fig. 3a). Thirty-nine of 40 OSCC and 57 of 59 OPMD serum samples did not show a standard amplifying curve for HPV 16 E6 using real-time PCR, but 1 OSCC and 2 OPMD serum samples had a late or deformed amplifying curve in two or three wells of the triplicate that was suspicious (Fig. 3b). In addition, 36 of 40 OSCC and all 59 OPMD tissue samples were negative for the standard amplifying curve of HPV 18 E6, but 4 OSCC tissue samples presented a late and deformed amplifying curve in two or three wells of the triplicate (Fig. 3c). Thirty-nine of 40 OSCC and 57 of 59 OPMD serum samples were negative for the standard amplifying curve of HPV 18 E6, but 1 OSCC and 2 OPMD serum samples had late and deformed amplifying curves in two or three wells of the triplicate (Fig. 3d). DNA sequence analysis was then performed on the suspicious samples, which found that all of the samples sequenced were negative for HPV16 and HPV18. Overall, 3 of 198 (1.52 %) and 7 of 198 (3.54 %) samples were false-positive for HPV16 E6 and HPV18 E6, respectively, using real-time PCR. Overall, none of the OSCC or OPMD cases were positive for HPV 16 or18 in our study.
Fig. 3

Amplification curves for HPV16 and HPV 18 with real-time PCR. a Detection of HPV16 E6 in tissue samples; b Detection of HPV16 E6 in serum samples; c Detection of HPV18 E6 in tissue samples; d Detection of HPV18 E6 in serum samples

Discussion

In the past few decades, there has been speculation worldwide about the role of HPV in the pathogenesis of HNSCC. The most commonly detected HPV, HPV16, accounts for 90 % of the HPV DNA-positive cases in HNSCC, followed by HPV18 and other high-risk subtypes [16]. However, the detection rate of HPV in OSCC and OPMD varies widely and remains controversial [8, 17]. This variation may due to differences in the types of sample, detection methods or geographic locations [8, 18]. Therefore, confirming the HPV infection rate in OSCC and OPMD cases may contribute to the study of carcinogenesis in the oral cavity [19, 20]. In this study, we used real-time PCR to detect HPV16 and HPV18 in paired tissue and serum samples of Chinese OSCC and OPMD patients [21]. We conducted complementary analyses to verify the results of the real-time PCR with DNA sequencing. We found that none of the patients with OSCC or OPMD demonstrated existence of high-risk HPV16 or HPV18. The absence of HPV DNA in our sample implies that HPV infection may not be common in Chinese patients with OSCC and OPMD.

A critical step in malignant transformation is the integration of high-risk HPV DNA into the human cellular genome, followed by the expression of the oncoproteins E6 and E7, which promote tumor progression [21]. In a previous study, although the reported detection rate of high-risk HPV DNA in OSCC was 6.6 %, HPV mRNA was only detected in 5.9 % [22]. These findings indicated that the mRNA or oncoproteins of HPV E6 and E7 were less commonly found than the DNA, as the presence of HPV in the genome differed from the HPV-related etiology [23, 24]. The gold standard to identify the presence of HPV was therefore suggested to be detecting HPV DNA [25].

Yadav et al. showed that the HPV DNA detection limit for conventional PCR was 200 copies, whereas for real-time PCR, which has a higher sensitivity, detecting HPV DNA required only 1 copy [26]. Lingen et al. detected high-risk HPV DNA in 9.8 % of OSCC cases using consensus primer PCR, but the positive rate was 6.6 % using real-time PCR [22]. Scapoli et al. found the detection rate of HPV16 to be 2 % in OSCC with real-time PCR [27]. Real-time PCR shows a higher sensitivity and specificity than conventional PCR assays [12, 22, 26]. In the current study, we utilized real-time PCR and found that 3 of 198 samples showed late and deformed amplifying curves of HPV 16 E6 and 7 of 198 samples had late and deformed amplifying curves of HPV 18 E6. To rule out false positives, we performed subsequent sequencing and found that the rate of false positives using real-time PCR to detect HPV16 E6 and HPV18 E6 DNA was 1.52 and 3.54 %, respectively. Ha et al. found a 2 % false-positive rate for real-time PCR using the minimum criteria of HPV DNA copy number, which was similar to our results [12].

The population has also been considered to be another factor affecting rate diversification. Several countries have revealed a zero detection rate of OSCC, including India [2830], Brazil [31], Japan [32] and Mozambique [33]. Other reported detection rates have been 1.54 % in Thailand [34], 6.6 % in America [22], 5 % in Mexico [35], 39.4 % in Spain [36] and 66.7 % in Sudan [37]. Studies performed in China have yielded varied results using conventional PCR assays, ranging from 2.2 to 74 % [3842]. However, real-time PCR data for OSCC has not been reported in China. Our study revealed a zero detection rate of HPV16 and 18 in OSCC by combining real-time PCR and DNA sequencing, which was a reliable method and provided further understanding of HPV infection in Chinese patients.

HPV infection has been identified in cancers of the cervix [43], vulva [44], vagina [44], anus [44], penis [45] and oropharynx [46]. It is widely accepted that OPSCCs, especially tonsillar cancers, are frequently associated with HPV infection [17]. The recent reported prevalence of HPV in OPSCC was approximately 60-70 % [47], but the corresponding rate was substantially lower and significantly varied in OSCC [8, 17]. HPV prevalence in OPSCC has been suggested to be an independent prognostic factor [47]. HPV-positive OPSCC has been shown to be distinct from HPV-negative OPSCC with regard to prognosis [4850]. However, there have been no direct correlations between HPV infection and oral carcinogenesis [23, 27, 51].

HPV has been detected not only in cancer but also in premalignant lesions, such as in lesions of the cervix and breast [9, 52, 53]. In contrast, there was a lack of HPV in premalignant lesions of the colon [5456]. Interestingly, Ha et al. demonstrated a low prevalence (1.1 %) of HPV16 in OPMD [12]. Similarly, we detected no presence of HPV 16 and 18 in Chinese patients with OPMD.

Conclusion

Overall, we demonstrated a prevalence rate of 0 % of HPV 16 and 18 in Chinese patients with OSCC and OPMD. Our data suggests that high-risk HPV16 and HPV18 infection may not be common in Chinese patients with OSCC and OPMD. Combining real-time PCR and DNA sequence for HPV DNA detection is an effective strategy to eliminate false positives.

Abbreviations

HNSCC: 

head and neck squamous cell carcinoma

HPV: 

human papillomavirus

OPSCC: 

oropharynx squamous cell carcinoma

OSCC: 

oral squamous cell carcinoma

Declarations

Acknowledgements

The authors thank the Department of Oral Pathology at the Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, for its support in diagnosis. This study was also supported by the General Program of the National Natural Science Foundation of China (no. 30872887), the Natural Science Foundation of Shanghai Municipality (no. 15ZR1424700) and the National Clinical Key Specialized Subject Construction Project ([2013]544-03).

Authors’ contributions

XJC extracted the DNA, performed real-time PCR, and drafted the manuscript. KS extracted the DNA and collected tissue and serum samples. WWJ designed the study and reviewed the manuscript drafts. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Oral Mucosal Diseases, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

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© Chen et al. 2016