Detection of High-Risk Human Papillomavirus in Oral Cavity Squamous Cell Carcinoma Using Multiple Analytes and Their Role in Patient Survival

Abstract

Purpose: Accurate detection of human papillomavirus (HPV) in oral cavity squamous cell carcinoma (OSCC) is essential to understanding the role of HPV in disease prognosis and management of patients. We used different analytes and methods to understand the true prevalence of HPV in a cohort of patients with OSCC with different molecular backgrounds, and we correlated HPV data with patient survival.

Methods: We integrated data from multiple analytes (HPV DNA, HPV RNA, and p16), assays (immunohistochemistry, polymerase chain reaction [PCR], quantitative PCR [qPCR], and digital PCR), and molecular changes (somatic mutations and DNA methylation) from 153 patients with OSCC to correlate p16 expression, HPV DNA, and HPV RNA with HPV incidence and patient survival.

Results: High prevalence (33% to 58%) of HPV16/18 DNA did not correlate with the presence of transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 positive and only 6% were both HPV DNA and HPV RNA positive. Most tumors with relatively high copy number HPV DNA and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA, and HPV RNA, either alone or in combination, did not correlate with patient survival. Nine HPV-associated genes stratified the virus-positive from the virus-negative tumor group with high confidence ( P < .008) when HPV DNA copy number and/or HPV RNA were considered to define HPV positivity, and not HPV DNA alone, irrespective of copy number ( P < .2).

Conclusion: In OSCC, the presence of both HPV RNA and p16 is rare. HPV DNA alone is not an accurate measure of HPV positivity and therefore may not be informative. HPV DNA, HPV RNA, and p16 do not correlate with patients' outcome.

Fig 1

Human papillomavirus (HPV) genome organization and locations of different primers (either consensus or type specific) or probes used in the study to detect HPV DNA and RNA. The numbers before and after the arrows represent the corresponding nucleotide number in the HPV genome. ddPCR, droplet digital polymerase chain reaction; qPCR, quantitative PCR; RT-PCR, real-time PCR.

Fig 2

p16 and human papillomavirus (HPV) DNA in oral cavity squamous cell carcinoma (OSCC). (A) Representative images of immunohistochemical staining of p16 in positive and negative OSCC tissue sections; cervical tissue was used as a positive control. (B) Relative polymerase chain reaction (PCR) amplification efficiency and sensitivity of consensus and type-specific primers for detection of HPV using HPV16 or HPV18 individual positive control cell lines (UMSCC-47 and Hep2). (C) Representative HPV DNA PCR in oral cavity tumors with both consensus and type-specific primers. OT, oral tongue.

Fig 3

Detection of HPV DNA and RNA. (A i-vi) HPV16/18 assays using quantitative polymerase chain reaction (qPCR) and (Bi-iii) droplet digital PCR (DDPCR) in OSCC. (A i,iv, B i) Standard curves were obtained by using cloned HPV16/18 plasmids. (A ii,v, B ii) Data were subsequently obtained by using the positive (UMSCC-47 and Hep2) and negative (UPCI:SCC29B and UPCI:SCC40) cell line DNA to count HPV DNA in (A iii,vi,Biii) oral cavity tumors. (C) HPV16 (top panel) and HPV18 (bottom panel) E6 and E7 mRNA expression in tumors using qPCR. Horizontal dotted lines: threshold lines for negative samples. BM, buccal mucosa; Ct, cycle threshold; OT, oral tongue.

Fig 4

(A) Kaplan-Meier survival plots linking tumors with various attributes such as grade, stage, p16 immunohistochemistry (IHC), HPV DNA, and HPV RNA. (B) Clustering of nine methylated genes stratifying the HPV-positive from the HPV-negative group of tumors along with the HPV-associated pathways in HPV-positive tumors. Cyto, cytoplasmic (staining); MDSCC; moderately-differentiated squamous cell carcinoma; Nuc, nuclear staining; PDSCC, poorly-differentiated squamous cell carcinoma; WDSCC, well-differentiated squamous cell carcinoma.

Fig A1

Increasing amount of genomic DNA from cell lines used as positive or negative controls for human papillomavirus16 (HPV16) and HPV18 quantitative polymerase chain reaction (qPCR). HPV16 qPCR using negative control (A) Hep2 and (B) UPCI:SCC029B. HPV18 qPCR using negative control (C) UMSCC-47 and (D) UPCI:SCC029B. Error bars are drawn using data from three independent experiments. Ct, cycle threshold.

Fig A2

Amplification efficiency of human papillomavirus16 E6 (HPV16E6) and HPV18L1 primers measured by polymerase chain reaction amplification of serially diluted HPV16/18 cloned plasmid copies. M, marker; N, negative control.

Fig A3

Human papillomavirus (HPV) polymerase chain reaction (PCR) performed by using different sets of consensus or type-specific primers with oral cavity squamous cell carcinoma tumor DNA. PCR for oral cavity tumors with (A) MY09/11, (B) HPV16E6, (C) HPV18L1 using cell lines as positive and negative controls, (D) HPV16E6 for batch 2 (15 tumors), (E) HPV CPI-II using cell lines as positive controls, and (F) HPV16L1 using cell lines for positive controls. BM, buccal mucosa; L, DNA ladder; N1, UPCI:SCC029B DNA (300 ng); N2, no template control (NTC); OT, oral tongue; P1, positive control cervical DNA sample 1; P2, positive control cervical DNA sample 2; P3, UMSCC-47 DNA (HPV16-positive cell line).

Fig A4

Inhibition of amplification reactions for detecting human papillomavirus (HPV) in polymerase chain reactions at high concentrations of tumor genomic DNA (used with PGMY09-11 primer) spiked with HPV-positive UMSCC-47 DNA.

Fig A5

The effect of amplification cycles on polymerase chain reactions. The genomic DNAs used for positive control (UMSCC-47) and negative control cell lines were 63.0 ng and 300 ng, respectively. dNTP, deoxynucleotide triphosphates; NTC, no template control.

Fig A6

Positive and negative cell line DNA used for threshold in droplet digital polymerase chain reaction experiment. OTSCC, oral tongue squamous cell carcinoma.

Fig A7

Kaplan-Meier survival analysis with tumors from patients according to habits, age, and nodal status. Overall survival (OS) percentages for patients who were (A) positive v negative for any habit, (B) positive for tobacco chewing v no habit, (C) positive for alcohol consumption and tobacco chewing v no habit, (D) positive for alcohol consumption and smoking v no habit, (E) age older than 40 v age younger than 40 years, and (F) their nodal status.

Fig A8

Kaplan-Meier survival analysis with (A-H) human papillomavirus (HPV) DNA and (I-J) HPV RNA. (A) Overall survival (OS) with DNA polymerase chain reaction (PCR). (B) Disease-free survival (DFS) with DNA PCR. (C) OS with DNA qPCR. (D) DFS with DNA qPCR. (E) OS with DNA ddPCR. (F) DFS with DNA ddPCR. (G) OS with HPV positive in PCR+qPCR+ddPCR vs HPV negative in PCR+qPCR+ddPCR. (H) DFS with HPV positive in PCR+qPCR+ddPCR vs HPV negative in PCR+qPCR+ddPCR. (I) DFS with HPV RNA, and (J) DFS with p16 IHC.

Fig A9

Kaplan-Meier survival analysis of tumors with (A-B) high copy number human papillomavirus (HPV) DNA and/or HPV RNA and (C-D) HPV-negative tumors with mutations in significant genes. DFS, disease-free survival; Mut, mutation; OS, overall survival; WT, wild-type.

Fig A10

Mutational frequency in tumors with mutations in three commonly mutated (Mut) genes. WT, wild-type.