OF ORAL CANCER, CERVICAL CANCER PATIENTS AND NORMAL INDIVIDUALS
Dissertation submitted to
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY
In partial fulfilment for the Degree of MASTER OF DENTAL SURGERY
BRANCH VI
ORAL PATHOLOGY AND MICROBIOLOGY
APRIL 2016
Acknowledgement
blessings.
My sincere gratitude to my post graduate teacher, Dr. K. Ranganathan, MDS, MS (Ohio), Ph.D, Professor and Head of
Department of Oral and maxillofacial pathology, Ragas Dental College and Hospital for providing me the insight that made me do this study. It was a great privilege to work under his guidance.
I owe my heartfelt gratitude to my guide, Dr. Umadevi K. Rao, Professor, Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, for her valuable guidance, constant support and encouragement. I will always be grateful for all the dedicated efforts and pain that she took in completing my work.
I offer my sincere thanks to Dr. Elizabeth Joshua, Professor, Department of Oral and maxillofacial pathology Ragas Dental College and Hospital for her guidance and support.
I earnestly thank Professor, Dr. T. Rooban, Department of Oral and Maxillofacial Pathology, Ragas Dental College for his valuable advices that helped me to improve this study.
I am extremely grateful to Dr. N. Lavanya, Dr. C. Lavanya, Department of Oral and Maxillofacial Pathology, Ragas Dental College for their support and motivation. I also thank Dr. Kavitha and Dr. Sudarsan for supporting me.
I extend my sincere gratitude and respect to Mrs Kavitha Wilson, Geneticist and Lab Manager for her technical guidance whenever needed.
I am grateful to Ms. Aarthi, Biostatistician, for her assistance with statistics. I wish to extend my thanks to the technician, Mr Rajan for his support throughout the study. I also thank Mrs Vasanthi, attender for her prayers, help and support during my post-graduate course.
I wish to acknowledge the support and help provided by Dr. Madan Mohan, Department of Oral and Maxillofacial Surgery, Karpaga Vinayaga Institute of Dental Sciences, Dr. Rajaraman, Surgical Oncologist, Government Royapettah Hospital and Dr. C. S. Mani, Surgical Oncologist, Kumaran Hospital in collecting the samples. I also acknowledge the technical support provided by Ms. Prescila, Technical head of Shrimpex Biotech Service Pvt. Ltd.
I acknowledge gratefully the help of my batch-mates Dr. Angaiyarkkanni, Dr. Deepasri, Dr. Joseph, Dr. Malarvizhi and
encouragement and support.
I am obliged to thank my parents, Mr. I. Bose and Mrs. R.S.T. Nirmala, for their love, prayers and their support in my decisions.
I thank my dear sister, Malathy and my niece, Avanthika, for their constant love and understanding.
A special thanks to my friends for being there for me always. I thank Dr. Mahalakshmi, Dr. Suhasini, Dr. Bharghavi and Dr. Nishanthini for their moral support and motivation in every juncture of my life.
The association of Human Papilloma Virus (HPV) in cervical and oropharyngeal cancer has been established. There is growing evidence that HPV contributes to oral cancer along with alcohol and tobacco use. In our study we investigated the presence of HPV 16 in the whole mouth fluid of oral cancer patients, cervical cancer patients and normal individuals.
AIM:
To quantify the expression of HPV 16 in the whole mouth fluid of oral cancer patients, cervical cancer patients and normal individuals by quantitative real time polymerase chain reaction (qPCR).
MATERIALS AND METHODS:
DNA was extracted from the whole mouth fluid of 20 patients with oral cancer, 10 patients with cervical cancer and 10 normal individuals. The DNA was extracted and quantified by quantitative real time polymerase chain reaction.
RESULTS:
Out of 40 samples, in 8 patients (40%) with oral cancer, 4 patients (40%) with cervical cancer and 7 (70%) normal individuals, HPV 16 was isolated in the whole mouth fluid. The mean HPV 16 viral load was highest in
CONCLUSION:
HPV 16 was detected in the whole mouth fluid of a subset of oral cancer patients, cervical cancer patients and normal individuals in this present study. The presence and effect needs to be studied to establish the etiologic role of HPV 16 in the pathogenesis of a subset of oral cancers.
KEYWORDS: HPV 16, oral cancer, cervical cancer, whole mouth fluid, PCR.
2. AI M AND OBJE CT IVES 4 3. MATE RI ALS AND METHO DS 5 4. REVIE W O F LITE RAT URE 21 5. RESULTS 5 3 6. DISCUSSIO N 60 7. SUMMARY AND CONCL USIO N 73 8. BIBLIO GRAPHY 7 5 9. ANNEXURES
I . I N S T I T U T I O N A L R E V I E W B O A R D A P P R O V A L F O R M I I . D I S S E R T A T I O N P R O T O C O L
I I I . C A S E S H E E T I V . C O N S E N T F O R M
V . V A L I D A T I O N S H E E T F O R H P V 1 6 P R I M E R S V I . H P V 1 6 D N A Q U A N T I T A T I O N – F I N A L R E S U L T S V I I . A M P L I F I C A T I O N P L O T
V I I I . S T A N D A R D C U R V E I X . A B B R E V I A T I O N S
X . P L A G I A R I S M C H E C K F O R M
XI. D E P A R T M E N T D E C L A R A T I O N F O R M
Introduction
1
It has been estimated that about 14 million new cancer cases and 8 million cancer deaths occurred worldwide in 2012. India, which is home to
about 17% of the global population, accounts for 7.8% of global cancer burden and 8.3% of global cancer deaths. In India the five most common cancers in both sexes are the cancers of breast, uterine cervix, lips and oral cavity, lung and colorectum. Cervical cancer is the second most common cancer in Indian women which contributes to more than one fifth of all new cervical cancer cases diagnosed worldwide1. Head and neck squamous cell carcinoma is the sixth most common malignancy worldwide which includes malignancies of oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, paranasal sinuses and salivary glands. In India, head and neck squamous cell carcinoma accounts for 40% of all malignancies. Oropharyngeal squamous cell carcinoma and oral squamous cell carcinoma are the most common types of head and neck squamous cell carcinoma, accounting for 263,900 new cases and 128,000 deaths worldwide in 20082. In India, oral cancer accounts for one-third burden of the world in terms of incidence as well as death. Oral cancer is the second most common cancer among Indian men and fifth most common cancer among Indian women as estimated by International Agency for Research on Cancer in 20121.
Human Papilloma Viruses (HPVs) are a heterogeneous group of DNA viruses. More than 100 different types of HPV exist and at least 15 types are thought to have oncogenic potential4. HPV contribute to 99.7% of cervical
2
cancer cases worldwide. Based on their association with cervical cancer and
precursor lesions, HPV can be of three types. Low-risk HPV types include 12 HPV types namely 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 and CP6108.
High-risk HPV types include 15 types namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. Probable high-risk types include types 26, 53 and 663. The most common types in women without cervical abnormalities are HPV 16, 42, 58, 31, 18, 56, 81, 35, 33, 45 and 52. HPV 16/18 accounts for 70% of all cervical cancers, 41 to 67% of high-grade squamous intraepithelial lesions and 16 to 32% of low-grade squamous intraepithelial lesions. Other types of HPV like HPV 31, 33, 35, 45, 52 and 58 accounts for 20% of the cervical cancers worldwide4.
HPV-associated head and neck squamous cell carcinoma is an entity
with peculiar clinical and molecular characteristics. More than 90%
HPV-associated head and neck squamous cell cancers are caused by HPV 165. Among head and neck squamous cell carcinoma, HPV is closely associated with oropharyngeal squamous cell carcinoma. It is estimated that tumors in the oropharynx are five times more likely to be HPV-positive than those in the oral cavity, larynx, or hypopharynx6. The best established risk factors for oral cancer are tobacco chewing and smoking. However, it has been estimated that nearly 20% of the patients develop malignancies without any etiologic risk factors. There is growing evidence that along with alcohol and tobacco, HPV contributes significantly to oral cancer7.
3
Oral HPV infection had been identified in the healthy individuals and the relationship of cervical HPV infection and oral HPV infection remains unclear. The oral exfoliated cells harbor HPV DNA and HPV has been detected in the whole mouth fluid of oral cancer patients which may allow early cancer detection and monitoring of disease progression. The present study was conducted to quantify and compare HPV 16 in the whole mouth fluid of oral cancer patients, cervical cancer patients and normal individuals by quantitative real time polymerase chain reaction.
Aim and Objectives
4 AIM:
To evaluate the expression of Human Papilloma Virus type 16 (HPV 16) in the whole mouth fluid of oral cancer patients, cervical cancer
patients and normal individuals by quantitative real time polymerase chain reaction (qPCR).
OBJECTIVES:
To evaluate the presence of HPV 16 in the whole mouth fluid of oral cancer patients, cervical cancer patients and normal individuals by qPCR.
To compare the viral load in the whole mouth fluid of patients with oral cancer and normal individuals.
To compare the viral load in the whole mouth fluid of patients with cervical cancer and normal females.
To compare the viral load in the whole mouth fluid of patients with oral cancer and cervical cancer.
HYPOTHESIS (NULL):
HPV 16 is not detectable in the whole mouth fluid of oral cancer patients, cervical cancer patients and normal individuals.
Materials and Methods
5 STUDY DESIGN:
This cross sectional study was conducted over a period of one year (from 2014 to 2015) in which whole mouth fluid was obtained from patients in each group and analyzed by qPCR for HPV-16 DNA.
INCLUSION CRITERIA:
Patients with a histopathological diagnosis of primary oral or cervical cancer were included in the study.
EXCLUSION CRITERIA:
Patients with a history of prior malignancy, autoimmune disease, hepatitis, immunodeficiency states and patients who were under any treatment for oral cancer or cervical cancer were excluded from the study.
STUDY GROUPS:
Group I: (n=20) Patients who were diagnosed to have oral cancer and who have not been initiated on any therapy.
Group II: (n=10) Patients who were diagnosed to have cervical cancer and who have not been initiated on any therapy.
6 CONTROL GROUP:
Group III: (n=10) Individuals who were apparently healthy.
STUDY SETTING:
The study was approved by the Institutional Review Board of Ragas Dental College and Hospital (Annexure I). The patients who attended Ragas Dental College and Hospital and Government Royapettah Hospital participated in the study. DNA extraction and qPCR procedure was conducted in collaboration with Shrimpex Biotech Service Pvt. Ltd.
A thorough oral examination was performed and demographic details of the patients including name, age, gender, past medical history, habits history, family history and oral lesions were recorded in preformatted case sheets (Annexure II). Informed consent was then obtained from all the patients (Annexure III). Clinical staging of oral cancer was done by TNM (Tumor- node-metastasis) classification64. FIGO (International Federation of Gynecology and Obstetrics) system was followed for staging of cervical cancer. The FIGO staging is as follows:
Stage 0 – Carcinoma in situ
Stage I – Carcinoma confined to cervix
Stage II – Carcinoma beyond cervix though not to the pelvic sidewall or lower third of the vagina
7
Stage III – Carcinoma involves the lower third of the vagina with no extension to the pelvic sidewall
Stage IV- Carcinoma with extension beyond true pelvis or biopsy proven to involve the mucosa of the bladder or the rectum
Unstimulated saliva which is considered as representative of the whole mouth fluid was obtained from patients in each group by spitting method as described by Navazesh et al8. The collected saliva sample was immediately transferred to the ice box maintained at 40C. The samples were then stored in cryofreezer at -700C until further analysis.
METHODOLOGY:
Armamentarium:
For patient examination and sample collection:
Gloves
Mouth mask
10 ml sample collection container labelled with sample names
For DNA extraction and qPCR
2 ml micro centrifuge tube
Pipettes
Pipette tips
8
Vortex (Figure 1)
Table top centrifuge (Figure 2)
0.2 ml thin wall 8 stripped PCR tubes, 96 well plate
Real Time PCR Instrument- Eppendorf Realplex™ (Figure 3)
SALIVA COLLECTION:
Unstimulated saliva was collected by spitting method as described by Navazesh et al8. The subjects were instructed to tilt their heads forward, keep their eyes open and make as few movements as possible, including swallowing. Then the subjects were asked to expectorate the saliva which collected behind their closed lips at the end of each minute for every ten minutes.
PROTOCOL FOR THE EXPERIMENT
DNA extraction
DNA quantification
Preparation of premix
Placement of the prepared mix in the thermocycler
Programming the PCR machine
Interpretation of the graphs
9 DNA EXTRACTION
DNA extraction was done using DNA extraction kit (Swift saliva kit™) by spin column method.
Contents of Swift saliva kit™ (Figure 4):
S. No Kit components Quantity (for 50 preparations)
1 Lysis buffer 35 ml
2 Binding buffer (conc.) 10 ml
3 Buffer SE (Elution buffer) 5 ml
4 SMS (Shrimpex Mini Spin™)column 50
5 Collection tubes 50
The saliva samples were taken from the deep freezer and kept for thawing till they reach room temperature. The protocol followed for the DNA extraction is as follows:
1. 1 or 2 ml of saliva sample was added into a 2 ml micro centrifuge tube and the tubes were centrifuged at the rate of 13,400 rpm for 3 minutes.
2. The supernatant was discarded, 600 µl of lysis buffer was added into the pellet and homogenized properly with the help of vortex.
3. The tubes were centrifuged at the rate of 13,400 rpm for 3 minutes.
10
4. 350 µl of the clear supernatant was then transferred to another 1.5 ml tube and 350 µl of binding buffer was added and vortexed few times to mix properly.
5. 700 µl of lysate was loaded on to the SMS column and the tubes were centrifuged at 13,400 rpm for 30 seconds and then the flow through was discarded.
6. 600 µl of 70 % ethanol was added to the SMS column and centrifuged at 13,400 rpm for 30 seconds and then the flow through was discarded.
7. Again 600 µl of 70 % ethanol was added to the SMS column and centrifuged at 13,400 rpm for 30 seconds and then the flow through was discarded.
8. The SMS column was placed into a new collection tube and centrifuged at 13,400 rpm for 2 minutes. Empty spin for the elution of residual ethanol was done.
9. The flow through was discarded and the SMS column was placed into a new 1.5 µl micro centrifuge tube.
10. 40-50 µl of elution buffer was added and after 1 or 2 minutes it was centrifuged at 13,400 rpm for 1 minute.
11. The column was then discarded.
DNA QUANTIFICATION
The extracted DNA was then quantified by spectrophotometer (NanoDrop™) and the purity of the DNA was assessed. DNA has an
11
absorbance in the range of 230 to 320 nm. Therefore absorbance in this range allows the measurement of DNA concentration. The sample purity was determined by the ratios 260/280 nm and 260/230 nm. A good quality DNA should have A260 value (absorbance value) in the range of 0.1 to 1, A260/A280 ratio 1.7 to 2 and A260/A230 ratio >1.5.
Before making sample measurement, a blank sample (water) was measured.
1) Blank sample was loaded onto the lower measurement pedestal with the sampling arm of the spectrophotometer open.
2) The sampling arm was closed. The sample column is automatically drawn between the upper and lower pedestals.
3) The spectral measurement was made using operating software on the computer connected to the spectrophotometer. (Figure 8)
4) If the spectrum was a straight baseline then the blank was wiped from both pedestals and saliva sample was loaded. If the spectrum was not in straight baseline then steps 1, 2 and 3 were repeated until flat spectrum was obtained.
5) Then the saliva sample was pipetted onto the lower measurement pedestal and steps 1, 2 and 3 were repeated.
QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR)
The extracted DNA was quantified using qPCR based on TaqMan principle. The TaqMan assay is composed of a forward primer, reverse primer
12
and a dual labelled probe, which is complementary to template strand of the DNA present in the reaction mixture. The probe has a 5'-reporter dye and a downstream, 3'-quencher dye. GenoRime Real-Time HPV16 Kit™ was used to detect the HPV type 16 genome which targets the E7 region. The kit can quantify samples with DNA concentrations of 2 ng/ µl to 200 ng/µl and the amplicon size was 83bp nucleotides.
Properties of probes and primers used:
Forward Primer
No. of bases - 25
Molecular weight - 7813
GC content - 48%
Tm - 65°C Reverse Primer
No. of bases - 23
Molecular weight - 7057
GC content - 52%
Tm - 65°C Hydrolysis Probe
No. of bases - 28
Molecular weight - 9725
GC content - 61%
Tm - 75°C
13
Reporter dye - FAM (6-carboxy fluorescein) Quencher dye - BHQ (Black hole quencher)
After primer and probe designing, the specificity of the amplicon was verified by using the BLAST (Basic Local Ali gnment Search Tool) algorithm.
Contents of GenoRime Real Time HPV 16 Kit™: (Figure 5)
S. No Kit components Quantity
1 qPCR Master mix 2 x 500 µl
2 HPV16 Primer & Probe mix 1 x 200 µl
3 DNase free water 1 x 500 µl
4 Standard & Positive control
(2.5×108 per µl of positive control)
1 x 50 µl
Negative template control (NTC)
PCR grade water was added as negative template control. To confirm the absence of contamination, negative control was included every time the PCR was run.
Positive control:
The positive control consisted of synthetic HPV 16 DNA containing 83bp nucleotides. The positive control was used in four dilutions in every run to confirm that the assay for detection of the target gene is working well.
14 PCR protocol:
All reagents were completely thawed before use. The tubes were centrifuged to collect all the liquid in the tube.
1) Preparation of premix: The amount of reagent to be added to the premix was calculated as follows
10 µl qPCR Master Mix x N 2 µl HPV16 Primer probe mix x N 3 µl water x N
(where N denotes the number of reactions to set up per assay)
If number of samples (n) including controls = 1 to 14, then N = n + 1 If number of samples (n) including controls > 15, then N = n + 2 2) The contents of the premix were mixed by inverting the centrifuge tube
five times and then the tubes were centrifuged to collect all the contents at the bottom of the tube.
3) An aliquot of 15 µl of premix was added to each labelled tube.
4) 5 µl of PCR grade water, 5 µl of positive control and 5 µl of sample was added to appropriately labelled separate tubes.
5) The tubes were sealed immediately after adding NTC, positive control and samples to reduce the chances of cross contamination.
6) The contents were spun briefly in micro-centrifuge.
7) The prepared PCR tubes were placed immediately in Real-time thermocycler.
15
In our study qPCR was run in two batches. In the first batch (test run) 6 samples, 1 positive control and 1 negative template control were run and in the second batch 40 samples, 4 standards and 1 negative template control were run.
Programming the Eppendorf realplex® software 2.2: (Figure 9)
1) In the Plate Layout window, the following set up parameters was specified.
Filter 520 nm : FAM Sample volume: 20 μl Probe : TaqMan
Background : AxygenTM – 20 µl
2) The wells loaded with samples were selected and name of the sample and the target to be detected was assigned.
3) PCR program in the navigator window at the upper left corner of the screen was selected and the following parameters were set. (Figure 10) Lid temperature: (37-110oC). The set lid temperature is maintained constant during the program run.
Simulate Mastercycler gradient: Gradient function is used to vary the temperature distribution across the block. Simulate Mastercycler gradient is a program set up where the thermal block can be programmed such that each of the 12 columns have a different temperature.
16
Thermal sample protection: It is used to maintain the thermal block at 20oC during the lid heating phase. It reduces thermal load on the samples and minimizes the probability of nonspecific products. Thermal block is a multichannel metal block present in the lid, where water is heated with an exterior element.
Impulse: High heating rate (pulse) is achieved during the first temperature control step. It is used to reduce nonspecific PCR products.
Temperature mode:
a) Standard mode: It is the block temperature control mode for standard applications. This setting is used for sample volumes between 20µl to 50 µl or if the fast mode results in weak amplification.
b) Fast mode: This setting is used for reaction volumes <20µl and for templates with low G/C content.
c) Safe mode: This setting is used for reaction volumes >50µl.
Switch off lid at low temperature: It is a program where the lid heater switches off automatically if the block temperature is <15 oC.
Program Segment Temperature (0C)
Time Cycles Heat
activation
1 950C 2 min 1
PCR Cycle
2 950C 10 sec
38
3 600C 10sec
17
4) Real-time PCR data was acquired through the 520 nm FAM channel for HPV 16 during the 60ºC incubation step.
Optimization of the reaction:
The standards were diluted to generate a standard plot.
1) 90 µl of PCR grade water was pipetted into 4 tubes and labeled 1-4 2) 10 µl of positive control template was pipetted into tube 1
3) Vortexed thoroughly
4) Pipette tip was changed and 10 µl was pipetted from tube 1 into tube 2
5) Vortexed thoroughly
6) Steps 4 and 5 were repeated to complete the dilution series up to 2 dilutions.
Now the tube 1, 2, 3 and 4 contains 2.5×108, 2.5×107, 2.5×106, 2.5×105 copies/ml of positive control respectively.
Interpretation of the amplification plot: (Annexure VII)
PCR amplification curve is obtained by plotting the number of cycles against the intensity of fluorescent signals generated. The fluorescence signal correlates with the initial amount of DNA during the exponential phase of the amplification. There are four phases in the amplification plot. They are:
1. Linear ground phase: It indicates the baseline fluorescence at the beginning of the PCR cycle.
18
2. Early exponential phase: A threshold is reached and the fluorescence of the amplified product will be greater than the background fluorescence.
3. Log-linear phase: There is optimal amplification of PCR product and with each cycle there is doubling of fluorescence signals.
4. Plateau phase: The reagents in the reaction mixture will become limited.
Threshold: It refers to the level of signal that reflects a statistically significant increase over the calculated baseline signal. The threshold is automatically set at 10 times the standard deviation of the fluorescence value of the baseline.
Cq: The cycle at which the fluorescent signal of the reaction rises above the threshold is known as the Quantification cycle (Cq). This Cq value has a linear correlation corresponding to the starting amount of template given.
Noiseband is an algorithm of Eppendorf Realplex® software version 2.2, which is used to calculate Cq value.
Baseline: It refers to the signal level during the initial cycles of PCR which can be equated to the background or noise of the reaction.
Interpretation of standard curve: (Annexure VIII)
A standard curve was constructed by plotting the Cq value against the logarithmic value of known starting amount of target molecule and its subsequent dilutions. A difference in the value of Cq between two dilutions of
19
approximately three should be obtained. At an efficiency of 1, the number of target doubles in one PCR cycle. Ideally the plot must be linear with slope - 3.32, Efficiency 1, R2 =1 and the Y-intercept between 33-37 cycles.
Slope: It is a measure of reaction efficiency. The slope is calculated by linear regression. PCR efficiency of 100% corresponds to a slope of -3.32.
Efficiency of 100% implies that the template doubles after each cycle during exponential amplification.
Efficiency: Efficiency is derived from slope using the following formula:
Efficiency = [10-1/slope]-1
R2 (Correlation coefficient): It refers to the coefficient of determination which is a measure of accuracy of dilution and precision of pipetting. It implies how well the data fits the standard curve. It reflects the linearity of the standard curve.
Y-intercept: It corresponds to the theoretical limit of detection of the reaction.
It indicates the sensitivity of the assay.
The sample was considered as positive when the sample exhibits a growth within 38 cycles. A specimen was considered negative for HPV 16 virus if curves do not cross the threshold line within 38 cycles.
Quantification of qPCR assay:
Absolute quantification was done using standards of known concentration and similar composition to the target amplicon. Then the standard curve was used to determine the starting amount of each unknown template based on its threshold value.
20 STATISTICAL ANALYSIS:
Data entry, database management and analysis were done using SPSS® software version 21. The demographic details comprising of age, gender, past medical history, habits history, and family history of the study groups were tabulated.
Chi-square test was applied to find out the statistical significance of the HPV 16 positivity in oral cancer patients with respect to the habit of smoking, tobacco chewing or alcohol consumption and TNM staging. Kruskal-Wallis test was used to compare the mean HPV 16 viral load among the study groups.
A p value of ≤ 0.05 was considered as statistically significant.
Review of Literature
21 HPV-induced carcinogenesis:
Human Papilloma Virus (HPV) is a DNA virus that infects the human epithelial cells with different anatomic site preferences. Oral mucosa has histologic features and properties similar to that of cervical mucosa. Hence, cervical cancer and HPV associated head and neck squamous cell carcinoma share similar mechanisms of carcinogenesis induced by HPV. The HPV genome has three domains: a noncoding upstream regulatory region, an early region compromising of E6, E7, E1, E2, E4, and E5 that encode proteins required for regulation of viral DNA replication and viral gene expression and a late region encoding the L1and L2 viral capsid proteins. The prefix E and L relates to the position of open reading frame within the genome, and also the timing of expression relative to viral replication. HPV DNA integrates into the host genome and induces carcinogenesis through two viral oncogenes namely E6 and E7. The E7 oncoprotein induces cell proliferation by disrupting pRb (Retinoblastoma tumor suppressor) family which mediates transcriptional repression of genes involved in cell cycling. E7 can activate cyclin dependent kinase and inhibits cyclin-dependent kinase inhibitors like p21 and p27. E7 protein also induces p16 which is a tumor suppressor protein in normal cells.
The E6 oncoprotein targets the degradation of the p53 tumor suppressor protein via an ubiquitin-mediated process, resulting in cell cycle arrest and apoptosis. The high-risk HPV E7 protein binds to pRb with higher affinity when compared to the low-risk HPV. HPV can also induce cancer when the
22
viral DNA is in episomal form. HPV positive tumors are characterized by the loss of pRb and Cyclin D1 expression and by over-expression of p16 whereas the HPV-negative tumors over-express pRB and Cyclin D1 and under-express p166. The most common method for detection of HPV is the polymerase chain reaction (PCR). The PCR primers that can be used to detect HPV, target either the L1 region or the E6/E7 region. The commonly used consensus primers targeting the L1 region include GP5+/GP6+ primer set, MY09/MY07 primer set and PGMY09/11 primers35.
HPV and Cervical cancer:
Cervical cancer is the one of the most common malignancies among Indian women accounting for 15-51% of all cancers affecting females. A strong etiologic association has been established between high-risk HPV infections and cervical cancer. In India, 85-90% of cervical cancers are squamous cell carcinoma and HPV 16 is the most prevalent type accounting for 70-90% of cervical cancer. In Indian women, the peak of infection with HPV16 reaches at 26-35 years in contrast to 18-25 years in western countries9.
Bosch et al (1995) confirmed the etiologic role of HPV in cervical cancer worldwide. They analyzed more than 1000 invasive cervical cancer specimens obtained from 32 hospitals across 22 countries and performed PCR based assays. HPV DNA was detected in 93% of the specimen and there was no significant variation in HPV positivity among countries. HPV 16 was the
23
most common type in all countries except Indonesia. 50% of the specimens were HPV 16 positive whereas 14% were HPV 18 positive10.
Cuschieri et al (2004) investigated the prevalence of multiple HPV types in 3444 liquid based cytology samples obtained from women who attended a screening program in Scotland. Real time polymerase chain reaction was done using GP5+/GP6+ consensus primers for screening and linear array assay for genotyping HPV. HPV DNA was detected in 20% of the samples. High-risk HPV types (77%) were more prevalent than low risk types (27%). Multiple high-risk HPV types were detected in 3% of the samples negative for cervical cancer and in 33%, 42% and 40% of the samples with borderline, mild and high grade cervical cancer respectively. Multiple high-risk HPV types (25%) were more frequently found in young women of age <25. HPV 16 was detected in 6% of the women and it was detected more frequently in women with high grade cervical cancer (49%) 11.
Sowjanya et al (2005) assessed the genotype distribution of high-risk HPV types in 41 cervical cancer specimens obtained from a cancer hospital in Hyderabad and also in 185 cervicovaginal swabs obtained from women who participated in a rural community screening program. Hybrid Capture assay and PCR-Line blot assay were performed. High-risk HPV types were detected in 88% of the cervical cancer specimen, 10% of the clinician-collected cervicovaginal samples and 7% of the self-collected samples. HPV 16 (67%) and HPV 18 (19%) were the most predominant HPV types detected in cervical
24
cancer specimens whereas HPV 52(30%) and HPV 16 (18%) were the most common types detected in cervical swabs. This study suggested that self- collected cervical samples can be used as an alternative for HPV testing in India12.
Dunne et al (2007) analyzed self-collected vaginal samples from 1921 females aged 14 to 59 years who participated in National Health and Nutrition Examination Surveys (2003–2004) to determine the prevalence of HPV
infection in United States. Roche line blot assay was performed using L1 consensus primers. HPV was detected in 27% (95% confidence interval
CI, 23.3-30.9) of the samples with greater prevalence among females aged 20 to 24 years (34%). HPV 62 (3%), HPV 84 (3%), HPV 53(3%), HPV 89 (2%) and HPV 61 (2%) were the most common types detected13.
Smith et al (2007) conducted a meta-analysis on studies published between the year 2002 to 2006, to study the HPV prevalence in invasive cervical cancer and high-grade squamous intraepithelial lesions. HPV 16/18 was attributed to 70% of invasive cervical cancer and 52% of high-grade squamous intraepithelial lesions. 55% of invasive cervical cancer was associated with HPV 16 and 15% with HPV18 infection. Combined HPV 16/18 prevalence was slightly higher in Europe, North America and Australia (74–77%) than in Africa, Asia and South/Central America (65–70%). The next most common HPV types were HPV 31, 33, 35, 45, 52 and 58. This study suggested that vaccine against HPV 16/18 can effectively prevent two third of
25
invasive cervical cancer and half of high-grade squamous intraepithelial lesions worldwide14.
Bae et al (2008) conducted a meta-analysis on data published between 1995 and 2007 to estimate the overall prevalence of HPV in Korean women.
The overall prevalence of HPV was 23.9% (95% CI: 23.8-24.1%) in women with normal cytology and 95.8% (95% CI: 95.4-96.2%) in women with cervical cancer. HPV 16 was the most predominant type irrespective of cervical disease. HPV 16 and 18 accounted for 65% of invasive cervical cancer, 48% of high grade lesions and 24% of low grade lesions. The prevalence HPV types 58 and 52 were found to be higher in cervical cancer in Korean women when compared with other countries15.
Dursun et al (2009) investigated the cervical smears obtained from 507 women attending a clinic in Turkey between 2004 and 2008. Real time polymerase chain reaction was performed using GP5 and GP6 primers. HPV DNA was detected in 20% of the women with normal cervical cytology and 36% of the women with abnormal Pap smears. The HPV types prevalent in cytologically normal women were HPV 16 (36%), HPV 6 (22%), HPV 18 (13%) and multiple type HPVs (8.9%). The most common HPV types in cytologically abnormal women were HPV 16 (35%), HPV6 (19%) and HPV18 (8%) 16.
26
Hariri et al (2011) analyzed self-collected cervico-vaginal swab sample from 4150 females from the Unites States (14-54 years of age) who participated in National Health and Nutrition Examination Surveys (2003–
2006). HPV prevalence was estimated by linear array genotyping assay which uses L1 consensus polymerase chain reaction with PGMY09/11 primers. HPV was detected in 43% of the females and the prevalence increased with age.
The most common types were for HPV 62(7%), HPV 53(6%) and HPV 16(5%) 17.
Li et al (2011) performed a meta-analysis on data published from 1990 to 2010 to estimate the HPV type-specific prevalence in invasive cervical cancer. HPV 16 and HPV 18 were the most common types accounting for 73% of invasive cervical cancer. The prevalence of HPV 16/18 was between 70 and 76% in all world regions except Asia. A variation in type distribution was observed across Asia. In Western/Central Asia, 82% of invasive cervical cancer was HPV 16/18-associated compared to only 68% in Eastern Asia.
HPV type 58 was most commonly observed in Eastern Asia compared elsewhere. The prevalence of multiple HPV infections has increased from 4%
(in 1990-1999) to 16% (in 2006-2010). Increased sensitivity of the HPV DNA detection tests was considered to be the factor responsible for this rise18.
Hopenhayn et al (2014) analyzed data from seven cancer registries in the United States to estimate HPV type prevalence in invasive cervical cancer cases which were diagnosed between 1994 and 2005. DNA was extracted
27
from formalin fixed paraffin-embedded tissue specimen and subjected to linear array and if inadequate, re-tested with INNO-LiPA Genotype test. HPV was detected in 91% of the cases with HPV 16 accounting for 51%, HPV 18 for 16% and other HPV types like 45, 33, 31, 35 and 52 for 24% of the cases.
HPV was found to be negative in women of older age, in more advanced stage tumors and in tumors that were adenocarcinomas19.
Denny et al (2014) performed a cross-sectional epidemiological study to assess HPV prevalence and type distribution in women with invasive cervical cancer in Ghana, Nigeria, and South Africa. DNA probe hybridization and DNA enzyme immune assay were performed on paraffin-embedded tissue blocks. HPV was positive in 95% of squamous cell carcinoma and 77% of adenocarcinoma. The most prevalent HPV types were HPV 16 (51%), HPV 18 (17%), HPV 35 (9%), HPV 45 (7%), HPV 33 (4%) and HPV 52 (2%). The prevalence of single and multiple HPV infections was higher among Human Immunodeficiency Virus positive women (98%) 20.
HPV in Oral and Oropharyngeal cancer:
The most common location of HPV associated head and neck squamous cell carcinoma is the oropharynx. In 2012, the International Agency for Research on Cancer stated that HPV 16 causes cancer of the oropharynx21. It has been estimated that 25.6% of oropharyngeal squamous cell carcinoma worldwide are HPV-related. 75% of the head and neck squamous cell
28
carcinoma burden occurs in men. The prevalence of HPV infection in head and neck squamous cell carcinoma worldwide shows large variations22.
Syrjanen K et al (1983) were the pioneers in stating that HPV may be involved in the development of oral squamous cell carcinoma. Their study evaluated 40 oral squamous cell carcinoma biopsy specimens with the help of light microscopy and subjected the specimens to immune-peroxidase staining to detect the HPV antigens. Histopathologic features changes suggestive of HPV infection were present in 40% of the cases and HPV positive nuclei were present in 20% of the cases. They concluded that HPV might be the agent involved in at least certain types of oral squamous cell carcinoma, although further confirmation with other techniques is needed23.
Herrero et al (2003) conducted a multicenter case-control study of cancer of the oral cavity and oropharynx in nine countries from April 1996 to December 1999. 1670 patients were included in the study from cancer centers in Italy, Spain, Northern Ireland, Poland, India, Cuba, Canada, Australia and Sudan. HPV DNA was detected in biopsy specimens and oral exfoliated cells using polymerase chain reaction and HPV 16 L1, E6, E7 antibodies were detected in plasma using Enzyme Linked Immunosorbent Assay (ELISA).
HPV was most prevalent in cancer of the tonsil (25%). HPV DNA was detected in biopsy specimen of 4% of oral cancer and 18% of oropharyngeal cancer and in exfoliated cells of 4% of oral cancer patients and 9% of oropharyngeal cancer patients. HPV was not detected in 90% of the patients
29
with HPV positive tumor specimen. This observation suggested that HPV status of exfoliated cells may not accurately reflect the tumor HPV status.
HPV detection in biopsy specimens was less common among tobacco smokers than nonsmokers (OR-Odds Ratio, 0.4; 95% CI, 0.2 to 0.9). In tumor samples
obtained from India, HPV was less prevalent among tobacco chewers (OR, 0.5; 95% CI, 0.1 to 2.0) than among non-chewers. HPV DNA was
prevalent in oropharyngeal cancer in stages III-IV (22%) compared to oropharyngeal caner in stages 0-I (7%). 27% of the patients with HPV positive oral cancer and 52% of the patients with HPV positive oropharyngeal cancer were seropositive for HPV 16 L1 antibodies. 13% of the patients with HPV positive oral cancer and 65% of the patients with HPV positive oropharyngeal cancer were seropositive for HPV 16 E6/E7 antibodies. Hence, they stated that antibodies to HPV E6/E7 can serve as a minimally invasive marker of HPV associated cancers24.
Kreimer et al (2005) from France systematically reviewed the studies published after 1995 that employed polymerase chain reaction-based method to detect HPV in head and neck squamous cell carcinoma biopsies. HPV was detected in more than one-third (36%) of the oropharyngeal squamous cell carcinoma, in 24% of oral squamous cell carcinoma and in 24% of laryngeal squamous cell carcinoma. HPV 16 accounted for 31% of HPV positive oropharyngeal squamous cell carcinoma and 16% of oral squamous cell carcinoma. HPV 18 was less frequently observed in oropharyngeal squamous
30
cell carcinoma (1%) when compared with oral squamous cell carcinoma (8%).
They stated that this variation in type distribution of HPV with site may be due to the difference in immunologic response. The prevalence of HPV in the oropharyngeal squamous cell carcinomas was greater in North America (47%) and Asia (46%) when compared to Europe (28%). The prevalence of HPV in oral squamous cell carcinomas was similar in Europe and North America (16%) but greater in Asia (33%). They concluded that misclassification of advanced oropharyngeal squamous cell carcinomas as oral squamous cell carcinomas could have inflated the prevalence of HPV infection in oral cancers in Asia25.
Koppikar et al (2005) investigated head and neck squamous cell carcinoma biopsy specimens obtained from Tata Memorial Hospital in Mumbai during the year April 1999-April 2003. Their study included 83 oral squamous cell carcinoma, 19 other head and neck squamous cell carcinoma specimen and exfoliated buccal cells obtained from 102 individuals were taken as controls. The frozen tissue specimens were subjected to polymerase chain reaction using primers targeting L1 region. 15% of males and 27% of females harbored HPV DNA. HPV was present in 34% of the oral squamous cell carcinoma specimen and in 33% of oropharyngeal cancer. Multiple HPV infections were present in 14% and HPV 16, HPV 18 were detected in 6% of the tumors. HPV was more frequently detected in patients with chewing habits alone (30%) whereas no HPV DNA was detected in patients who had the habit
31
of only smoking. Hence they have concluded that HPV virus along with other carcinogens could play a role in oral carcinogenesis26.
D’Souza et al (2007) performed a case–control study on 100 patients diagnosed with oropharyngeal cancer and 200 control patients without cancer who attended a hospital in Baltimore during the period 2000-2005. HPV-16 DNA was detected in formalin fixed, paraffin-embedded tissue specimen using in situ hybridization and in oral specimen obtained with a cytology brush by means of multiplex polymerase chain reaction using PGMY09/11 primers targeting the L1 region. Antibodies to HPV 16 L1 protein, E6 and E7 oncoproteins were detected in the serum using ELISA. 13% of the patients with oropharyngeal cancer did not have any history of tobacco or alcohol use.
57% of the patients were seropositive for HPV16 L1 region whereas 67% of the patients were seropositive for E6 and E7 region. Oral HPV16 infection was detected in 32% of the patients and HPV 16 DNA was detected in 72% of the tumors. A high lifetime number of oral-sex or vaginal-sex partners were associated with HPV-16 positive oropharyngeal cancer. Irrespective of the tobacco and alcohol use, the association between HPV positivity and oropharyngeal cancer was increased27.
Anaya-Saavedra et al (2008) from Mexico in their case-control study investigated the association of HPV in patients with oral squamous cell carcinoma and various risk factors. A personal interview, demographic, clinical details and incisional biopsies were obtained from 62 patients with
32
oral squamous cell carcinoma who attended a hospital in Mexico during the year September 2003-July 2006. Oral specimen obtained with cytobrush from patients who were free of cancer or any pre-malignant conditions were regarded as controls. The samples were subjected to polymerase chain reaction using two sets of primers (MY09/11 and GP5+/6+). Among the patients with oral squamous cell carcinoma 53% were males with median age of 60 years and 47% were females with median age of 70 years. 74% of the cases presented at advanced stages (stage III and IV) and 49% showed well differentiated carcinoma. HPV prevalence was 44% in cases and 17% in controls. The most frequent types detected in cases were HPV-16 (56%) and HPV-18 (19%). Oral squamous cell carcinoma patients had 6-fold increased risk of oral high-risk HPV infection (OR = 6.2; 95% CI: 2.98–12.97) when compared with the controls. 56% of high-risk HPV positive patients with oral squamous cell carcinoma did not have the habit of tobacco or alcohol use in contrast to 28% of high-risk HPV negative cases. 16% of the oral squamous cell carcinoma patients did not use tobacco or alcohol or did not have high- risk HPV which suggested that unidentified risk factors still exist which contributes to carcinogenesis28.
Termine et al (2008) performed meta-analysis from January 1988-2007 which included studies that used paraffin-embedded
biopsy specimen of head and neck squamous cell carcinoma to detect HPV. Majority of the studies had small to medium sample size (<100) which
33
showed a wide variation in HPV prevalence (1 to 100%) whereas the larger sample sized studies showed a lower HPV prevalence (1% to 49%). The overall prevalence of HPV DNA was 35%, HPV prevalence in oral squamous cell carcinoma was 38% and 24% in other head and neck squamous cell carcinomas. High-risk HPV was the most prevalent type. Polymerase chain reaction based studies revealed higher prevalence of oral squamous cell carcinoma (40%) when compared to in situ hybridization based studies (30%).
Hence, they concluded that correct distinction of head and neck squamous cell carcinoma by tumor site and use of more sensitive detection methods should be considered essential for accurate assessment of prevalence of HPV29.
Shukla et al (2009) in their review stated that the prevalence of HPV in oral squamous cell carcinoma differs in different geographical regions within Indian subcontinent. The HPV 16 infection was found to be reported in 27% of oral cancer from North India and 25% to 31% from the western part.
Multiple HPV infection was observed in about 14% of cases. HPV prevalence in southern India was highly variable where the overall frequency of HPV infection was 74% while 41% showed multiple HPV infection and 42%
showed HPV 16 infection9.
Ang et al (2010) in their retrospective analysis investigated the effect of HPV status on survival of patients with stage III or IV oropharyngeal squamous cell carcinoma. The patients who were included in the study were a part of the clinical trial conducted by radiation therapy oncology group from
34
July 2002 to May 2005. Formalin fixed, paraffin-embedded tumor specimens were obtained and subjected to in situ hybridization to detect HPV 16 DNA and immunohistochemistry to evaluate the expression of p16. HPV was detected in 64% of oropharyngeal squamous cell carcinoma. HPV positive oropharyngeal squamous cell carcinoma was more frequently detected in males (86%) and patients who never smoked (29%). Patients with HPV positive tumor had better 3-year survival (95% CI, 77.2 to 87.6) when compared to HPV-negative tumor (95% CI, 48.1 to 66.1). Also an agreement was observed between the presence of p16 expression and presence of HPV DNA in tumor. Hence, they stated that p16 expression is a good surrogate marker for tumor HPV status 30.
Chaturvedi et al (2011) determined the prevalence of HPV in oropharyngeal cancer tissue specimen obtained from three population-based cancer registries in the surveillance, epidemiology, and end results residual tissue repositories program during the period 1984-2004. The procedures that were carried out on 271 oropharyngeal squamous cell carcinoma formalin fixed paraffin-embedded tissue specimen were: reverse line blot hybridization for HPV genotyping, real time- polymerase chain reaction to evaluate HPV16 viral load and HPV 16 E6/E7 mRNA expression, in situ hybridization to detect HPV 16 DNA and immunohistochemistry to evaluate HPV E7 protein expression. HPV prevalence was estimated across four calendar periods:
1984 to 1989, 1990 to 1994, and 1995 to 1999. The prevalence of HPV was
35
44% by reverse line blot hybridization, 30% by HPV 16 viral load, 35% by HPV16 E6/E7 oncogene expression and 28% by in situ hybridization. HPV prevalence increased over calendar time irrespective of the HPV detection assay used. An improvement in the overall survival of patients with HPV positive oropharyngeal squamous cell carcinoma was observed whereas the survival of HPV negative patients remained unchanged. Their study estimated an increase in the overall incidence of oropharyngeal squamous cell carcinoma by 28% during 1988 to 2004 and in prevalence of HPV positive oropharyngeal squamous cell carcinoma from 16% during 1984-1989 to 73% during 2000 to 2004. Also, there was a decline in the incidence of HPV negative oropharyngeal squamous cell carcinoma by 50%. They concluded that if this incidence trend continues, then the annual number of HPV positive oropharyngeal squamous cell carcinoma will surpass the annual number of cervical cancers by the year 2020 in the United States31.
Mehanna et al (2013) from the United Kingdom analyzed the trends of HPV prevalence with respect to time and region by conducting a systematic review which included studies published from 1996 to 2010. The overall HPV prevalence in oropharyngeal squamous cell carcinoma was 48% and in non- oropharyngeal squamous cell carcinoma was 22%. The prevalence of HPV was 40% in Europe, 60% in North America, and 33% in other regions. HPV 16 was the most common type detected in 96% of oropharyngeal squamous cell carcinoma. Increase in prevalence of HPV positive oropharyngeal
36
squamous cell carcinoma from 41% (before 2000) to 72% (2005 onward) was observed. This increase was evident in Europe and in North America. They also observed a decline in the prevalence of non-oropharyngeal squamous cell carcinoma over time from 22% (before 2000) to 6% (2005 onward). There was no significant difference in the prevalence of HPV detected by techniques of low or medium sensitivity and techniques of high or very high sensitivity.
So, they concluded that this change in the prevalence was not a result of improvements in sensitivity and performance of the detection techniques32.
Patel et al (2014) from Gujarat estimated the prevalence of HPV 16 and 18 infections in 97 oral squamous cell carcinoma and 52 cervical cancers.
Type-specific polymerase chain reaction was performed on frozen specimens using consensus primers located within L1 region. Among patients with oral cancer, 87% of the patients were males with mean age of 47 years and 13%
were females with mean age of 46 years. Buccal mucosa (40%) was the most common site involved followed by tongue (21%). 89% of the patients had tobacco habits. HPV was not detected in any of the oral squamous cell carcinoma specimens whereas 60% of the cervical cancer specimens were HPV-positive. Among cervical cancer patients, 90% had HPV 16 and 10%
had HPV 18 infection. Hence, they suggested that HPV 16 and 18 do not play an important role in oral carcinogenesis7.
37 Detection of HPV:
Polymerase chain reaction is a highly sensitive and cost effective method for HPV detection. However, polymerase chain reaction methods have low specificity and cannot distinguish the episomal form from integrated form of HPV DNA. Also, standard polymerase chain reaction techniques cannot identify whether the HPV is derived from tumor or healthy mucosa. HPV DNA in tumor tissue can also be detected by in situ hybridization. In situ hybridization is highly specific and can distinguish episomal and integrated HPV DNA. It has been observed that polymerase chain reaction based studies reveal a higher rate of prevalence of HPV when compared to in situ hybridization based studies. HPV specific IgG antibodies in serum can also be used as a biomarker to determine previous or current HPV infection status6.
Smeets et al (2007) from Netherlands evaluated several assays that can be performed on fresh frozen paraffin-embedded tissue specimens to detect HPV. The assays analyzed were: polymerase chain reaction using general primer (GP)5+/6+, viral load analysis, HPV 16 DNA fluorescence in situ hybridization, HPV 16 E6 mRNA real time-polymerase chain reaction, p16 immunostaining and on corresponding serum samples of antibodies against HPV 16 proteins L1,E6 and E7. E6 mRNA expression by real time- polymerase chain reaction showed 100% sensitivity and specificity and was considered superior when single assays were compared. They also
38
demonstrated that fluorescence in situ hybridization was very specific but less sensitive33.
Kornegay et al (2003) from Spain assessed the accuracy and reproducibility of polymerase chain hybridization for HPV DNA detection
using the PGMY primers. 109 samples including 29 HPV-negative (19 cervical samples collected with cryobrush and 10 buffer controls) and 80 HPV-positive samples (47 cervical samples and 33 synthetic samples) were
tested blindly by three laboratories. Intralaboratory agreement ranged from 86% to 98% for HPV DNA detection. Interlaboratory reliability of PGMY for HPV DNA positivity and HPV typing was very good, with levels of agreement of >95% and kappa values of >0.87. They concluded that HPV testing can be accomplished reliably with polymerase chain reaction by using a standardized written protocol and quality-controlled reagents34.
Morris (2005) from Australia in their review stated that HPV polymerase chain reaction strategies directed at the E6/E7 region is preferable to L1 since E6 and E7 are the oncogenic regions of HPV. Also, E6 and E7 are retained after infection whereas E2 and L1 can be deleted as the disease progresses. Hence, L1 primers used for screening will detect HPV in women with cervical intraepithelial neoplasia while advanced lesions may be missed.
It was concluded that the selection of E6/E7 may provide the optimum choice for polymerase chain reaction to detect HPV35.
39 Oral HPV infection in normal individuals:
Oral HPV infection can be acquired by sexual transmission or by mother to child transmission or by autoinoculation. Oral HPV prevalence increases with age and with number of sexual partners. It is detected in 3–5%
of adolescents and 5–10% of adults. Oral HPV infection is also increased in men, HIV-infected individuals and current tobacco users5. The prevalence of oral HPV varies from 0 to 70% in normal oral mucosa, 0 to 85% in potentially malignant oral disorders and 0 to 100% in oral malignances. This variation could be due to the difference in ethno-geographic origin of patients, sample collected (buccal scraping, swab, biopsy or mouthwash), sample size and molecular techniques used in different studies36.
D'Souza et al (2009) in their study determined whether the oral sexual behaviors were associated with oral HPV infection. Oral rinse samples were collected from 210 students recruited during the year 2007 and from 332 control patients attending a clinic in Baltimore during 2000-2006. Multiplex polymerase chain reaction using PGMY09/11 primers was performed followed by line-blot hybridization to identify the HPV types. HPV prevalence was 5% among the control patients and 3% among the students of age range 18–23 years. The odds of developing oral HPV infection increased in control patients who were current smokers (OR, 3.9; 95% CI, 1.2-12.7) and who reported to have >10 oral sex partners (OR, 5.2; 95% CI, 1.1-25). Among college students, the odds of developing oral HPV infection increased with
40
increase ≥6 recent oral sex (OR, 7.9; 95% CI, 1.1-59) or open-mouthed kissing (OR, 17; 95% CI, 1.5-198). Hence, they concluded that oral sex contact may play a role in transmission of HPV37.
Kreimer et al (2010) conducted a systemic review of the literature which included studies between 1997 and 2009 that detected HPV DNA in oral specimens of healthy individuals. In 18 studies that were included, HPV DNA was detected by PCR based assays in a total of 4,581 healthy individuals. HPV 16 was more prevalent in developing countries (4%) when compared to developed countries (0.7%) and HPV 16 accounted for 28% of all oral HPV infections. HPV prevalence was similar in men and women. It was found that the HPV prevalence was inversely proportional to the sample size.38
Gillison et al (2012) conducted a cross-sectional study as a part of the National Health and Nutrition Examination Survey 2009-2010 to determine the prevalence of oral HPV infection in the United States. Oral rinse samples were obtained from 5579 participants of age 14 to 69 years. HPV DNA was detected by multiplex PCR and genotyping was done by line-blot hybridization. The prevalence of oral HPV infection was 7%. HPV 16 was the most prevalent type accounting for 1% of HPV infections. Men had higher prevalence (10%) than women (4%) for any oral HPV infection. HPV prevalence was more among individuals with more lifetime or recent number of sex partners and among those who had oral sex at a younger age. Oral HPV infection was 3-fold higher in men when compared to women. Among men,
41
based on age a bimodal distribution of HPV was observed with peak prevalence at 55-64 years. Association of smoking and oral HPV was higher among women than men39.
Pickard et al (2012) analyzed oral rinse samples obtained from 1000 students at The Ohio State University, aged 18 to 30 years. Multiplex
PCR was performed on the samples using PGMY primers targeting L1 region.
Oral HPV prevalence was 2% and HPV 16 prevalence was 0.2%. Participants ever having consumed alcohol, participants with 5 or more lifetime open mouth kissing or lifetime oral sex partners were associated with HPV infection. This study suggested that oral sexual contact play a role in transmission of oral HPV infection40.
Du et al (2012) from Sweden performed a study which enrolled 408 females and 82 males, 15–23 years of age, who visited a clinic in
Stockholm. Oral rinse samples were obtained from all the participants and cervical samples were collected from the females. Oral HPV prevalence was 9% whereas the cervical prevalence was 74%. The oral HPV prevalence was 9% in females and 10% in males. HPV 16 was detected in 2.9% of oral samples and 38% of cervical samples. HPV 18 was detected in 0.2% of oral samples and 14% of cervical samples. Oral HPV infection was more frequent among females with cervical HPV infection (17%) than without (4%) cervical HPV infection. HPV types commonly detected in the cervical tract like HPV- 16, 18, 39, 51, 56, 59, 82 were also observed in the oral cavity but only fewer
42
HPV types were detected in the oral compared with the genital tract. This study emphasized the importance of investigation to determine if the current HPV vaccines also prevent oral HPV infection41.
Antonsson et al (2013) from Australia ascertained the prevalence of oral HPV infection in oral rinse samples obtained from 307 students from Australian University who were of age 18–35 years. DNA was extracted from the samples and PCR was performed using GP5+/6+ primers. HPV was detected in 2.3% of samples (43% HPV-18, 14% HPV-16, -67, -69 and -90).
HPV was more prevalent among males (86%) and among patients who had oral sex (83%), and who reported to be diagnosed with HIV (86%). 33% of the patients who reported to have received Gardasil vaccine were all HPV negative42.
Oral HPV infection in women with cervical cancer:
Saini et al (2010) in their cross-sectional study analyzed the buccal swabs obtained from 70 women diagnosed with cervical cancer and their children. HPV prevalence was determined using Hybrid Capture 2 high-risk HPV detection system which is a nucleic acid hybridization assay. It was observed that 75% of the high-risk HPV positive women belonged to age group 50-59 years. High-risk HPV was detected only in 6% of the women with cervical cancer and 3% of their children. There was no association of
