Hamzan, Nurul Izzati
(2020)
Development of real-time loopmediated isothermal amplification and PCR assays for detection of human papillomavirus 16 in oral squamous cell carcinoma.
PhD thesis, Universiti Sains Malaysia.
Abstract
Human papillomavirus genotype 16 (HPV-16) involvement in the
development of oral squamous cell carcinoma (OSCC) has been well-documented. Its
detection is crucial to classify OSCC into positive and negative cases; as this affects
prognosis. The conventional method of detection for high-risk HPV relies upon p16
immunohistochemistry (IHC) as a surrogate marker which has drawbacks on its
lengthiness, low specificity level (46 to 78%), broad range of staining intensity cut-off
value (range 5 to 75%), requirement of expertise and costly. Thus, in an effort to
improve the diagnostic test for HPV-related oral cancer, we have developed a realtime
LAMP (qLAMP) assay for rapid, sensitive, specific and quantitative detection of
HPV-16 in OSCC. The first phase was focused on the development of qLAMP and
PCR assays, while in the second phase the developed assays were evaluated using
OSCC clinical samples (tissue, n=63; saliva, n=13; and blood, n=59) and healthy
(saliva, n=50). The hematoxylin and eosin (H&E) and p16 IHC staining using
formalin-fixed paraffin embedded (FFPE) tissues was done to evaluate the tumour
histological grading and determine high-risk HPV positivity, respectively. The Kappa
value was determined between two raters for p16 staining. The sensitivity, specificity,
positive predictive value (PPV), negative predictive value (NPV) and accuracy of
qLAMP were evaluated, in comparison with PCR and p16 IHC. It was found that the
developed qLAMP assay successfully amplified HPV-16, and no cross-reaction with
other HPV strains, respiratory viruses and oral bacterial. The positive amplification
starts as early as 21:18 minute and the whole process can be completed within one
hour. The LOD for qLAMP and PCR assays were 4.68 X 101 and 4.68 X 103 copies
per microliters, respectively. The developed qLAMP assay detected HPV-16 positivity
in three tissue (4.7%) and saliva (23%) samples from OSCC patients, while the PCR
assay detected two (3.17%) HPV-16 positives in tissue and one (7.69%) in saliva
samples, with the HPV-16 viral load ranging from 4.68 X 101 to 4.68 X 104. The
sensitivity, specificity, PPV, NPV and accuracy of qLAMP assay towards p16 IHC
were 100%. The sensitivity, specificity, PPV, NPV and accuracy of in-house PCR
assay were 67%%, 100%, 100%, 98% and 98%, respectively. p16 IHC staining
showed three positivity of tissues with the H score ranged from 40 to 225% and welldifferentiated
grade. Very good agreement (қ = 1.0) was found between two raters for
evaluation of p16 IHC staining. In conclusion, the developed qLAMP was highly
sensitive and specific, and rapid for the detection of HPV-16 in OSCC. This study is
novel as it is the first report describing the use of both tissue and saliva as the sample
matrix for detection of HPV-16 in OSCC and the detection platform using real-time to
quantify the viral load of the infection in comparison to the current available HPV-16
detection kit.
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