Human Papillomavirus DNA Detection by Droplet Digital PCR in Formalin-Fixed Paraffin-Embedded Tumor Tissue from Orophary
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ORIGINAL RESEARCH ARTICLE
Human Papillomavirus DNA Detection by Droplet Digital PCR in Formalin‑Fixed Paraffin‑Embedded Tumor Tissue from Oropharyngeal Squamous Cell Carcinoma Patients Camila Marques Schiavetto1 · Priscila Marinho de Abreu2 · Sandra Ventorin von Zeidler2 · Laís Machado de Jesus1 · Raiany Santos Carvalho3 · Maria Thereza Cirino1 · Adriana Cruvinel Carloni1 · Cristina Oliveira1 · Cristovam Scapulatempo‑Neto1,4 · Gisele Caravina de Almeida4,5 · Nei Soares de Menezes5 · André Lopes Carvalho1 · Rui Manuel Reis1,6,7 · Ana Carolina de Carvalho1 Accepted: 9 November 2020 © Springer Nature Switzerland AG 2020
Abstract Introduction High-risk human papillomavirus infection impacts staging and prognosis of oropharyngeal squamous cell carcinomas (OPSCCs). Determination of HPV status in tumor tissue by p16-immunohistochemistry (p16-IHC) can be challenging; therefore, complementary methodologies could be useful in a clinical setting. Objective To test for accuracy and clinical relevance of HPV-DNA detection in formalin-fixed and paraffin-embedded (FFPE) tumor samples by droplet digital PCR (ddPCR). Materials and Methods Fifty OPSCCs were tested for p16-IHC status followed by HPV-16/18 DNA detection/quantification in FFPE-recovered DNA using ddPCR. Accuracy for HPV status determination and association with patient information were also evaluated. Results 32.0% (16/50) of the cases were p16-IHC positive (p16 +), 42.0% (21/50) had detectable levels of HPV-16 DNA, and none were positive for HPV-18 DNA. A higher median viral load of HPV-16 DNA was observed in p16 + cases (p 5 copies/ng)
HPV-16 DNA P16-IHC/HPV-16 (> 10 copies/ng) DNA (no cutoff)
Copy number/ng of DNA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
60 51 50 46 63 72 65 66 56 53 65 45 79 60 51 59 56 57 79 53 49 44 40 50 54 44 53 53 52 59 65 49 69 64 54 68 42 74 53 55 56 72 63 69 59 60 42
M M M M M M M F M M F M M M F M M M M M M M M M M M M M M M M M M M M M M M F F M M M M M M F
− − − − − − + + − + − − − − + − − − − + − − + − + + + + + − − − − − − − − − + + − − − − − + +
n/a n/a n/a n/a n/a n/a + + n/a + n/a n/a n/a n/a − n/a n/a n/a n/a + n/a n/a n/a n/a + + + + n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a + + n/a n/a n/a n/a n/a n/a +
− − − − − − − + − + − − − − − − − − − + + + + − + + + + − − − − − − − − − − + + − − − − − − +
− − − − − − − + − + − − − − − − − − − + − + + − − + + + − − − − − − − − − − + + − − − − − − +
0 0 0 0 0 0.48 0.68 8800 0 2452 2.6 0 0.68 0.72 0.50 0 0 0.46 0 6900 6 10 50 0 6.6 948 552 986 0 0 0 0 0 0 0.48 0 0 0 1268 346 0 0 0 0 0 0 488
− − − − − + + + − + + − + + + − − + − + + + + − + + + + − − − − − − + − − − + + − − − − − − +
−/− −/− −/− −/− −/− −/+ +/+ +/+ −/− +/+ −/+ −/− −/+ −/+ +/+ −/− −/− −/+ −/− +/+ −/+ −/+ +/+ −/− +/+ +/+ +/+ +/+ +/− −/− −/− −/− −/− −/− −/+ −/− −/− −/− +/+ +/+ −/− −/− −/− −/− −/− +/− +/+
C. M. Schiavetto et al.
Table 2 (continued) Patient
Age, years
Gender
p16-IHC
HPV E6/ HPV-16 E7 mRNA DNA (no c
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