Eur Arch Otorhinolaryngol DOI 10.1007/s00405-017-4662-0
LETTER TO THE EDITOR
Microarray technology for identification of human papilloma virus subtype in oropharyngeal squamous cell carcinoma Soung Min Kim1
Received: 18 June 2017 / Accepted: 28 June 2017 Ó Springer-Verlag GmbH Germany 2017
To the Editor, I have read with great concerns a recently published by Qureishi et al., entitled ‘‘Current and future techniques for human papilloma virus (HPV) testing in oropharyngeal squamous cell carcinoma’’ [1]. Although this review article was well written and provided a great deal of HPV-testing in oropharyngeal squamous cell carcinoma (OPSCC) information, I would like to add a few additional recommendations based on recently updated microarray technology. In Qureishi et al.’s article [1], the current techniques and the clinical applicability of emerging techniques including p16 immunohistochemistry (IHC), HPV DNA in situ hybridization (ISH), and HPV polymerase chain reaction (PCR), are introduced as the commonest tests for HPV detection in OPSCC. To date, there is no established standard method for detecting HPV as Qureishi et al. said. Detection methods vary not just in design, but in detection targets, such as HPV DNA, HPV RNA, viral oncoproteins, cellular proteins, and HPV-specific serum antibodies [2]. Among these, HPV DNA detection with PCR is known to show acceptable sensitivity and specificity for the detection of HPV 16 and 18 [3]. A DNA chip kit (MY-HPV chip kitÒ, Mygene Co., Seoul, Korea) could be shown as the example for HPV
microarray testing. This is a genotypic test that can identify the types of HPV by reacting to a spotted oligonucleotide chip with 19 kinds of HPV-specific sequences, including a high-risk group (16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56, 58) and a low-risk group (6, 11, 34, 40, 42, 43, 44). In this DNA chip method, DNA isolation, amplification by PCR, hybridization, washing, and scanning were sequentially performed to obtain results (Fig. 1) [4]. A 500–1000 ll extracted sample was transferred to a 1.5 ml tube by a serum separator or pipet. The supernatant was removed by centrifuging at 6000–9000 rpm for 10 min. After a 1 ml washing buffer was added, followed by centrifuging twice at 6000–9000 rpm for 5 min, the supernatant was removed, and the precipitate was dried. The mixtures of precipitate and extraction DNA were incubated in a 50 °C water bath for more than 3 h and then heated at 90–100 °C for 20 min. After centrifugation at 14,000 rpm for 10 min, a separated total amount of 150 ll of supernatant was used to conduct PCR. The first and second PCRs were performed
This comment refers to the article available at doi:10.1007/s00405-017-4503-1. & Soung Min Kim [email protected]; [email protected] 1
Department of Oral and Maxillofacial Surgery, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea
Fig. 1 Flow diagrams of HPV DNA chip (MY-HPV chip kitÒ, Mygene Co., Seoul, Korea) analysis, including DNA isolation, amplification by PCR, hybridization, washing, and scanning
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Eur Arch Otorhinolaryngol Table 1 The summary of first and second PCR with a reagent, condition and cycles 1st PCR—2.5% agarose (450, 250 bp)
2nd PCR—2.5% agarose (150–200 bp)
Reagent
Volume
Condition
PCR premix 1 (450 bp) premix 3 (250 bp)
7.0 ll
50 °C/3 m
DNA
5.0 ll
95 °C/15 m
Cycles
Reagent
Volume
Condition
cycles
1
PCR premix 2 (150 bp)
6.5 ll
95 °C/5 m
1
1
1st PCR product
2.0 ll
95 °C/30 s
20
50 °C/40 s 72 °C/30 s Taq polymerase
0.3 ll
95 °C/30 s
35
Taq polymerase
0.3 ll
D.W
16.2 ll
Total
25.0 ll
72 °C/3 m
1
55 °C/30 s 72 °C/30 s D.W
12.7 ll
Total
25.0 ll
72 °C/3 m
1
PCR polymerase chain reaction, D.W distilled water, m minutes, s seconds, bp base pair, °C celsius
sequentially, as described in Table 1. The second PCR product was denatured at 95 °C for 5 min. The hybridization buffer was incubated at 43 °C for 10 min. These mixtures of PCR product and 35 ll hybridization buffer were inserted into each well of a slide and placed in water. Hybridization was performed at 43 °C for 4 h (Table 1). The hybridized slides are washed, and the hybridization seal was removed. After washing, the chip scanners were checked to see if they were coupled to each HPV-types probe; we determined the HPV subtypes by analyzing the results. When the HPV DNA chip was positive, we used the HPV-types noted by the HPV DNA chip results. If the HPV DNA chip was negative and the PCR result was positive, we regarded it as ‘‘other type’’ HPV-positive. Lastly, if both the PCR and HPV DNA chip results were negative, we concluded that the sample was HPV-negative. Detection of HPV can now move towards a more directed, clinically relevant and standardized approach. HPV DNA microarray detection with PCR pointed to its incomparably sensitivity. These methods can detect HPV at levels well below one viral copy genome per cell although detection of HPV DNA using PCR lacks specificity and HPV E6/E7 antibody detection lacks sensitivity [2, 5]. Thus, multimodality detection strategies are recommended to utilize the strengths of individual assays in combination to optimize the overall reliability of HPV detection. Since two HPV vaccines for cervical cancer, a quadrivalent and bivalent types that use virus-like particles (VLPs), were currently used in the medical commercial market in 2010, sampling techniques for HPV include microscopy, ELISA, Southern blot, Dot blot, hybrid capture, DNA microarray, and ligase chain reaction for probe amplification had been introduced [6, 7]. Although a standard procedure has not yet been generally accepted, both PCR and ISH are well validated, and gene expression by DNA microarray has recently gained acceptance as a high throughput method [8].
123
Recently, many analytical results by DNA microarray have been shown a clear and understandable analysis of HPV-associated OPSCC [3, 4]. The DNA microarray method has proper accuracy for detecting known HPV subtypes simultaneously, but has also limitations detecting new subtypes. I suggest that a DNA microarray method has proper accuracy for detecting known HPV subtypes in OPSCC. Acknowledgements This study was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI15C0689) and by the International Research & Development Program of the National Research Foundation of Korea (NRF2015K1A3A9A01028230). Compliance with ethical standards Conflict of interest There are no conflicts of interest in this article. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Review Board (IRBS-D2017006) at Seoul National University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
References 1. Qureishi A, Mawby T, Fraser L, Shah KA, Møller H, Winter S (2017) Current and future techniques for human papilloma virus (HPV) testing in oropharyngeal squamous cell carcinoma. Eur Arch Otorhinolaryngol 274(7):2675–2683 2. Westra WH (2014) Detection of human papillomavirus (HPV) in clinical samples: evolving methods and strategies for the accurate determination of HPV status of head and neck carcinomas. Oral Oncol 50(9):771–779 3. Lim YK, Choi JH, Park S, Kweon OJ, Park AJ (2016) Comparison of three different commercial kits for the human papilloma virus genotyping. J Clin Lab Anal 30(6):1110–1115 4. Kim SM, Kwon IJ, Myoung H, Lee JH, Lee SK (2017) Human papillomavirus (HPV) in Korean oral cancer patients using microarray technology. Human Pathol (submitted)
Eur Arch Otorhinolaryngol 5. Combes JD, Franceschi S (2014) Role of human papillomavirus in non-oropharyngeal head and neck cancers. Oral Oncol 50(5):370–379 6. Snow AN, Laudadio J (2010) Human papillomavirus detection in head and neck squamous cell carcinomas. Adv Anat Pathol 17(6):394–403 7. Feller L, Wood NH, Khammissa RA, Lemmer J (2010) Human papillomavirus-mediated carcinogenesis and HPV-associated oral
and oropharyngeal squamous cell carcinoma. Part 2: human papillomavirus associated oral and oropharyngeal squamous cell carcinoma. Head Face Med 6:15 8. Kim SM (2016) Human papilloma virus in oral cancer. J Korean Assoc Oral Maxillofac Surg 42(6):327–336
123
LETTER TO THE EDITOR
Microarray technology for identification of human papilloma virus subtype in oropharyngeal squamous cell carcinoma Soung Min Kim1
Received: 18 June 2017 / Accepted: 28 June 2017 Ó Springer-Verlag GmbH Germany 2017
To the Editor, I have read with great concerns a recently published by Qureishi et al., entitled ‘‘Current and future techniques for human papilloma virus (HPV) testing in oropharyngeal squamous cell carcinoma’’ [1]. Although this review article was well written and provided a great deal of HPV-testing in oropharyngeal squamous cell carcinoma (OPSCC) information, I would like to add a few additional recommendations based on recently updated microarray technology. In Qureishi et al.’s article [1], the current techniques and the clinical applicability of emerging techniques including p16 immunohistochemistry (IHC), HPV DNA in situ hybridization (ISH), and HPV polymerase chain reaction (PCR), are introduced as the commonest tests for HPV detection in OPSCC. To date, there is no established standard method for detecting HPV as Qureishi et al. said. Detection methods vary not just in design, but in detection targets, such as HPV DNA, HPV RNA, viral oncoproteins, cellular proteins, and HPV-specific serum antibodies [2]. Among these, HPV DNA detection with PCR is known to show acceptable sensitivity and specificity for the detection of HPV 16 and 18 [3]. A DNA chip kit (MY-HPV chip kitÒ, Mygene Co., Seoul, Korea) could be shown as the example for HPV
microarray testing. This is a genotypic test that can identify the types of HPV by reacting to a spotted oligonucleotide chip with 19 kinds of HPV-specific sequences, including a high-risk group (16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56, 58) and a low-risk group (6, 11, 34, 40, 42, 43, 44). In this DNA chip method, DNA isolation, amplification by PCR, hybridization, washing, and scanning were sequentially performed to obtain results (Fig. 1) [4]. A 500–1000 ll extracted sample was transferred to a 1.5 ml tube by a serum separator or pipet. The supernatant was removed by centrifuging at 6000–9000 rpm for 10 min. After a 1 ml washing buffer was added, followed by centrifuging twice at 6000–9000 rpm for 5 min, the supernatant was removed, and the precipitate was dried. The mixtures of precipitate and extraction DNA were incubated in a 50 °C water bath for more than 3 h and then heated at 90–100 °C for 20 min. After centrifugation at 14,000 rpm for 10 min, a separated total amount of 150 ll of supernatant was used to conduct PCR. The first and second PCRs were performed
This comment refers to the article available at doi:10.1007/s00405-017-4503-1. & Soung Min Kim [email protected]; [email protected] 1
Department of Oral and Maxillofacial Surgery, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea
Fig. 1 Flow diagrams of HPV DNA chip (MY-HPV chip kitÒ, Mygene Co., Seoul, Korea) analysis, including DNA isolation, amplification by PCR, hybridization, washing, and scanning
123
Eur Arch Otorhinolaryngol Table 1 The summary of first and second PCR with a reagent, condition and cycles 1st PCR—2.5% agarose (450, 250 bp)
2nd PCR—2.5% agarose (150–200 bp)
Reagent
Volume
Condition
PCR premix 1 (450 bp) premix 3 (250 bp)
7.0 ll
50 °C/3 m
DNA
5.0 ll
95 °C/15 m
Cycles
Reagent
Volume
Condition
cycles
1
PCR premix 2 (150 bp)
6.5 ll
95 °C/5 m
1
1
1st PCR product
2.0 ll
95 °C/30 s
20
50 °C/40 s 72 °C/30 s Taq polymerase
0.3 ll
95 °C/30 s
35
Taq polymerase
0.3 ll
D.W
16.2 ll
Total
25.0 ll
72 °C/3 m
1
55 °C/30 s 72 °C/30 s D.W
12.7 ll
Total
25.0 ll
72 °C/3 m
1
PCR polymerase chain reaction, D.W distilled water, m minutes, s seconds, bp base pair, °C celsius
sequentially, as described in Table 1. The second PCR product was denatured at 95 °C for 5 min. The hybridization buffer was incubated at 43 °C for 10 min. These mixtures of PCR product and 35 ll hybridization buffer were inserted into each well of a slide and placed in water. Hybridization was performed at 43 °C for 4 h (Table 1). The hybridized slides are washed, and the hybridization seal was removed. After washing, the chip scanners were checked to see if they were coupled to each HPV-types probe; we determined the HPV subtypes by analyzing the results. When the HPV DNA chip was positive, we used the HPV-types noted by the HPV DNA chip results. If the HPV DNA chip was negative and the PCR result was positive, we regarded it as ‘‘other type’’ HPV-positive. Lastly, if both the PCR and HPV DNA chip results were negative, we concluded that the sample was HPV-negative. Detection of HPV can now move towards a more directed, clinically relevant and standardized approach. HPV DNA microarray detection with PCR pointed to its incomparably sensitivity. These methods can detect HPV at levels well below one viral copy genome per cell although detection of HPV DNA using PCR lacks specificity and HPV E6/E7 antibody detection lacks sensitivity [2, 5]. Thus, multimodality detection strategies are recommended to utilize the strengths of individual assays in combination to optimize the overall reliability of HPV detection. Since two HPV vaccines for cervical cancer, a quadrivalent and bivalent types that use virus-like particles (VLPs), were currently used in the medical commercial market in 2010, sampling techniques for HPV include microscopy, ELISA, Southern blot, Dot blot, hybrid capture, DNA microarray, and ligase chain reaction for probe amplification had been introduced [6, 7]. Although a standard procedure has not yet been generally accepted, both PCR and ISH are well validated, and gene expression by DNA microarray has recently gained acceptance as a high throughput method [8].
123
Recently, many analytical results by DNA microarray have been shown a clear and understandable analysis of HPV-associated OPSCC [3, 4]. The DNA microarray method has proper accuracy for detecting known HPV subtypes simultaneously, but has also limitations detecting new subtypes. I suggest that a DNA microarray method has proper accuracy for detecting known HPV subtypes in OPSCC. Acknowledgements This study was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI15C0689) and by the International Research & Development Program of the National Research Foundation of Korea (NRF2015K1A3A9A01028230). Compliance with ethical standards Conflict of interest There are no conflicts of interest in this article. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Review Board (IRBS-D2017006) at Seoul National University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
References 1. Qureishi A, Mawby T, Fraser L, Shah KA, Møller H, Winter S (2017) Current and future techniques for human papilloma virus (HPV) testing in oropharyngeal squamous cell carcinoma. Eur Arch Otorhinolaryngol 274(7):2675–2683 2. Westra WH (2014) Detection of human papillomavirus (HPV) in clinical samples: evolving methods and strategies for the accurate determination of HPV status of head and neck carcinomas. Oral Oncol 50(9):771–779 3. Lim YK, Choi JH, Park S, Kweon OJ, Park AJ (2016) Comparison of three different commercial kits for the human papilloma virus genotyping. J Clin Lab Anal 30(6):1110–1115 4. Kim SM, Kwon IJ, Myoung H, Lee JH, Lee SK (2017) Human papillomavirus (HPV) in Korean oral cancer patients using microarray technology. Human Pathol (submitted)
Eur Arch Otorhinolaryngol 5. Combes JD, Franceschi S (2014) Role of human papillomavirus in non-oropharyngeal head and neck cancers. Oral Oncol 50(5):370–379 6. Snow AN, Laudadio J (2010) Human papillomavirus detection in head and neck squamous cell carcinomas. Adv Anat Pathol 17(6):394–403 7. Feller L, Wood NH, Khammissa RA, Lemmer J (2010) Human papillomavirus-mediated carcinogenesis and HPV-associated oral
and oropharyngeal squamous cell carcinoma. Part 2: human papillomavirus associated oral and oropharyngeal squamous cell carcinoma. Head Face Med 6:15 8. Kim SM (2016) Human papilloma virus in oral cancer. J Korean Assoc Oral Maxillofac Surg 42(6):327–336
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