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Histopathologic features of high risk HPV-associated oral epithelial dysplasia
Vol. 117 No. 1 January 2014
LETTERS TO THE EDITOR Histopathologic features of high risk human papillomavirus-associated oral epithelial dysplasia To the Editor: We read with great interest the article by McCord et al. titled “Association of high-risk human papillomavirus infection with oral epithelial dysplasia.”1 We reported similar findings in our recent analysis of 20 cases of human papillomavirus (HPV)-associated oral epithelial dysplasias.2 In our experience, specific light microscopic features strongly predict HPV-driven oral epithelial dysplasia and by applying these criteria, 100% of our cases were positive for high risk HPV and p16. The most characteristic feature is the number of apoptotic and karyorrhectic cells throughout the full thickness of the epithelium; the latter are similar to the mitosoid cells seen in Heck disease and referred to as mitotic figures and mitotic-like structures by McCord et al.1 It is unclear whether the dyskeratotic cells to which McCord et al. alluded may have represented similar apoptotic cells. The mean number of apoptotic and karyorrhectic cells within two high-power fields (magnification 600) was 19.3 (median 18; range 6-40) and 1.8 (median 1; range 0-7) in HPV vs. non-HPV cases, respectively (P < .0001). While McCord et al. noted that p16 positivity involved “at least the deep half of the epithelial thickness in all cases,” our cases showed full thickness p16 positivity up to the parakeratin layer similar to what is demonstrated in Fig. 1B. The pattern of positivity for high risk HPV and p16, and for the H and E appearance of severe dysplasia ended abruptly in our cases, similar to Fig. 1B and C, suggesting clonality. These findings are similar to those illustrated by Daley et al., although not all their cases were positive for HPV and p16 studies were not performed.3 Although variable numbers of koilocytes were noted in each of our cases, their presence was not a reliable feature. Although 2 of the 20 dysplasia cases in our study were associated with invasive squamous cell carcinomas, the behavior of HPV-associated oral epithelial dysplasia remains unclear. We concur with McCord et al. that further large-scale studies are indicated to compare the behavior and prognosis of HPV-and nonHPV-associated cases of oral epithelial dysplasia. Mark A. Lerman, DMD Sook-Bin Woo, DMD Oral Medicine, Infection, and Immunity Harvard School of Dental Medicine 120
188 Longwood Avenue, Boston, MA 02115 United States
REFERENCES 1. McCord C, Xu J, Xu W, et al. Association of high-risk human papillomavirus infection with oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115:541-549. 2. Woo SB, Cashman EC, Lerman MA. Human papillomavirusassociated oral intraepithelial neoplasia. Mod Pathol. 2013. http:// dx.doi.org/10.1038/modpathol.2013.70 [Epub ahead of print]. 3. Daley T, Birek C, Wysocki GP. Oral bowenoid lesions: differential diagnosis and pathogenetic insights. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90:466-473. http://dx.doi.org/10.1016/j.oooo.2013.05.024
Histopathologic features of high risk HPV-associated oral epithelial dysplasia In Reply: We thank Drs. M.A. Lerman and S.B. Woo for their interest in our article and comments in their letter, “Histopathologic features of high-risk HPV-associated oral epithelial dysplasia.” We are excited to learn of their findings and analyses of human papillomavirus (HPV) associated oral epithelial dysplasia. The two studies, Woo et al. and our study, used a different approach to identify cases of high-risk HPV associated oral epithelial dysplasia (OED).1,2 Woo et al. selected cases diagnosed as epithelial dysplasia with marked apoptosis from their biopsy service and found that all cases were positive for high risk HPV by in situ hybridization and showed strong and continuous immunostaining for p16. We used p16 immunostaining to screen for high risk HPV associated epithelial dysplasias. p16 positive cases were examined for HPV by in situ hybridization. The high-risk HPV associated dysplasias were found to have a distinctive histologic appearance. Therefore the two studies independently arrived at the link among biologically significant infection by high risk HPV, strong and continuous p16 staining of the lesion and a distinctive histologic appearance that results from the viral infection. The histologic appearance of high risk HPV associated OED can be distinguished from HPV-negative OED in 2 respects. First, HPV associated lesions show full thickness dysplasia throughout the lesion, as described in both papers. Second, individual abnormal epithelial cells are present through the entire thickness of the epithelium. Woo et al. referred to this finding as marked karyorrhexis and apoptosis. We hesitate to use
OOOO Volume 117, Number 1
the term apoptosis without confirmation from specific biochemical assays.3 All cases of HPV positive OED in our study showed abnormal epithelial cells varying from cells with contracted, densely eosinophilic cytoplasm to large cells with pale cytoplasm and fragmented nuclear material, as well as cells with a giant nucleus or multiple nuclei. We used a descriptive approach to report on these appearances in our paper. In this respect, the detailed illustrations of the abnormal epithelial cells in Fig. 2 of Woo et al. are most helpful. We agree that high risk HPV infection is associated with strong, continuous p16 staining of the dysplastic epithelium. The slight disagreement in the reported thickness of the stained epithelium (‘at least the deep half’ compared to ‘more than 2/3’) could be due to difference in staining protocol. HPV associated oral lesions result from infection of basal cells. Replication of viral DNA allows virus to be transmitted to the progeny of infected basal cells that populate the suprabasal layers.4 We feel that discussion of clonality would require the use of genetic markers. HPV associated OED are uncommon lesions in our biopsy service. We would be interested to participate in large-scale studies to improve our understanding of HPV and non-HPV associated epithelial dysplasia. Christina McCord, DDS Grace Bradley, DDS, MSc Faculty of Dentistry, University of Toronto 124 Edward Street, Toronto ON M5G 1G6, Canada REFERENCES 1. McCord C, Xu J, Xu W, et al. Association of high-risk human papillomavirus infection with oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115:541-549. 2. Woo SB, Cashman EC, Lerman MA. Human papillomavirusassociated oral intraepithelial neoplasia. Mod Pathol. 2013; [Epub ahead of print]. 3. Galluzzi L, Vitale I, Abrams JM, et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 2012;19: 107-120. 4. IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans. Human Papillomaviruses. Lyon, France: IARC; 2007:: 48-51. http://dx.doi.org/10.1016/j.oooo.2013.06.005
Proper size of the 3-dimensional periodontal ligament stem cell (3D PDLSC) sphere is vital for cell viability To the Editor: In a recent issue of Oral Surgery, Oral Medicine, Oral Pathology, and Oral Radiology, we read with great
LETTERS TO THE EDITOR
121
interest the article by Singhatanadgit et al.,1 titled “Osteogenic potency of a 3-dimensional scaffold-free bonelike sphere of periodontal ligament stem cells in vitro.” The authors have shown a novel culture technique using a polypropylene tube to produce osteogenic spheres. On cross referencing, however, we fervently disagree with their interpretation concerning the size of the 3-dimensional periodontal ligament stem cell (3D PDLSC) sphere which may lead to inappropriate concept regarding stem cell-based bone engineering. The authors stated that samples in osteogenic medium had a desirable diameter of approximately 3.0 0.2 mm, compared with that in control culture media which had a relatively smaller diameter of 2.0 0.2 mm. Additionally, they compared the spheres in their study with that produced in a 96-well plate,2 and concluded that their spheres with much greater size (diameters of several mm) were better for osteogenic differentiation of the encapsulated stem cells. However, based on the previous literatures, we find that spheres in millimeter-scale are far beyond the diffusion limit of oxygen, thus not favorable for cell survival and viability.3,4 Several studies also focused on the proper size of stem cell-laden spheres in this field. Man et al.5 fabricated platelet-rich plasma and adipose-derived stem cell-laden alginate microspheres with appropriate radius of 176 mm (diameter of 352 mm) to investigate their angiogenic and osteogenic potentials. Besides, Zhou et al.6 produced alginate-fibrin microbeads with suitable diameters of several 100 mm, which can degrade rapidly and release the cells in a few days. Taken together, a small size of the 3D PDLSC sphere is vital for inner cell survival. First of all, the influx of nutrients and the outflux of metabolic wastes would be easier due to the small size and high surface area of the microspheres. Secondly, the release of proteins from spheres is related both to diffusion distances and 3 dimensional sphere size.7 Small size would facilitate release of growth factors and intense signals, which is important for initiating proper response of surrounding tissue in vivo. Finally, we would like to congratulate the authors for their contributions to encapsulation technology, which is innovative to stem cell-based bone engineering. Lin Xiang, PhD Li Ma, PhD Tianlu Wang, PhD Na Wei, PhD Ping Gong, PhD Faculty, State Key Laboratory of Oral Diseases and Dental Implant Center West China Hospital of Stomatology Sichuan University, No. 14, Third Section Renmin Nan Road, Chengdu Sichuan 610041, China
LETTERS TO THE EDITOR Histopathologic features of high risk human papillomavirus-associated oral epithelial dysplasia To the Editor: We read with great interest the article by McCord et al. titled “Association of high-risk human papillomavirus infection with oral epithelial dysplasia.”1 We reported similar findings in our recent analysis of 20 cases of human papillomavirus (HPV)-associated oral epithelial dysplasias.2 In our experience, specific light microscopic features strongly predict HPV-driven oral epithelial dysplasia and by applying these criteria, 100% of our cases were positive for high risk HPV and p16. The most characteristic feature is the number of apoptotic and karyorrhectic cells throughout the full thickness of the epithelium; the latter are similar to the mitosoid cells seen in Heck disease and referred to as mitotic figures and mitotic-like structures by McCord et al.1 It is unclear whether the dyskeratotic cells to which McCord et al. alluded may have represented similar apoptotic cells. The mean number of apoptotic and karyorrhectic cells within two high-power fields (magnification 600) was 19.3 (median 18; range 6-40) and 1.8 (median 1; range 0-7) in HPV vs. non-HPV cases, respectively (P < .0001). While McCord et al. noted that p16 positivity involved “at least the deep half of the epithelial thickness in all cases,” our cases showed full thickness p16 positivity up to the parakeratin layer similar to what is demonstrated in Fig. 1B. The pattern of positivity for high risk HPV and p16, and for the H and E appearance of severe dysplasia ended abruptly in our cases, similar to Fig. 1B and C, suggesting clonality. These findings are similar to those illustrated by Daley et al., although not all their cases were positive for HPV and p16 studies were not performed.3 Although variable numbers of koilocytes were noted in each of our cases, their presence was not a reliable feature. Although 2 of the 20 dysplasia cases in our study were associated with invasive squamous cell carcinomas, the behavior of HPV-associated oral epithelial dysplasia remains unclear. We concur with McCord et al. that further large-scale studies are indicated to compare the behavior and prognosis of HPV-and nonHPV-associated cases of oral epithelial dysplasia. Mark A. Lerman, DMD Sook-Bin Woo, DMD Oral Medicine, Infection, and Immunity Harvard School of Dental Medicine 120
188 Longwood Avenue, Boston, MA 02115 United States
REFERENCES 1. McCord C, Xu J, Xu W, et al. Association of high-risk human papillomavirus infection with oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115:541-549. 2. Woo SB, Cashman EC, Lerman MA. Human papillomavirusassociated oral intraepithelial neoplasia. Mod Pathol. 2013. http:// dx.doi.org/10.1038/modpathol.2013.70 [Epub ahead of print]. 3. Daley T, Birek C, Wysocki GP. Oral bowenoid lesions: differential diagnosis and pathogenetic insights. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90:466-473. http://dx.doi.org/10.1016/j.oooo.2013.05.024
Histopathologic features of high risk HPV-associated oral epithelial dysplasia In Reply: We thank Drs. M.A. Lerman and S.B. Woo for their interest in our article and comments in their letter, “Histopathologic features of high-risk HPV-associated oral epithelial dysplasia.” We are excited to learn of their findings and analyses of human papillomavirus (HPV) associated oral epithelial dysplasia. The two studies, Woo et al. and our study, used a different approach to identify cases of high-risk HPV associated oral epithelial dysplasia (OED).1,2 Woo et al. selected cases diagnosed as epithelial dysplasia with marked apoptosis from their biopsy service and found that all cases were positive for high risk HPV by in situ hybridization and showed strong and continuous immunostaining for p16. We used p16 immunostaining to screen for high risk HPV associated epithelial dysplasias. p16 positive cases were examined for HPV by in situ hybridization. The high-risk HPV associated dysplasias were found to have a distinctive histologic appearance. Therefore the two studies independently arrived at the link among biologically significant infection by high risk HPV, strong and continuous p16 staining of the lesion and a distinctive histologic appearance that results from the viral infection. The histologic appearance of high risk HPV associated OED can be distinguished from HPV-negative OED in 2 respects. First, HPV associated lesions show full thickness dysplasia throughout the lesion, as described in both papers. Second, individual abnormal epithelial cells are present through the entire thickness of the epithelium. Woo et al. referred to this finding as marked karyorrhexis and apoptosis. We hesitate to use
OOOO Volume 117, Number 1
the term apoptosis without confirmation from specific biochemical assays.3 All cases of HPV positive OED in our study showed abnormal epithelial cells varying from cells with contracted, densely eosinophilic cytoplasm to large cells with pale cytoplasm and fragmented nuclear material, as well as cells with a giant nucleus or multiple nuclei. We used a descriptive approach to report on these appearances in our paper. In this respect, the detailed illustrations of the abnormal epithelial cells in Fig. 2 of Woo et al. are most helpful. We agree that high risk HPV infection is associated with strong, continuous p16 staining of the dysplastic epithelium. The slight disagreement in the reported thickness of the stained epithelium (‘at least the deep half’ compared to ‘more than 2/3’) could be due to difference in staining protocol. HPV associated oral lesions result from infection of basal cells. Replication of viral DNA allows virus to be transmitted to the progeny of infected basal cells that populate the suprabasal layers.4 We feel that discussion of clonality would require the use of genetic markers. HPV associated OED are uncommon lesions in our biopsy service. We would be interested to participate in large-scale studies to improve our understanding of HPV and non-HPV associated epithelial dysplasia. Christina McCord, DDS Grace Bradley, DDS, MSc Faculty of Dentistry, University of Toronto 124 Edward Street, Toronto ON M5G 1G6, Canada REFERENCES 1. McCord C, Xu J, Xu W, et al. Association of high-risk human papillomavirus infection with oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115:541-549. 2. Woo SB, Cashman EC, Lerman MA. Human papillomavirusassociated oral intraepithelial neoplasia. Mod Pathol. 2013; [Epub ahead of print]. 3. Galluzzi L, Vitale I, Abrams JM, et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 2012;19: 107-120. 4. IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans. Human Papillomaviruses. Lyon, France: IARC; 2007:: 48-51. http://dx.doi.org/10.1016/j.oooo.2013.06.005
Proper size of the 3-dimensional periodontal ligament stem cell (3D PDLSC) sphere is vital for cell viability To the Editor: In a recent issue of Oral Surgery, Oral Medicine, Oral Pathology, and Oral Radiology, we read with great
LETTERS TO THE EDITOR
121
interest the article by Singhatanadgit et al.,1 titled “Osteogenic potency of a 3-dimensional scaffold-free bonelike sphere of periodontal ligament stem cells in vitro.” The authors have shown a novel culture technique using a polypropylene tube to produce osteogenic spheres. On cross referencing, however, we fervently disagree with their interpretation concerning the size of the 3-dimensional periodontal ligament stem cell (3D PDLSC) sphere which may lead to inappropriate concept regarding stem cell-based bone engineering. The authors stated that samples in osteogenic medium had a desirable diameter of approximately 3.0 0.2 mm, compared with that in control culture media which had a relatively smaller diameter of 2.0 0.2 mm. Additionally, they compared the spheres in their study with that produced in a 96-well plate,2 and concluded that their spheres with much greater size (diameters of several mm) were better for osteogenic differentiation of the encapsulated stem cells. However, based on the previous literatures, we find that spheres in millimeter-scale are far beyond the diffusion limit of oxygen, thus not favorable for cell survival and viability.3,4 Several studies also focused on the proper size of stem cell-laden spheres in this field. Man et al.5 fabricated platelet-rich plasma and adipose-derived stem cell-laden alginate microspheres with appropriate radius of 176 mm (diameter of 352 mm) to investigate their angiogenic and osteogenic potentials. Besides, Zhou et al.6 produced alginate-fibrin microbeads with suitable diameters of several 100 mm, which can degrade rapidly and release the cells in a few days. Taken together, a small size of the 3D PDLSC sphere is vital for inner cell survival. First of all, the influx of nutrients and the outflux of metabolic wastes would be easier due to the small size and high surface area of the microspheres. Secondly, the release of proteins from spheres is related both to diffusion distances and 3 dimensional sphere size.7 Small size would facilitate release of growth factors and intense signals, which is important for initiating proper response of surrounding tissue in vivo. Finally, we would like to congratulate the authors for their contributions to encapsulation technology, which is innovative to stem cell-based bone engineering. Lin Xiang, PhD Li Ma, PhD Tianlu Wang, PhD Na Wei, PhD Ping Gong, PhD Faculty, State Key Laboratory of Oral Diseases and Dental Implant Center West China Hospital of Stomatology Sichuan University, No. 14, Third Section Renmin Nan Road, Chengdu Sichuan 610041, China