Histological subtypes and characteristic structures of HPV-associated oropharyngeal carcinoma; study with Japanese cases
© Fujimaki et al.; licensee BioMed Central Ltd. 2013
Received: 12 November 2013
Accepted: 10 December 2013
Published: 19 December 2013
Human papillomavirus-associated oropharyngeal carcinoma (HPV-OPC) is clinicopathologically distinct entity from the HPV-unassociated one (nHPV-OPC). This study aimed to determine the relationship between histological subtypes of OPC and HPV status for Japanese cases and to identify histological structures of HPV-OPC.
66 OPC cases were categorized into conventional squamous cell carcinoma (SCC) and the variants. Conventional SCC was subcategorized into keratinizing (KSCC), non-keratinizing (NKSCC), and hybrid SCC (HSCC). HPV status of all cases was determined using p16-immunohistochemistry and HPV-DNA ISH.
Two histological subtypes, NKSCC and HSCC, tended to be HPV-OPC and KSCC tended to be nHPV-OPC with statistical significance. Two histological structures, abrupt keratinization, defined in the text, and comedo-necrosis among non-maturing tumor island, were observed for 58.1% and 38.7% of HPV-OPC, and tended to exist for HPV-OPC with statistical significance.
This study showed the association of NKSCC/HSCC with HPV-OPC in Japanese cases, and two histological structures, abrupt keratinization and comedo-necrosis among non-maturing island, were considered characteristic histological features of HPV-OPC.
The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1816432541113073.
KeywordsHuman papillomavirus-associated oropharyngeal carcinoma Non-keratinizing squamous cell carcinoma Abrupt keratinization Comedo-necrosis among non-maturing island
Human papillomavirus (HPV)-associated oropharyngeal carcinoma (HPV-OPC) has been recognized as a distinct clinicopathologic entity of head and neck cancer. HPV-OPC has been reported to be distinct from HPV-unassociated oropharyngeal carcinoma (nHPV-OPC) on a molecular, epidemiological, and clinical basis[2, 3]. From a clinical perspective, HPV-OPC is likely to be more radio-/chemo-sensitive, with a longer recurrence-free period, more positivity in regional lymph nodes, and with better prognosis than nHPV-OPC[4–7]. As for the pathological view, p53 overexpression/mutation are found at significantly lower frequency for HPV-OPC than for nHPV-OPC[1, 8].
In terms of histopathology, there have been several papers on some histological subtypes/variants that are more likely to be HPV-associated[9–15]. For example, Chernock et al. reported that non-keratinizing SCC (NKSCC) and hybrid SCC (HSCC) were more likely to be HPV-positive than keratinizing SCC (KSCC)[9, 10]. However, there has been no study using Japanese cases, or no study focusing on characteristic histopathological structures for HPV-OPC so far. In Japanese OPC cases, the impact of several risk factors other than HPV infection, such as tobacco/alcohol exposure, may be different from that in Western countries. For example, in Western countries, patients with HPV-OPC are reported to have less tobacco exposure but more marijuana exposure than those with nHPV-OPC[3, 5]. It is important to see the tendency of histological subtypes of HPV-OPC in Japanese cases with the different background risk factors.
In this study, we classified our HPV-OPC and nHPV-OPC cases by histological subtypes and determined if there are some histological features of concern, in order to determine the specificity/sensitivity of NKSCC, KSCC, HSCC for HPV-OPC with Japanese OPC cases and to identify histological structures characteristic for HPV-OPC.
In this study, HPV status for each case was judged by p16 immunohistochemistry (IHC)/in situ hybridization (ISH). Although there have been studies which propose genomic amplification of human telomerase gene or C-MYC as helpful screening methods for HPV-associated cancer/high-grade lesions in cases of uterine cervix[16, 17], a gold standard method for detecting HPV-associated OPC has not been established at present. Several studies have mentioned the reliability and accessibility of p16 IHC and ISH for HPV DNA[18–21]. In this study, we used the combined detection method of HPV ISH and p16 IHC according to Singhi et al..
Clinical features of oropharyngeal SCCs
(n = 66)
(n = 31)
(n = 35)
Assessment of histopathology of oropharyngeal SCC
Immunohistochemistry (IHC) for p16
IHC for p16 was performed using 4-μm-thick tissue sections from formalin-fixed paraffin-embedded tissues and with DAKO EnVision™ + Kit (DAKO Corp., Carpenteria, CA) following the manufacturer’s instructions.
In situ hybridization (ISH) for high-risk HPV-DNA
For ISH, a catalyzed signal amplification method for biotinylated probes (DAKO GenPoint, Carpenteria, CA) was used. Briefly, 5-μm tissue sections underwent deparaffinization, heat-induced target retrieval in citrate buffer (DAKO Target Retrieval Solution, Carpenteria, CA), and digestion using Proteinase K (DAKO, Carpenteria, CA). Slides were subsequently hybridized with a biotinylated HPV16/18-type-specific probe (DAKO, Carpenteria, CA). Signal amplification was performed by consecutive application of streptavidin-HRP complex and biotinyl tyramide. Visualization of positive hybridization signals was performed by incubation with the chromogenic substrate diaminobenzidine. Interpretation of staining was performed without knowledge of p16 IHC positivity or tumor origin. In some cases where there was discordance between p16 IHC and HPV 16/18 status (i.e. in p16 IHC-positive, ISH-negative cases), we performed analysis with a more extended panel of ISH probes that included types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 (DAKO, Carpenteria, CA) in order to check for other high-risk HPV associations.
As controls, squamous cell carcinoma of the tonsil was hybridized with a positive control (Human DNA, DAKO, Carpenteria, CA) biotinylated DNA probe, and with a negative control (Plasmid DNA, DAKO, Carpenteria, CA) biotinylated DNA probe.
To determine the association of each of the histological subtypes/histological structures with HPV status, and to see the relationship of patients’ age (43-59/60-87) and sex (F/M) to histological subtypes of OPC, cross-tabulation analyses were performed using Fisher’s exact test. The statistical analyses were performed using PASW Statistics 18.0 (SPSS, Chicago, IL).
This study was approved by the ethics committee of Juntendo University School of Medicine.
Histopathological features of HPV-OPC
Histological classification of oropharyngeal carcinomas
Number of cases
Abrupt keratinization in oropharyngeal carcinomas
Number of cases
Comedo-necrosis among non-maturing tumor island in oropharyngeal carcinomas
Number of cases
The present study showed that most (87% of all cases and 90% of biopsy cases) OPC cases were conventional SCC, and were subcategorized into 36.7% KSCC, 31.7% NKSCC, and 18.3% HSCC, where NKSCC and HSCC were more likely to be HPV-OPC, and most KSCC was nHPV-OPC with statistical significance. These results for Japanese OPC cases are in accordance with those of Chernock et al.. In the study by Chernock et al., they compared the frequency of each of the three subtypes and the result of ISH for HPV-DNA and p16 IHC, and their result of ISH seemed to be more similar to our present data than that of p16 IHC.
In the present Japanese study, no significant difference in clinical data, including risk factors, was seen between HPV-OPC and nHPV-OPC groups. In Western countries, patients with HPV-OPC have different epidemiological data including less tobacco exposure, more marijuana exposure, and lower age than those with nHPV-OPC[3, 5]. It seems interesting that, despite the different clinical background, there are still similar tendencies of histological subtypes between Japanese and Western HPV-OPC.
A few researchers have reported the association of papillary, basaloid, and undifferentiated SCC (lymphoepithelial SCC) with HPV-OPC[11–15]. In this study, there was also a tendency of the former two variants to be HPV-OPC, although with a small sample size.
When investigating the histopathological features of HPV-OPC, some histological structures were noteworthy. Abrupt keratinization, cancer pearl formation/squamous maturation in an abrupt fashion among non-mature tumor island, or at a peripheral site of tumor nest, were not usually observed in our daily pathological practice for squamous cell carcinoma of any other organ/tissue. Although comedo-necrosis is not rare in many carcinomas including keratinizing SCC, comedo-necrosis among non-maturing SCC cells is unusual. Hence, we analyzed whether these two structures are characteristic histological structures for HPV-OPC or not. This study suggested that these two histological structures, abrupt keratinization and comedo-necrosis among non-maturing tumor island, are very specific for HPV-OPC.
Our study showed that NKSCC and HSCC are statistically more likely to be HPV-associated carcinoma, while KSCC is statistically more likely to be nHPV-OPC, which was also true for Japanese patients. Two histological structures, abrupt keratinization and comedo-necrosis among non-maturing island, were considered to be very specific pathological structures for HPV-OPC.
Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
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