Abstract
Introduction
Human papillomavirus (HPV) is among the reported etiologies of laryngeal squamous cell carcinoma (LSCC). Understanding the impact of HPV on LSCC may help reduce its incidence. This study investigates the association between HPV and LSCC as well as the roles for different immunohistochemical stains in HPV detection.
Methods
A total of fifty-two formalin-fixed, paraffin-embedded tissue samples of LSCC, diagnosed between 2005 and 2015, were obtained from the archives of the Pathology Department. The samples were stained and processed to evaluate the relationship of HPV to LSCC.
Results
Patients had a mean age of 65.02 ± 14.341 years. By polymerase chain reaction (PCR), high-risk strains of HPV were detected in 15.4% of tissue samples. HPV-16 was found in 75% of the positive samples for an overall prevalence of 13.5%. P16 immunostaining was positive in 15.4% of cases while cyclin B and cyclin E were positive in 65.4% and 76.9%, respectively. Over half of the cases were histologically graded as moderately-differentiated, 28.8% as well-differentiated, and 15.4% as poorly-differentiated.
Discussion
The prevalence of HPV-positive LSCC was lower than previously stated in the literature. HPV-16 was the most commonly detected subtype, in concordance with the findings of multiple other studies. HPV-positive LSCC trended with higher histologic grade. P16 and cyclin E immunohistochemical stains were of limited use in identifying HPV in LSCC. In contrast, cyclin B had a high sensitivity which could be used to rule out HPV in LSCC.
Electronic supplementary material
The online version of this article (10.1007/s12105-020-01150-2) contains supplementary material, which is available to authorized users.
Keywords: Laryngeal squamous cell carcinoma, Human papilloma virus, Cyclin-dependent kinase inhibitor p16, Cyclin B, Cyclin E, PCR
Introduction
Laryngeal cancer comprises 30–40% of head and neck malignancies and 1% of all malignant tumors. In 2018, 177,422 newly diagnosed cases of laryngeal cancer were registered. Cancers of the larynx caused 100,000 deaths globally and accounted for 1% of all cancer deaths [1].
Etiologic factors for the development of laryngeal squamous cell carcinoma (LSCC) include air pollution, alcohol, and smoking [2]. Increasingly, studies have associated human papillomavirus (HPV) to the development of LSCC and suggested the virus may be among the most important causes of these malignancies. The reported percentages of HPV-positive LCSSs has varied, however [3]. HPV infection is causative of numerous cancer types, including head and neck cancers, and its potential oncogenic role in laryngeal cancer development has been well recognized in recent years [4].
HPV is the most common sexually transmitted infection (STI) in the United States, and over 290 million women are infected with HPV at any given time [5]. HPV transmission is associated with open-mouth kissing and oral sex, with increased transmission as compared to vaginal sexual intercourse [6]. The consequences of HPV infection include 528,000 cervical cancer cases and 266,000 cervical cancer-related deaths each year [7].
Immunohistochemistry (IHC) techniques can be used to assess various cell cycle proteins that may serve as important diagnostic or prognostic markers in tumors. For example p16, a cyclin dependent kinase inhibitor, is a surrogate marker of HPV-E7 degradation of retinoblastoma protein and used to detect HPV in oropharyngeal cancers [8]. Cyclin B1 is a subunit of cyclin dependent kinase 1 which is essential for the G2 phase-mitosis transition. Its overexpression is related to multiple human cancers including head and neck squamous cell carcinoma [9]. Cyclin E is a component of the retinoblastoma pathway expressed in G1-S cells. It has been extensively studied in human tumors due to activating mutations in regulatory pathways [10].
Identifying HPV in LSCC may lead to a decrease its incidence and mortality. This study aims to explore the prevalence of high-risk HPV in LSCC in Northern Jordan and assess the ability of different immunohistochemical (IHC) stains to identify the virus in tumor samples.
Materials and Methods
Sample Collection
A total of fifty-two (52) formalin-fixed, paraffin-embedded samples with histologically-proven diagnoses of LSCC were obtained from the archives of the Pathology Department at King Abdullah University Hospital from the period 2005 to 2015. Twenty-six (26) control cases with histologic diagnoses of benign laryngeal lesion were also obtained and matched for age and sex (case: control ratio 2:1). All samples were reviewed, and a consensus diagnosis was confirmed by three pathologists. The study was performed on archival material and the samples were coded and processed anonymously.
Immunohistochemical Staining Protocol
Four micrometre sections were produced from the 52 paraffin blocks, each representing a different LSCC specimen. The sections were de-waxed in two changes of xylene, hydrated through a graded series of ethanol alcohol, and rehydrated in distilled water. Antigen retrieval was performed in a PT-link - Instrument (Dako, Glostrup, Denmark) using a high pH-citrate buffer for 20 minutes. Endogenous peroxidase activity was blocked by incubation with 2.5% hydrogen peroxide for 10 minutes. Slides were then washed with phosphate buffer saline twice, and the following primary antibodies were applied: anti-cyclin B1 antibody (mouse monoclonal [v152] to cyclin B1), protein G purified (diluted 1:200 for 45 minute at room temperature); anti-cyclin E1 antibody (rabbit polyclonal to cyclin E1), protein A purified (diluted 1:200 for 45 minutes at room temperature); anti-P16 ARC antibody (rabbit polyclonal to P16 ARC), immunogen affinity purified (diluted 1:200 for 45 minutes at room temperature); anti-HPV antibody (mouse monoclonal [K1H8] to HPV), tissue culture supernatant (pre-diluted/ready to use). All markers were from Abcam Company, Cambridge, MA, USA. After the application of antibodies, slides were washed twice with PBS, and signal visualization was carried out using the Envision Dual Link System HRP Kit, K8000 (Dako, lostrub, Denmark). The slides were washed with distilled water and counterstained with Mayer’s hematoxylin. The immunohistochemical (IHC) stains were performed on the cancerous lesions and the normal adjacent squamous epithelium if applicable.
PCR Protocol
Extraction of DNA was performed via the QIAamp DNA Formalin Fixed Paraffin Embedded (FFPE) Tumor Tissue kit from QIAGEN and followed by real-time polymerase chain reaction (PCR) for detection of high-risk HPV genotypes. FFPE tissue samples were treated with 1 ml xylene and absolute ethanol twice. Afterwards, the samples were treated with lysis buffer and proteinase K at 56 °C for 15 minutes. The ethanol was added as a binding solution and the whole solution transferred into the filter. Screening was performed on the extracted FFPE using the Rotor-Gene 6000 cycler based on Taqman probe from QIAGEN. The quality of the extracted DNA was assayed versus an internal control in the same sample to assure the quality and quantity.
The study was approved by the Jordan University of Science and Technology Institutional Review Board. Statistical analysis was carried out using IBM SPSS version 24. A p value ≤ 0.05 was considered statistically significant.
Results
A total of 52 cases, 43 males and 9 females, of laryngeal squamous cell carcinoma (LSCC) were included in this study. The enrolled patients had a mean age of 65.02 ± 14.341 years. The median age of the population; 65 years, was similar to its mean with a range of 76 years (24–100). A total of 32 patients (61.5%) were 60 years or older at the time of diagnosis and 38.5% of patients were below the age of 60 (Table S1).
HPV Detection by PCR
High-risk strains of HPV were detected in eight cases yielding a positivity rate of 15.4%. Meanwhile, the positivity rate in the control group was zero (p = 0.032). Out of the high-risk HPV-HPR positive cases, six specimens were positive for HPV-16 (75%), one was positive for HPV-18 (12.5%), and one was positive for both HPV-16 and HPV-33 (12.5%) (Figure S1). HPV-PCR positive cases had a mean age of 64.63 ± 4.579 years. There was no statistically significant age difference between HPV-PRC positive and HPV-PCR negative cases, although the negative tumors had a slightly higher mean age (65.09 ± 14.467 years).
HPV Immunohistochemical Stain
All cases were tested for HPV by IHC. Only one case that was positive by PCR showed immunoreactivity by IHC, yielding a sensitivity of 12.5%. The positive specimen demonstrated a HPV-16 genotype by PCR. HPV-IHC had no false positive results giving a specificity and positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 86.3%. When examining the staining pattern, the single positive case showed no expression in the adjacent normal epithelium. Furthermore, no cases showed positive uptake with negative results or with no normal adjacent epithelium.
Other Immunohistochemical Stains
The three additional IHC stains, p16, cyclin B1, and cyclin E1, showed variable outcomes. p16 was positive in eight cases (15.4%) of which two were HPV-PCR-positive: one for HPV-16 and the other for both HPV-16 and HPV-33. Meanwhile, cyclin B1 and cyclin E1 were positive in 65.4% and 76.9% of samples, respectively. Six cases were positive for cyclin B1 and HPV-PCR and seven were positive for cyclin E1 and HPV-PCR with HPV genotypes of HPV-16, -18, and combined − 16 and − 33 detected. Table S2 demonstrates the previous results in addition to the sensitivity, specificity, PPV, and NPV of each to evaluate the capability of these IHCs in predicting HPV status.
The expression of these markers was also assessed in relation to histologic grade: well-, moderately-, and poorly-differentiated. Cyclin B1 showed increased expression with high histological grade, reaching 100% of poorly differentiated tumours, while expression of neither p16 or cyclin E1 expression showed correlation.
Normal Adjacent Squamous Epithelium
Overall, normal adjacent squamous epithelium (NASE) was positive for p16 in 10 cases and negative in 25 cases. No NASE was present for evaluation in 17 cases. Among the 8 LSCCs that were p16 positive in the tumor cells, 4 also showed expression in NASE, 2 were negative in NASE, and 2 cases lacked NASE for evaluation.
Regarding cyclin B1 expression, 34.6% of cases showed NASE positivity, 32.7% were negative, and 32.7% lacked NASE for evaluation. Among the 34 specimens with cyclin B1 expression in tumor cells, half were also NASE positive, 20.6% were negative, and 29.4% lacked NASE. Only one case was negative for cyclin B1 in tumor cells but positive in NASE. Ten cases were negative for cyclin B1 in tumor cells and NASE, and 7 cases lacked NASE for evaluation.
Cyclin E1 was positive in 76.9% of LSCCs in tumor cells. Of these, NASE was positive in 65% of cases, negative in 5%, and not present in 30%. Among the remaining LSCCs negative for cyclin E1, 58.3% were also negative in NASE. The remainder lacked NASE. There were no cases negative for cyclin E1 in the tumor that showed expression in NASE.
Tumor Differentiation
Of all LSCCs, 15 (28.8%) were well-differentiated, 29 (55.8%) were moderately differentiated, and 8 (15.4%) were poorly differentiated. Patients with well-, moderately-, and poorly-differentiated tumours had mean ages of 63.20, 66.03, and 64.75 years, respectively.
The eight HPV-PCR positive cases comprised one well-differentiated (12.5%), four moderately-differentiated (50%), and three poorly-differentiated tumors (37.5%). The well-differentiated tumor had a combined HPV-16 and − 33 genotype. Among the four moderately-differentiated cases, three had a HPV-16 genotype and one had a HPV-18 genotype. All poorly-differentiated tumors had a HPV-16 genotype (Table S3). Table S3 summarizes these findings along with IHC expression.
According to these findings, HPV-positive LSCCs were more likely to be poorly-differentiated. As shown in Figure S2, 37.5% of HPV-PCR positive cases were poorly-differentiated compared to 11.4% of HPV-negative cases. Meanwhile, 31.8% of well-differentiated LSCCs were HPV-PCR positive and 12.5% were HPV-PCR negative.
Discussion
HPV Prevalence
The reported prevalence of HVP in LSCC varies widely across studies, ranging from 0 to 75% [11, 12]. In a meta-analysis of fifty-five studies, however, the overall HPV prevalence was reported at 28% (95% CI 23.5–32.9) in laryngeal cancers [2]. In the current study, the prevalence of HPV positive LSCC fell below the overall prevalence at 15.4%. Compared to studies focused in the Middle East, these results are lower than those reported in Iran and Turkey at 25% and 26%, respectively, while significantly higher than the 3% reported in Egypt in the year 2018 [2].
There are several theories regarding the range of HPV positivity in LSCC including sample contamination, high frequency of false positives, and geographic variation [3, 13]. Another consideration is the disparity in techniques used to detect HPV DNA as seen in the different detection rates between quantitative PCR and ELISA based techniques [14]. In this study, the difference of HPV-positivity rates between PCR and IHC also varied with 1% of samples (1 case) showing HPV by IHC as opposed to 15.4% (8 cases) by PCR. This agrees with previous studies implying the superiority of PCR over IHC for diagnosing and detecting HPV [15]. Thus, PCR is indicated as the initial test for its improved sensitivity while IHC proved to be of limited value in this sample.
HPV Genotype
In the current study, HPV-16 was the most frequently encountered genotype and identified in 87.5% of HPV-positive cases. This finding corroborates earlier studies reporting HPV-16 as the most common type in laryngeal cancer cases globally and in the Middle East [2, 3]. The dominance of HPV-16 and HPV-18, which was encountered in a single case in our study, in oral cancers was similar to those focused in the Middle East region [16]. All HPV genotypes were high-risk; no low-risk types were detected in our cohort. As this study focused on malignant tumors, this finding was expected and in agreement with results reported in multiple studies [2, 17].
Differentiation
HPV-positive tumors appeared as moderately-differentiated squamous cell carcinomas in half of the cases. This is consistent with previous studies reporting that HPV-positive laryngeal carcinoma tends to present as moderately-differentiated [18]. Moreover, the current study showed that HPV-positive LSCCs were more likely to be associated with higher histologic grades as compared to HPV-negative cases. This finding is in agreement with previous reports linking HPV-positivity to poorly-differentiated lesions [19].
P16 Immunohistochemical Staining
By IHC, p16 is considered positive when “block” staining occurs. This is defined as strong nuclear and cytoplasmic expression in continuous 10–20 cell segments involving the basal and parabasal layers [20]. Overexpression of p16 is a well-established surrogate marker for high-risk HPV in oropharyngeal squamous cell carcinoma (OPSCC) [18]. In this study of LSCCs, p16 expression was not a reliable marker for HPV positivity, as only 25% of HPV-positive cases by PCR showed positive p16 staining. This finding contrasts studies of OPSCC that state a strong correlation between HPV infection and p16 overexpression and attribute the minimal number of p16-positive cases with HPV-negative results to genetic perturbations [21, 22]. In other studies evaluating HPV and p16 expression in LSCC, wide variation is reported. While some studies agree with our finding and report limited correlation, others report correlations greater than 65% [23]. This might be owed to differences in interpretation of p16 positivity.
The 25% sensitivity of p16 IHC indicates that false positive staining compared to PCR results is seen in 75% of HPV-related LSCC cases, making this stain unreliable for confirming HPV involvement in laryngeal cancers. This finding contradicts previously reported studies that imply that high-level p16 is an excellent biomarker for high-risk HPV infections and cancers [24].
Cyclin B1 Immunohistochemical Stain
Cyclin B1 directs the transition from G2 to M phase. Its overexpression has been related to oral, esophageal, breast, colon, and prostate carcinomas [25]. In laryngeal cancers, cyclin B1 is expressed predominantly in the cytoplasm whereas it is expressed in the nucleus in normal mucosa [26]. In this study, cyclin B1 was expressed in all cases of poorly-differentiated LSCCs. Moderately-differentiated and well-differentiated tumors were positive for cyclin B1 in 62.1% and 53.3% of cases, respectively. This aligns with previous reports indicating high cyclin B1 expression among poorly-differentiated tumors as compared to other histologic grades and suggests an association with more aggressive behavior [26, 27].
Cyclin B1 was previously reported to be HPV-type dependent and overexpressed in high-risk types, most specifically the HPV-18 genotype [28, 29]. In our study, only a single confirmed case of HPV-18 was identified which was cyclin B1 positive. A more detailed study focusing on the HPV-18 genotype could help investigate the role of cyclin B1 in HPV-18 associated laryngeal carcinogenesis.
The high sensitivity, 87.5%, can be used to rule out HPV in LSCC if cyclin B1 staining is negative. However, a positive test result would be of minimal benefit due to the low-moderate specificity of cyclin B1; 38.6%. With a 100% detection rate in poorly-differentiated tumors, cyclin B1 may be used to evaluate the disease grade and severity.
CyclinE1 Immunohistochemical Stain
Cyclin E plays a role in the G1-S transition and is related to malignant transformation. In this study, cyclin E1 was expressed in 65.4% (34/52) of LSCC cases. It was further expressed in 75% of HPV-positive cases and 77.3% of HPV-negative cases. These findings show cyclin E1 expression is of equivocal significance for detection of HPV in LSCC. Some studies reported dysregulated expression of cyclin E1 in LSCC and found a closer relationship between its expression and disease size, stage, and differentiation [30].
Limitations
To the best of our knowledge, this is the first study evaluating HPV prevalence in LSCC in Jordan. We also used a variety of biomarkers in a trial to determine oncogenic activity. One of the limitations of this study is that HPV DNA presence does not provide sufficient evidence for HPV-induced carcinogenesis, and thus these findings mandate further investigation.
IHC stains used in this study are repeatedly linked to overall survival, tumor size, and location. Knowing this data could have helped gain a greater insight into the relation and provide recommendations accordingly.
PCR is a widely used, highly sensitive and specific indicator of HPV in laryngeal cancer. We acknowledge, however, the higher chances of false positive results when compared to in-situ hybridization due to the inferiority of PCR in differentiating between biologically irrelevant and clinically significant HPV. Nevertheless, the majority of studies use PCR techniques to determine the prevalence of HPV in LSCC enabling comparison. Using a mixture of both techniques is recommended in future studies to minimize the chances of type I and type II errors.
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Funding
This research is funded by Deanship of Research at Jordan University of Science and Technology. Proposal Number: 20150359.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
Ethical approval was obtained from Jordan University of Science and Technology IRB Committee Proposal 20150359.
Footnotes
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Supplementary Materials
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Electronic supplementary material 3 (TIFF 111 kb)