HPV integration begins in the tonsillar crypt and leads to the alteration of p16, EGFR and c-myc during tumor formation

The prevalence of human papillomavirus (HPV) infection is high in the oropharyngeal mucosal regions, of which the tonsil is the most commonly affected. There may be a link between HPV and the pathogenesis of tonsillar cancer (TC), because of common anatomical characteristics between cervical and tonsillar cancer. We aimed to clarify whether HPV directly affects the oncogenesis and biologic behavior of TC by making a comparison between infection prevalence, physical status and viral loading numbers, and clinicopathologic prognostic factors. To compare HPV-related molecules between TC and tonsillitis (CFT), p16, survivin, HIF-1alpha, skp-1, cyclin A, cyclin B1, c-myc and EGFR were investigated. We observed a significant difference in HPV prevalence between 52 TCs and 69 CFTs (73.1% vs. 11.6%), and most of the HPVs were type 16 (87.2%) and nonepisomal (94.1%). Most TCs associated with HPV arose from the tonsillar crypts, and tended to be inverted and poorly differentiated. Compared with HPV-negative TC, HPV-positive TC showed a strong association with p16 overexpression (p<0.0001), and an inverse association with EGFR amplification (p=0.0478). HPV-16 integration status was strongly associated with c-myc amplification (p=0.034) and HIF-1alpha overexpression (p=0.022). HPV-16 integration could be directly related to tonsillar carcinogenesis initially in tonsillar crypts, followed by cell cycle aberration such as p16 overexpression related to the G1-S phase.     

Heterogeneity of 18F‐FDG PET combined with expression of EGFR may improve the prognostic stratification of advanced oropharyngeal carcinoma

The Ang's risk profile (based on p16, smoking and cancer stage) is a well‐known prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). Whether heterogeneity in ¹⁸F‐fluorodeoxyglucose (FDG) positron emission tomographic (PET) images and epidermal growth factor receptor (EGFR) expression could provide additional information on clinical outcomes in advanced‐stage OPSCC was investigated. Patients with stage III–IV OPSCC who completed primary therapy were eligible. Zone‐size nonuniformity (ZSNU) extracted from pretreatment FDG PET scans was used as an index of image heterogeneity. EGFR and p16 expression were examined by immunohistochemistry. Disease‐specific survival (DSS) and overall survival (OS) served as outcome measures. Kaplan–Meier estimates and Cox proportional hazards regression models were used for survival analysis. A bootstrap resampling technique was applied to investigate the stability of outcomes. Finally, a recursive partitioning analysis (RPA)‐based model was constructed. A total of 113 patients were included, of which 28 were p16‐positive. Multivariate analysis identified the Ang's profile, EGFR and ZSNU as independent predictors of both DSS and OS. Using RPA, the three risk factors were used to devise a prognostic scoring system that successfully predicted DSS in both p16‐positive and ‐negative cases. The c‐statistic of the prognostic index for DSS was 0.81, a value which was significantly superior to both AJCC stage (0.60) and the Ang's risk profile (0.68). In patients showing an Ang's high‐risk profile (N = 77), the use of our scoring system clearly identified three distinct prognostic subgroups. It was concluded that a novel index may improve the prognostic stratification of patients with advanced‐stage OPSCC.     

Tumour-infiltrating lymphocytes predict for outcome in HPV-positive oropharyngeal cancer

Background:

Human papillomavirus (HPV)-positive oropharyngeal cancer (OPSCC) is associated with improved survival compared with HPV-negative disease. However, a minority of HPV-positive patients have poor prognosis. Currently, there is no generally accepted strategy for identifying these patients.

Methods:

We retrospectively analysed 270 consecutively treated OPSCC patients from three centres for effects of clinical, pathological, immunological, and molecular features on disease mortality. We used Cox regression to examine associations between factors and OPSCC death, and developed a prognostic model for 3-year mortality using logistic regression analysis.

Results:

Patients with HPV-positive tumours showed improved survival (hazard ratio (HR), 0.33 (0.21–0.53)). High levels of tumour-infiltrating lymphocytes (TILs) stratified HPV-positive patients into high-risk and low-risk groups (3-year survival; HPV-positive/TIL_high=96%, HPV-positive/TIL_low=59%). Survival of HPV-positive/TIL_low patients did not differ from HPV-negative patients (HR, 1.01; P=0.98). We developed a prognostic model for HPV-positive tumours using a ‘training'' cohort from one centre; the combination of TIL levels, heavy smoking, and T-stage were significant (AUROC=0·87). This model was validated on patients from the other centres (detection rate 67% false-positive rate 5.6% AUROC=0·82).

Interpretation:

Our data suggest that an immune response, reflected by TIL levels in the primary tumour, has an important role in the improved survival seen in most HPV-positive patients, and is relevant for the clinical evaluation of HPV-positive OPSCC.     

Extracapsular spread and adjuvant therapy in human papillomavirus-related, p16-positive oropharyngeal carcinoma

BACKGROUND:

Extracapsular spread (ECS) is commonly used to justify adjuvant chemotherapy in patients with head and neck cancer. The role of ECS as a prognosticator and adjuvant therapy determinant in surgically resected, human papillomavirus‐related oropharyngeal squamous cell carcinoma (OPSCC), however, has never been determined.

METHODS:

Of 210 oropharynx patients in a prospective transoral laser microsurgery database, 152 patients who had p16‐positive primary OPSCC and pathologically positive necks were eligible for the study. ECS was measured from routine reporting (ECS_report) and by using a novel histologic grading system (ECS_graded). Proportional hazards models and matched analyses were used to compare the impact of ECS and adjuvant therapy on disease‐free survival (DFS). Patients with and without graded ECS were matched for T‐stage, surgical margins, and adjuvant therapy.

RESULTS:

At a median follow‐up of 43 months, the presence of ECS was not associated with poorer DFS in multivariate analyses (ECS_report: hazard ratio [HR], 3.42; 95% confidence interval [CI], 0.45‐25.88; P = .23; ECS_graded: HR, 2.54; 95% CI, 0.88‐7.34; P = .09). T‐stage and high‐grade ECS, ie soft tissue metastasis (STM_graded) were prognostic. Overall and in the presence of ECS or even STM, adjuvant CRT was not associated with better DFS over radiotherapy alone (HR, 0.25; 95% CI, 0.06‐1.13; P = .07). In addition, matched analyses demonstrated no significant reduction in DFS for the presence of ECS versus the absence of ECS or reduced DFS for the administration of adjuvant radiotherapy alone versus CRT in ECS‐positive patients.

CONCLUSIONS:

Routinely reported ECS was not prognostic in this study. Adjuvant CRT versus radiotherapy alone produced no improvement in DFS for ECS‐positive patients. The authors propose that de‐escalated adjuvant therapy should be considered for patients with p16‐positive OPSCC who undergo surgery and that routinely reported ECS should not be used to justify adjuvant chemotherapy. Cancer 2012;3519–3530. © 2011 American Cancer Society.     

EGFR及p16蛋白在鼻咽癌中的异常表达

为了探讨鼻咽癌组织（ＥＧＦＲ）和ｐ１６蛋白的表达与鼻咽癌发生发展及２者表达的相互关系,应用ＳＰ免疫组化法对６４例鼻咽癌组织及６０例癌旁组织进行ＥＧＦＲ和ｐ１６蛋白的检测,结果显示,鼻咽癌组织ＥＧＦＲ阳性率为７１．８８％（４６／６４）,与分化程度有关（Ｐ〈０．０１）,而与临床分期无关（Ｐ〉０．０５）,癌旁组织ＥＧＦＲ阳性率为２６．６７％（１６／６０）,其中正常粘膜阳性率为１７．５０％（７／４０）,非     

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous‐cell‐carcinoma (OPSCC). Although HPV⁺ OPSCC metastasize faster than HPV^? tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV⁺ OPSCC remain elusive. In this study, we examined whether expression of HPV16‐E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16‐E6E7 expression leads to an increase in the number of stationary (CD44^high/EpCAM^high) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44^high/EpCAM^low) CSC pool. This increase in migratory CSCs could also be confirmed in HPV⁺ OPSCC. Differentially expressed miRNAs from HPV16‐E6E7 positive CD44^high/EpCAM^low CSCs were validated by RT‐qPCR and in situ hybridization on HPV16⁺ OPSCCs. These experiments led to the identification of miR‐3194‐5p, which is upregulated in primary HPV16⁺ OPSCC and matched metastasis. MiR‐1281 was also found to be highly expressed in HPV⁺ and HPV^? metastasis. As inhibition of this miRNA led to a markedly reduction of CD44^high/EpCAM^low cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR‐1281 and miR3194‐5p may represent promising targets to block metastatic spread of OPSCC.     

Diagnostic accuracy of p16INK4a immunohistochemistry in oropharyngeal squamous cell carcinomas: A systematic review and meta‐analysis

The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta‐analysis to determine the diagnostic accuracy of p16^INK4a immunohistochemistry (IHC) to identify HPV‐induced OPSCC. We identified all studies that performed p16^INK4a IHC (index test) and HPV E6/E7 mRNA detection using an amplification‐based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16^INK4a IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16^INK4a IHC, HPV DNA PCR, HPV DNA ISH and p16^INK4a IHC/HPV DNA PCR combined testing was 94% (95%‐confidence interval (CI) 91–97%), 98% (CI 94–100%), 85% (CI 76–92%) and 93% (CI 87–97%), respectively. The pooled specificity was 83% (CI 78–88%), 84% (CI 74–92%), 88% (CI 78–96%) and 96% (CI 89–100%), respectively. p16^INK4a IHC/HPV DNA PCR combined testing was as sensitive as either p16^INK4a IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16^INK4a IHC is highly sensitive but moderately specific to diagnose HPV‐transformed OPSCC when used as a single test. Combined p16^INK4a IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV‐induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials.     

Correlation between human papillomavirus and p16 overexpression in oropharyngeal tumours: a systematic review

Background:

A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diagnostic measures and definition of p16 overexpression. We evaluated means of p16 and HPV diagnostics, and quantified overexpression of p16 in HPV-positive and -negative OP-SCCs by mode of immunohistochemical staining of carcinoma cells.

Methods:

PubMed, Embase, and the Cochrane Library were searched from 1980 until October 2012. We applied the following inclusion criteria: a minimum of 20 cases of site-specific OP-SCCs, and HPV and p16 results present. Studies were categorised into three groups based on their definition of p16 overexpression: verbal definition, nuclear and cytoplasmatic staining between 5 and 69%, and ⩾70% staining.

Results:

We identified 39 studies with available outcome data (n=3926): 22 studies (n=1980) used PCR, 6 studies (n=688) used ISH, and 11 studies (n=1258) used both PCR and ISH for HPV diagnostics. The methods showed similar HPV-positive results. Overall, 52.5% of the cases (n=2062) were HPV positive. As to p16 overexpression, 17 studies (n=1684) used a minimum of 5–69% staining, and 7 studies (n=764) used ⩾70% staining. Fifteen studies (n=1478) referred to a verbal definition. Studies showed high heterogeneity in diagnostics of HPV and definition of p16. The correlation between HPV positivity and p16 overexpression proved best numerically in the group applying ⩾70% staining for p16 overexpression. The group with verbal definitions had a significantly lower false-positive rate, but along with the group applying 5–69% staining showed a worse sensitivity compared with ⩾70% staining.

Conclusions:

There are substantial differences in how studies diagnose HPV and define p16 overexpression. Numerically, p16 staining is better to predict the presence of HPV (i.e. larger sensitivity), when the cutoff is set at ⩾70% of cytoplasmatic and nuclear staining.     

口咽鳞癌组织中HPV16感染和p16蛋白表达的临床意义

目的:检测口咽鳞癌中HPV16感染及p16表达率,并分析临床意义。方法:采用原位杂交法检测60例口咽鳞癌组织中HPV16的表达,免疫组化法检测95例口咽鳞癌组织p16的表达情况,分析HPV16与p16的相关性,总结上述两种蛋白在口咽癌中的临床意义。结果:口咽鳞癌组织中HPV16感染率为48.3%（29/60）,p16阳性表达率为52.6%（50/95）,60例标本中HPV16感染与p16表达显著相关（P<0.001）,用p16表达预测HPV16感染的正确率可达90%（54/60）,跟国内外报道一致。p16阳性组相比p16阴性组,吸烟、饮酒、非嚼槟榔、T3+T4、有淋巴结转移、III-IV临床分期和高分化者所占比例更低,但不具有统计学差异(P>0.05)。平均随访时间38个月,随访率65.3%（62/95）,62例口咽鳞癌病人中p16阳性组和p16阴性组的3年总体生存率和无瘤生存率分别为70.4%对比40.0%（P=0.008）和63.0%对比25.7%（P=0.004）。总体生存率多因素分析提示,p16阴性病人死亡风险是p16阳性病人的2.12倍(HR 0.471,95%CI 0.201-1.103,P=0.083),无瘤生存率多因素分析提示,p16阴性病人死亡风险是p16阳性病人的2.36倍(HR 0.432,95%CI 0.201-0.891,P=0.024),p16与口咽癌的预后密切相关。结论:p16表达和HPV16表达存在紧密相关性,p16可以替代HPV16来预测预后,p16蛋白阳性口咽癌患者具有非吸烟、非饮酒、嚼槟榔和肿瘤分化程度低的趋势,检测p16表达对口咽癌病人预后判断有重要的提示意义。     

Intensity‐modulated radiotherapy outcomes for oropharyngeal squamous cell carcinoma patients stratified by p16 status

BACKGROUND:

Patients with oropharyngeal squamous cell carcinoma (OPSCC) treated with intensity‐modulated radiotherapy (IMRT) were stratified by p16 status, neck dissection, and chemotherapy to correlate these factors with outcomes.

METHODS:

A total of 112 patients with OPSCC treated with IMRT from 2002 to 2008 were retrospectively analyzed. All patients received RT to 66‐70 Gray. Forty‐five of the tumors were p16 positive (p16+), 27 were p16 negative (p16?), and 41 had unknown p16 status. Sixty‐two patients had postradiation neck dissections. Nine patients with p16? tumors and 28 patients with p16+ tumors received chemotherapy. The distribution of T, N, and stage grouping among the p16+ and p16? patients was not significantly different, and 87.5% patients had stage III/IV disease.

RESULTS:

The median follow‐up was 26.3 months. For patients with p16+ tumors, p16? tumors, and the overall cohort, the actuarial 3‐year locoregional progression‐free survival rate was 97.8%,73.5%, and 90.5% respectively (P = .006) and the disease‐free survival rate was 88.2%, 61.4%, and 81.7%, respectively (P = .004). Patients with p16+ tumors had an 89.5% and 87.5% pathologic complete response (CR) on neck dissection with and without chemotherapy, respectively. In contrast, patients with p16? tumors had a 66.7% and 25.0% pathologic CR on neck dissection with and without chemotherapy, respectively.

CONCLUSIONS:

In this series, p16 status was found to be a significant predictive biomarker and patients with p16+ tumors had much better outcomes than patients with p16? tumors. Further investigation is warranted to determine whether less intense therapy is appropriate for selected patients with p16+ OPSCC, whereas more aggressive strategies are needed to improve outcomes in patients with p16? disease. Cancer 2010. © 2010 American Cancer Society.     

HPV16 semiquantitative viral load and serologic biomarkers in oral and oropharyngeal squamous cell carcinomas

A considerable subset of oropharyngeal squamous cell carcinomas (SCCs) are positive for human papillomavirus (HPV); however, delineating etiologically-associated HPV infections from SCCs with concurrent HPV infection unrelated to tumorigenesis is challenging. Viral load assessment in biopsy specimens may help facilitate such differentiation. HPV16 viral load and serologic markers were assessed among oral and oropharyngeal cases from a multinational study conducted by the International Agency for Research on Cancer (IARC). HPV16 viral load, measured semiquantitatively by PCR-enzyme immunoassay, was dichotomized as high or low based on the median optical density value. Serologic antibodies to HPV16 virus-like particles (VLPs) and to HPV16 E6 and E7 proteins were measured by ELISA. Compared to HPV DNA-negative cases (n = 852), HPV16 DNA-positive cases with high viral load (n = 26) were significantly more likely to originate in the oropharynx (odds ratio [OR], 12.0; 95% confidence interval [CI], 5.2-27.5) and, after adjustment for tumor site (AdjOR), have antibodies against HPV16 VLPs (AdjOR, 14.6; 95% CI, 6.0-35.6), E6 (AdjOR, 57.6; 95% CI, 21.4-155.3) and E7 (AdjOR, 25.6; 95% CI, 9.3-70.8). HPV16 DNA-positive cases with low viral load (n = 27) were more commonly oropharyngeal (OR, 2.7; 95% CI, 1.1-6.2) and seropositive for HPV16 VLPs (AdjOR, 2.7; 95% CI, 1.1-6.9), E6 (AdjOR, 3.0; 95% CI, 0.7-14.0) and E7 (AdjOR, 3.5; 95% CI, 0.7-16.3), compared to HPV DNA-negative cases; the associations, however, were neither as strong nor as significant as the associations for high viral load. As there appears to be a strong association between HPV16 serologic markers and viral load, in the absence of data on serologic markers, HPV16 viral load may be used to help delineate the subset of HPV16 DNA-positive oral and oropharyngeal cancers that may be the consequence of HPV infection.     

口咽鳞癌HPV感染与p16表达及放疗预后相关性研究

目的 探讨口咽鳞癌HPV感染与p16表达及放疗预后相关性,以及p16是否有预测放疗预后的价值。方法 对1999—2008年间本院42例初治口咽鳞癌组织标本采用PCR检测HPV-DNA,采用免疫组织化学法检测p16表达情况。对HPV、p16不同状态肿瘤根治性放疗后局部区域CR及HPV感染与p16表达相关性比较行χ²检验,Kaplan-Meier法计算OS并Logrank法检验。     

Impact of EGFR and EGFR ligand expression on treatment response in patients with metastatic colorectal cancer

Up to 50% of patients with colorectal cancer (CRC) have either synchronous or metachronous hepatic metastases in the course of their disease. Patients with metastatic CRC (mCRC) whose tumors express wild-type KRAS benefit from treatment with monoclonal antibodies (such as cetuximab or panitumumab) that target the epidermal growth factor receptor (EGFR). However, the therapeutic response to these antibodies is variable, and further predictive models are required. The present study examined whether expression of different EGFRs or their ligands in tumors was associated with the response to cetuximab treatment. Tumor tissues, collected during liver resection in 28 patients with mCRC, were analyzed. The protein expression levels of EGFR/ErbB1, ErbB2, ErbB3 and the EGFR ligands heregulin and amphiregulin were determined using Luminex 200^® and enzyme-linked immunosorbent assays. Computed tomography or magnetic resonance imaging was performed 4 weeks before and 6–8 weeks after treatment with cetuximab. Response to treatment was assessed using the response evaluation criteria for solid tumors (RECIST). The association between the protein expression levels of different EGFRs and their ligands with RECIST criteria was then analyzed to determine whether these protein levels could predict the treatment response to cetuximab. A total of 12 patients exhibited a partial response, 9 exhibited stable disease and 7 exhibited progressive disease after cetuximab therapy according to RECIST. The expression levels of EGFRs (EGFR/ErbB1, ErbB2 and ErbB3) and their ligands (heregulin and amphiregulin) were not significantly associated with the response to cetuximab therapy. Therefore, the present study indicated that EGFR or EGFR ligand expression did not predict treatment response in patients with CRC with liver metastases following cetuximab therapy.     

Survey on human papillomavirus/p16 screening use in oropharyngeal carcinoma patients in the United States

BACKGROUND:

Patients with human papillomavirus (HPV)‐positive oropharyngeal carcinoma (OC) have better prognosis than patients with HPV‐negative OC. The objective of the current study was to assess how different practices across the United States treat patients with OC with respect to screening for HPV DNA or p16.

METHODS:

Five hundred forty‐two randomly selected radiation oncologists were sent an 11‐question survey by email regarding the use of HPV/p16 screening in OC. The questionnaire addressed demographics of the practice, intensity‐modulated radiotherapy (IMRT) use, screening practices for HPV DNA or p16, which year this began, the use of HPV or p16 data to direct patient care, and future plans for its use if it had not already been instituted.

RESULTS:

One hundred ninety‐two responses (39.6%) were received. Thirty‐five percent of respondents (67 of 188) reported screening for HPV DNA routinely, whereas 4.8% of respondents (9 of 188) reported screening for p16. Of the physicians who did not use screening techniques, 37.2% (44 of 118 respondents) reported future plans to institute these screening techniques, 20% (9 of 45 respondents) stated plans to institute these techniques in the next 6 months, 55.5% (25 of 45 respondents) stated plans to institute these techniques within 6 months to 1 year, and 22.2% (10 of 45 respondents) stated plans to institute these techniques within 1 to 2 years. Academic physicians were more likely to use screening techniques (62.7%; P < .001) compared with private practitioners (31.4%). Only 12.4% of respondents reported using HPV or p16 data to direct care.

CONCLUSIONS:

Approximately 40.4% of radiation oncology practices that responded to a survey in the United States screened for HPV DNA or p16 in OC, whereas only 12.4% used it to further direct care. This number appears to be growing rapidly. Clinical trials to further elucidate how HPV or p16 status should direct care in OC are warranted. Cancer 2010. © 2010 American Cancer Society.     

Correlation of p27 protein expression with HER-2/neu expression in breast cancer

Strong expression of human epidermal growth factor receptor 2 (HER-2)/neu in breast cancer has been associated with poor prognosis. Reduced expression of p27(Kip1), a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in breast cancer. In this study, we provide a correlation between these two important prognostic markers in patients with breast cancer. Breast tumor screening using immunohistochemistry indicated that downregulation of p27 correlated with HER-2/neu overexpression in studying 11 normal breast tissues and 51 primary breast carcinomas. We found HER-2/neu protein overexpression in 20 (41%) of 49 breast cancers and low p27 protein expression in 47 (92%) of 51 breast cancers. All 20 (100%) of the tumors that overexpressed HER-2/neu had low levels of p27 protein product; this correlation was statistically significant (P = 0.035). Decreasing p27 expression correlated with increasing HER-2/neu activity. Our results suggest that one function of the HER-2/neu product is to downregulate p27 expression in breast cancer. This study may be significant in selecting patients for HER-2/neu antibody therapy in the future. Mol. Carcinog. 30:169--175, 2001.