p16INK4a overexpression predicts translational active human papillomavirus infection in tonsillar cancer

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin‐dependent kinases p16^INK4a. In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type‐specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence‐labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT‐PCR were performed, and for p16^INK4a detection, immunohistochemistry was performed. With 21/39 (53%) HPV‐positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16^INK4a staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16^INK4a expression was of statistical significance (p = 0.02). Kaplan‐Meier estimations revealed better survival outcome for patients with HPV‐positive tumors with detectable E6/E7 mRNA and p16^INK4a overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16^INK4a staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16^INK4a as a surrogate marker with the potential to impact the standard of care of HPV DNA‐positive head and neck carcinomas.     

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。     

P16和CyclinE蛋白在食管鳞状细胞癌中的表达及意义

目的探讨P16和cyclinE蛋白在食管鳞状上皮、增生上皮和癌变上皮中表达状况及其与鳞状细胞癌发生、发展和转移的相关性。方法采用SP免疫组织化学方法,检测食管鳞癌72例,粘膜上皮增生21例,正常对照13例和淋巴结转移癌15例中P16和cyclinE基因蛋白的表达。结果P16在正常上皮组无阳性表达,增生组为23.81%,鳞癌组为38.89%;高分化鳞癌组阳性表达率(41.67%)显著高于低分化鳞癌组(27.27%)(P<0.05);有淋巴结转移组(16.67%)和无淋巴结转移组(42.85%)比较具有显著性差异(P<0.05);原发癌组阳性率显著高于淋巴结转移癌组(P<0.05)。cyclinE在正常上皮组无阳性表达,增生上皮组为33.3%,鳞癌组为51.4%;高分化鳞癌组阳性表达率(44.44%)显著低于低分化鳞癌组(81.81%)(P<0.05);淋巴结转移组cyclinE阳性表达率(79.17%)和淋巴结无转移组(48.57%)比较有显著性差异(P<0.05)。P16和cyclinE表达与肿瘤部位和浸润深度相关性不明显。结论在食管癌中P16和cyclinE基因蛋白的表达与病理分化程度有关;检测P16和cyclinE对食管癌早期诊断有指导作用。     

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.     

Human papillomavirus tumor infection in esophageal squamous cell carcinoma

The association between human papillomavirus (HPV) and esophageal squamous cell carcinoma (ESCC) has been recognized for over three decades. Recently, multiple meta-analyses have drawn upon existing literature to assess the strength of the HPV-ESCC linkage. Here, we review these analyses and attempt to provide a clinically-relevant overview of HPV infection in ESCC. HPV-ESCC detection rates are highly variable across studies. Geographic location likely accounts for a majority of the variation in HPV prevalence, with high-incidence regions including Asia reporting significantly higher HPV-ESCC infection rates compared with low-incidence regions such as Europe, North America, and Oceania. Based on our examination of existing data, the current literature does not support the notion that HPV is a prominent carcinogen in ESCC. We conclude that there is no basis to change the current clinical approach to ESCC patients with respect to tumor HPV status.     

p16(INK4a) is a useful marker of human papillomavirus integration allowing risk stratification for cervical malignancies

The present study was conducted to assess utility of p16(INK4a) immunopositivity as a surrogate marker for genomic integration of high-risk human papillomavirus infection (hrHPV). A total of 29 formalin-fixed, paraffin-embedded cervical low-grade squamous intraepithelial lesions (LSILs), 27 high-grade squamous intraepithelial lesions (HSILs) and 53 invasive squamous cell carcinomas (SCCs), histologically-diagnosed between 1st January 2006 to 31st December 2008 at the University of Malaya Medical Centre were stained for p16(INK4a) (CINtec Histology Kit (REF 9511, mtm laboratories AG, Heidelberg, Germany). Immunopositvity was defined as diffuse staining of the squamous cell cytoplasm and or nucleus (involving > 75% of the intraepithelial lesions or SCCs). Staining of basal and parabasal layers of intraepithelial lesions was pre-requisite. One (3.4%) LSIL, 24 (88.9%) HSIL and 46 (86.8%) SCC were p16(INK4a) immunopositive. All normal squamous epithelium did not express p16(INK4). p16(INK4a) expression was significantly lower (p<0.05) in LSIL compared with HSIL and SCC with no difference in expression between HSIL and SCC.The increased p16(INK4a) immunopositivity in HSIL and SCC appears in line with the integrated existence of the hrHPV and may provide more insightful information on risk of malignant transformation of cervical squamous intraepithelial lesions than mere hrHPV detection.     

Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma

Background:

Autophagy is a programmed cell survival mechanism that has a key role in both physiologic and pathologic conditions. The relationship between autophagy and cancer is complex because autophagy can act as either a tumour suppressor or as a tumour promoter. The role of autophagy in oral squamous cell carcinoma (OSCC) is controversial. Several studies have claimed that either a high or low expression of autophagy-related proteins was associated with poor prognosis of OSCCs. The aims of the study were to compare autophagy in OSCCs, verrucous hyperplasias, and normal oral mucosas, and to inspect the prognostic role of autophagy in OSCCs.

Methods:

We used the autophagosome marker, LC3B, and autophagy flux marker, p62/SQSTM1 (p62), by using immunohistochemistry, and examined p62 mRNA by RNA in situ hybridization, to evaluate autophagy in 195 OSCCs, 47 verrucous hyperplasias, and 37 normal oral mucosas. The prognostic roles of LC3B and p62 protein expressions in OSCCs were investigated.

Results:

We discovered that the normal oral mucosa exhibited limited LC3B punctae and weak cytoplasmic p62 staining, whereas the OSCCs exhibited a marked increase in LC3B punctae and cytoplasmic p62 expression. The expression pattern of LC3B and cytoplasmic p62 of the verrucous hyperplasias were between normal oral mucosas and OSCCs. The normal oral mucosas, verrucous hyperplasias, and OSCCs presented no differences in nuclear p62 expression and the p62 mRNA level. p62 mRNA expression was elevated in a minority of cases. High p62 mRNA expression was associated with high p62 protein expression in the cytoplasm. Increased LC3B punctae, high cytoplasmic p62, and low nuclear p62 expressions in OSCCs were associated with aggressive clinicopathologic features and unfavourable prognosis. In addition, low nuclear p62 expression was an independent prognostic factor for overall and disease-specific survival rates. Furthermore, we disclosed that high cytoplasmic p62 expression accompanied with either a low or high LC3B expression, which indicated autophagy impairment under basal or activated autophagic activity, was associated with aggressive behaviour in advanced OSCCs.

Conclusions:

We suggested that autophagy was altered during cancer initiation and progression. Autophagy impairment contributed to cancer progression in advanced OSCCs.     

HPV感染及p53与Cdc2蛋白表达对喉癌术后复发的预测价值

目的检测人乳头状瘤病毒(human papilloma virus,HPV)及p53和Cdc2蛋白表达与喉鳞状细胞癌发生及复发的关系。方法选取2003-01-01-2011-12-31唐山市协和医院92例喉鳞状细胞癌手术切除标本,采用PCR-反向点杂交方法检测喉癌组织中HPV感染情况,SP免疫组化方法检测p53和Cdc2蛋白表达,收集临床病理资料并进行随访。结果随访>2年的喉癌患者中有18例出现复发(复发组),74例无复发(未复发组)。92例喉癌组织中HPV的感染率为19.57%(18/92);喉癌组织中p53和Cdc2蛋白的阳性表达率分别为60.87%(56/92)和65.22%(60/92)。HPV感染、p53及Cdc2蛋白表达与喉癌的分化程度、分期均无明显相关性,P值均>0.05;HPV阳性和阴性喉癌患者术后复发率分别为0(0/18)和24.32%(18/74),差异有统计学意义,χ2=4.007,P=0.045;喉癌复发组和未复发组中的p53蛋白表达率分别为72.22%(13/18)和58.11%(43/74),差异无统计学意义,χ2=0.691,P=0.406;喉癌复发组和未复发组中Cdc2蛋白表达率分别为88.89%(16/18)和59.46%(44/74),差异有统计学意义,χ2=4.307,P=0.038。喉癌组织中HPV的感染与p53(χ2=0.316,P=0.574)及Cdc2(χ2=0.574,P=0.487)蛋白表达均无相关性。结论喉癌组织中存在着一定程度的HPV感染及p53、Cdc2蛋白质的表达,但三者与喉癌的分期及分化程度无关;HPV阳性患者术后复发率低于HPV阴性患者;Cdc2蛋白表达阳性者术后复发率高于阴性患者。HPV感染、p53及Cdc2 3种因素可能参与了喉癌的发生、发展及复发过程。     

食管鳞状细胞癌中p16和cyclinD1的表达及其临床意义

 目的　探讨p16、cyclinD1蛋白在食管鳞状细胞癌（ESCC）组织中的表达及其与临床病理之间的关系。方法　应用免疫组织化学法,对55例手术切除的原发ESCC患者的肿瘤组织蜡块进行p16和cyclinD1蛋白的检测。结果　55例ESCC患者中p16蛋白表达率为49.1 %（27／55）。p16蛋白缺失与淋巴结转移有一定的相关性（P＜0.05）;p16表达阳性者5年生存率为71.4 %（10／14）。55例ESCC中cyclinD1蛋白表达率为74.5 % （41／55）,cyclinD1阳性表达患者的5年生存率低于阴性表达患者。结论　ESCC患者的p16蛋白表达缺失较常见, p16蛋白的失表达与cyclinD1基因蛋白过表达在食管癌发生及转移过程中可能有协同作用,可作为判断预后的参考指标之一。     

No role for human papillomavirus in esophageal squamous cell carcinoma in China

Certain regions of China have high rates of esophageal squamous cell carcinoma (ESCC). Previous studies of human papillomavirus (HPV), a proposed causal factor, have produced highly variable results. We attempted to evaluate HPV and ESCC more definitively using extreme care to prevent DNA contamination. We collected tissue and serum in China from 272 histopathologically‐confirmed ESCC cases with rigorous attention to good molecular biology technique. We tested for HPV DNA in fresh‐frozen tumor tissue using PCR with PGMY L1 consensus primers and HPV16 and 18 type‐specific E6 and E7 primers, and in formalin‐fixed paraffin‐embedded tumor tissue using SPF₁₀ L1 primers. In HPV‐positive cases, we evaluated p16^INK4a overexpression and HPV E6/E7 seropositivity as evidence of carcinogenic HPV activity. β‐globin, and thus DNA, was adequate in 98.2% of the frozen tumor tissues (267/272). Of these, 99.6% (95% confidence interval (CI) = 97.9–100.0%) were negative for HPV DNA by PGMY, and 100% (95% CI = 98.6–100%) were negative by HPV16/18 E6/E7 PCR. In the corresponding formalin‐fixed paraffin‐embedded tumor specimens, 99.3% (95% CI = 97.3–99.9%) were HPV negative by SPF₁₀. By PGMY, 1 case tested weakly positive for HPV89, a noncancer causing HPV type. By SPF₁₀, 2 cases tested weakly positive: 1 for HPV16 and 1 for HPV31. No HPV DNA‐positive case had evidence of HPV oncogene activity as measured by p16^INK4a overexpression or E6/E7 seropositivity. This study provides the most definitive evidence to date that HPV is not involved in ESCC carcinogenesis in China. HPV DNA contamination cannot be ruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringent tissue procurement and processing protocols.     

Comparison of human papillomavirus in situ hybridization and p16 immunohistochemistry in the detection of human papillomavirus‐associated head and neck cancer based on a prospective clinical experience

BACKGROUND:

Human papillomavirus (HPV) is a causative agent in a subset of head and neck squamous cell carcinomas (HNSCCs). These HPV‐related cancers have a clinicopathologic profile that diverges from HPV‐negative HNSCCs. Accordingly, HPV testing may soon become integrated into standard pathologic assessment of HNSCCs.

METHODS:

Data were prospectively collected for all patients with head and neck carcinomas who had undergone HPV testing at the Johns Hopkins Hospital as part of clinical care during a 57‐month period. HPV testing consisted of concurrent HPV16 in situ hybridization (ISH) and p16 immunohistochemistry (IHC). Wide spectrum HPV ISH was reserved for p16‐positive cases that were HPV‐16 negative.

RESULTS:

HPV analysis was performed on 256 head and neck carcinomas in an effort to predict clinical outcomes (56%), localize primary tumor origin (21%), establish tumor classification (9%), determine patient eligibility for vaccine trials (8%), or satisfy patient curiosity (5%). A total of 182 (71%) tumors were HPV positive. HPV positivity correlated with oropharyngeal site (82% vs 9%) and male sex (77% vs 48%). p16 positivity was present in all 176 HPV16‐positive cases, and in 19 of 80 (24%) cases that were HPV‐16 negative. In 6 (32%) discordant cases, p16 expression was because of the presence of another HPV type.

CONCLUSIONS

A feasible strategy that incorporates p16 IHC and HPV ISH is able to detect HPV in a high percentage of oropharyngeal carcinomas. HPV status is frequently requested by the oncologist to estimate clinical outcome, and used by pathologists to establish tumor classification and determine site of tumor origin. Cancer 2010. © 2010 American Cancer Society.     

FOXC1蛋白在喉鳞状细胞癌中表达及临床意义

目的：探讨FOXC1蛋白在人喉鳞癌组织中的表达及其与淋巴结转移、分化程度等临床病理参数间的关系。方法：采用免疫组织化学法检测FOXC1蛋白在喉鳞癌（LSCC）及癌旁正常切缘组织中的表达,并结合临床病理学特征进行统计学分析。结果：FOXC1蛋白阳性表达定位于喉鳞癌细胞的胞核或胞浆,其在LSCC组及对照组中的阳性表达率分别为96.8%和27.1%（P〈0.05）,FOXC1浆表达与颈部淋巴结转移、分化程度及分型关系密切,而与性别、年龄、临床分期等无明显关系;FOXC1核表达与分型相关,与其它临床病理学参数间无明显相关。结论：FOXC1蛋白在喉鳞癌组织中阳性表达率明显高于癌旁正常切缘组织,它可能在喉鳞癌的侵袭、转移过程中发挥重要作用。FOXC1可能成为判定喉癌外科切缘及分子靶向治疗的新靶标。     

喉鳞状细胞癌中PIN1基因扩增及表达异常

目的：研究PIN1基因在喉癌组织中的表达情况。方法：采用差异PCR、核型分析方法检测喉鳞癌及癌旁对照组织中PIN1基因扩增情况;采用RT-PCR检测同批组织中PIN1 mRNA表达情况;采用免疫组织化学、Western Blot方法检测PIN1蛋白表达情况。结果：与癌旁对照组织相比,40例喉癌组织中有9例（9/40,22.5%）存在PIN1基因扩增,27例（27/40,67.5%）PIN1 mRNA过表达,26例（26/40,65%）PIN1蛋白过表达（P〈0.05）。40例中17例原代培养成功,可制备中期染色体标本,其中3例（3/17,17.65%）存在19号染色体增加。结论：喉鳞状细胞中PIN1基因拷贝数增加,PIN1 mRNA和蛋白质过表达,可能与喉癌发生发展有关。