Immunohistochemical detection of X-linked inhibitor of apoptosis in head and neck squamous cell carcinoma

The X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP group of structurally related caspase inhibitors. Experimental and clinical evidence implicates XIAP in resistance to cancer therapy and in clinical aggressiveness of certain tumors. We examined the expression of XIAP in head and neck squamous cell carcinoma (SCC). Four-micrometer sections from 59 routinely processed specimens of head and neck SCC were subjected to citrate-based antigen retrieval, followed by incubation with monoclonal anti-XIAP antibody (BD Biosciences, San Jose, Calif) and EnVision Plus reagents (Dako, Carpinteria, Calif). Granular cytoplasmic staining was considered positive; the extent and intensity of staining were recorded. Normal squamous epithelium was either nonstaining (n=22), displayed generally weak basal staining (n=9), or moderate basal staining (n=1). Squamous dysplasia or carcinoma in situ was either nonstaining (10 of 18 cases) or displayed generally weak staining (8 of 18 cases). Varying degrees of XIAP positivity were found in 41 (69.5%) of 59 carcinomas. Most of the nonstaining and weakly staining carcinomas were well or moderately differentiated. In contrast, intense and extensive staining was most frequently found in poorly differentiated carcinomas. In keratinized tumor nests, staining was strongest peripherally and became diminished in central keratinized zones. New parameters of tumor aggressiveness are needed for more effective triaging of patients to appropriately aggressive therapies. The present findings suggest that the potent apoptotic inhibitor XIAP may be such a biomarker in head and neck SCCs, of resistance to apoptosis-inducing therapies, and, possibly, of responsiveness to a new class of XIAP-suppressive drugs presently in clinical trials for other malignancies or in preclinical development.     

Molecular epidemiology of DNA repair gene polymorphisms and head and neck cancer

Although tobacco and alcohol consumption are two common risk factors of head and neck cancer (HNC), other specific etiologic causes, such as viral infection and genetic susceptibility factors, remain to be understood. Human DNA is often damaged by numerous endogenous and exogenous mutagens or carcinogens, and genetic variants in interaction with environmental exposure to these agents may explain interindividual differences in HNC risk. Single nucleotide polymorphisms (SNPs) in genes involved in the DNA damage-repair response are reported to be risk factors for various cancer types, including HNC. Here, we reviewed epidemiological studies that have assessed the associations between HNC risk and SNPs in DNA repair genes involved in base-excision repair, nucleotide-excision repair, mismatch repair, double-strand break repair and direct reversion repair pathways. We found, however, that only a few SNPs in DNA repair genes were found to be associated with significantly increased or decreased risk of HNC, and, in most cases, the effects were moderate, depending upon locus-locus interactions among the risk SNPs in the pathways. We believe that, in the presence of exposure, additional pathway-based analyses of DNA repair genes derived from genome-wide association studies (GWASs) in HNC are needed.     

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Lima L M C, de Souza L R, da Silva T F, Pereira C S, Guimarães A L S, de Paula A M B & Carvalho H A
(2012) Histopathology  60, 489–496
DNA repair gene excision repair cross complementing‐group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors Aims: To evaluate the associations of excision repair cross complementing‐group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2–19.7, P = 0.027], which was also high in patients with advanced stage (OR=5.04, 95% CI: 1.07–23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40–33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07–23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.     

The fibroblast growth factor receptor 4 (FGFR4) Arg388 allele correlates with survival in head and neck squamous cell carcinoma

BACKGROUND: The increased expression of the fibroblast growth factor receptor 4 (FGFR4) has been identified in many human cancers. Recently, a single nucleotide polymorphism changing the sense codon 388 from glycine to arginine was identified in the FGFR4 gene. The FGFR4 Arg(388) allele was found to be associated with a poor prognosis for positive node breast cancer, high-grade soft-tissue sarcoma, colon carcinoma, and head and neck squamous cell carcinoma (HNSCC). METHODS: We decided to verify the impact of the FGFR4 Arg(388) allele on survival as well as its association with histoclinical data in 75 cases of HNSCC. The FGFR4 Arg(388) allele was detected by PCR-RFLP and DNA sequencing. RESULTS: The FGFR4 Arg(388) allele was detected in 42.5% of the tumors (37% heterozygous Gly/Arg and 5.5% homozygous Arg/Arg). The presence of at least one Arg allele was significantly correlated with reduced overall survival after 24 months of follow-up. The cases involving the Arg allele presented an increased mortality risk of 2.2 if compared to the non-carrier cases. CONCLUSION: The FGFR4 Arg(388) allele is associated with a shortened survival.     

DNA microarray reveals ZNF195 and SBF1 are potential biomarkers for gemcitabine sensitivity in head and neck squamous cell carcinoma cell lines

Gemcitabine is a potential chemotherapy drug for treatment of head and neck squamous cell carcinoma (HNSCC), however, the poor or partial response of HNSCC patients to gemcitabine demonstrated the urgent need for gemcitabine biomarkers to improve the therapy. In present work, 10 HNSCC cell lines were employed to figure out the biomarkers for gemcitabine sensitivity. The sensitivities of these 10 cell lines to gemcitabine and the basal expression of these cell lines was investigated, the correlation between gemcitabine response (IC50 dose) and gene expression was investigated by Pearson correlation and FDR estimation. The top seven positive genes responsible for gemcitabine sensitivity were validated by qPCR in these 10 HNSCC cell lines, while only two genes (SBF1 and ZNF195) were expression-correlated to gemcitabine response. Furthermore, ZNF195 expression was closely associated with gemcitabine sensitivity in the subsequent independent validation in cell lines from various types of cancer. Our work might provide potential biomarkers for gemcitabine sensitivity in HNSCC and various type of cancer.     

Association of the GSTP1 and NQO1 polymorphisms and head and neck squamous cell carcinoma risk

The GSTP1 and NQO1 have been reported to be associated with an increased risk for smoking related head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to determine the effect of these metabolic gene polymorphisms on the risk of HNSCC. The study population included 294 histologically confirmed HNSCC cases and 333 controls without cancer. Genotyping analysis of the GSTP1 Ile105Val and NQO1 Trp139Arg genes was performed by polymerase chain reaction-based techniques on DNA prepared from peripheral blood. The Mantel-Haenszel chi2 test was used for statistical analysis. The allele frequencies of the GSTP1 and NQO1 polymorphisms were not statistically significant between cases and controls. In analyzing the association between smoking amounts and genetic polymorphisms, GSTP1 and NQO1 polymorphisms were associated with cigarette smoking amounts in cases. G allele containing genotypes in GSTP1 and T allele containing genotypes in NQO1 were associated with a tobacco dose-dependent increase in risk of HNSCC and these genotype distributions were statistically significant (p<0.05). We found that the GSTP1 105Val allele and NQO1 139Arg allele were associated with tobacco dose-dependent increase in risk of HNSCC. GSTP1 and NQO1 genotype polymorphisms may play an important role in the development of smoking related HNSCC.     

Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (including rs25487, rs861539, rs7003908, rs28383151, rs13181, and rs2228001) in DNA repair genes (XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3) interacted with AFB1, and the gene-environmental interactive role in the risk of HCC using hospital-based case-control study (including 1486 HCC cases and 1996 controls). Genotypes of DNA repair genes were tested using TaqMan-PCR technique. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 2.08 for medium AFB1 exposure level and OR = 6.52 for high AFB1 exposure level]. Increasing risk of HCC was also observed in these with the mutants of DNA repair genes (risk values were from 1.57 to 5.86). Furthermore, these risk roles would be more noticeable under the conditions of two variables, and positive interactive effects were proved in the followed multiplicative interaction analysis. These results suggested that DNA repair risk genotypes might interact with AFB1 in the risk of HCC.     

HPV相关的头颈部鳞状细胞癌的研究进展

随着吸烟、饮酒危险因素的相应控制,头颈部鳞状细胞癌的发病率有所下降,但是一部分由高危型HPV16/18感染引起的头颈部鳞癌的发病率却呈明显的上升趋势。HPV相关的头颈部鳞癌因特殊的致病因素而表现出独特的基因表达谱和生物学特性,且对放化疗敏感、预后良好以及有更高的生存率,因此治疗方式也有区别于其他头颈部鳞癌。本文就近些年来关于HPV相关的头颈部鳞癌流行病学特点、致癌机制以及诊断与防治作一综述。     

Human papillomavirus detection in fine needle aspiration cytology of lymph node metastasis of head and neck squamous cell cancer

BackgroundCurrently, testing on HPV in oropharyngeal squamous cell carcinoma (OPSCC) is performed on histological material. However, in a certain percentage of the cases who present with lymph node metastases no primary tumor can be identified and only fine needle aspiration cytology (FNAC) is available for analysis.ObjectivesPurpose of this study was to assess HPV status on FNAC and to validate it using histological material of the same patients.Study designPatients with cervical metastasis from OPSCC or cancer of an unknown primary tumor (CUP), diagnosed between 2007 and 2012 were included. In 6 of the 47 patients, no primary tumor could be identified. HPV detection and genotyping was performed in both FNAC slides scrapings and formalin fixed paraffin embedded (FFPE) histological material from the same patients, using the HPV SPF₁₀-LiPA₂₅ assay. HPV PCR analysis on FFPE material was considered the reference standard for HPV status of each case.ResultsCompared with HPV negative cases (n = 22), significantly more HPV positive cases (n = 25) presented initially with cervical metastasis (27% vs 56% respectively; p = 0·047). The HPV PCR assay on FNAC material showed a high sensitivity (96%; 95% CI 86.6–97.4) and specificity (100%; 95% CI 85.1–96.7) using the reference standard of HPV PCR analysis on FFPE material of the same patients.ConclusionIn this study, testing on HPV in FNAC of cervical lymph node metastases of SCC is validated. It provides a valuable alternative for testing of HPV on histological material from patients with oropharyngeal squamous cell carcinoma or cancer of an unknown primary tumor.     

Expression of Wnt gene family and frizzled receptors in head and neck squamous cell carcinomas

Genes of the Wnt and Frizzled class, expressed in HNSCC tissue and cell lines, have an established role in cell morphogenesis and differentiation, and also they have oncogenic properties. We studied Wnt and Fz genes as potential tumor-associated markers in HNSCC by qPCR. Expression levels of Wnt and Fz genes in 22 unique frozen samples from HNSCC were measured. We also assessed possible correlation between the expression levels obtained in cancer samples in relation to clinicopathologic outcome. Wnt-1 was not expressed in the majority of the HNSCC studied, whereas Wnt-5A was the most strongly expressed by the malignant tumors. Wnt-10B expression levels were related with higher grade of undifferentiation. Related to Fz genes, Fz-5 showed more expression levels in no-affectation of regional lymph nodes. Kaplan–Meier survival analyses suggest a reduced time of survival for low and high expression of Wnt-7A and Fz-5 mRNA, respectively. qPCR demonstrated that HNSCC express Wnt and Fz members, and suggested that Wnt and Fz signaling is activated in HNSCC cells.     

头颈部鳞癌的微卫星不稳定性和杂合性丢失的初步研究

目的:探讨头颈部鳞癌的微卫星不稳定性(MSI)及杂合性丢失(LOH)。方法:选择来自3、5、6、8、9、13、17和18号染色体的15个微卫星标志对36例头颈部鳞癌标本和相应的外周血进行微卫星分析。结果:36例头颈部鳞癌中,27.8%(10/36)分别有1-8个位点存在MSI,MSI发生率较高的位点为:D17S520(22.9%)、D6S105(16.7%)和D8S264(13.9%)。在9p21-p22和3p14等处存在一定的LOH。微卫星异常的检出率与肿瘤分期、分级无相关性。结论:提示MSI是头颈部鳞癌中较为常见的遗传学变化,染色体9p21-p22和3p14区域可能存在与头颈部鳞癌有关的抑癌基因。     

Core classification of head and neck squamous cell carcinomas: Correlations between morphology,DNA ploidy and HPV infection

Aims

The study intended to reveal whether HPV infection is reflected by nuclear morphology and DNA cytometry parameters in head and neck squamous cell carcinomas (HNSCC).

Methods

In total, 39 HNSCC were selected for reanalysis by histomorphology applying the core classification, DNA cytometry and HPV detection. For the core classification, HE sections were assessed by a score system to evaluate the nuclear size, the mitosis size, their variabilities and the presence of tripolar or tetrapolar mitoses. HPV was analyzed by consensus PCR followed by a hybridization method for virus typing. Static DNA cytometry was applied on single cell suspension focusing particularly on the parameters DNA modal value, DNA index peak, DNA index mean, 2c deviation index and 5c exceeding rate. Statistical analysis was done by T-test or Fisher's exact test.

Results

The analysis revealed that HPV positive HNSCC had significantly smaller nuclei than HPV negative cases. Increasing values of the nuclear size and mitosis size were significantly associated with higher indices of the DNA cytometry analysis.

Conclusions

The study confirms that the core classification can provide information on the ploidy of HNSCC and that HPV positive tumors represent a distinct morphological and genetic carcinoma subtype.     

Molecular predictors of clinical outcome in patients with head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) involves the upper aerodigestive tract and can destroy the structure and function of organs involved in voice, speech, taste, smell and hearing, as well as vital structures necessary for survival. HNSCC has long been a treatment challenge because of the high rate of recurrences and of advanced disease at the time of diagnosis. Molecular identification of tissue biomarkers in diagnostic biopsy specimens may not only identify patients at risk for developing HNSCC but may also select patients that may benefit from more aggressive treatment modalities. Several biomarkers studied to date such as the proteins p53, cyclin D1, p16, Cox-2 enzyme, epidermal growth factor and vascular endothelial growth factor receptors, matrix metalloproteinases and the Fhit marker for genomic instability could be manipulated for the therapeutic benefit of these patients. This review presents the most updated information on molecular biomarkers with the greatest prognostic potential in HNSCC and discusses some factors that contribute to the controversy concerning their prognostic importance.     

Expression of integrins and E-cadherin in squamous cell carcinomas of the head and neck

Integrins and cadherins are cell adhesion molecules suggested to play an important role in malignant progression and tumour differentiation. Our aim was to characterise the pattern of expression and the relations between integrin beta1, beta4, beta6 and E-cadherin and the different histopathological features important when judging tumour differentiation, using a well-defined scoring system. Formalin-fixed paraffin-embedded pre-irradiation biopsies from 85 patients with head and neck squamous cell carcinomas (HNSCC) were stained and evaluated for the expression of integrin beta1, beta4 and beta6 and E-cadherin. The integrins were upregulated in carcinomas compared to the adjacent mucosa and E-cadherin was downregulated. However, differences were found within the tumour: Expression of E-cadherin was lost and the three integrins were upregulated at the tumour borders, compared to central parts of the tumour biopsy. Expression of the integrins did not correlate with tumour or histopathological parameters, whereas expression of E-cadherin was correlated with high degree of keratinisation, high nuclear maturation and few mitoses - factors that characterise well-differentiated carcinomas -and E-cadherin can therefore be considered as a marker of differentiation. Furthermore, loss of adhesion expressed by low E-cadherin and integrin beta4 correlated with the presence of nodal metastases at the time of diagnosis.     

Approximately 580 Kb surrounding the MYC gene is amplified in head and neck squamous cell carcinoma of Indian patients

Amplification of the MYC gene is reported to be associated with the development of head and neck squamous cell carcinoma (HNSCC). However, there are no data concerning whether the amplification is confined to the MYC gene itself or spans distant 5' and/or 3' regions of this gene in HNSCC as seen in different lymphomas, colon carcinoma, and uterine cervical carcinoma (CaCx). In this study, we analyzed the alterations (amplification/rearrangement) in the 580 Kb surrounding of the MYC gene to understand the status of this locus in primary HNSCC of Indian patients. The MYC alterations were analyzed by Southern blot using the pal-1/MYC/MLVI4 probes. The alterations in the MYC locus (adjacent region of the c-myc gene) were then correlated with the various clinicopathological parameters. The overall amplification involving the MYC locus was seen in 46% of the samples. The MYC gene, pal-1 region, and MLVI4 region were amplified in about 38%, 24%, and 20% of the samples, respectively. Some samples showed co-amplification encompassing pal-1-MYC-MLVI4 or pal-1-MYC or MYC-MLVI4 regions. No significant association was observed between the amplification in the MYC locus and the different clinicopathological parameters except for tumor differentiation. Thus, it seems that, similar to other tumors, the MYC gene may be activated by amplification in its surrounding 5' and/or 3' region in HNSCC.     

The clinicopathological roles of alpha-B-crystallin and p53 expression in patients with head and neck squamous cell carcinoma

AIMS: The aim of the study was to investigate the expression of alpha-B-crystallin and p53 in head and neck squamous cell carcinoma (HNSCC). METHODS: Alpha-B-crystallin and p53 expressions from 118 HNSCC were studied by immunohistochemistry and correlated with clinicopathological parameters. RESULTS: Alpha-B-crystallin expression was seen in 28% (n = 33) of HNSCC. All except one poorly differentiated HNSCC were negative for alpha-B-crystallin. p53 expression was seen in 63% (n = 73) of HNSCC and was more common in moderately/poorly differentiated HNSCC (p = 0.034). The proportion of cases with positive staining for either alpha-B-crystallin or p53 was different in different anatomical locations in the head and neck. Patients with HNSCC having a high portion of tumour cells expressing p53 had a shorter survival than the other groups (p = 0.032). CONCLUSION: The expression of p53 and alpha-B-crystallin were related to the differentiation and site of the HNSCC. Alpha-B-crystallin was not a prognostic marker for HNSCC.     

Molecular discrimination of multiple primary versus metastatic squamous cell cancers of the head/neck and lung

Patients with squamous cell carcinoma (SqCa) arising in the head and neck (H/N) commonly develop solitary pulmonary metastases that mimic the clinical, radiographic, and pathologic presentation of new primary lung SqCa. Primary pulmonary and metastatic SqCas cannot be differentiated from each other histologically. However, distinguishing multiple independent primary neoplasms from a primary H/N SqCa with pulmonary metastasis has prognostic significance due to its impact on tumor stage, the most important determinant of prognosis. Since genomic instability is a common feature of cancer, we hypothesized that independently-arising neoplasms in an individual patient would exhibit measurable genomic variation, enabling discrimination of tumor lineage and relatedness. In this study, we describe a molecular approach for analysis of genetic variation among multiple tumors from a single patient that does not rely on collection of normal tissue, and which can be performed with minimal tumor samples. Genomic DNA from H/N and lung SqCas from individual patients were analyzed by microsatellite PCR to identify discordant allelic variation. This method is rapid, sensitive, does not require constitutional DNA for comparison, and can be applied to the analysis of archival tumor DNA. Our results demonstrate that microsatellite PCR can identify discordant genetic variation among multiple tumors from a single patient, facilitating the molecular discrimination of metachronous primary SqCa versus solitary pulmonary metastasis from a H/N primary SqCa.     

头颈部鳞癌中抑癌基因PTEN的研究进展

The FTEN, having a dual specificity phosphatase activity, is the first tumor suppressor gene that possess phosphatase activity hitherto. Many researches have suggested that FTEN play a major role in the tumorgenesis. In clinical, the head and neck squamous cell carcinoma(HNSCC) is one of the most common ma-lignant tumors. In this review, advances in the research of FTEN and the relationship between the PTEN and HNSCC are discussed.     

Human papillomavirus in metastatic squamous carcinoma from unknown primaries in the head and neck: A retrospective 7 year study

Goal: Human Papillomavirus (HPV) is found to be increasingly implicated in head and neck cancers. The objective of this study was to determine the primary site of origin of HPV positive squamous carcinomas metastatic to lymph nodes of the neck.Methods: Surgical pathology records from January 1, 2000 to July 31, 2007 were used to identify surgically removed neck lymph nodes with the diagnosis of metastatic squamous carcinoma. Specimens in formalin-fixed, paraffin-embedded blocks were examined for HPV (+) by analyzing sequencing data generated by PCR and immunostaining for the expression of the p16INK biomarker, which is overexpressed if Rb is not present. H & E stained slides were also reviewed for histological classification. The available retrospective demographics were extracted from the charts to determine trends of confounding factors.Results: Of the 43 patient samples, 41 contained adequate DNA to test for HPV. The mean age of the 41 patients was 62 years. All of the patients smoked and 39/41 patients consumed alcohol. The overall HPV (+) incident rate was 27% (11/41) by PCR with strongly diffuse or strong focal p16 staining. 9 of the 34 males and 2 of the 7 females had HPV (+) carcinomas. The average age of the 2 HPV (+) females was 44, compared to the HPV (−) females who averaged 70. The average age of the HPV (+) males was 56 compared with the average age 55 of the HPV (−) males. HPV (+) carcinomas appeared to arise from multiple sites in the oropharynx, particularly the tonsils and tongues, including unknown primaries. By histological exam, most metastatic HPV(+) squamous carcinomas were poorly differentiated (basaloid) microscopically and grossly cystic.Conclusion: The 27% HPV (+) squamous cancers metastatic to neck lymph node originated from multiple sites in the oropharynx. The HPV (+) female population, although a total of only 2, tended to be much younger than the HPV (−) ones, whereas the HPV (+) male population was similar in age to the HPV (−) male population.