Genotypic analysis of tumor suppressor genes PTEN/MMAC1 and p53 in head and neck squamous cell carcinomas

Objectives: Tumor suppressor gene mutations in both p53 and PTEN/MMAC1 genomic DNA have been detected in many types of cancer. The purpose of this study was to investigate the presence and importance of PTEN/MMAC1 mutations in squamous cell carcinomas. Methods: Exons of each gene were amplified after polymerase chain reaction (PCR) using genomic DNA derived from cell lines of squamous cell carcinoma of the head and neck (SCCHN) and snap-frozen biopsy specimens from primary established head and neck tumors. The amplified and purified DNA was then sequenced directly. Result: As anticipated, point mutations of the p53 gene were found in 80% of cell lines examined. A single base mutation in codon 151 was found in six of 10 cell lines studied. PTEN/MMAC1 gene mutations were found in neither the cell lines tested nor the tumor biopsy samples. Conclusion: This study, as well as a large volume of data, confirms that mutations of the p53 gene are frequent events in head and neck cancer cell lines. Although PTEN/MMAC1 gene mutations have been found in a variety of carcinomas, this gene was not found to be mutated in SCCHN cell lines or in primary squamous cell carcinomas of the head and neck. This information is useful for further studies of mutations in these cell lines. Laryngoscope, 108:1553–1556, 1998     

ras Mutations and expression in head and neck squamous cell carcinomas

Mutational activation and overexpression of the family of ras proto-oncogenes have been associated with many human tumors. The role of mutations of H-ras, K-ras, and N-ras, as well as expression of the respective protein products (p21s) in normal mucosa, dysplastic mucosa, and squamous cell carcinomas (SCCs) of the head and neck has not been fully described. In our study, 51 tumors (40 paraffin embedded and 11 fresh frozen) were examined to determine if mutational activation of ras is an important molecular event in head and neck SCC. Analyses of codons 12, 13, and 61 of H-ras, K-ras, and N-ras revealed no mutations, suggesting that mutational activation of ras is not important in the majority of head and neck SCCs. Immunocytochemistry (ICC) was used to define the expression of H-ras, K-ras, and N-ras in normal mucosa, dysplastic mucosa, and SCC of the head and neck and to determine if expression of ras family members correlated with early or late events in the development of SCC. Expression of p21^N-ras in nine samples of histologically normal head and neck mucosa revealed moderate staining in the basal proliferative layers with progressively less staining as cells matured. The most superficial layers of normal mucosa failed to express p21^N-ras. A low level of p21^H-ras was expressed in all layers of normal mucosa while K-ras was not expressed. ICC of SCC tumor sections revealed cytoplasmic expression of N-ras in nine of nine tumors, H-ras in five of nine tumors, and K-ras in one of nine tumors. Expression of H-ras, K-ras, and N-ras in head and neck SCC was not related to histologic differentiation or TNM staging; however, p21^N-ras was overexpressed in seven of nine tumors. Furthermore, the pattern of N-ras expression in dysplastic lesions revealed expression in all layers of the mucosa in contrast to normal mucosa, which expresses p21^N-ras primarily in the basal proliferative layer. The change in p21^N-ras expression pattern in dysplastic mucosa and its overexpression in the majority of tumors suggest that loss of control of N-ras expression may be an early step in carcinogenesis of head and neck SCC.     

c-myc oncogene copy number in squamous carcinoma of the head and neck.

PURPOSE: Altered resident cellular genetic sequences (oncogenes) may result in malignant transformation, maintenance of tumor growth, and metastatic propensity. In this pilot study, we have elected to probe c-myc oncogene in evaluating specimens from human squamous cell carcinoma. MATERIALS AND METHODS: Samples were obtained from 24 patients with squamous cell carcinoma of the head and neck. The ratio of tumor DNA values to that of control DNA was used to estimate the c-myc copy number. RESULTS: Data from material obtained from eight patients was analyzed to the point of c-myc copy number. Tumors varied from stage II through IV. Five originated in the oral cavity and three in the larynx. Analysis of primary tumors demonstrated that two of eight had increased c-myc copy numbers. Histologically positive neck specimens were encountered in five of the study patients. Three demonstrated elevated c-myc copy numbers, two of which had had increased copy number at the primary site. CONCLUSION: This study confirms that c-myc amplification can be present in squamous cell carcinoma of the head and neck. c-myc Amplification may also be present in neck metastasis. Oncogene amplification in neck metastasis may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification.     

Prevalence of RAS oncogene mutation in head and neck carcinomas.

RAS genes encode for a protein (p21) known to play an important role in the regulation of normal signal transduction and cell growth. Activation of RAS genes have been strongly implicated in the pathogenesis of cancer in cell line studies, animal models and in human tumors. RAS genes have been shown to be mutated in 10 to 15% of human solid tumors but the frequency of mutation varies widely depending on the tumor type. The prevalence of RAS mutation has not been well-established in head and neck squamous cell carcinomas (SCC). The purpose of our study was to screen a relatively large number (50) SCC tumors using a gene amplification technique, the polymerase chain reaction (PCR). H-RAS gene mutation is identified by diagnostic restriction length polymorphism, created by introducing specific mismatched primers in the PCR. The first 20 tumors were also amplified and directly sequenced for K-RAS codon 12 and 13. Four of the 50 screened tumors were positive for H-RAS codon 12 mutation. All tumor DNA screened normal at codon 61 and the first 20 tumors were also normal at K-RAS codon 12 and 13. The prevalence of RAS mutations appears to be low in head and neck squamous cell carcinomas. Tumors positive for point mutation in the H-RAS gene revealed some unusual clinical characteristics.     

DNA amplification for the in vitro detection of Candida albicans in head and neck squamous cell carcinomas

DNA was extracted from whole cells of Candida albicans and digested with HindIII restriction enzyme. After electrophoresis in a segment of the lane containing between 800 and 1200 base pairs (bp) of DNA fragments, a 1.1-kilobase (kb) fragment was found that hybridizes to biopsied tumor cells from head and neck squamous cell carcinomas (SCC). From the nucleotide sequence of the putative gene locus, primers were synthesized for use in a polymerase chain reaction (PCR) with DNA extracted from 18 SCC of the upper aerodigestive tract. After 30 cycles of amplification all tumors were found to contain sufficient amplified DNA to be detected in polyacrylamide or agarose gels. In contrast, template DNA from lymph nodes and malignant lymphomas failed to generate positive signals under these conditions. However, samples of DNA obtained from head and neck SCC cells in vitro, Candida glabrata, and Candida parapsilosis after PCR were found to contain homologous sequences. Application of this technique to head and neck SCC biopsies may help to identify quickly the presence of concurrent candidal species.     

Genetic analysis of head and neck squamous cell carcinoma and surrounding mucosa

OBJECTIVE: Comparative genomic hybridization was performed on head and neck squamous cell carcinoma and surrounding mucosa to determine whether common chromosomal aberrations could be detected that would predispose an individual to developing a second primary tumor. DESIGN: Biopsy specimens were taken from 19 patients with squamous cell carcinoma of the head and neck, 3 samples from each person: 1 specimen from the tumor site and 1 each from 1 and 5 cm from the macroscopic tumor margin. Samples were snap frozen in liquid nitrogen. A portion of each distant sample and tissue taken from immediately adjacent to the site of the tumor specimen were sectioned and stained with hematoxylin-eosin, either to search by light microscopy for tumor cells or signs of dysplasia in the distant samples, or to determine whether the tumor specimen had substantial non-tumor cell content. Tissue adjacent to the tumor biopsy site was used because the biopsy specimens were relatively small. Comparative genomic hybridization was performed on all samples. SUBJECTS: Nineteen patients with newly diagnosed carcinomas of the head and neck. RESULTS: The tumor biopsy specimens showed no substantial nontumor cell content, and the distant specimens were all histologically normal. The tumors showed multiple mutations: mean (SD) number of deletions, 5.4 (4.3); amplifications, 5.2 (4.6). Deletion of chromosome 3p was seen in 13 of 19 cases and was associated with amplification of 3q in 10 cases. No mutations were seen in the distant biopsy specimens. CONCLUSIONS: Frequently occurring chromosomal aberrations were seen in the tumor cells, suggesting a key role for these mutations in tumor development. Screening histologically normal upper aerodigestive tract mucosa with comparative genomic hybridization does not provide information on early genetic events that predispose a patient to developing a second primary tumor.     

Occult nodal metastasis in head and neck carcinoma patients treated with chemoradiotherapy

Background and aimNeck lymph node metastasis plays an important role in the prognosis of patients with squamous cell carcinoma of the head and neck. The aim of this study was to evaluate the occult nodal metastasis in patients with head and neck squamous cell carcinoma (HNSCC) treated with chemo radiotherapy.MethodsIn this 5-year prospective study, patients with recurrent head and neck squamous cell carcinomas (HN-SCC) after primary treatment with chemoradiotherapy or radiotherapy that candidate for surgery were enrolled. In total, 50 patients with squamous cell carcinomas of the head and neck with N0 neck were included in the study. Age, initial location of recurrent tumor, T staging in primary and recurrent tumors, neck condition (N0 or N+), and pathology report for neck metastasis, number of affected lymph nodes and duration of tumor recurrence were examined.ResultsOut of 50 patients with mean age of 57.04 ± 14.4 years, 13 were female (26%) and 37 (74%) were male. In terms of primary tumor size, 52% (26 patients) were in T2 stage. The primary and recurrent tumor was located in the oral cavity in 33 patients (66%). Nine 0f 50 patients (18%) had occult metastases.ConclusionIt seems that END surgery is necessary for treatment the occult lymph node neck metastasis of recurrent head and neck cancers with N0 neck. Therefore, it is possible that END surgery has reduced cervical recurrence in these patients.     

Molecular pathogenesis of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) represents 6% of all cancers. The overall 5-year survival rate for patients with this type of cancer is among the lowest of the major cancer types and has not improved dramatically during the last decade. The pathological staging, in particular the nodal stage, is the most important factor in HNSCC. The lack of progress in head and neck oncology emphasizes the importance of molecular genetic studies to define alterations that may correlate with tumor behavior. The molecular alterations observed in HNSCC are mainly due to oncogene activation and tumor suppressor gene inactivation, leading to deregulation of cell proliferation. These alterations include gene amplification and overexpression of oncogenes such as ras, myc, EGFR and cyclin D1, and mutations and deletions leading to p16 and TP53 tumor suppressor genes inactivation. This article reviews the molecular changes commonly observed in HNSCC. The biological function of these markers and the potential clinical application are discussed. Advances in the understanding of the molecular basis of HNSCC will help in the identification of new molecular markers that could be used for a more accurate diagnosis and assessment of prognosis and may open the way for novel approaches to treatment and prevention.     

Expression of c-myc oncoprotein in laryngeal squamous cell carcinoma

Objective c-myc seems to play a pivotal role in normal growth and development as well in cellular transformation and carcinogenesis. Overexpression of the c-myc oncogene has been observed in many hematopoetic and solid tumors. The role of c-myc protein in squamous cell carcinoma of the head and neck in general and laryngeal squamous cell carcinomas (LSCCs) in particular is far from clear. The aim of this study was to evaluate the relations between the level of c-myc protein in LSCCs and the clinicopathological data of patients, DNA ploidy and the SG₂M phase index (PI).

Material and Methods The c-myc protein level was evaluated immunohistochemically in tumor specimens from 50 patients with LSCC. The DNA index and SG₂M PI were determined by means of flow cytometry.

Results We found c-myc protein in 34 (68%) tumors. Expression of c-myc protein was demonstrated to be frequent in non-metastatic cases (p=0.016). There was no association between c-myc protein level and age, primary tumor size, histological grading or type of cancer. In 13 (26%) cases we observed DNA aneuploid tumors. The mean value of the SG₂M PI was 22.5%. Expression of c-myc protein was not related to SG₂M PI or DNA ploidy.

Conclusions We have shown that c-myc oncoprotein may be involved in the genesis of LSCC. Our findings suggest that the detectability of c-myc protein is associated with a lower metastatic potential. The c-myc oncogene is probably not as important in laryngeal cancers compared to other cancers. Further investigations must be performed to establish the value of predicting nodal metastases in LSCC.     

Variability of genetic alterations in different sites of head and neck cancer

OBJECTIVE: Tumors arising from different sites of the head and neck area have different clinical behavior. However, most of the studies on genetic alterations in head and neck squamous cell carcinomas do not make a distinction between the sites within this area. The objective of this study is to compare the genetic alterations in three different sites of the head and neck (larynx, oropharynx, and hypopharynx). STUDY DESIGN: Prospective study. METHODS: Thirty-eight laryngeal, 29 oropharyngeal, and 37 hypopharyngeal carcinomas were studied. DNA from tumor and healthy tissue was evaluated for amplification of the oncogenes at 11q13 region (CCND1, FGF3, FGF4 and EMS1) and of the oncogenes MYC and ERBB1; for integration of the human papillomavirus (HPV) types 6b and 16; for loss of heterozygosity (LOH) at p53 and NAT2; and for the cellular DNA content. RESULTS: FGF3 and FGF4 showed a significantly higher frequency of amplification in hypopharyngeal tumors (P =.006 and P =.0002, respectively). CCND1 amplification had a nearly statistically significant (P =.072) higher frequency of amplification in hypopharyngeal tumors. Aneuploid tumors were found in a significantly lower proportion in the larynx (P =.03) compared with the other sites. For the other genetic alterations, no significant differences among the three sites were found. CONCLUSIONS: These results suggest that cancers originating from different sites in the head and neck may have different tumor biology. Therefore, they should be considered as different entities.     

头颈鳞癌血管生成与颈淋巴结转移相关性研究

目的 :探讨头颈鳞癌血管生成与其颈淋巴结转移的关系以及血管内皮生长因子(VEGF)在头颈鳞癌血管生成中的作用。方法 :应用免疫组化SABC法检测 5 8例头颈鳞癌组织中微血管密度 (IMVD)及VEGF的表达。结果 :5 8例头颈鳞癌组织中IMVD为 2 3.93± 8.77,肿瘤分化程度 ,高与中、高与低间 ,IMVD差异有显著性意义 (均P <0 .0 5 ) ;中与低间 ,差异无显著性意义 (P >0 .0 5 )。颈淋巴结转移组IMVD(2 7.92± 9.11)明显高于非转移组 (2 0 .6 9± 7.0 8) ,其差异有显著性意义 (P <0 .0 1)。癌组织中VEGF表达与瘤内IMVD呈正相关 (rs=0 .4 87,P <0 .0 1)。结论 :瘤内IMVD可作为预测头颈鳞癌颈淋巴结转移的一个重要指标 ;VEGF可促进头颈鳞癌血管生成。     

Immunohistochemical identification of squamous carcinoma of the head and neck with monoclonal antibody SQM1

Frozen tissue sections of biopsies from head and neck squamous cancer lesions were examined for immunohistochemical staining with a recently developed monoclonal antibody, designated as SQM1 antibody and directed against the surface membrane of squamous carcinoma cells. SQM1 antibody stained selectively squamous carcinoma, while normal mucosa and cells of the stroma were non-reactive. Positive staining of tumor was found in 33/35 specimens obtained from several major sites of the head and neck area and was observed in primary manifestations and lymph node metastases as well as in recurrences. The most consistent reactivity was seen with carcinomas of the tongue. Well differentiated squamous carcinomas contained a higher proportion of SQM1 positive tumor cells than poorly differentiated carcinomas. We suggest that the SQM1 antibody may aid in the immunohistochemical identification of squamous carcinoma of the head and neck area.     

Pharmacodiagnostic value of the HER family in head and neck squamous cell carcinoma

Two protooncogene products, EGFR (Her-1, c-erbB-1) and HER2 (Her-2/neu, c-erbB-2), have been reported to be frequently overexpressed in head and neck squamous cell carcinoma (HNSCC). In order to identify patients who may benefit from targeted cancer treatment for these two molecules, we determined the expression status of EGFR and HER2 in 129 HNSCC tumor specimens. Two pharmacodiagnostic kits (EGFR pharmDx and HercepTest) were used to identify HNSCC tumors that overexpress EGFR or HER2. Overexpression of EGFR was detected in 42.6% of the tumor specimens, while HER2 was only rarely expressed (overexpression was observed in just 3.1% of all cases). Given the necessity of new therapeutic modalities for patients suffering from HNSCC, treatment EGFR signaling inhibitors appears to be warranted, whereas therapeutic intervention with HER2 inhibitors seems to be inappropriate in this tumor type.     

Mutation of p53 in Squamous Cell Cancer of the Head and Neck: Relationship to Tumor Cell Proliferation

Rapid proliferation of squamous cell carcinomas of the head and neck (SCCHN) during therapy may contribute to treatment failure. We have investigated the presence of p53 abnormalities in patients with SCCHN as a correlate of proliferation rate and other pathologic and clinical variables. p53 Mutation, as determined by polymerase chain reaction and single-strand conformation polymorphism analysis of microdissected frozen sections of tumor biopsies, was significantly associated with a high labeling index, as determined by in vivo infusion of IUdR and BrdU (P = 0.017). p53 Protein expression was detected by immunohistochemistry with two different antibodies, followed by quantitative image analysis. Many cases exhibited strong p53 protein expression in the absence of mutations within the conserved region of the gene, and expression was not related to proliferation. The presence of p53 mutations was related to tumor differentiation in this group of patients.     

Head and Neck Cancer: The Importance of Oxygen

Objectives To use recently introduced polarographic technology to characterize the distribution of oxygenation in solid tumors, explore the differences between severe hypoxia and true necrosis, and evaluate the ability to predict treatment outcomes based on tumor oxygenation. Study Design Prospective, nonrandomized trial of patients with advanced head and neck cancer, conducted at an academic institution. Methods A total of 63 patients underwent polarographic oxygen measurements of their tumors. Experiment 1 was designed to determine whether a gradient of oxygenation exists within tumors by examining several series of measurements in each tumor. Experiment 2 was an analysis of the difference in data variance incurred when comparing oxygen measurements using oxygen electrodes of two different sizes. Experiment 3 compared the proportion of tumor necrosis to the proportion of very low (≤2.5 mm Hg) polarographic oxygen measurements. Experiment 4 was designed to explore the correlation between oxygenation and treatment outcomes after nonsurgical management. Results No gradient of oxygenation was found within cervical lymph node metastases from head and neck squamous cell carcinomas (P > .9). Tumor measurements achieved with larger (17 μm) electrodes displayed smaller variances than those obtained with smaller (12 μm) electrodes, although this difference failed to reach statistical significance (P = .60). There was no correlation between tumor necrosis and the proportion of very low (≤2.5 mm Hg) oxygen measurements. There was a nonsignificant trend toward poorer locoregional control and overall survival in hypoxic tumors. Conclusions Hypoxia exists within cervical lymph node metastases from head and neck squamous carcinomas, but the hypoxic regions are distributed essentially randomly. As expected, measurements of oxygen achieved with larger electrodes results in lowered variance, but with no change in overall tumor mean oxygen levels. Polarographic oxygen measurements are independent of tumor necrosis. Finally, oxygenation as an independent variable is incapable of predicting prognosis, probably reflecting the multifactorial nature of the biological behavior of head and neck cancers.     

erbB-2/Her-2 gene amplification and overexpression in parotid gland tumors

Summary Proto-oncogenes represent a family of normal cellular genes that were identified on the basis of their similarity to genetic sequences with known tumorigenic + or transforming potential. Accumulating evidence links alterations in either the structure, copy number, or expression of one or another of these genes to neoplasia. One such gene, called erbB-2/Her-2 was found amplified in an adenocarcinoma of the human salivary gland and has also been found associated with primary human breast cancer. Patients with multiple copies of the gene have had a shorter overall survival. In the present study, 21 tumors of the parotid gland were examined by Southern and Northern blot hybridization for amplification and possible overexpression of the erbB-2/Her-2 oncogene. Normal parotid gland tissue was used as negative control. The parotid gland lesions comprised 7 pleomorphic adenomas, 5 squamous cell carcinomas, 4 cases of chronic fibrotic sialadenosis, 3 mucoepidermoid carcinomas as well as 1 lymphoma and 1 cystadenolymphoma. Gene amplification was found in 1 of the pleomorphic adenomas, with 2 tumors showing a significant overexpression of the erbB-2/Her-2 oncogene. Because 3–5% of all pleomorphic adenomas undergo malignant transformation, close follow-up of patients is currently underway.     

Cell kinetic markers in cutaneous squamous and basal cell carcinoma of the head and neck

IntroductionProliferation markers play a significant role in the biologic behavior of tumors. Geminin is a known inhibitor of the cell cycle and DNA replication and has not been previously reported in cutaneous basal and squamous cell carcinomas of the head and neck.ObjectivesWe aimed to investigate proliferation markers ki67, MCM2, and geminin in head and neck cutaneous basal and squamous cell carcinomas.MethodsForty cases of each tumor were immuostained with ki67, MCM2, and geminin followed by assessment of labeling indices (LIs). MCM2/ki67- and geminin/ki67-ratios were also determined; t-test was used for statistical analysis (p < 0.05).ResultsThere was no significant difference in ki67 (p = 0.06) and MCM2 (p = 0.46) between cutaneous basal and squamous cell carcinomas; however, geminin LI was significantly higher in squamous cell carcinomas compared to cutaneous basal cell carcinomas (p < 0.001). Only geminin/ki67 showed a significant difference between the two tumors with the ratio showing significantly higher numbers in squamous cell carcinomas (p = 0.015).ConclusionsGeminin could be regarded as an effective factor in the pathogenesis of head and neck cutaneous cutaneous basal cell carcinomas and squamous cell carcinomas and may be one of the responsible elements in the difference between the biologic behavior of these tumors.     

Indications and pitfalls of immunohistochemistry in head and neck cancer

Immunohistochemistry (IHC) has been employed in the differential diagnosis of tumors.ObjectiveTo assess the use of IHC in cases of head and neck tumor.MethodThis is a retrospective study of the cases included in the Cancer Registry of the institution.ResultsIHC was used in 76 (11%) of 704 pathology tests. Most cases were carcinomas (85.80%), and 83.66% of them were squamous cell carcinomas. All tests were done with diagnostic purposes. The most frequently used antibodies were 34BE12 (37.18%), AE1/AE3 (35.9%), 35BH11 (28.21%), CD45 (25.64%), CD20 (24.36%), CD30 (24.36%), CK7 (23.08%) and CD3 (23.08%).ConclusionsIHC was used in 10.67% of the head and neck tumor cases submitted to pathology testing, mostly for carcinoma (5.26%). In the determination of squamous cell carcinoma, IHC accounted for 18.42% of all tumors.     

DNA-flow cytometry of head and neck carcinoma: the importance of uniform tissue sampling and tumor sites

Summary Flow cytometric DNA ploidy measurements using deparaffinized tumor specimens were performed on 46 squamous cell carcinomas of the head and neck, including 22 carcinomas of the oropharynx, 18 carcinomas of the larynx and six carcinomas of the oral cavity. Aneuploidy was found in 14 of these tumors with carcinomas of the larynx and oral cavity showing almost equal percentages of DNA aneuploidy (10/18 and 3/6, respectively). In contrast, only 1 of the oropharyngeal carcinomas was aneuploid. Accurate microscopy-controlled sampling of tumor tissue from the histological tissue blocks was found to be mandatory in order to obtain reliable ploidy measurements.