Immunohistochemical detection of epidermal growth factor receptor in laryngeal squamous cell carcinomas.

Laryngeal squamous cell carcinomas from 15 consecutive preoperatively irradiated patients were investigated for the expression of epidermal growth factor receptor (EGF receptor). The study was performed on frozen sections by means of the 5-layer APAAP technique employing an antibody recognizing the extracellular part of the EGF receptor. In sections from 9 of the patients with laryngeal squamous cell carcinoma, normal differentiated epithelia were included. Sections from 6 of these patients, in addition, contained dysplastic epithelia. Expression of EGF receptor-like material was demonstrated in the basal cell layer of normally differentiated laryngeal epithelial and in dysplastic epithelia. Fourteen of the squamous cell carcinomas proved EGF receptor positive. Nearly all cells in the poorly differentiated carcinomas showed positive staining with the antibodies. In moderately to well differentiated carcinomas a reduction in the extent of staining was seen in certain areas. Especially for the epithelial pearls, the staining reaction was localized to the undifferentiated cells in the periphery. This finding corresponds to the staining pattern observed in the basal cell layers of normal epithelial. The present investigation confirms the expression of EGF receptor-like material in normal laryngeal epithelial, dysplastic epithelial and squamous cell carcinoma. The staining pattern was similar to that observed in oral squamous cell carcinomas, predominantly varying inversely with cellular differentiation.     

Expression of epidermal growth factor receptor in laryngeal dysplasia and carcinoma

The immunoreactivity of epidermal growth factor receptor (EGFR) was studied in laryngeal biopsy specimens from 24 patients. The study comprised 5 cases of normal laryngeal mucosa, 5 cases of dysplasia, 7 cases of well differentiated squamous cell carcinoma, and 7 cases of poorly to moderately differentiated squamous cell carcinoma. EGFR was in general not expressed in normal and dysplastic epithelia, whilst all carcinomas showed a rather strong positive immunoreactivity. There was no significant difference in staining patterns between the well and poorly to moderately differentiated carcinomas. The results suggest that EGFR constitutes a component of neoplastic, but probably not preneoplastic, laryngeal disease. The study failed to reveal any difference in staining pattern between different types of laryngeal squamous cell carcinoma.     

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.     

Involucrin in laryngeal dysplasia. A marker for differentiation

Involucrin is a major structural subunit of the cross-linked protein envelope that encases keratin in maturing squamous cells. Intracytoplasmic involucrin is identifiable via immunoperoxidase techniques as these cells migrate from the basal layer to the more superficial layers of the stratified epithelium. Normal squamous epithelia and mildly dysplastic epithelia show uniform staining in the suprabasal and superficial layers of the mucosa but show no staining in the basal layer. Moderate to severe dysplasias and invasive carcinomas demonstrate irregular or focal staining in all three layers. Thirty-three microscopic samples from 27 glottic laryngeal biopsy specimens were reviewed. The histochemically abnormal differentiation identified via involucrin staining correlated with accepted histologic criteria for dysplasia. Involucrin staining may provide objective information to assist the pathologist in differentiating degrees of dysplasia in laryngeal biopsy specimens.     

Overexpression of scatter factor and its receptor (c-met) in oral squamous cell carcinoma

Hypothesis: Scatter factor (SF) is a pleiotropic growth factor that recently has been shown to induce epithelial cell proliferation, random motility, and invasion via interaction with its receptor, a tyrosine kinase encoded by the c-met proto-oncogene. Studies involving a variety of solid tumors have suggested that overexpression of the SF/c-met ligand-receptor pair is associated with the acquisition of a malignant phenotype. We hypothesize that SF and c-met are overexpressed in epithelial malignancies of the head and neck including squamous cell carcinoma (SCC) of the oral cavity. Study Design: Immunohistochemical staining of randomly selected normal, dysplastic, and malignant oral tissues. Methods: Formalin-fixed, paraffin-embedded tissues were obtained from the Department of Oral Pathology at Shands Hospital (University of Florida), Gainesville, Florida. Examples of mild dysplasia, severe dysplasia, well-differentiated SCC, moderately differentiated SCC, and poorly differentiated SCC were randomly selected from the dictated reports of one of two staff oral pathologists. Histologically normal margins of each specimen served as normal controls. The tissues were immunohistochemically stained using commercially available antibodies against SF and c-met. Appropriate negative controls were run with each batch to ensure staining specificity. Evaluation of staining intensity was carried out using a computerized image analysis system. A one-way analysis of variance (ANOVA) with pairwise multiple-comparison procedures (Fisher method) was used to analyze the data. Results: Statistically significant differences (P < .0001) in the intensity of staining were noted between the malignant and normal and the malignant and dysplastic tissues for both SF and c-met. No differences were appreciated when staining of normal and dysplastic sections of the SF-stained tissue were compared. Conclusions: The results suggest that the SF/c-met ligand-receptor pair is overexpressed in SCC of the oral cavity.     

Immunohistochemical detection of glutathione S-transferase (GST) -pi in head and neck carcinoma and its changes by radiotherapy]

It is well known that the "placental form of glutathione S-transferase" (GST-pi) is present in high concentrations in most human carcinomas. However, its expression in head and neck carcinomas have not yet been reported. The author investigated the expression of GST-pi in the tissue of pharyngeal and laryngeal carcinomas by the immunohistochemical procedure, and the following results were obtained: 1) GST-pi was positive in 80% of laryngeal carcinomas (35 cases) and 52.8% of pharyngeal carcinomas (36 cases). As a rule, well differentiated squamous cell carcinomas showed stronger expression of GST-pi than poorly differentiated squamous cell carcinomas. 2) Although normal epithelia of the pharynx and larynx showed negative GST-pi, it should be noticed that 54.6% of precancerous epithelia (11 cases) showed positive GST-pi. 3) Most patients treated with radiotherapy showed the diminution of GST-pi expression after the irradiation. However, co-relation between the strength of initial GST-pi expression and the effectiveness of radiotherapy was not observed (p < 0.01).     

Prognostic significance of epidermal growth factor receptor in squamous cell carcinoma of the maxillary sinus

Summary The expression of epidermal growth factor receptor (EGFR) was determined immunohistochemically in two groups of maxillary sinus squamous cell carcinomas, one with recurrences at the primary site after combination therapy with radiotherapy, chemotherapy and/or surgery and one without local recurrences. Using a four-graded scale (-,+,++,+++), 9 of the 10 recurrent carcinomas had a staining intensity and proportion of stained cells of ++ or more. A comparable staining intensity was found in 9 of the 18 non-recurrent carcinomas. This difference is statistically significant (Fisher's exact probability test, P < 0.05). These results indicate that an increased expression of EGFR may influence the recurrence rate of squamous cell carcinoma of the maxillary sinus after combined therapy.     

Expression of focal adhesion kinase and phosphorylated focal adhesion kinase in squamous cell carcinoma of the larynx

OBJECTIVES: Focal adhesion kinase (FAK) is overexpressed in a variety of human cancers including those derived from the oral cavity. The purpose of this work is to determine the expression patterns of FAK and its activated form, FAK pY397, in squamous cell carcinoma of the larynx and to correlate FAK expression with tumor differentiation and clinical parameters. STUDY DESIGN: A retrospective study using archival tissue. METHODS: Thirty-five paraffin embedded tissue specimens of laryngeal carcinoma were obtained from the Department of Pathology at the University of Florida College of Medicine. Immunohistochemical staining of the specimens for FAK and activated phospho-FAK (FAK pY397) was performed. Intensity of staining, distribution of staining, and percentage of cells stained was determined by one pathologist. RESULTS: There was a statistically significant correlation between FAK staining intensity and tumor differentiation. Poorly differentiated tumors stained more intensely than moderately differentiated tumors (P <.001). There was no correlation between FAK pY397 staining and differentiation (P =.163). However, FAK pY397 staining was unexpectedly found in the nuclei of many specimens. FAK was present in the basal layer of cells within nontransformed squamous mucosa derived from tonsillectomy specimens and in blood vessels. A small amount of FAK pY397 was also localized to blood vessels in nontransformed squamous mucosa. CONCLUSION: FAK and phospho-FAK are overexpressed in squamous cell carcinoma of the larynx. FAK expression correlates with differentiation. Future investigations will examine the potential of FAK and FAK pY397 expression both as a prognostic indicator and a point of therapeutic inhibition.     

Cytokeratin 18 expression in squamous cell carcinoma of the head and neck

Cytokeratin (CK) expression was studied in squamous cell carcinomas of different subsites in the head and neck by using cryostat sections from 27 head and neck squamous cell carcinomas (HNSCCs) and 6 cell lines established from HNSCC. All tissues were analyzed immunohistochemically with a panel of monospecific anti-keratin monoclonal antibodies. Most carcinomas recapitulated the expression pattern of keratins present in the basal layer of normal epithelium from the site of tumor origin. Regional differences in the expression of simple-epithelial type of keratins in stratified (pseudostratified) epithelia were to a large extent repeated in corresponding carcinomas. In the present study, localization of various keratins were surveyed and CK 18 specific monoclonal antibodies were specifically used to distinguish SCCs of the larynx or hypopharynx from SCCs of the oral cavity. CK 18 staining of almost all tumor cells was detected in 11 of 12 SCCs of the larynx and hypopharynx, but was only detected sporadically in 3 of 9 SCCs of the oral cavity. The present results show that CK 18 typing might be useful for distinguishing sites of origin of various HNSCCs. Findings also indicate that CK 18 expression in SCC might be modulated by microenvironmental factors.     

Undifferentiated carcinoma of nasopharyngeal type of tonsil

A review of 2262 squamous cell carcinomas of the tonsillar region seen at the Institut Gustave-Roussy, Villejuif, France, from 1970 to 1986 showed 1837 well- and poorly differentiated squamous cell carcinomas and 425 undifferentiated squamous cell carcinomas. Eighteen patients with undifferentiated squamous cell carcinomas presented histologic characteristics of undifferentiated carcinomas of nasopharyngeal type. Radiosensibility and radiocurability (complete sterilization with 70 Gy administered) was found in this group with an excellent long-term control of local disease (77% at 10 years actuarial). Epstein-Barr virus-related serologic tests were performed for seven patients. Four of them presented the serologic anti-Epstein-Barr virus titer patterns, generally associated with undifferentiated carcinomas of nasopharyngeal type (1280 to 5120 for viral capsid antigen/IgG and 40 to 320 for viral capsid antigen/IgA). These observations confirm that undifferentiated carcinomas of the nasopharyngeal type may arise outside the nasopharynx.     

Glutathione S‐Transferase π in Squamous Cell Carcinoma of the Head and Neck

Objectives/Hypothesis Oxidative/reductive (redox) DNA damage from radical species such as nitric oxide (NO·) are increasingly being implicated in the development of cancer. Moreover, redox‐protective cellular mechanisms, such as glutathione S‐transferase, may determine cellular susceptibility to this redox‐mediated damage. Methods Formalin‐fixed, paraffin‐embedded tissue samples of 11 normal oral mucosa, 15 reactive/dysplastic lesions, and 131 head and neck squamous cell carcinomas (HNSCCs) were immunohistochemically stained using a polyclonal antibody against glutathione S‐transferase π (GST‐π). Slides were reviewed in a blinded fashion by the study pathologist (g.k.h.) and intensity was graded, noting the pattern of immunostaining. These staining characteristics were compared with those obtained using monoclonal antibodies against endothelial constitutive nitric oxide synthase (ecNOS) and nitrotyrosine, a marker of NO·'s pathological nitrosylation of proteins on serial sections of the same tissue. Patient charts were reviewed and clinical data collected. Results The expression of GST‐π was significantly increased in reactive/dysplastic and HNSCC samples compared with normal squamous mucosa (P < .001 for both). Furthermore, among the carcinomas the expression of GST‐π was significantly increased in higher‐grade lesions (P < .02). The expression of GST‐π correlated highly with the expression of ecNOS and nitrotyrosine (P < .0001 for both). Conclusions These findings demonstrate that GST‐π is upregulated in conjunction with the NO·‐generating ecNOS isoform and implicate GST‐π in protecting HNSCC from the cytotoxic effects of high concentrations of NO· found in the tumor bed.     

Cytomorphological Analysis of Keratinocytes in Oral Smears from Tobacco Users and Oral Squamous Cell Carcinoma Lesions——A Histochemical Approach

Aim To analyse the cytomorphological features of keratinocytes in smears obtained from the oral mucosa of tobacco users and from oral squamous cell carcinoma lesions.Methodology Oral smears were obtained from clinically,normal appearing mucosa of oral squamous cell carcinoma patients (n=20) and from the mucosa of smokers (n=20),and apparently healthy individuals (n=20) were used as controls.The smears were histochemically stained and cytomorphological assessment of the keratinocytes was carried out.One-way ANOVA (Analysis of Variance) was used for comparing the parameters among multiple groups and Tukey-HSD test was used to compare the mean values between groups.Results The mean nuclear area of keratinocytes from the mucosa of tobacco users was 46±2.57 and that of the oral squamous cell carcinoma lesion was 81.54±4.31.While there was a significant (P=0.001) reduction in the cellular area of keratinocytes from oral squamous cell carcinoma lesion when compared with those from oral smears of tobacco users.Conclusion Cytomorphometric analysis of keratinocytes can serve as a useful adjunct in the early diagnosis of oral squamous cell carcinomas.     

Cell proliferation and p27Kip1 expression in endophytic schneiderian papillomas

OBJECTIVE: To clarify epithelial cell proliferation and p27Kip1 expression along the stepwise histological changes from endophytic schneiderian papillomas to associated carcinomas. STUDY DESIGN: Retrospective investigation involved surgical specimens from 58 patients. METHODS: Immunohistochemical assessment involved the nuclear Ki67 antigen expressed in proliferating cells. Further, the cyclin-dependent kinase (cdk) inhibitor p27Kip1 was assessed. Binding of p27Kip1 to the cyclin E-cdk2 complex inhibits this kinase, which results in cell cycle arrest. The expression rates of both proteins were compared between nonpapillomatous nasal mucosa, endophytic schneiderian papillomas, carcinoma in situ, and invasive squamous cell carcinomas. Statistics involved the Wilcoxon and Mann-Whitney u tests. Significance was set at P <.05. RESULTS: Comparable cell proliferation rates were observed between non-papillomatous nasal mucosa and cylindrical cell papillomas. Significant increases in cell proliferation were found along the stepwise series of histological changes involving non-papillomatous nasal mucosa, columnar epithelium in inverted papillomas, transitional and squamous metaplasia in inverted papillomas, and dysplastic inverted papillomas (P <.05, respectively). A tendency toward increased cell proliferation in carcinoma in situ compared with dysplastic inverted papillomas was present; however, this was not statistically significant. The expression rates of p27Kip1 were comparable between non-papillomatous nasal mucosa and all histological subtypes within nondysplastic endophytic schneiderian papillomas. Significantly reduced p27Kip1 expression was found in surface cells in dysplastic compared with non-dysplastic inverted papillomas, as well as in the total number of cells in carcinoma in situ compared with dysplastic inverted papillomas (P <.05, respectively). CONCLUSIONS: Inverted papillomas but not cylindrical cell papillomas show increased cell proliferation compared with nonpapillomatous nasal mucosa. Stepwise increases in cell proliferation accompany the consecutive histological changes within inverted papillomas. Among them, increased cell proliferation along with the development of dysplasia and carcinoma in situ is associated with reduced p27Kip1 expression.     

Dendritic cells in precancerous lesions of the larynx

OBJECTIVES: Hyperplastic lesions of the laryngeal mucosa can eventually develop into squamous cell carcinoma The relationship between dendritic cell infiltration of head and neck cancers and prognosis is well known. Surprisingly, data regarding dendritic cell infiltration in precancerous lesions are not available today. It was the purpose of our study to extend these observations and to investigate in more detail the density and distribution of dendritic cells in pre-cancerous lesions. STUDY DESIGN: Retrospective survey by immunohistochemistry. METHODS: For this study we investigated paraffin-embedded tissue sections of 41 specimens. Histological diagnosis disclosed precancerous lesions of the larynx in 34 cases and in 7 cases, squamous cell carcinoma Immunohistochemical study was performed using antibodies against the cell surface markers S-100, HLA-DR, CD20, CD45 RO, CD45 RA, and Lag. Typical dendritic cell distributions of the immunostained specimens were photographed and measured on a quantitative basis. The medical histories of the patients were then analyzed retrospectively. RESULTS: HLA-DR+ cells could be detected in 14 of 16 cases in mild dysplastic lesions. The infiltration of the dysplastic lesions was sparse compared to cases with higher-graded dysplastic lesions. The distribution patterns of the dendritic cells in specimens with severe dysplastic lesions, but squamous cell carcinoma were extremely similar and markedly different from those in grades I and II specimens. Memory T lymphocytes (CD45 RO+) were detected more often in the group with severe dysplastic lesions (8 of 9 cases) than in the group with squamous cell carcinoma (3 of 8 cases). The inverse became evident for CD20 and CD45 RA immunolabeling. CONCLUSIONS: Few dendritic cells were found in the precancerous lesions. This may suggest that these early lesions (grades I and H) are not efficiently monitored by the immune system. Therefore they may develop into carcinomas unimpaired by cytotoxic T cells. As the degree of malignancy rises (grade III), more dendritic cells infiltrate the tumor.     

Expression of epidermal growth factor receptor in glottic carcinoma and its relation to recurrence after radiotherapy.

The expression of EGFR was determined immunohistochemically in two groups of patients with glottic carcinoma, one that recurred after a full course of radiotherapy and one that did not. Using a 4-graded scale (-,+,++, ) 80% (12/15) of the recurrent carcinomas had a staining intensity and proportion of stained cells of ++ or more. The same figure for non-recurrent carcinomas was 39% (7/18). The difference is statistically significant (chi-squared with Yates' correction, P less than 0.05). The results indicate that an increased expression of EGFR may influence the rate of recurrence of glottic squamous cell carcinoma after radiotherapy.     

Development of monoclonal antibodies with specificity to oral squamous cell carcinoma

A panel of five monoclonal antibodies have been produced that show binding when directed against human oral head and neck squamous cell carcinomas. These monoclonal antibodies are derived from antibody-secreting cells obtained using cell hybridization techniques. The activity of the antibody was tested in vitro against oral head and neck squamous cell carcinoma (n = 10). Control groups included normal oral mucosa (n = 7), hyperkeratosis (n = 5), mild dysplasia (n = 4), and severe dysplasia (n = 3). Results using immunoperoxidase staining confirmed the binding of the five monoclonal antibodies to human head and neck squamous cell carcinoma cells, with predominant reactivity (50% to 90%) in the majority of the specimens, compared with negative (0%) to weak (less than 5%) or focal (5% to less than 50%) reactivity in the control specimens.     

Chromosomal aneuploidy precedes morphological changes and supports multifocality in head and neck lesions.

OBJECTIVE: To identify chromosome changes associated with the transformation of dysplastic lesions and to verify evidence for multifocality in synchronous premalignant lesions associated with head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Chromosomal aneuploidy was evaluated in sections of formalin-fixed, paraffin-embedded tissues from 16 patients with HNSCC, including sites with normal squamous mucosa, dysplasia (low- and high-grade), and invasive tumor. METHODS: A panel of 6 centromeric probes (chromosomes 1, 3, 7, 8, 9, and 17) was analyzed in dual-color fluorescence in situ hybridization assays, using matched hematoxylin-eosin-stained sections for histologic correlation. RESULTS: Imbalances for most of the targets tested were found in 20 of 24 invasive carcinoma sites, mainly represented by gain in copy number per cell. However, cell populations with chromosome losses and gains in multimodal patterns were concomitantly observed in a number of tumors, indicating a high degree of chromosome instability. The detection of chromosomal aneuploidy precedes the malignant transformation as indicated by findings of monosomy and trisomy in normal squamous mucosa, and in low-grade and high-grade dysplasia sites. Loss of chromosomes 3 and 17 prevailed in low-grade dysplasias, and gain of chromosomes 7 and 8 were prevalent in high-grade dysplasias. Synchronous low-grade and high-grade dysplastic lesions displayed discordant molecular signatures, suggesting a multifocal origin. CONCLUSIONS: The interphase fluorescence in-situ hybridization (FISH) assay with centromeric may detect early changes in the progression of dyplastic epithelia to invasive carcinoma and supports the field cancerization theory of multifocality.     

Lectin-binding patterns and cell kinetics of head and neck squamous cell carcinomas

In order to elucidate the cell characteristics of head and neck squamous cell carcinomas, the cell kinetics and lectin binding patterns were compared with the histological classification and staging of the tumors, using surgically resected materials (maxillary sinus 10, oral cavity 21, pharynx 8, larynx 11). Eight biotinylated lectins (WGA, 1-PHA, ConA, UEA1, RCA1, SBA, DBA, PNA) were applied to the paraffin-embedded sections, and were visualized histochemically by the streptavidin-alkaline phosphatase method. The DNA contents of the isolated carcinoma cells obtained from the adjacent thick sections were evaluated using an epi-illumination cytofluorometer after propidium iodide staining. On lectin histochemistry, the binding pattern of WGA lectin was similar between carcinoma tissues and normal tissues, but the binding was more intense in well differentiated than less differentiated carcinomas. Lymph node metastasis was found to be related to the presence of cells with poor WGA-binding. In the binding patterns of the other lectins, RCA1, SBA and ConA were related to the differentiation of carcinomas, but they were not related to the TNM-classification. DNA cytofluorometry exhibited marked polyploidization, which progressed with the advancement of the clinical and pathological staging of carcinomas. However, the DNA ploidy pattern was not associated with the cell characteristics such as the degree of histological differentiation and the lectin-binding pattern, except that the appearance of aneuploidy had some relationship with the binding-patterns of UEA1 and 1-PHA.     

Expression of epidermal growth factor receptor in glottic carcinoma and its relation to recurrence after radiotherapy

expression of EGFR was determined immunohistochemically in two groups of patients with glottic carcinoma, one that recurred after a full course of radiotherapy and one that did not. Using a 4-graded scale (-, +,++,+++) 80% (12/15) of the recurrent carcinomas had a staining intensity and proportion of stained cells of ++ or more. The same figure for non-recurrent carcinomas was 39% (7/18). The difference is statistically significant (chi-squared with Yates' correction, P &<5). The results indicate that an increased expression of EGFR may influence the rate of recurrence of glottic squamous cell carcinoma after radiotherapy.     

Guanylate cyclase activity in normal and neoplastic squamous epithelia from the upper aero-digestive tract

Cyclic nucleotides (cAMP, cGMP) are suggested to participate in the regulation of cell growth and differentiation. Guanylate cyclase is the enzyme which catalyzes the synthesis of cGMP. The basal guanylate cyclase activity was slightly higher in well-differentiated squamous cell carcinomas than in normal mucosa, but was two-fold higher in papillomas. Poorly differentiated squamous cell carcinoma did not show any increased basal activity. Stimulation with nitroprusside (NP) resulted in a 20% increased activity for normal mucosa and a 30% increase for poorly differentiated carcinomas, whereas enzyme prepared from well-differentiated squamous carcinomas and papillomas showed a two-fold increase.