Human Laryngeal Squamous Cell Carcinoma Research Articles

Targeted therapy of human laryngeal squamous cell carcinoma in vitro by antisense oligonucleotides directed against telomerase reverse transcriptase mRNA

A number of different approaches have been developed to inhibit telomerase activity in human cancer cells. In this study, the effect of antisense oligonucleotides (ODNs) by targeting human telomerase reverse transcriptase (hTERT) mRNA in a laryngeal cancer cell line (Hep-2) was investigated. A 20mer antisense oligodeoxynucleotide targeting the most open part of hTERT mRNA (anti-hTERT) and a mismatched control sequence were synthesized. Cells were treated daily with oligonucleotides for up to 72 hours. hTERT mRNA expression was measured by the reverse transcription polymerase chain reaction (RT-PCR) assay; telomerase activity by the telomerase PCR ELISA assay kit (TRAP; Boehringer Mannheim, GmbH, Mannheim, Germany). Cell viability after administration of ODNs was determined using the MTT assay. Morphological changes were examined by haematoxylin and eosin staining. The cell cycle was analyzed using flow cytometry. It was found that antisense treatment induced a decrease in hTERT mRNA expression, telomerase activity, cell growth rate, cell viability, and an increase in apoptosis. The results suggest that inhibition of telomerase activity in Hep-2 cells by short-term antisense treatment against the mRNA of hTERT results in apoptotic cell death. The treatment with anti-hTERT may be useful as a treatment modality for laryngeal squamous carcinoma.

Activation and Abnormalities of Cell Cycle Regulating Factor in Head and Neck Squamous Cell Carcinoma Cell Lines: Abnormal Expression of CDKN2 Gene in Laryngeal Squamous Cell Carcinoma

Background: Cyclin-dependent kinase (CDK) inhibitors are family of molecules that regulate the cell cycle. The CDKN2, a CDK4 inhibitor, also called p16, has been implicated in human tumorigenesis. The CDKN2 inhibits the cyclin/CDK complexes which regulate the transition from G1 to S phase of cell cycle. There is a previous report that homozygous deletion of CDKN2 region on chromosome 9p21 was detected frequently in astrocytoma, glioma and osteosarcoma, less frequently in lung cancer, leukemia and ovarian cancer, but not detected in colon cancer and neuroblastoma. However, little is known about the relationship between CDKN2 and laryngeal cancer. Therefore this study was initiated to investigate the role of CDKN2 in human laryngeal squamous cell carcinoma development.1) Materials and methods: We used 5 human laryngeal carcinoma cell lines whether they have deletions or losses of CDKN2 gene expression by DNA-PCR or RT-PCR, respectively. We examined 8 fresh frozen human laryngeal cancer tissues to detect the loss of heterozygosity (LOH) of CDKN2. PCR was performed by using microsatellite markers of short arm of human chromosome 9 (D9S126, D9S144, D9S156, D9S161, D9S162, D9S166, D9S171, D9S200 and D9SIFNA). For informative cases, allelic loss was scored if the signal of one allele was significantly decreased in tumor DNA when compared to the same allele in normal DNA. Results: The CDKN2 DNA deletion was observed in 3 cell lines. The CDKN2 mRNA expression was observed in only one cell line, which was very weak. LOH was detected in 7 cases (87.5%). Conclusion: These results suggest that CDKN2 plays a role in the carcinogenesis of human laryngeal squamous cell carcinoma.

Expression of hypoxia inducible factor-1α and its relationship to apoptosis and proliferation in human laryngeal squamous cell carcinoma

To investigate the expression of hypoxia inducible factor-1 alpha (HIF-1alpha) and its relationship to apoptosis and proliferation in laryngeal squamous cell carcinoma (LSCC), immunohistochemical method was used to detect the expression of HIF-1alpha and PCNA. Tunnel technique was used to detect in situ cell apoptosis in LSCC. Our results showed that the expression of HIF-1alpha was related to the clinical stages of cancer and lymph node metastasis (P<0.05). The relationship between HIF-1alpha and PCNA was statistically significant (P<0.05) and no relationship was found between HIF-1alpha and apoptosis (P>0.05) It is concluded that HIF-1alpha plays a role in the carcinogenesis of laryngeal carcinoma and is correlated with proliferation, but bears no relationship with the apoptosis of tumor cells in LSCC.

Loss of caspase-8 activation pathway is a possible mechanism for CDDP resistance in human laryngeal squamous cell carcinoma, HEp-2 cells

Cisplatin (CDDP) is among the most widely used and most effective chemotherapeutic agent for many types of human cancer. Because killing cancer cells by chemotherapy is principally executed by apoptosis, a defective apoptotic program might acquire drug resistance. Flow cytometric Annexin V assay demonstrated that HEp-2 cells (human laryngeal cancer) were persistently resistant to CDDP as compared to HeLa cells (human uterine cervical cancer), despite the same histological type and wild-type p53 status. CDDP treatment caused steady induction of p53 protein in both cancer cell types, although it was more dramatic in CDDP-resistant HEp-2 cells, which was correlated well with p53 Ser15 phosphorylation, but not with the expression level of HPV type 18 E6 oncoprotein in these cells. Importantly, CDDP differently activated caspase cascades between HEp-2 and HeLa cells. CDDP activated the caspase-8 pathway through TNFR superfamily receptors such as Fas, but not caspase-9 in HeLa cells. On the other hand, the caspase-9 pathway was significantly activated in HEp-2 cells, although the activation of caspase-8 by CDDP was deficient. This different response to CDDP in caspase-8 activation was not related with the expression level of either Fas or FasL in these cells. We concluded from these results that loss of the caspase-8 activation pathway in HEp-2 cells was a possible mechanism for its resistance to CDDP-induced apoptosis. The caspase-8 pathway might play an important role in CDDP-induced apoptosis in HPV-positive human squamous cell carcinomas.

High frequency of p53 intronic point mutations in laryngeal squamous cell carcinoma*

Abstract Intronic point mutations are rare and totally unknown for human laryngeal squamous cell carcinoma (LSCC). To explore the relationship of p53 gene intronic mutation to the development of human LSCC, DNA was extracted from both tumor tissues and matched normal tissues of 55 patients with LSCC in northeast of China. Polymerase chain reaction amplification-single strand conformational polymorphism (PCR-SSCP) combined with silver staining and DNA direct sequencing were used to detect mutations in exons 7∼8 (p53E7 and p53E8) and introns 7∼8 (p53I7 and p53I8) of p53 gene. The p53E7 mutation was detected in 17 out of 55 patients, and the p53I7 mutation in 21 patients. No mutation was found at p53E8 or p53I8 site. The difference between tumor group and paired normal group on the rates of both p53E7 and p53I7 mutations was statistically significant. The rate of p53I7 mutations in tumor tissue was higher than that of normal tissue, and so was that of p53E7. Sequence analysis revealed that most p53I7 mutatio...

Correlation analysis between STK15 gene and laryngeal carcinoma

Objective: To explore the relationship between STK15 gene abnormal expression and laryngeal carcinoma. Methods: Tumor tissues and matched normal tissues were taken from 55 LSCC patients. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect STK15 expression in 110 specimens. Results: In 38 of the 55 cases (69.1%), the STK15 expression at the mRNA levels was higher than that of the paired normal tissue. The ratio of ADV (average density value) of STK15 gene to ADV of β-actin gene was 1.22±0.49 in the cancer tissue, and 0.99±0.54 in the paired normal tissue with a significant difference (t=4.539, P<0.01). Conclusion: There was obvious association between the STK15 overexpression and laryngeal carcinoma. It may serve as an alternative mechanism of activating the pathogenesis of human laryngeal squamous cell carcinoma.

Localization and level of expression of β-catenin in human laryngeal squamous cell carcinoma

Objective We studied the participation of β-catenin in the histologic differentiation of laryngeal squamous cell carcinomas. Study design and setting At the National Institute of Respiratory Diseases, a tertiary referral center, localization and level of expression of β-catenin were compared between normal epithelium (15 cases) and primary tumors in different degrees of differentiation (38 cases), using an immunohistochemical procedure. Results Cell membrane staining of β-catenin was observed in normal epithelium and in well and moderately differentiated carcinomas. Cytoplasmic redistribution was observed in poorly differentiated carcinomas. Loss of β-catenin correlated with tumor dedifferentiation. Conclusion Reduction of cell membrane β-catenin expression correlated with tumor dedifferentiation. Significance Loss of β-catenin may lead to diminishing the strength of the intercellular adhesion system, thereby promoting the invasive phenotype of the squamous cell carcinoma of the larynx.

The nuclear localization of NFkappaB and p53 is positively correlated with HPV16 E7 level in laryngeal squamous cell carcinoma.

The interaction between the HPV (human papilloma virus) 16 E7 and other cell growth factors, such as p53 and NFkappaB in laryngeal cancer is not clearly understood. The aim of this study was to examine the expression of these three proteins in tumor and non-tumor laryngeal tissues from patients with laryngeal squamous cell carcinoma. These three proteins were dominantly expressed in the nucleus and their levels were higher in the tumor tissue than in the non-tumor tissue, although the comparison between the tumor and non-tumor tissues of p53 staining did not reach significance. The intensity of the nuclear stain of E7 and p53 was stronger than that of p65, a subunit of NFkappaB. Correlation analysis revealed that there was a positive relationship between the level of HPV16 E7 and the expression of p65. The correlation between E7 and p53 was also significant, although to a lesser degree. The finding of nuclear localization of p65 suggests that NFkappaB is constantly activated in the laryngeal cancer cells, whereas the sequestration of p53 in the nucleus may represent a mutated form of p53, which is probably inactivated by HPV16 oncoproteins. In conclusion, this study suggests that the nuclear localization of NFkappaB and p53 may play a role in the development of human laryngeal squamous cell carcinoma infected with HPV16.

Changes in tumor hypoxia measured with a double hypoxic marker technique

Purpose: Development of a double hypoxic cell marker assay, using the bioreductive nitroimidazole derivatives CCI-103F and pimonidazole, to study changes in tumor hypoxia after treatments that modify tumor oxygenation. Methods and Materials: Both hypoxic markers were visualized by immunohistochemical techniques to detect changes in hypoxic fraction induced by carbogen breathing (95% O 2 and 5% CO 2) or hydralazine injection. The protocol was tested in a human laryngeal squamous cell carcinoma xenograft line. Quantitative measurements were derived from consecutive tissue sections that were analyzed by a semiautomatic image analysis system. Qualitative analysis was obtained by double staining of the two hypoxic markers on the same tissue section. Results: A significant correlation between the hypoxic fractions for the two markers, CCI-103F and pimonidazole, was found in air breathing animals. After carbogen breathing, the hypoxic fraction decreased significantly from 0.07 to 0.03, and after hydralazine treatment, the hypoxic fraction increased significantly. Reduction of hypoxia after carbogen breathing was most pronounced close to well-perfused tumor regions. Conclusions: With this method, employing two consecutively injected bioreductive markers, changes in tumor hypoxia can be studied. A significant reduction in hypoxia after carbogen breathing and a significant increase in hypoxia after hydralazine administration was demonstrated.

Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line.

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.

Establishment and characterization of human laryngeal squamous cell carcinoma cell lines

Six human laryngeal squamous cell carcinoma cell lines (SNU-46, -585, -899, -1066, -1076, -1214) established from Korean patients are reported. In vitro culture of six squamous cell carcinoma cell lines derived from primary tumors of the larynx. Description of the cell line phenotypes and determination of molecular characteristics. Six laryngeal squamous cell carcinoma cell lines were cultured. The cell phenotypes, including the histopathology of the primary tumors and in vitro growth characteristics, were determined. Molecular characterization was also performed, including DNA fingerprinting analysis and abnormalities of p15, p16, p53, and TGF-betaRII genes by polymerase chain reaction-based single strand conformation polymorphism and sequencing analysis. All cell lines grew as adherent cells; five lines grew as monolayers and one other line grew as stratifying colonies. All lines showed 1) high viability (75%-92%) with various doubling times (36-96 h); 2) absence of Mycoplasma and other bacteria; and 3) genetic heterogeneity by DNA profile analysis. p53 Mutations were found in three lines and p16 mutations were observed in five cell lines. TGF-betaRII mutations were found in two lines: one line had frameshift mutation and another line had a missense mutation at the kinase domain. These newly established and characterized laryngeal squamous cell carcinoma cell lines will be useful for investigating the biologic characteristics of laryngeal cancer.

Downregulation of p21/WAF1 is related to advanced and dedifferentiated laryngeal squamous cell carcinoma.

AIM: To analyse p21/WAF1 expression and its relation to p53, apoptosis, cell proliferation, clinicopathological characteristics, and patient survival in human laryngeal squamous cell carcinoma. METHODS: Primary tumours for analyses were...

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.

Vascular architecture and microenvironmental parameters in human squamous cell carcinoma xenografts: effects of carbogen and nicotinamide

Background and purpose: A better understanding of the vascular architecture and the microenvironmental parameters (VAMP) will allow the identification of tumours that can be more effectively treated by intensified fractionated radiotherapy or modifiers of blood flow and oxygenation or combinations of these approaches. Materials and methods: Proliferation (BrdUrd), vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (NITP) were studied in two human laryngeal squamous cell carcinoma tumour lines grown as xenografts in nude mice. The effects of carbogen and nicotinamide on these parameters were evaluated. Results: Carbogen treatment resulted in a decrease of the number of perfused blood vessels from 66% to 55% in one of the two tumour lines. In this tumour line nicotinamide prevented this reduction of tumour blood flow by carbogen. In both tumour lines the labelling index (LI) decreased after treatment with carbogen for 1 h, from 11–13% to 5–7%. Both tumour lines showed a drastic reduction of hypoxia by carbogen alone or by carbogen plus nicotinamide. Conclusions: In both laryngeal squamous cell carcinoma xenograft tumour lines carbogen was very effective in reducing diffusion limited hypoxia. Only in one of the two tested tumour lines carbogen also caused a reduction of tumour blood perfusion, which could be compensated for by nicotinamide. In addition, carbogen reduced tumour cell proliferation. The fact that differences in response to nicotinamide and carbogen were observed and that they can be studied in vivo provides a basis for further development of a ‘predictive profile’ which will guide the clinician to select the optimal treatment for individual patients or groups of patients.

Clinical Outcome and Tumour Microenvironmental Effects of Accelerated Radiotherapy with Carbogen and Nicotinamide

Experimental studies have shown an almost 2-fold increase in effectiveness if accelerated radiotherapy combined with carbogen and nicotinamide (ARCON) was compared with standard radiotherapy. This combination was chosen in order to overcome repopulation of clonogens during radiotherapy and to minimize tumour hypoxia. Analysis of microenvironmental parameters is required to identify tumours that can benefit from these new treatment approaches. In this study 124 patients with stage III or IV head and neck squamous cell carcinomas received ARCON treatment. Vascular architecture, perfusion, proliferation and oxygenation were studied in two human laryngeal squamous cell carcinoma xenograft lines and the effects of carbogen and nicotinamide were analysed. Loco-regional control for stage III-IV larynx carcinomas was 85%, for hypopharynx carcinomas 50% and for oral cavity and oropharynx carcinomas 65%. In the experimental studies, carbogen treatment resulted in one tumour line in a decrease of blood perfusion, which was reversed if nicotinamide was added. The other tumour line showed no perfusion changes after carbogen or nicotinamide treatment. Both tumour lines showed a drastic reduction of hypoxia after carbogen breathing only or carbogen breathing plus nicotinamide. The ARCON schedule results in high loco-regional tumour control rates. Analysis of tumour microenvironmental parameters showed differences in response to carbogen and nicotinamide between different tumour lines of similar histology and site of origin. This indicates that it may be advantageous to base the selection of patients for oxygenation modifying treatment on microenvironmental tumour characteristics.

Subadditive interaction of radiation and taxol in vitro

To examine the dependency of Taxol-radiation interactions on the scheduling of the two agents. The human laryngeal squamous cell carcinoma line SCC20 was used for this study. Cells were irradiated as subconfluent cultures using Cs-137 gamma rays at a dose rate of 1.75 Gy/min. Cultures were pretreated with Taxol (7.5 nM for 12 h, S.F. = 0.4) and then irradiated with graded doses followed by either immediate plating or holding for 6 h either in the absence or presence of 7.5 nM Taxol prior to plating for colony-forming ability. Experiments in which cells were irradiated and then exposed to 7.5 nM Taxol for both 12 and 18 h were also performed. Parallel-flow cytometric analyses of cell-cycle distribution of the various treated populations were carried out. The results indicate that pretreatment with Taxol induced a G2 block which was maintained during 6 h postirradiation holding either in the presence or absence of Taxol. No modification of radiosensitivity in the low-dose region was seen for cells treated with Taxol, irradiated, and plated immediately, with the resulting survival being compatible with an additive effect. However, for Taxol-pretreated cells held for 6 h postirradiation, either in the absence or presence of Taxol, the resulting survival reproducibly demonstrated a marked less than additive effect. This was particularly prominent for cells held in the presence of Taxol. Subsequent experiments in which Taxol was added to cells immediately postirradiation again demonstrated a less than additive effect of the two modalities. The results of this study are consistent with a dual mechanism of action involving Taxol-induced radiation resistance, possibly as a consequence of postirradiation holding in G2, and radiation-induced Taxol resistance through an as-yet-undefined mechanism.

Epidermal growth factor receptor immunoreactivity in human laryngeal squamous cell carcinoma

Epidermal growth factor receptor (EGFR) is a transmembrane protein receptor with tyrosine kinase activity. The protein has cysteine-rich sequence repeats in its extracellular ligand-binding domains. Elevated levels of EGFR are associated with malignant transformation of squamous cells and are observed in squamous cell carcinomas from the lung, head, neck, skin, cervix and esophagus. We examined the expression of EGFR in laryngeal squamous cell carcinoma (SCC) (N = 24) and non-neoplastic polyps (N = 7) using streptavidin-biotin immunohistochemistry and a monoclonal antibody (Serotec: MCA-571) to the EGFR protein. The carcinomas were classified as well differentiated (N = 2), moderately differentiated (N = 16) and poorly differentiated (N = 6). Tissues from metastatic tumor deposits in lymph nodes (N = 5) were also studied. Overexpression of EGFR was present, in the form of strong cytoplasmic immunostaining, in the majority of the SCC cases (n = 20; 83%) and in all of the metastatic tumor deposits. In contrast, although some of the vocal cord polyps showed weak (n = 2) to moderate (n = 5) immunoreactivity, none had evidence of strong EGFR immunoreactivity. The differences in EGFR immunoreactivity were significant between primary laryngeal SCC and vocal cord polyps (p = 0.0001; chi 2 test), and between metastatic laryngeal SCC and vocal cord polyps (p = 0.0001; chi 2 test). Laryngeal carcinoma cases which showed EGFR overexpression had a lower median survival period compared to those without overexpression In conclusion, a different extent of EGFR expression is demonstrated in laryngeal squamous cell carcinomas and non-neoplastic vocal cord polyps using an immunohistochemical method. Some trends in the prognostic value of EGFR immunoreactivity in laryngeal carcinomas appear to emerge in this study.

Nm23-H1 protein immunoreactivity in laryngeal carcinoma.

The nm23-H1 gene encodes a 17-kilodalton cytoplasmic and nuclear protein that has recently been shown to be reduced in a number of human carcinomas including breast, colorectal, lung, gallbladder, and biliary tract carcinomas. This study examines the immunohistochemical staining characteristics of the nm23-H1 protein in human laryngeal carcinomas and nonneoplastic laryngeal polyps, and attempts to determine if there is any relationship between reduction of nm23-H1 protein immunoreactivity and prognosis of patients with laryngeal carcinoma. Routine streptavidin-biotin immunohistochemistry with a polyclonal antibody was employed to study the expression of the nm23-H1 protein in laryngeal squamous cell carcinoma (SCC) (N = 22) and nonneoplastic polyps (N = 8). The carcinomas were classified as well differentiated (N = 2), moderately differentiated (N =15), and poorly differentiated (N = 5). Tissues from metastatic tumor deposits in lymph nodes (N = 5) were also studied. A semiquantitative immunostaining index was derived from the intensity and extent of staining of the cells. All laryngeal polyps showed intense immunostaining for the nm23-H1 gene product in the squamous epithelium. However, reduced immunoreactivity was found in nearly half of the SCC cases (N = 10; 46%), with the least staining intensity found in tumor metastases in lymph nodes (N = 4; 80%), and were associated with a shorter median survival of 14.3 months. In contrast, tumors that demonstrated moderate to strong nm23-H1 protein immunostaining were associated with a longer median survival period of 20.4 months. There is reduced expression of the nm23-H1 gene in human laryngeal SCC compared with nonneoplastic laryngeal polyps. Reduction in the intensity and extent of nm23-H1 protein immunostaining appears to correspond to reduced duration of patients survival.

Nm23‐H1 protein immunoreactivity in laryngeal carcinoma

BACKGROUND The nm23-H1 gene encodes a 17-kilodalton cytoplasmic and nuclear protein that has recently been shown to be reduced in a number of human carcinomas including breast, colorectal, lung, gallbladder, and biliary tract carcinomas. This study examines the immunohistochemical staining characteristics of the nm23-H1 protein in human laryngeal carcinomas and nonneoplastic laryngeal polyps, and attempts to determine if there is any relationship between reduction of nm23-H1 protein immunoreactivity and prognosis of patients with laryngeal carcinoma. METHODS Routine streptavidin-biotin immunohistochemistry with a polyclonal antibody was employed to study the expression of the nm23-H1 protein in laryngeal squamous cell carcinoma (SCC) (N = 22) and nonneoplastic polyps (N = 8). The carcinomas were classified as well differentiated (N = 2), moderately differentiated (N = 15), and poorly differentiated (N = 5). Tissues from metastatic tumor deposits in lymph nodes (N = 5) were also studied. A semiquantitative immunostaining index was derived from the intensity and extent of staining of the cells. RESULTS All laryngeal polyps showed intense immunostaining for the nm23-H1 gene product in the squamous epithelium. However, reduced immunoreactivity was found in nearly half of the SCC cases (N = 10; 46%), with the least staining intensity found in tumor metastases in lymph nodes (N = 4; 80%), and were associated with a shorter median survival of 14.3 months. In contrast, tumors that demonstrated moderate to strong nm23-H1 protein immunostaining were associated with a longer median survival period of 20.4 months. CONCLUSIONS There is reduced expression of the nm23-H1 gene in human laryngeal SCC compared with nonneoplastic laryngeal polyps. Reduction in the intensity and extent of nm23-H1 protein immunostaining appears to correspond to reduced duration of patient survival. Cancer 1996;77:2246-50.

Abnormalities in type IV collagen immunoreactivity in human laryngeal cancers

Abnormal patterns of expression of the basement membrane type IV collagen are observed in many human cancers. This study examines the immunohistological expression of type IV collagen in the basement membrane in human laryngeal squamous cell carcinomas (SCC) (n = 24). Non-neoplastic vocal cord polyps (n = 4) were used as controls. The formalin-fixed, paraffin-embedded tissues were sectioned and pretreated with protease prior to immunostaining for type IV collagen. There was a statistically significant difference in type IV collagen expression between laryngeal SCC and vocal cord polyps (p = 0.0001; chi 2 test with continuity correction). In laryngeal SCC (n = 24; 100%), type IV collagen distribution was discontinuous and irregular or absent around individual or groups of neoplastic cells. In contrast, all of the cases of vocal cord polyps (n = 4; 100%) displayed a continuous pattern of subepithelial basement membrane type IV collagen. This study has shown that abnormal distribution of type IV collagen occurs in laryngeal squamous cell carcinomas but not in non-neoplastic vocal cord polyps. This may be related to either abnormal synthesis or to the breakdown of the collagen and it may be of use as a potential biological marker in the study of laryngeal carcinogenesis.