Laryngeal Epithelium Research Articles

Age-related changes in sensory and secretomotor nerve endings in the larynx of F344/N rat

The aim of the present study was to define the age-related changes in sensory and secretomotor nerve endings in the larynx of F344/N rats. For this purpose, laryngeal tissue sections obtained from 12-, 24- and 35-month-old F344/N rats were compared with respect to the density, distribution and morphology of various types of sensory and secretomotor nerve endings immunoreactive for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP) and substance P (SP). Two distinct forms of PGP 9.5-immunoreactive motor end-plates were noted; the large sized motor end-plates localized in thyroarytenoid and cricoarytenoid muscles were degenerated in aged rats, while the small sized motor end-plates, localized predominantly in vocal muscles, did not show any age-related changes. CGRP- and SP-immunoreactive nerve fibres of the laryngeal glands did not show any age-related changes. Subepithelial laminar nerve endings immunoreactive to PGP 9.5 showed degeneration with ageing. Aggregates of terminal arborisations in the subepithelial region were smaller in aged animals. PGP 9.5-immunostained taste cells and well-developed subgemmal network were abundant in 12- and 24-month-old rats, but only a few were noted in aged rats. The total number of taste buds decreased significantly with ageing. CGRP- and SP-immunostained taste bud-nerve endings were noted in 12- and 24-month-old rats, but only rarely in 35-month-old rats. The laryngeal epithelium contained PGP 9.5-, CGRP- and SP-immunoreactive thin free nerve endings with many varicosities; their number and distribution were similar between 12- and 24-month-old rats, while only a few endings were observed in 35-month-old rats. Our results indicated that ageing is associated with the reduction of laryngeal sensory and secretomotor nerve endings.

The Ljubljana classification of epithelial hyperplastic laryngeal lesions: a morphometric evaluation of nuclear pleomorphism

Epithelial hyperplastic laryngeal lesions (EHLL) are associated, with a varying degree of "epithelial risk"- to develop invasive carcinoma. Several classifications have been proposed but none has received a total agreement. The 1999 Ljubljana classification distinguished four grades: simple, abnormal and atypical hyperplasia and in situ carcinoma (ISC). The first two grades are considered benign lesions; the ISC is the malignant lesion, while the atypical hyperplasia is considered a "risky lesion". This is characterized by alterations of epithelial cells towards malignancy, but not to the extent to be found in carcinoma cells. Such characteristics refer to cytomorphological (e.g., nuclear hyperchromatism, nucleoli, increased nuclear/cytoplasmic ratio) and architectural (e.g. stratification, orientation, maturation) features. In the Ljubljana scheme, nuclear pleomorphism is one of the most important features. We wanted to improve the importance of nuclear pleomorphism in the basal cells layer in different classes of EHLL using morphometrical analysis. We studied 8 cases of simple hyperplasia, 10 of abnormal hyperplasia, 10 of atypical hyperplasia and 8 of ISC using the software SAM (Shape Analytical Morphometry). The results were submitted to univariate statistical analysis. Nuclear dimensions (maximum diameter, perimeter and area) showed a progressive increase from simple to atypical hyperplasias to ISC, while abnormal hyperplasia showed the lowest values. On the contrary, analytical parameters related to nuclear contour irregularities and asymmetries showed their highest values in abnormal hyperplasia nuclei. There were no significant differences between atypical hyperplasia and ISC, while it was possible to differentiate abnormal hyperplasia from the others. In conclusion basal nuclei of atypical hyperplasia and ISC are similar so that other cytological and morphological architectural parameters are necessary to distinguish the two lesions. Abnormal hyperplasia seems to be the biological category of 'proliferative " benign laryngeal epithelium; simple hyperplasia refers to "stable" - irritative epithelium.

Expression of Gap Junction Proteins (Connexin 26, 30, 32, 43) in Normal Mucosa, Hyperkeratosis and Carcinoma of the Human Larynx

Introduction: Gap junction proteins (connexins = Cx) form transmembrane channels and mediate the transfer of small molecules and ions between the cytoplasm of adjacent cells. Most tissues express several Cx isoforms. The precise combination might play an important role in the maintenance of cell differentiation. Human carcinogenesis is accompanied by aberrant expression and function of Cx. While the larynx is a target organ for many tumor promoters, no data on Cx expression in laryngeal mucosa are available. The goal of the study was to observe the expression of different Cx (Cx26, -30, -32 and -43) in the normal mucosa, hyperkeratoses and carcinomas of the human larynx. Method: The immunofluorescence method was performed in normal (n = 7) and dysplastic (n = 6) laryngeal mucosa and in squamous cell carcinoma (n = 7) using affinity-purified polyclonal rabbit antibodies against the 4 Cx isoforms and FITC-conjugated secondary antibodies. Results: The immunofluorescence staining of the normal human vocal fold’s epithelium showed the expression of Cx26 and Cx30 in the parabasal and intermediate layers, whereas Cx43 was localized in the basal, parabasal and lower intermediate layers. Cx epitopes could not be found in the upper layers. The precanceroses showed a similar expression of the Cx compared to normal laryngeal epithelium. Due to the higher degree of staining observed in dysplastic specimens, a hyperexpression of Cx26, -30 and -43 could be assumed. The squamous cell caricnomas were characterized by inhomogeneous staining for Cx26, -30 and -43. Regions of intensive expression alternated with regions of no expression. Cx32 could not be observed by immunofluorescence staining in laryngeal tissue. Conclusion: In immunohistochemical terms, there was no alteration of the expression of Cx isoforms during carcinogenesis in the laryngeal epithelium. These results do not exclude a loss of functional intercellular gap junction communication by posttranslational modifications of Cx isoforms or disturbed Cx integration into the gap junction channel. Further studies should investigate potential defective gap junctional intercellular communication in cancer cells based on molecular studies.

PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells

Laryngeal papillomas are caused by infection of the laryngeal epithelium by human papillomavirus type 6 or type 11 (HPV-6/-11). Previous studies in our laboratory have demonstrated an increase in PI3 kinase levels in papilloma tissue. However, activation of the downstream effector of PI3 kinase, protein kinase B (PKB/Akt), was reduced. This observation was explained by the elevated expression of the phosphatase and tensin homologue (PTEN), a recently characterized tumour suppressor, in papilloma tissue. Recent investigation of the possible functional roles of PTEN during papilloma development has now indicated that the level of tyrosine(705)-phosphorylated signal transducer and activator of transcription 3 [PTyr(705)STAT3] could be inversely correlated to that of PTEN as well. In vitro phosphatase assays suggested the presence of an increased level of a PTyr(705)STAT3 phosphatase in papilloma extract. Immunodepletion of PTEN from papilloma extracts resulted in a reduction of the PTyr(705)STAT3 phosphatase activity. Transfection of PTEN cDNA into HeLa cells attenuated STAT3 phosphorylation at Tyr(705) in a dose-dependent manner. This attenuation of STAT3 phosphorylation was independent of the STAT3 kinase. Interestingly, introduction of a lipid phosphatase mutant of PTEN (G129E) resulted in heightened PTyr(705)STAT3 phosphatase activity, relative to that obtained from wild-type PTEN transfection. These data indicate that PTEN negatively regulates STAT3 activation in HPV-infected papilloma cells. Induction of PTEN and reduction of activated STAT3 might be a result of a host defence mechanism or a virus-directed strategy to alter normal epithelial differentiation programming.

Human papillomavirus in larynx.

The core of the present clinical and basic research knowledge of laryngeal human papillomavirus (HPV) infection is described. Review. A computer-aided search of MEDLINE database supplemented by hand searches of key journals was conducted. One of the tumor-promoting factors in the larynx is the HPV found both in normal laryngeal epithelium and in laryngeal tumors. The most important manifestation of laryngeal HPV infection is laryngeal papillomatosis, a rare disease caused by HPV types 6 and 11. In laryngeal carcinogenesis, the role of HPV remains uncertain. The means of transmission of HPV are partly unknown, and the course of laryngeal HPV infection is unpredictable and variable. Treatment of laryngeal papillomatosis is based on surgery, especially on CO2 laser and shaver. Alpha-interferon is the drug of choice in patients whose response to surgery is poor. However, neither interferon nor other antiviral drugs are able to eradicate the virus from laryngeal mucosa. Little is known about immunological mechanisms involved in laryngeal HPV infection, but in defense against HPV cellular immunity is considered a more important mechanism than humoral immunity. A good experimental model of HPV infection is lacking in which the entire viral life cycle can take place. Organotypic cell cultures (collagen rafts) are useful, but the rate-limiting step in this method is the difficulties in culturing HPV-positive epithelial cells. Although laryngeal papillomatosis is clinically well defined, the mechanisms and treatment modalities of laryngeal HPV infection need further investigations.

Detection of cytokeratins in normal and malignant laryngeal epithelia by means of reverse transcriptase-polymerase chain reaction.

Cytokeratins (CKs) are a subgroup of intermediate filament proteins that take part in forming the cytoskeleton. The epithelial cells in different organs express distinct CKs, and this expression may be modified during malignant transformation. Here we describe the use of reverse transcriptase-polymerase chain reaction followed by Southern blotting to determine the profile of expression of CKs in both normal and malignant laryngeal samples. Thirty-six samples were subjected to histologic examination and classified as 17 squamous cell carcinomas, 3 dysplastic lesions, and 16 normal samples. CK8 and CK19 were expressed in almost all samples, both cancerous and normal, and were therefore used to verify the integrity of RNA. Expression of CK2, CK9, and CK20 was not detected in any of the samples, normal or cancerous. CK15 and CK18 showed low sensitivity for detection of cancer (36.4% and 45.5%, respectively). CK10 showed relatively high sensitivity (91%), but only moderate specificity (69.2%). Only CK17 showed both high sensitivity and specificity (91% and 92.3%, respectively; positive predictive value, 91%). We propose that CK17 may be considered a promising candidate to use as a molecular marker for malignant transformation in laryngeal squamous cell carcinoma.

Cell Biology of Laryngeal Epithelial Defenses in Health and Disease: Preliminary Studies

Esophageal epithelium has intrinsic antireflux defenses, including carbonic anhydrases (CAs I to IV) that appear to be protective against gastric reflux. This study aimed to investigate the expression and distribution of CA isoenzymes in laryngeal epithelium. Laryngeal biopsy specimens collected from the vocal fold and interarytenoid regions were analyzed by Western blotting and immunofluorescence. Carbonic anhydrases I and II were expressed by the majority of samples analyzed. In contrast, CA III was differentially expressed in the interarytenoid samples and was not detected in any vocal fold samples. The expression of CA III was increased in esophagitis as compared to normal esophageal tissue. Carbonic anhydrase I and III isoenzymes were distributed cytoplasmically in the basal and lower prickle cell layers. The laryngeal epithelium expresses some CA isoenzymes and has the potential to protect itself against laryngopharyngeal reflux. Laryngeal tissue may be more sensitive to injury due to reflux damage than the esophageal mucosa because of different responses of CA isoenzymes.

Similarities and differences in lectin cytochemistry of laryngeal and tracheal epithelium and subepithelial seromucous glands in cases of sudden infant death and controls

BACKGROUNDIt has been speculated that non-specific defence mechanisms of the epithelium and subepithelial seromucous glands play a role in the larynx and lungs in cases of sudden infant death.METHODSThe larynx...

Expression of melanoma inhibitory activity in melanoma and nonmelanoma tissue specimens

Melanoma inhibitory activity (MIA) is a small soluble protein secreted by malignant melanoma cells and chondrocytes. Prior studies suggested that MIA expression was relatively tissue-specific, making it a potentially useful marker for melanoma. The current investigations sought to more clearly define the range of tumor/tissue-types where MIA is expressed, compared with expression of 4 other potential melanoma marker genes (tyrosinase melanoma antigen recognized by T cells [MART-1/MelanA], gp100, and melanoma growth-stimulatory activity [MGSA/Gro alpha]). Expression of these genes was assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry in 23 melanoma tumor specimens and in 25 additional nonmelanoma or nonmalignant specimens. MIA, tyrosinase, and MGSA were expressed in most melanoma specimens. Specificity was highest for MART-1, followed by MIA and tyrosinase. Increasing the number of cycles of amplification from 35 to 40 increased sensitivity but decreased specificity of most markers, though MIA was relatively robust. MIA mRNA was also detected in carcinomas of the colon, ovary, kidney, and head/neck, as well as in normal laryngeal epithelium. Although MIA discriminated melanoma from nonmelanoma at least as well as tyrosinase, no single mRNA marker had accuracy greater than 71%, raising potential concern about application of these particular mRNA markers to the minimal disease setting. HUM PATHOL 31:1381-1388.

Laryngeal mucosa of head and neck cancer patients shows increased DNA damage as detected by single cell microgel electrophoresis

Major risk factors for cancer of the oral cavity, pharynx and larynx are smoking and excess alcohol consumption. Since long-term survival rates of head and neck cancer patients have not substantially been improved, new preventive strategies including the use of cancer chemopreventive agents have to be developed. With the aim of developing biomarkers which can verify the efficacy of chemopreventive interventions, a standardised alkaline microgel electrophoresis (MGE) assay was applied as a sensitive and rapid tool to detect DNA damage on a single cell level. Macroscopically normal laryngeal mucosa biopsies obtained by surgery from head and neck cancer patients ( n=29) and from hospital controls ( n=22) were analysed by MGE in a pilot study. As compared to controls, cells from head and neck cancer patients showed a significantly elevated DNA damage without any further genotoxic treatment ( P<0.01). We conclude that this increased background DNA damage in laryngeal epithelia could result from genetic alterations caused by smoking and alcohol leading, in accord with the field cancerisation hypothesis, to a gradual decrease of genomic stability and malignancy. MGE should now be explored as a rapid screening method in larger clinical studies: (i) to identify high-risk subjects carrying cells with decreased genomic stability and (ii) to verify the efficacy of chemopreventive regimens to prevent or slow down the development of head and neck cancer in high-risk persons.

Preliminary experiences with contact endoscopy of the larynx.

Nine patients with laryngeal polyps, four with Reinke's edema, three with leukoplakia, one with papilloma and one with malignant tumor were studied by means of laryngeal contact endoscopy during microlaryngoscopy. This technique allowed in vivo and in situ visualization of the superficial layer of the laryngeal epithelium after staining with methylene blue. Cell structures evaluated were the size and color of the nuclei, the nucleus/cytoplasm ratio, nuclear and cytoplasmic contours, the presence of nucleoli, mitoses and keratoses, as well as the microvascular network of the mucosa and superficial cellular changes from normal to pathological. The normal squamous epithelium of the vocal cord showed a homogeneous cellular population with regular nuclear and cytoplasmic morphological characteristics and a uniform nucleus-to-cytoplasm ratio. Specific cellular epithelial patterns and several alterations of the vascular distribution were found in different pathological conditions. Cytological pictures obtained at contact endoscopy were consistent with histological findings in all the patients studied.

Calbindin D28k-immunoreactive afferent nerve endings in the laryngeal mucosa.

The distribution of the calbindin D28k in the laryngeal sensory structures was studied by immunohistochemistry, immunoelectronmicroscopy, and double immunofluorescence with calretinin-immunoreactivity. Moreover, origin of the nerve endings were observed using retrograde tracer, fast blue. Immunoreactivity for calbindin D28k was found in the various types of nerve endings in the larynx, namely, laminar nerve endings, nerve endings associated with the taste buds, intraepithelial nerve endings, and endocrine cells. The laminar endings with calbindin D28k-immunoreactivity were observed in the subepithelial connective tissue. In some endings, terminals were expanded. The laminar endings were also observed in the perichondrium of the epiglottic cartilage. In the epiglottic and arytenoid epithelia, thick nerve fibers with calbindin D28k-immunoreactivity ascending to taste buds and intragemmal nerve fibers were also observed. Within the epithelial layer, intraepithelial free nerve endings with calbindin D28k-immunoreactivity were observed. Furthermore, diffuse endocrine cells were observed within the laryngeal epithelium. By immunoelectron microscopy, immunoreaction products in the endings mentioned above were localized in the cytoplasm of the axon terminals and nerve fibers which contained with numerous mitochondria. Out of the 100 laminar endings, 18 endings were immunopositive for both calbindin D28k and calretinin, 33 were positive for calbindin D28k but negative for calretinin, and 49 were positive for only calretinin in the double immunofluorescence microscopy. The nerve fibers associated with the taste buds and the free nerve endings, which immunostained for calbindin D28k, were not stained with antibody against calretinin. After injection of the fast blue in the laryngeal mucosa, fast blue-labeled cells were mainly observed in the nodose ganglia. Of the total number of labeled cell in the nodose and dorsal root ganglia at the level C1 to Th2, 65.1% occurred in nodose ganglia (572/879, n = 6). In the nodose ganglia, 79.7% of labeled cells (456/572) were immunoreacted for calbindin D28k. The distribution of calbindin D28k-immunoreactivity may be differnt from that of calretinin. It is suggested that calbindin D28k have regulatory role on intracellular calcium concentration in the laryngeal sensory corpuscles.

Prognostic significance of the Ca(2+) binding protein S100A2 in laryngeal squamous-cell carcinoma.

We investigated by immunocytochemistry the expression of the Ca(2+) binding protein S100A2 in 62 cases of laryngeal squamous-cell carcinoma (SCC). S100A2 was detected in 18/19 (95%) low-grade tumors and in 22/43 (51%) high-grade tumors, which were partially keratinizing. The remaining 21/43 (49%) high-grade tumors were non-keratinizing, anaplastic tumors and clearly S100A2-negative. In normal laryngeal squamous epithelium and in laryngeal SCC, S100A2 expression was strictly associated with that of cytokeratins 14 (P = 0.0002) and 17 (P = 0.0021), suggesting an association of S100A2 expression and cell commitment to squamous differentiation. A correlation was found between S100A2 tumor positivity and longer relapse-free (P = 0.0005) and overall (P = 0.0095) survival.

Immunohistochemical study of chondroitin-6-sulphate and tenascin in the larynx: a loss of chondroitin-6-sulphate expression accompanies squamous cell carcinoma invasion.

Chondroitin 6-sulphate is a glycosaminoglycan component of both cell membrane and basement membrane proteoglycans. In vitro it can inhibit tenascin, a molecule critical for epithelial cell migration during development and in wound healing. The immunohistochemical expression of chondroitin-6-sulphate and tenascin has been examined in 143 laryngeal biopsies from 38 patients, with particular attention to changes occurring with squamous cell carcinoma invasion. All tissues were formalin-fixed and paraffin-embedded. An avidin-biotin complex immunoperoxidase technique was used. Immunostaining for chondroitin-6-sulphate was seen in the basement membrane and/or cell membranes of basal and suprabasal cells of the laryngeal epithelium. Immunostaining of cell or basement membrane was seen at least focally in 67 of 71 (94 per cent) biopsies with no atypia, in 39 of 45 (87 per cent) biopsies with mild/moderate atypia, and in 16 of 16 (100 per cent) biopsies with severe dysplasia or carcinoma in situ (CIS); but in only 2 of 18 biopsies with invasion, although in neither of these was chondroitin-6-sulphate immunostaining seen at the actual site of invasion. Tenascin immunostaining was seen along the basement membrane in all biopsies. Those with CIS or invasion showed, in addition, strong tenascin staining of the adjacent stroma. The loss of chondroitin-6-sulphate immunostaining concurrent with squamous cell carcinoma invasion in the larynx suggests that loss of a chondroitin-6-sulphate-containing proteoglycan, or a change in proteoglycan side-chain composition, is a critical step in laryngeal epithelial tumour invasion.

High-risk human papillomavirus types and squamous cell carcinoma in patients with respiratory papillomas

Respiratory papillomas (RPs) are benign, virally induced tumors of the larynx and respiratory epithelium that may obstruct the airway and tend to recur frequently. RPs are thought to be the result of infection with the human papillomaviruses (HPVs) types 6 and 11. We surveyed archival RP specimens to determine whether there were correlations of HPV type with patient characteristics or clinical course. Paraffin-embedded papilloma specimens of 45 different patients were analyzed. We assessed HPV types using the polymerase chain reaction with E6 consensus primers, hybrid capture assays (high or low risk), and dot blot hybridization of generic E6 PCR products with E6 type-specific oligonucleotide probes. The presence and type of HPV were correlated with patient data from a retrospective chart review. We found that RPs may have either low- or high-risk HPV types and some contain multiple HPV types. Respiratory infection with high-risk HPV apparently introduces a long-term risk of squamous cell carcinoma development, even in the absence of conventional cofactors. Low-risk HPV infection may also act in association with these cofactors to promote carcinogenesis. Our data also show a racial imbalance in RP that may indicate a difference in genetic resistance and/or susceptibility to HPV infection and the development of RP. (Otolaryngol Head Neck Surg 1999;120:698-705.)

Immunohistologic and molecular genetic studies of the effect of glutathione-S-transferases on the development of squamous epithelial carcinomas in the area of the head-neck

While cigarette smoking and alcohol consumption are the major risk factors for the development of head and neck carcinomas, it is assumed that genetic factors contribute to risk. The aim of this study was to characterize the influence of the carcinogen metabolizing glutathione-S-transferases on susceptibility to head and neck carcinomas. Polymorphisms at GSTM1, M3, T1 and P1 gene loci were determined in 398 head and neck cancer patients and 216 controls using polymerase chain reaction and restriction enzyme digestion. The epithelial distribution of the GSTs was determined by immunohistochemical methods. The GSTM1 A/B genotype was less frequent in all tumor groups compared with controls. The GSTM3 B/B genotype was reduced only in the laryngeal cancer group whereas GSTP1 A/A showed significant differences between pharyngeal cancer patients and controls. Accordingly, GSTM3 was expressed only in the cilia of the laryngeal respiratory epithelium. In contrast, GSTP1 was distributed throughout all outer layers of the squamous cell epithelium. While GSTM1 seems to influence susceptibility to all head and neck cancers, GSTM3 and P1 reflected site-specific differences. Thus GSTM3 appears to be associated with altered risk only to laryngeal cancer whereas GSTP1 is likely to influence pharyngeal cancer risk.

Epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA) and Ki-67 antigen in laryngeal epithelial lesions.

The epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA) and Ki-67 immunostaining in paraffin sections from 154 cases of laryngeal squamous cell carcinoma, 25 specimens of normal and hyperplastic laryngeal epithelium, and 21 preneoplastic lesions was examined. The difference of EGFR expression and PCNA score between laryngeal cancer and dysplastic lesions vs normal and hyperplastic epithelium was significant. There was significant difference in Ki-67 score between malignant vs premalignant lesions and normal and hyperplastic epithelium. There was significant correlation between PCNA score and histopathological grading of the tumour. The highest PCNA expression was detected in stage G3. Our findings have shown that PCNA and Ki-67 staining can be used as a marker of cell proliferative activity in laryngeal epithelial lesions. EGFR, PCNA and Ki-67 expression correlate with severity of laryngeal lesions. PCNA immunostaining can aid in estimating the histological grade of malignant lesions.

Irregular expression of hyaluronan and its CD44 receptor is associated with metastatic phenotype in laryngeal squamous cell carcinoma.

The distributions of hyaluronan (HA) and its CD44 receptor were studied in 24 normal, 27 dysplastic samples of laryngeal epithelium and in 172 squamous cell carcinomas (LSCC), using a specific probe prepared from cartilage proteoglycan (bHABC, biotinylated hyaluronan binding complex) and a monoclonal antibody (Hermes 3). HA and CD44 were expressed similarly in all normal and about 90% of dysplastic and neoplastic laryngeal epithelia. In the normal epithelium HA and CD44 were homogeneously distributed throughout the epithelium, whereas the most superficial layers were negative. This was in contrast to the picture in dysplastic epithelium and well-differentiated invasive carcinomas, which were entirely HA and CD44 positive. Local areas with a low signal for HA and CD44 were present in 11% and 22% of the samples with dysplasia, and in 27% and 28% of those with carcinoma, respectively. The presence of this staining irregularity was associated with poor differentiation of the carcinoma, a significantly elevated mitotic index and a high frequency of nodal spreading and metastases. Furthermore, the irregular staining showed a trend towards poor disease-free survival, suggesting that an altered metabolism of HA is a common feature in LSCC and is associated with an aggressive growth pattern.

Ki-67 immunostaining and prognosis in laryngeal cancer.

The expression of the antigen defined by the Ki-67 antibody in paraffin sections from 154 biopsies of laryngeal squamous cell carcinomas was examined. There was a significant difference in Ki-67 expression between the control group and the patients with cancer (P < 0.001). There was no significant difference in the Ki-67 score between the patients with recurrence and the patients with a satisfactory outcome after treatment. There was no significant correlation between the Ki-67 score and the patient's age and sex, T and N stage and site of the tumour. The survival time of patients with a Ki-67 score > 30 was shorter than patients with a Ki-67 score < or = 30 but the difference did not reach statistical significance (P = 0.055). Multivariate analysis indicated that the only important prognostic factor was the existence of lymph node metastases. We could not confirm the value of Ki-67 as a prognostic factor in laryngeal cancer. Ki-67 score may assist in differentiating malignant from benign laryngeal epithelium.

Subchronic inhalation toxicity study of caprolactam (with a 4-week recovery) in the rat via whole-body exposures.

This study was designed to assess the potential subchronic inhalation toxicity of caprolactam when administered as a 3-micron aerosol from an aqueous solution to Sprague-Dawley CD rats (10/sex/group) via whole-body exposure. The study was enhanced with the inclusion of motor activity measurements and a functional observational battery to assess the neurotoxic potential of caprolactam. The rats were exposed at least 65 times over a 13-week period for 6 h per day, 5 days per week, to target concentrations (3 microns, mass median aerodynamic diameter) of 0, 25, 75, and 250 milligrams per cubic meter (mg/m3). An additional 10 animals/sex/group were similarly exposed and then held for a 4-week recovery period. Exposure levels were determined gravimetrically six times daily; one daily sample was analyzed by high-pressure liquid chromatography. No deaths were observed in the study during the exposure or recovery periods. Treatment-related responses such as labored breathing and nasal discharge were seen during many of the exposures. Similar responses as well as moist rales were seen during the nonexposure periods during the 13 weeks of exposure. However, these responses abated during the 4-week recovery period. There were no clearly treatment-related responses observed with ophthalmoscopic examinations, body weight measurements, food consumption measurements, neurobehavioral evaluations, clinical pathology evaluations, organ weight measurements, or macroscopic pathology examinations. Microscopic findings that were considered related to exposure to the test material were seen in the nasoturbinal tissues (hypertrophy/hyperplasia of goblet cells in the respiratory mucosa and intracytoplasmic eosinophilic material in epithelial cells of the olfactory mucosa) of the two higher-exposure group animals and in the laryngeal tissues (squamous/squamoid metaplasia/hyperplasia of the pseudostratified columnar epithelium covering the ventral seromucous gland) of all three exposure group animals. These changes were considered to be adaptive responses to an irritant (caprolactam). The keratinization of the metaplastic epithelium in the larynx was considered to be an adverse effect. By the end of the 4-week recovery period, there was complete regression of the keratinization in the larynx, but recovery of the adaptive nasoturbinal effects had not completely resolved. In conclusion, the whole-body exposure of Sprague-Dawley rats to caprolactam as a respirable aerosol for 6 h/day, 5 days/week, for 13 weeks at gravimetrically determined levels of 24, 70, and 243 mg/m3 resulted in respiratory tract effects (laryngeal) at the highest exposure level with complete recovery within 4 weeks postexposure. The results indicate that the no-observed-adverse-effect level for caprolactam is 70 mg/m3, based on upper respiratory effects, with 243 mg/m3 representing a no-observed-effect level for systemic toxicity, neurotoxicity, and lower respiratory tract effects.