Role of ancillary techniques in profiling unclassified laryngeal malignancies.

Laryngeal biopsies, contrary to biopsies from many other sites of the body, very often contain minute amounts of tumour tissue that may consist of morphologically undifferentiated tumour only. In haematoxylin- and eosin-stained sections, there may be no indicative features of what specific tumour entity that is present. In the larynx, particularly small round cell neoplasms, primary or metastatic, often cause a diagnostic dilemma and where an incorrect diagnosis can induce substantial clinical consequences for the patient (e.g., primary neuroendocrine carcinomas vs metastatic variants, certain sarcomas). If sufficient/representative material has been obtained, the application of immunohistochemistry and/or molecular techniques should in virtually every case reveal the true nature of the malignancy. In cases with sparse amount of material, and therefore a limited number of sections to be cut, a careful and thoughtful stepwise approach is necessary to ascertain a reliable diagnosis, or at least guide the clinician to the most likely diagnoses. With today's advanced and widely available technology with an abundance of markers to discriminate different tumours, the use of the term "undifferentiated" should be largely unnecessary. In the exceptional, and indeed exceedingly rare cases, when a classification is not possible, even after repeat biopsy, we suggest that the laryngeal neoplasm is better termed "unclassified malignant neoplasm" rather than "undifferentiated malignant neoplasm".

Salivary acinic cell carcinoma: reappraisal and update.

Epidemiologic and clinicopathologic features, therapeutic strategies, and prognosis for acinic cell carcinoma of the major and minor salivary glands are critically reviewed. We explore histopathologic, histochemical, electron microscopic and immunohistochemical aspects and discuss histologic grading, histogenesis, animal models, and genetic events. In the context of possible diagnostic difficulties, the relationship to mammary analog secretory carcinoma is probed and a classification is suggested. Areas of controversy or uncertainty, which may benefit from further investigations, are also highlighted.

Primary, combined, atypical carcinoid and squamous cell carcinoma of the larynx: a new variety of composite tumour.

OBJECTIVE: We report the first case of a laryngeal composite tumour consisting of a squamous cell carcinoma combined with an atypical carcinoid. METHODS: Case report and review of the literature concerning laryngeal composite tumours. RESULTS: Primary laryngeal carcinoma is the most common malignancy of the upper aerodigestive tract. The vast majority are of the squamous cell type. Primary neuroendocrine neoplasms represent a rare, heterogeneous subset of laryngeal malignancies, comprising typical carcinoid, atypical carcinoid, small cell carcinoma and paraganglioma. Primary combined neuroendocrine and squamous cell carcinoma of the larynx is even more rarely encountered, with only 14 publications of this so-called composite tumour to date. In each case, the neuroendocrine component has been small cell carcinoma. CONCLUSION: The treatment of primary neoplasms comprising more than one histological type is tailored to the most biologically aggressive tumour. Accurate diagnosis of the histological nature of laryngeal composite tumours is imperative to ensure optimal therapy.

Eosinophilic colitis.

Eosinophilic colitis is a rare chronic inflammatory bowel condition of unknown etiology. We report a case of cecal volvulus causing obstruction in a patient with eosinophilic colitis. A 48-year-old lady presented with abdominal pain, constipation, and abdominal distension. Clinically and radiologically, she was diagnosed to have cecal volvulus. Preoperative colonoscopic reduction failed. At laparotomy, a right hemicolectomy with primary anastomosis was undertaken. Histology of the resected specimen showed diffuse eosinophilic infiltration suggesting eosinophilic colitis. To the best of our knowledge, this association has been never reported.

Intranasal myopericytoma. A tumour with perivascular myoid differentiation: the changing nomenclature for haemangiopericytoma.

We present a case report of a patient who developed a sinonasal myopericytoma treated by surgical excision through a lateral rhinotomy. Some aggressive features on pre-operative computed tomography scanning and the complexity of recent changes in the histological nomenclature for these tumours led to consideration of adjuvant therapy. The close histological relationship between myopericytoma, myofibromatosis, solitary myofibroma and infantile haemangiopericytoma is discussed. This group of lesions constitute a single morphological spectrum with differentiation towards perivascular myoid cells (pericytes). Currently myopericytoma is the most appropriate and accepted term embracing all these entities. A review of the literature has been reassuring in identifying these tumours as benign but with a reasonably high rate of local recurrence (17 per cent). The treatment of choice is surgical excision with further excisions for local recurrence.

The Ljubljana classification: a practical strategy for the diagnosis of laryngeal precancerous lesions.

There is no internationally accepted classification of epithelial hyperplastic laryngeal lesions (EHLL). The majority of current classifications follow criteria similar to those commonly used for cervical epithelial lesions. However, the different etiology of laryngeal cancer and its particular clinical and histologic features necessitate a grading system more appropriate to this region. The Ljubljana classification of EHLL was devised in 1971 to cater to this requirement. Detailed criteria for histologic grading in this classification were formulated by a working group on EHLL of the European Society of Pathology in 1999. The system recognizes four grades: simple and abnormal hyperplasia are benign categories; atypical hyperplasia ("risky" epithelium) is potentially malignant, and carcinoma in situ actually malignant. The main features by which the proposed grading system differs from other classifications are: 1. the distinction between benign and potentially malignant lesions; 2. the positive separation of carcinoma in situ from atypical hyperplasia; 3. the lack of prognostic significance for any surface keratin layer. The eventual outcome of EHLL patients so graded justifies the proposal for separating the lesions into a benign group, showing malignant transformation in only 0.9% of cases, from a potentially malignant group showing malignant transformation in 11% of cases. For diagnostically difficult cases, supplementary techniques such as those using morphometry, immunohistochemical and molecular biology are advised to improve the accuracy of diagnosis and predictions of their biological behavior.

Apoptosis and phagocytosis of tissue-dwelling eosinophils in sinonasal polyps.

OBJECTIVE: Sinonasal polyps contain numerous tissue-dwelling eosinophils, but the mechanisms causing their accumulation, functional activities, and resolution are largely unknown. STUDY DESIGN: Nasal polyp tissue from 14 patients was evaluated for cellular expression of CD95, CD68, and annexin-V, for the degree of apoptosis, and for phagocytosis of eosinophils. MATERIAL AND METHODS: Histological sections were immunostained as single stains for CD95, CD68, and annexin-V, and as an immunostaining for CD68 combined with a modified Vital New Red staining. The latter staining is specific for eosinophils. Other sections were stained by terminal d-UTP nick end labeling (TUNEL) assay and routinely stained for H&E. Evaluation of the amount of stained cells was performed by counting the average number in 10 randomly chosen high-power fields. The TUNEL positivity was in all cases confirmed with apoptotic morphology. RESULTS: The inflammatory infiltrate consisted of numerous eosinophils but also a considerable amount of lymphocytes, mast cells, and macrophage-like CD68+ cells. CD95 was frequently expressed on eosinophils, on numerous other inflammatory cells, and also on morphologically apoptotic cells. annexin-V-positive eosinophils were not as frequent as CD95+ cells, but numerous annexin-V-positive eosinophils were found. CD68+ cells approximately equalled the number of eosinophils. The number of cells phagocytosing eosinophils varied between polyps. Apoptosis of eosinophils (as evaluated by TUNEL combined with apoptotic morphology) was a common finding in six of the polyps. CONCLUSIONS: Previous in vitro and ex vivo findings of CD95 on eosinophils are now supported by demonstration of CD95 on eosinophils in this in vivo study. This investigation revealed a switch of the membrane-bound phosphatidylserine of apoptotic cells, which is a novel observation. The study has demonstrated apoptosis of tissue-dwelling eosinophils, and that CD68+ macrophage-like cells phagocytose eosinophils within the sinonasal polyps.

Criteria for grading in the Ljubljana classification of epithelial hyperplastic laryngeal lesions. A study by members of the Working Group on Epithelial Hyperplastic Laryngeal Lesions of the European Society of Pathology.

AIMS: To validate histological criteria for the grading of epithelial hyperplastic laryngeal lesions (EHHL) (dysplastic laryngeal lesions), we used a system that had been devised and tested in Ljubljana, Slovenia over many years and was felt to be more appropriate to laryngeal pathology than is the commonly-used model of intraepithelial neoplasia in the cervix. METHODS AND RESULTS: Vocal cord biopsies of 45 patients with a broad spectrum of EHLL were reviewed. Detailed histological criteria were formulated for each of the four grades of EHLL in the Ljubljana classification, comprising simple hyperplasia (benign spinous layer augmentation), abnormal hyperplasia (benign basal and parabasal layer augmentation), atypical hyperplasia (risky for malignancy) and carcinoma in situ (actually malignant, but without invasion). CONCLUSIONS: Using these criteria a high degree of concordance of histological diagnoses of grading levels for the Ljubljana classification was achieved between the pathologists of the Working Group. The system was found to be more precise for routine diagnostic work than the others in vogue. The different grades of the Ljubljana classification correspond to significantly different levels yielded in each grade by the semiobjective methods of quantitative morphometry and immunohistochemistry.

Ventilator-associated sinusitis: a review.

A common foreign body of the nose in intensive care, the nasotracheal tube, has for 20 years been cited as a cause of bacterial infection of the paranasal sinus. High frequencies of bacterial culture positivity have occurred in several studies. However, the state of critically ill patients has to be evaluated before conclusions about cause of infection can be made. Nosocomial colonization with intensive care unit flora, in combination with use of antibiotics, precludes the use of procedures that are standard in office practice and microbiological diagnostics. New methods of sampling and quantitative culturing for the specific purpose of intensive care antral diagnostics, in combination with endoscopic inspection, have enlarged our knowledge of sinusitis. Among patients ventilator-treated for > or = 1 week, the occurrence of bacterial sinusitis is < 10%. For 80% of the examined antra there were similar inflammatory reactions without clinical signs of infection. Sporadically in these, cultures of antral specimens were positive for bacteria, which, by definition, would represent colonization.

RANTES is more prevalent in bacterial than in nonbacterial maxillary sinusitis: and P-selectin is preferentially up-regulated in diseased mucosae.

OBJECTIVE: To investigate the relationship of the clinical appearance, histological characteristics, bacterial culturing, and messenger RNA (mRNA) expression of RANTES, interleukin 6, and interleukin 12, as well as the occurrence of endothelial adhesion molecules, in inflammatory diseased maxillary sinus mucosa in critically ill patients. DESIGN: Prospective case series. SETTING: General intensive care unit and neurosurgical intensive care unit of a tertiary care hospital. SUBJECTS: Seven critically ill patients, nasotracheally intubated or tracheotomized, who received ventilator treatment for more than 7 days and treatment with antibiotics. INTERVENTIONS: Bilateral biopsy specimens of antral mucosa were obtained at sinoscopy. Reverse transcriptase-polymerase chain reaction was used to detect the cytokine mRNAs in situ on paraformaldehyde-fixed tissue, and intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and P-selectin were analyzed by immunochemistry on frozen sections. Sampling of secretion and tissue from the antra was performed for bacterial culturing. RESULTS: Macroscopic and histological appearance varied and showed moderate to pronounced inflammation in 6 antra. All 4 bacterially infected antra showed mRNA RANTES (P=.005). No correlation was found for interleukin 6 and interleukin 12. Up-regulation of P-selectin in all cases and sparse expression of vascular cell adhesion molecule 1 indicate that the inflammation is chronic but nonallergic in type. CONCLUSION: We find an indication that RANTES is more prevalent in bacterial sinusitis.

Tumour growth fraction and apoptosis in salivary gland acinic cell carcinomas. Prognostic implications of Ki-67 and bcl-2 expression and of in situ end labelling (TUNEL).

bcl-2 protein and Ki-67 (MIB-1) were studied in 32 acinic cell carcinomas (ACCs), all with a minimum of 5 years' clinical follow-up. Tumour apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and by morphological criteria. Five patients died of their disease. Patients with stage I tumours had significantly better survival compared with other stages (P < 0.05). Patients with MIB-1-negative tumours had significantly better survival than patients with MIB-1-positive tumours (P = 0.05). This study confirms a previous report that MIB-1 is an independent prognostic factor for survival in patients with ACC. Stage I tumours had high expression of bcl-2 protein, but there was no difference when compared with other stages. TUNEL positivity was most prevalent in stage I tumours, compared with stages II, III, and IV (P < 0.05), probably indicating more apoptosis. This could imply a capacity of stage I tumours ('early tumours') for early selection of tumour cells for elimination by apoptosis. There was no significant difference between expression of bcl-2 and TUNEL, between these parameters and clinical outcome, or between any parameter and morphological subclassification. We conclude that MIB-1 has prognostic value in ACC. Clinical staging, bcl-2, and TUNEL are also potentially useful as prognostic markers.

Basaloid squamous cell carcinoma of the maxilla.

The study reports the first case of basaloid squamous cell carcinoma (BSCC) involving both the oral mucosa and the tuberosity area of the maxilla. The tumour showed many histological similarities to cases previously reported, though mitoses were not frequent. The immunoreactivity for cytokeratin, S-100, vimentin, Ki-67, p53, c-erbB-2 and bcl-2 was also investigated. Immunostaining for the bcl-2 protein showed a high extent of positive cells, although only a moderate staining intensity. Staining for c-erbB-2 was negative. The pathological findings and the immunoreactivity may indicate that BSCC is not as high a grade carcinoma as previously suggested. Additional studies are thus clearly needed to confirm or reject this impression.

Apoptosis in epithelial hyperplastic laryngeal lesions.

The Fas receptor appears to be commonly expressed in all morphological types of epithelial laryngeal hyperplasia (HP). Fas-mediated apoptotic cell death would thus be a possible phenomenon in these lesions. We observed more anti-apoptotic bcl-2 protein in epithelia with simple HP compared to the more advanced types of HP. It is suggested that in simple HP there is not yet a need for an early selection for cell death. The observed overexpression of metallothionein (MT) in the basal layers of simple HP would also support such a theory. These basal cells are dividing, non-apoptotic cells, which have not yet been selected for death. All 20 cysteine residues in MT are involved in metal binding, interfering with the intracellular redox balance, and thereby possibly inhibiting certain apoptotic signals. MIB-1 positivity was found only in the atypical HP, CIS, and invasive carcinomas. Intuition suggests that high labelling would be associated with poor prognosis. The degree of apoptosis, evaluated by TUNEL, did not show any differences between different types of epithelia. Although TUNEL is sensitive and rather specific, we emphasise that all TUNEL positive cells have apoptotic type morphology, confirming good and appropriate use of the technique.

Prognostic significance of DNA ploidy in mucoepidermoid carcinoma.

Thirty-four mucoepidermoid carcinomas were studied retrospectively with regard to histological and clinical parameters. In 28 of the tumors DNA patterns were also assessed using flow cytometry. Twenty-two of the 28 tumors (79%) were DNA diploid and 6 (21%) DNA aneuploid. Two tumors (7%) showed intratumoral DNA as indicated by different stemlines in specimens investigated from different parts of the tumor. DNA ploidy correlated significantly with cervical lymph node status (P < 0.01), but not with tumor size or histological grade. The mean S-phase value was 2.7% and was significantly higher in aneuploid samples than in diploid ones (P < 0.05). The recurrence rate was significantly lower for patients with stage I and II tumor compared with those with stage III and IV disease (P < 0.01). Five aneuploid tumors showed significantly higher recurrence rates (5/6) than the diploid ones (1/22) (P < 0.01). In univariate analysis for survival, only N stage tumor (P < 0.05) and tumor DNA ploidy (P < 0.0003) had significant prognostic influence. Thus, DNA ploidy seems to be a valuable parameter for evaluating the biological behavior of mucoepidermoid carcinomas of the salivary glands.

Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts.

Acridine orange (AO) is a lysosomotropic weak base, a metachromatic fluorochrome, and a photosensitizer, as well. Living cells that are exposed for a short period of time to this compound at low concentration, and under ordinary culture conditions, accumulate the drug within their acidic vacuolar compartment, giving rise to a mainly red, granular fluoresence upon excitation with blue light. When AO-loaded cells are irradiated with intense blue light, AO soon starts to leak from late endosomes and lysosomes, partially shifting the fluorescence to a green, nuclear and diffuse cytosolic, one. This AO-relocalization is a consequence of photo-oxidation of the lysosomal membranes, which initially results in disruption of their proton-gradients and later, in leakage into the cytosol of a host of hydrolytic enzymes--as was here demonstrated by immunocytochemistry--which are capable of causing cellular damage. Most fibroblasts survived minor photo-oxidation, with a period of reparative autophagocytosis. Severe photo-oxidation, which resulted in severe lysosomal damage, caused cellular necrosis; whereas moderate stress, resulting in only partial lysosomal leakiness lead to apoptosis with TUNEL-positive nuclei and shrunken cytoplasm. The findings of the present study show that photo-oxidative damage to the membranes that surround the acidic vacuolar compartment, is an event that results in release of proteolytic and DNA-fragmenting enzymes into the cytosol, which may induce either necrosis, apoptosis, or reparable sublethal damage, depending on the magnitude of lysosomal rupture. Furthermore, the results strongly suggest that proteases and endonucleases of lysosomal origin may induce apoptosis if relocalized from the acidic vacuolar compartment into the cytosol.

Fas receptor is expressed in human lung squamous cell carcinomas, whereas bcl-2 and apoptosis are not pronounced: a preliminary report.

We report a pilot study on the Fas receptor (APO-1, CD95) in vivo in 15 human squamous cell (non-small) carcinomas and ten normal bronchial specimens. The principal aim was to investigate whether the so-called death receptor, Fas, is present in these tumours. Ligation of Fas promptly induces apoptosis, particularly in T Jurkat cells in vitro, and expression of Fas on human cancer would thus theoretically be of great interest. The immunoreactivity for the anti-apoptotic protein Bcl-2 was also investigated, and the degree of apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and conventional morphological criteria. Fas was present in all initial tumours but absent in control tissue, that is in the potential precursor cells of bronchial epithelium (P = 0.001). Fas was not detectable after radiotherapy (P = 0.03). We propose that radiotherapy induces an early selection of tumour cells rather than a down-regulation of Fas. Both Bcl-2 and apoptosis (TUNEL) were generally expressed at a modest level. In agreement with other studies, we did not find any significant correlation between Bcl-2 and prognosis, or between Bcl-2 and TUNEL. Hence, in this preliminary report, we have demonstrated Fas receptor in human squamous cell carcinomas in vivo. This is a novel finding, and the apparent absence of Fas after radiotherapy may have important therapeutic implications.

Metallothionein and Fas (CD95) are expressed in squamous cell carcinoma of the tongue.

Metallothionein (MT) is a chelator present in myoepithelial cells, whilst the Fas-receptor (APO-1, CD95) has been described primarily in human T Jurkat cells. 20 cases of carcinoma of the tongue were investigated immunocytochemically with regard to MT, Fas and Bcl-2. In normal oral squamous epithelium, MT is located in the basal/parabasal dividing cells only. In well-differentiated nests of carcinomas, MT is observed almost entirely in peripherally located cells. In situ end-labelling indicates apoptosis in the centre of these nests, but not in the peripheral areas. Less-differentiated areas show more general MT-positivity, but little apoptosis. All 24 tumours are Fas-positive, but normal epithelia are mainly negative (P < 0.0001). Bcl-2 protein was sparse in the tumours compared with MT and Fas (P < 0.0001). We thus suggest that MT, possibly due to its chelating properties, may contribute to delaying cells entering apoptosis, both in normal epithelium near the base and in less-differentiated regions of carcinoma. Moreover, Fas may be present in cells of human malignancies, as well as those of established malignant cell lines.

Oxidant-induced apoptosis: a consequence of lethal lysosomal leak?

When macrophage-like J-774 cells are subjected to limited oxidative stress, such as exposure to hydrogen peroxide in a moderate bolus dose, some of their lysosomes rupture-as here assayed by the acridine orange relocalization test-secondary to intralysosomal, iron-catalysed, oxidative reactions. The resultant leakage into the cytosol of hydrolytic enzymes, such as cathepsin-D (as shown here), may initiate a slow degradation/fragmentation process of an apoptotic type within cells still having intact plasma membranes. In contrast, severe oxidative stress also results in extensive lysosomal rupture but leads to necrosis. The chelation of (normally occurring) intralysosomal low-molecular weight iron, by endocytotic uptake of desferrioxamine, largely prevents oxidative stress-induced apoptosis whereas lysosomal iron-loading, by endocytotic uptake of complexed ferric iron, considerably enhances the process. We conclude that oxidant-mediated and iron-catalysed lysosomal rupture leads to decompartmentalization of lysosomal enzymes which in turn may initiate and promote the apoptotic process.