Assessment of preoperative ultrasonography of the neck and elective neck dissection in patients with oral squamous cell carcinoma.

Estimating the value of our preoperative workup in the treatment of patients with clinically N0 (cN0) squamous cell carcinoma of the oral cavity. Retrospective analysis. Results of preoperative palpation, ultrasound (US) and ultrasound-guided fine needle aspiration cytology (FNAC) were compared to the histological findings after unilateral or bilateral elective selective neck dissection of level I-III (SND I-III) in patients with cN0 squamous cell carcinoma of the oral cavity. Occult metastases were detected by in 50 (22%) out of the 224 cN0 patients. No metastases were found beyond level III in extended neck dissections. T1N0M0 tumors and T2N0M0 tumors metastasized in 8 out of 77 cases (10%) and 32 out of 112 (29%) cases, respectively. Staging of the cN0 neck by palpation and US (+/-ultrasound-guided FNAC) missed occult lymph node metastases in 22% of the patients with oral cavity squamous cell carcinoma. The use of SND I-III therefore still is warranted. Frozen section sampling seemed to be redundant in this selected group of patients, because no additional metastases were found in extended neck dissection specimens. It might not be necessary to perform elective neck dissection in patients with T1 tumors.

The prediction of mandibular invasion by squamous cell carcinomas with the expression of osteoclast-related cytokines in biopsy specimens.

Destruction of bone by tumour is caused by osteoclasts rather than by tumour cells directly. Tumour cells of invasive oral squamous cell carcinomas (SCC) release osteoclast-related cytokines and cytokines activate osteoclasts. The purpose of this study was to investigate the possibility of predicting mandibular invasion by SCC by analysis of the expression of osteoclast-related cytokines in biopsy specimens of SCC, adjacent or fixed to the mandible. Thirty-five biopsy specimens from the pathology archives were examined from patients who had been treated for SCC, adjacent or fixed to the mandible. The patients were divided into those with and without medullary invasion. The expression of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-11 was studied by immunohistochemical analysis. No significant differences were found in expression of TNF-alpha, IL-6 and IL-11 between biopsy specimens with or without medullary invasion. Quantification of the density of tumour-infiltrating lymphocytes was not reproducible. In conclusion, the expression of TNF-alpha, IL-6 and IL-11 in biopsy specimens of SCC, adjacent or fixed to the mandible, is not an appropriate method for predicting the presence of medullary invasion of the mandible.

No high-risk HPV detected in SCC of the oral tongue in the absolute absence of tobacco and alcohol--a case study of seven patients.

INTRODUCTION: In recent years, there has been an increase in the number of patients with squamous cell carcinoma (SCC) of the mobile tongue in the absence of tobacco and alcohol. The literature suggests that human papilloma virus (HPV) may be involved in the development of SCC of the head and neck, especially in non-smoking and non-drinking patients. In the oral cavity, however, the presence of the HPV genome has been reported with various percentages. This may be due to misclassification of the oropharyngeal tongue as oral cavity subsite and the use of various detection methods. MATERIALS AND METHODS: Therefore, we evaluated the role of HPV in seven consecutive non-smoking and non-drinking patients (2003-2006) with a SCC located at the oral, mobile tongue using in situ hybridization and SPF(10)Line Blot 25 polymerase chain reaction assays. RESULTS: No HPV was detected in these specimens. To further determine whether a relationship exists between HPV and SCC in the absence of tobacco and alcohol, subsequent studies at specific locations are necessary.

Assessment of mandibular invasion of squamous cell carcinoma by various modes of imaging: constructing a diagnostic algorithm.

The aim of this study was to determine the optimal combination of preoperative examination methods to predict mandibular invasion by squamous cell carcinoma of the oral cavity. Data were gathered prospectively but evaluated retrospectively. The preoperative results of clinical examination, conventional radiography, bone single photon emission computed tomography (SPECT), computed tomography and magnetic resonance imaging were compared to the histological results of resection specimens from 67 patients with tumours, adjacent or fixed to the mandible, histologically confirmed as squamous cell carcinoma. The examination methods with acceptable sensitivity and specificity were selected and diagnostic algorithms were constructed using all possible combinations. The preferred diagnostic algorithm was found to be either computed tomography or magnetic resonance imaging, followed by a bone SPECT in cases where the first scan is negative. A negative bone SPECT rules out mandibular invasion (100% sensitivity). This algorithm accurately predicted mandibular invasion in 85% of the patients, without yielding false negative results. In this study group, application of such an algorithm would have resulted in a reduction of the number of unnecessary mandibular resections by 50%. The suggested, preferred, diagnostic algorithm shortens the preoperative screening process, avoiding unnecessary costs, as well as considerably reducing the number of unnecessary mandibular resections.

Biological behavior of CIN lesions is predictable by multiple parameter logistic regression models.

OBJECTIVES: Progression and regression of premalignant cervical lesions cannot be predicted using conventional cytomorphological or histomorphological parameters. However, markers such as human papillomavirus (HPV) or makers indicating proliferation, genetic instability and chromosomal aberration may be of predictive value assessing short-term biological behavior of cervical intraepithelial neoplasia. In this paper, we have studied the usage of logistic regression models with Ki-67 labeling index (LI), chromosome index for chromosome 1 (CI#1) and aneusomy for chromosome 1 in cervical smears to predict progressive and regressive behavior of premalignant cervical lesions. METHODS: Retrospectively, the intake smears of 42 women showing regression in follow-up and of 31 women showing progression in follow-up were assessed. RESULTS: A multiparameter logistic regression model containing the parameters Ki-67 LI, CI#1 and the fraction of cells with four copies of chromosome 1 per nucleus appeared to be the best predicting model, overall correct classification of 93.2% (area under the receiver operating characteristic curve 0.96 +/- 0.02). After cross-validation, the model correctly classified 66 of 73 samples (90.4%). Moreover, the model predicted biological behavior perfectly assessing the smear taken subsequently to the intake smear of 46 women. CONCLUSION: Although measuring parameters indicating proliferation and chromosome 1 aberration is laborious, this study demonstrates that short-term progressive and regressive behavior is highly predictable using a model combing these parameters. We also showed that in the triage management of high-risk human papillomavirus-positive women with minimally abnormal smears applying a model as such can be useful.

A case of imported paracoccidioidomycosis: an awkward infection in The Netherlands.

Paracoccidioidomycosis is an important endemic mycosis in South America. In Europe the disease is very rare and only found as infections in travelers to Latin America. We report here the first case encountered in the Netherlands for which the appropriate diagnosis was not attained for several months. A Dutch 60-year-old man presented with a painful ulceration in the buccal mandibular vestibular mucosa of three months duration. While his medical history was uneventful, he had worked, until 8 years prior to his presentation, as a carpenter for 25 years in the jungles of Peru and Ecuador. An aberrant chest radiograph, CT-scan of the lungs and increased erythrocyte sedimentation rate were suggestive of sarcoidosis or a bronchiolitis obliterans organizing pneumonia. There was no improvement in the patient's symptoms despite the use of budesonide and prednisone medication, as well as tuberculosis prophylaxis with isoniazide and rifampicin, and local use of miconazole. Quite to the contrary, as an irritated, irregular hyperemic mucosa and gingiva with ulceration were noticed during this period of time. These precipitated an incisional biopsy through which a mixed inflammatory cellular infiltrate and large yeast cells were found on histopathologic examination. Based on the patient's travel history and the multiple budding yeastlike cells revealed in the biopsy tissue, the diagnosis of paracoccidioidomycosis was finally made. This was supported by the isolation of Paracoccidioides brasiliensis in culture. Antimycotic oral therapy with itraconazole was started and continued for 15 months. At two and five year follow-ups, the patient was asymptomatic. In Europe, it may be expected that diseases that are endemic in other areas will be seen more frequently in countries where the diseases are not routinely encountered. It is most likely that the use of corticosteroid medication, with its inherent immunosuppressive effect, resulted in the reactivation of an infection acquired many years before in Latin America. The etiologic agent then disseminated from the initial focal point to cause the ensuing oral mucous membrane lesions. The importance of the patient's prolonged residence in Latin America was overlooked. The very long latency of endemic mycoses emphasizes the need for a meticulous history which should include not only recent trips, but also past residence in foreign countries.

Vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways.

Two separate pathways leading to vulvar carcinoma have been suggested. First, a human papillomavirus (HPV)-dependent pathway, in which premalignant stages of vulvar cancer are the classic vulvar intraepithelial neoplasia (VIN) lesions. Second, an HPV-independent pathway, associated with differentiated VIN III lesions and/or lichen sclerosus. To obtain insight into the mechanisms underlying these pathways, we determined the relationship between HPV DNA and the expression of p14(ARF) and p16(INK4A) in non- and (pre)malignant vulvar lesions. Seventy-three archival samples of non- and (pre)neoplastic vulvar lesions were selected and tested for hr-HPV DNA using a broad-spectrum HPV detection/genotyping assay (SPF(10)-LiPA) and the expression of p14(ARF) and p16(INK4A). The prevalence of HPV increased with the severity of the classic VIN lesions; in VIN I no hr-HPV was detected, in VIN II 43%, and in VIN III 71% of the samples were hr-HPV-positive. Roughly the same was true for the expression of p14(ARF) and p16(INK4A). The simultaneous expression of p14(ARF) and p16(INK4A) was highly associated with the presence of hr-HPV DNA. Hr-HPV was detected in only a single case of the differentiated VIN III lesions, whereas no expression of p14(ARF) was found and 16(INK4A) was present in only two cases. All 16 samples of vulvar cancer were hr-HPV DNA- negative, although in respectively 63% and 25%, p14(ARF) and p16(INK4A) was expressed. No relation was found between hr-HPV and the expression of p14(ARF) and p16(INK4A) in the 20 nonneoplastic vulvar lesions. Our results provide further evidence that vulvar squamous cell carcinoma is a multifactorial disease that develops from two different pathways. First, an HPV-dependent pathway with a remarkable resemblance to CIN lesions and cervical carcinoma and second, an HPV-independent pathway in which differentiated VIN III lesions that are hr-HPV-negative may be precursors.

Down regulation of estrogen receptor expression is an early event in human papillomavirus infected cervical dysplasia.

PURPOSE: To study the alterations in hormonal sensitivity in relation to proliferative activity during the development of cervical dysplasia in women infected with high-risk human papillomavirus (hr-HPV). METHODS: Three to five biopsies of the cervix of eight patients were taken at colposcopy. Dysplasia was detected in 22 of the 32 biopsies, and 20 of these 22 biopsies contained hr-HPV. The labeling index (LI) as well as the intensity of staining of the MIB-1-, estrogen receptor (ER)-, and progesterone receptor (PR)-expression was assessed in each biopsy, including normal epithelium directly adjacent to the dysplastic lesions. RESULTS: Statistical analysis showed a significant increase in the MIB-1 LI with increasing severity of the dysplasia. The ER LI and ER intensity of staining in dysplastic lesions, as well as in morphologically normal epithelium directly adjacent to the dysplasia, showed a significant inverse relation with the severity of the dysplasia. The PR LI and intensity of staining did not differ between normal epithelium and dysplasia. The ER/MIB-1 ratio (including the ER LI and ER intensity of staining), and the PR/MIB-1 ratio (intensity of staining only) in dysplastic lesions showed a significant inverse relation with the severity of the dysplasia, while no alterations in these ratios were observed in morphologically normal epithelium adjacent to the dysplasia. CONCLUSION: Down regulation of ER expression may be the first alteration to take place in normal epithelium during the development of cervical dysplasia in women infected with hr-HPV. The significant decrease in the ER/MIB-1-, and PR/MIB-1-ratio in progressively dysplastic lesions indicates a loss of normal growth control by sex steroid hormones, which is not observed in normal epithelium.

Thymic cyst in the neck.

A rare case of a thymic cyst in the neck containing both thymus and parathyroid tissue in a 7-year-old boy is presented. The clinical presentation, diagnostic evaluation, surgical management and histopathological features are described. The embryology of cervical thymic cysts and the differential diagnosis of cystic neck masses in children are briefly reviewed. The diagnosis is seldom made preoperatively. Surgical resection is the treatment of choice for definitive diagnosis, resolution of symptoms and cure.

[Premalignant lesions of the oral mucosa. Prognosis, treatment and follow-up]. / Premaligne slijmvliesafwijkingen in de mondholte. Prognose, behandeling en vervolg.

To consider the value of prognostic factors in the development of a squamous cell carcinoma from a leukoplakia of the oral mucosa, a retrospective study was performed. Clinical and histological data of 104 patients with oral leukoplakia were analyzed. Leukoplastic lesions with dysplasia in the initial biopsy (n = 38) had been treated by excision (n = 28), by laser evaporation (n = 6) or a combination of these treatments (n = 4). Non-dysplastic lesions (n = 66) had been excised (n = 48), evaporised (n = 17) or treated by excision as well as laser evaporation (n = 1). During follow-up of maximal 6 years (mean 3.6 years), 12 patients had developed an infiltrative squamous cell carcinoma at the site of the primary lesion, 2 within a period of 24 months. No relation could be found between on the one hand size (p > 0.2), clinical aspect (p > 0.2), location (p > 0.45), and primary treatment (p > 0.15) of the lesion, and on the other hand the risk of developing a squamous cell carcinoma. Only a relation could be found between (the intensity of) dysplasia and the development of a squamous cell carcinoma (p < 0.001). It was concluded that because of the high risk of developing a squamous cell carcinoma, patients with a dysplastic mucosal oral lesion should be followed during a prolonged time.

Juxtacortical chondrosarcoma of the mandible: report of a unique case and review of the literature.

The clinical, radiographic, and pathological features of a juxtacortical chondrosarcoma of the mandible in a 25-year-old Caucasian woman are presented and the differential diagnostic aspects are discussed. To the best of our knowledge, this rare tumor has not previously been reported in the maxillofacial skeleton. Ten years after surgery there is no evidence of local recurrence or metastatic disease. As the prognosis of juxtacortical chondrosarcoma of the long bones seems to be relatively favorable compared with the usual central chondrosarcoma of similar grade of malignancy, this might also be expected for a similar tumor presentation in the maxillofacial skeleton.

Aggressive psammomatoid ossifying fibroma of the inferior turbinate and lateral nasal wall.

OBJECTIVE: Fibroosseous lesions are rare entities of the nose and paranasal sinuses, the terminology and classification of which are still confusing. Psammomatoid or aggressive (juvenile) ossifying fibroma is a benign messenchymal tumor usually met in the young age (5-15 years), in the sinonasal tract, bearing distinctive histomorphologic features and a tendency towards locally aggressive behaviour. METHODOLOGY: We report here a rare case of an aggressive psammomatoid ossifying fibroma of the inferior turbinate and the lateral nasal wall, with obstruction of the nasolacrimal duct, in a 68-year-old woman. RESULTS: Diagnosis was based on physical examination, CT scan imaging and histopathological examination. Treatment consisted of endoscopic intranasal resection of the tumor accompanied by removal of the lateral nasal wall. CONCLUSIONS: Fibro-osseous tumors of the nose and paranasal sinuses require aggressive surgical approach in order to avoid recurrence. Complete surgical excision may not always be possible mainly due to the tumor's extent and location.

A half-yearly chest radiograph for early detection of lung cancer following oral cancer.

In a retrospective analysis of 339 patients, treated with a curative intention for a squamous cell carcinoma of the oral cavity between 1988-1996, the value of a half-yearly routine chest radiograph during the first 2 years of follow-up, was evaluated. Metastatic or primary lung malignancies developed in 18 (5.3%) of the 339 patients in the first 2 years of the follow-up. In 11 (3.2%) patients these malignancies were detected on routine chest radiographs while in 7 (2.0%) patients these were detected on chest radiographs taken because of pulmonary complaints. The mean survival after detection of the pulmonary malignancy was 8 months. All patients died within 21 months, independent of pathology or therapy. No correlation was found between stage, location or differentiation of the primary tumour. As a result of this study it can be concluded that there is no benefit for the patient for a 6-month thoracic radiograph in routine follow-up for patients with oral squamous cell carcinoma. It gives false certainty and burdens the health care system.

The synergistic value of focus score and IgA% score of sublabial salivary gland biopsy for the accuracy of the diagnosis of Sjögren's syndrome: a 10-year comparison.

OBJECTIVE: Increasing the accuracy of the diagnosis of Sjögren's syndrome (SS) by placing emphasis on objective findings such as the presence of anti-Ro and anti-La autoantibodies and abnormal salivary gland tissue (SGT) histology is a current issue. In order to obtain optimal disease sensitivity and specificity of SGT findings, histological and immunohistological SGT examinations were compared. The first describes the extent of the lymphocytic infiltrate as a focus score (LFS), whereas the latter describes the composition of the infiltrate as a percentage of IgA-containing plasma cells (IgA%). METHODS: Both the LFS and IgA% score were assessed in 279 SGT biopsies taken from patients with symptoms suggestive of SS. In case histological conclusions did not match immunohistological conclusions patients were assigned to so-called mismatch groups. Patients in the mismatch groups were further classified using objective, serological parameters [rheumatoid factor (RF), anti-Ro, anti-La, anti-nuclear antibodies, gammaglobulin level]. RESULTS: In 249 samples (89%), LFS and IgA% resulted in the same conclusion. Within this group a total of 63 SGT samples (25%) were characteristic for SS showing LFS >1.0 and IgA% <70. In the mismatch groups after serological classification, both false positive as well as false negative scores were observed less frequently for IgA% as compared with LFS (50 vs 75% and 25 vs 50%, respectively). CONCLUSIONS: Additional immunohistological SGT examination provides greater disease sensitivity and specificity than histological SGT examination alone, thereby increasing accuracy of SS diagnosis.

The effect of Replens on vaginal cytology in the treatment of postmenopausal atrophy: cytomorphology versus computerised cytometry.

BACKGROUND: After the menopause decreased concentrations of oestrogen may result in insufficient maturation of the vaginal epithelium, which can lead to a range of vaginal discomforts. This state of vaginal atrophy may be treated with oestrogen replacement treatment. Replens, a non-hormonal alternative to oestrogen replacement treatment has been shown to be effective in relieving symptoms related to vaginal atrophy in previous studies. AIMS: To study the effect of Replens on the maturation of the vaginal epithelium and morphology of the vaginal cells and to compare the results of a recently developed cytomorphometric method with manual assessment of the degree of maturation in vaginal smears. METHODS: Vaginal smears from 38 postmenopausal women suffering from symptoms related to vaginal atrophy were analysed manually and by cytomorphometry. The maturation value (MV) and the percentages of (para)basal, intermediate, and superficial cells (maturation index; MI) were measured by both methods before and after treatment with Replens. Cytomorphometry also measured mean cellular area, mean nuclear area, and mean area ratio. RESULTS: A correlation was shown between the two methods in the assessment of percentages of (para) basal and intermediate cells and MV. Cytomorphometric data showed a significant increase in mean cellular area, indicating a positive effect of Replens on the maturation of the vaginal epithelium. Changes in nuclear area and ratio between nuclear and cellular areas were not significant. Treatment with Replens did not influence MI or MV, as assessed by the two methods. CONCLUSIONS: Replens did have an effect on vaginal morphology. The automated procedure may be useful for the assessment of maturation in vaginal smears and is more sensitive to small (subvisual) changes.

alpha-Catenin expression pattern and DNA image-analysis cytometry have no additional value over primary histology in clinical stage I nonseminomatous testicular cancer.

OBJECTIVE: To determine whether the alpha-catenin expression pattern and DNA content have additional value over primary tumour histology, including information on vascular invasion and tunica albuginea invasion, in detecting occult metastasis in patients with clinical stage I nonseminomatous germ cell tumours of the testis (NSGCT). PATIENTS AND METHODS: Fifty consecutive patients with clinical stage I NSGCT underwent retroperitoneal lymphadenectomy (RPLND) between 1986 and 1992. The orchidectomy specimens were histopathologically reviewed and immunohistochemically stained with mouse monoclonal anti-alpha-catenin antibody. The presence of an aberrant or negative staining in >10% of the malignant cells was defined as abnormal; in all other cases tumours were classified as normal. Furthermore, intact nuclei were isolated from 50 microm thick paraffin sections of the primary tumour, Feulgen stained, and analysed with an image-analysis system. RESULTS: Of the 50 patients, 14 had positive retroperitoneal nodes (stage IIa, 28%), one pathologically staged I patient developed a lung metastasis (stage IV) within 3 months of RPLND. Univariate analysis showed that the presence of embryonal cell carcinoma, vascular invasion and tunica albuginea invasion were predictive for occult metastases. In multivariate logistic regression analysis only vascular and tunica albuginea invasion were significant. All 11 patients with no embryonal cell carcinoma in the primary tumour were classified as having pathological stage I disease. Also, the tumours which were DNA-diploid (three) or DNA-polyploid (two) were pathologically stage I. In screening for occult metastases the DNA content and the alpha-catenin expression pattern had no additional value. CONCLUSION: Vascular and tunica albuginea invasion have prognostic value in identifying patients with clinical stage I NSGCT at high risk for occult retroperitoneal disease. In contrast, the absence of embryonal cell carcinoma could predict all patients at low risk for metastasis. The DNA-ploidy also identified patients at low risk. Other DNA-analyses and the alpha-catenin expression pattern provided no additional information. Further studies are recommended to identify patients who are at low or high risk for metastasis.

Patterns of proliferation related to vasculature in human head-and-neck carcinomas before and after transplantation in nude mice.

PURPOSE: The predictive potential of tumor cell kinetic parameters may be improved when they are studied in relation to other microenvironmental parameters. The purpose of this investigation was to quantitatively categorize human tumor samples according to proliferation patterns. Second, it was examined whether these characteristics are retained after xenotransplantation. METHODS AND MATERIALS: Fifty tumor samples from head-and-neck cancer patients were immunohistochemically stained for Ki-67 and vessels. Also, parts of the samples were transplanted into nude mice. Tumors were categorized according to previously described patterns of proliferation. Vascular and proliferation patterns were analyzed using an image processing system. RESULTS: The 50 tumors were categorized into four patterns of proliferation by visual assessment: marginal (6), intermediate (10), random (21), and mixed (12). One tumor could not be classified. These patterns were quantified by calculating the Ki-67 labeling index in distinct zones at increasing distance from vessels yielding good discrimination and significant differences between patterns. The probability of growth after xenotransplantation was significantly higher for tumors with a labeling index and vascular density above the median value compared to tumors with both parameters below the median (82% vs. 35%). Fifty percent of the tumors retained their proliferation patterns after xenotransplantation. CONCLUSION: The categorization by proliferation pattern previously described by others was reproduced quantitatively and spatially related to the vascular network using a computerized image processing system. The combination of quantitative and architectural information of multiple microenvironmental parameters adds a new dimension to the study of treatment resistance mechanisms. Tumor models representative of the various patterns can be used to further investigate the relevance of these architectural patterns.

Improved prediction of metastasis in tongue carcinomas, combining vascular and nuclear tumor parameters.

BACKGROUND: Predicting the presence of metastasis, based on tumor or tumor-related characteristics is of utmost importance. The authors studied the significance of tumor DNA features and tumor-related angiogenesis to predict the occurrence of metastasis in squamous cell carcinomas (SCCs) of the tongue. METHODS: Paraplast blocks from resection specimens of 20 metastasized and 20 nonmetastasized SCCs of the tongue with a minimum follow-up of 24 months were used. Tissue sections were stained with anti-CD34 monoclonal antibodies for vessel visualization, and according to Feulgen to stain DNA. Using image analysis, data from both stainings were computed for each of the 40 carcinomas. A logistic regression model to predict the presence of metastasis, based on vascular and nuclear morphology features, was developed. RESULTS: The intratumor variation of chromatin condensation and the percentage vessels smaller than 5 microm in diameter were selected for the model. The model correctly predicted metastasis in 90% of patients and excluded metastasis correctly in 75% of nonmetastasized tumors. Taking into account the prevalence of metastasis in SCC of the tongue of between 30% and 60%, this means a predictive value for a negative outcome of between 95% and 83%. CONCLUSIONS: The proposed model shows an improvement of predictive values compared with previous models with single parameters. Therefore, a multiparameter model appears to predict the multiparameter process of metastasis better.

Quantification of tumour vascularity in squamous cell carcinoma of the tongue using CARD amplification, a systematic sampling technique, and true colour image analysis.

The aims of this study of head and neck tissue samples were to develop an immunohistochemical protocol based on the catalysed reporter deposition (CARD) technique to enhance staining results for use in automated true colour image analysis, to assess the reproducibility of systematic tissue sampling in the angiogenic hot spot selection, and quantification of microvessel density (MVD) and other vessel characteristics. The latter data were compared between six metastasised tongue squamous cell carcinomas, vs. four non-metastasised. In comparison to the standard immunohistochemical protocol with anti-CD34 antibodies, CARD amplification resulted in both more intensely stained and larger numbers of vessels. Averaging the 10 most vascularised fields of the 40 to 60 systematically sampled fields in a tissue section resulted in an overall acceptable interobserver reproducibility for most assessed vessel parameters (r> or =0.76 and p< or =0.01). The percentage vessels with diameter <5 microm was significantly higher in the non-metastasised tongue carcinomas (p=0.02). However, for a number of tumours the effect of tissue sampling was significant. We conclude that CARD amplification is needed for reliable segmentation of vessels by image analysis systems, and that tumour heterogeneity is a limiting factor for all procedures in which tumour vascularity is assessed in a single tissue section. Figures on http://www.esacp.org/acp/2001/22-4/hannen.htm.

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.