Alfred Böcking

Cytologic and DNA-cytometric early diagnosis of oral cancer

Objective. The aim of this prospective study was to report on the diagnostic accuracy of conventional oral exfoliative cytology taken from white‐spotted, ulcerated or other suspicious oral lesions in our clinic. In addition we checked DNA‐image cytometry as an adjuvant diagnostic tool. Our hypothesis is that DNA‐aneuploidy is a sensitive and specific marker for the early identification of tumor cells in oral brushings. Study design. 251 cytological diagnoses obtained from exfoliative smears of 181 patients from macroscopically suspicious lesions of the oral mucosa and from clinically seemingly benign oral lesions which were exisiced for establishing histological diagnoses were compared with histological and/or clinical follow‐ups of the respective patients. Additionally nuclear DNA‐contents were measured after Feulgen restaining using a TV image analysis system. Results. Sensitivity of our cytological diagnosis on oral smears for the detection of cancer cells was 94.6%, specificity 99.5%, positive predictive value 98.1% and negative predictive value 98.5%. DNA‐aneuploidy was assumed if abnormal DNA‐stemlines or cells with DNA‐content greater 9c were observed. On this basis the prevalence of DNA‐aneuploidy in smears of oral squamous cell carcinomas in situ or invasive carcinomas was 96.4%. Sensitivity of DNA‐aneuploidy in oral smears for the detection of cancer cells was 96.4%, specificity 100%, positive predictive value 100% and negative 99.0%. The combination of both techniques increased the sensivity to 98.2%, specificity to 100%, positive predictive value to 100% and negative to 99.5%. Conclusions. Brush cytology of all visible oral lesions, if they are clinically considered as suspicious for cancer, are an easily practicable, cheap, non‐invasive, painless, safe and accurate screening method for detection of oral precancerous lesions, carcinoma in situ or invasive squamous cell carcinoma in all stages. We conclude that DNA‐image cytometry is a very sensitive, highly specific and objective adjuvant tool for the early identification of neoplastic epithelial cells in oral smears.

Diagnostic accuracy of effusion cytology

The aim of this investigation was to report on the diagnostic accuracy of conventional effusion cytology. Cytological diagnoses of 300 pleural effusions and 300 ascites were compared with clinical and/or histological follow‐ups of the respective patients. Sensitivity of our cytological diagnoses on pleural effusions was 50.0%, specificity 97.0%, positive predictive value 95.7%, and negative predictive value 86.4%. Sensitivity in ascitic effusions was 62.4%, specificity 98.0%, positive predictive value 100.0%, and negative predictive value 88.3%; 5.8% of diagnoses for pleural and 4.4% for peritoneal effusions were suspicious or doubtful. The overall false‐positive rate was 0.5%, while the false‐negative rate was 31.5%. False‐negative results were due to sampling errors in 71% of pleural and 73% of peritoneal effusions and to screening errors in 29% and 27%, respectively. Our data and those from the literature show that diagnostic accuracy of effusion cytology is still unsatisfactory and should be improved. Therefore, the use of different adjuvant methods is recommended. Diagn. Cytopathol. 1999;20:350–357.

Diagnostic value of nucleolar organizer regions (AgNORs) in brush biopsies of suspicious lesions of the oral cavity

Objective: The aim of this retrospective study was to report on the diagnostic accuracy of AgNOR‐analysis as an adjunctive diagnostic tool of conventional oral exfoliative cytology taken from suspicious lesions in our clinic. Study design: Cytological diagnoses obtained from brush biopsies of macroscopically suspicious lesions of the oral mucosa from 75 patients (final diagnoses: 53 histologically proven squamous cell carcinomas, 11 leukoplakias and other inflammatory oral lesions) and from 11 patients with normal mucosa as a negative control group were compared with histological and/or clinical follow‐ups. Five smears were doubtful and seven suspicious for tumor cells in the cytologic report. Number of AgNORs were counted in 100 squamous epithelial cell‐nuclei per slide after silver‐restaining. Results: Sensitivity of our cytological diagnosis alone on oral smears for the detection of squamous carcinomas was 92.5%, specificity 100%, positive predictive value was 100% and negative 84.6%. The best cut‐off value of the mean number of AgNOR dots per nucleus distinguishing benign from malignant cells was 4.8. The percentage of nuclei with more than three AgNORs had a cut‐off level of 70%. Applying these methods to twelve doubtful or suspicious cytological diagnoses we were able to correctly establish the diagnosis of malignancy in ten cases of histologically proven cancers and to reveal benignity in two histologically proven cases. Thus we achieved a positive and negative predictive value of 100% each. Conclusions: Smears from brushings of visible oral lesions, if clinically considered as suspicious for cancer, are an easily practicable, non‐invasive, painless, safe and accurate screening method for detection of oral cancerous lesions. We conclude that AgNOR‐analysis may be a useful adjunct to other methods in routine cytological diagnosis of oral cancer that can help to solve cytologically suspicious or doubtful cases. Colour figures can be viewed on http://www.esacp.org/acp/2003/25‐3/remmerbach.htm.

Methylation Assay for the Diagnosis of Lung Cancer on Bronchial Aspirates: A Cohort Study

Purpose: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16INK4a), retinoic acid receptor β2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. Experimental Design: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). Results: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16INK4a, and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. Conclusions: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.

Hemodynamic Support by Left Ventricular Assist Devices Reduces Cardiomyocyte DNA Content in the Failing Human Heart

Background— Whether adult cardiomyocytes have the capacity to regenerate in response to injury and, if so, to what extent are still issues of intense debate. In human heart failure, cardiomyocytes harbor a polyploid genome. A unique opportunity to study the mechanism of polyploidization is provided through the setting of hemodynamic support by left ventricular assist devices. Hence, the cardiomyocyte DNA content, nuclear morphology, and number of nuclei per cell were assessed before and after left ventricular assist device support. Methods and Results— In 23 paired myocardial samples, cardiomyocyte ploidy was investigated by DNA image cytometry, flow cytometry, and in situ hybridization. Nuclear cross-sectional area and perimeters were measured morphometrically, and the binucleated cardiomyocytes were counted. The median of the cardiomyocyte DNA content and the number of polyploid cardiomyocytes both declined significantly from 6.79 c to 4.7 c and 40.2% to 23%, whereas a significant increase in diploid cardiomyocytes from 33.4% to 50.3% and in binucleated cardiomyocytes from 4.5% to 10% after unloading was observed. Conclusions— The decrease in polyploidy and increase in diploidy after left ventricular assist device suggest a numeric increase in diploid cardiomyocytes (eg, through cell cycle progression with completion of mitosis or by increased stem cells). The cardiac regeneration that follows may serve as a morphological correlate of the recovery observed in some patients after unloading.

Aberrant promoter methylation of p16INK4a, RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer

Aberrant promoter methylation of normally unmethylated CpG‐islands offers a promising tool for the development of molecular biomarkers. We investigated bronchial aspirates of patients admitted for suspected lung cancer with regard to the prevalence of aberrant methylation of potential marker genes. Applying quantitative methylation specific PCR (QMSP) we analyzed bronchial aspirates from 75 patients with primary lung cancer and 64 bronchial aspirates of patients diagnosed with benign lung disease for promoter methylation of 3 candidate marker genes (p16INK4a, RARB2 and SEMA3B). Hypermethylation of p16INK4a detected 18/75 (24%) cases with primary lung cancer and was present predominantly in squamous cell carcinomas (14/25; 56%). RARB2 QMSP at an assay threshold greater than 30 was found in 42/75 (56%) patients with lung cancer without relation to histological subtype. Patients with benign lung disease showed methylation of p16INK4a and a RARB2 QMSP at an assay threshold greater than 30 in 0/64 (0%) and 8/64 (13%) cases, respectively. Combining the 2 methylation markers, p16INK4a and RARB2, yielded a sensitivity of 69% and a specificity of 87% for the diagnosis of pulmonary malignancy. In contrast, SEMA3B displayed frequent promoter methylation (around 90%) both in bronchial aspirates of tumor and nontumor cases and thus was not suited as a biomarker. The results of this study indicate that QMSP analysis of p16INK4a and RARB2 may aid the diagnosis of primary lung cancer in bronchial aspirates. In particular, detection of p16INK4a methylation by QMSP may serve as a highly specific marker of pulmonary squamous cell carcinoma.

Diagnostic and prognostic use of DNA image cytometry in cervical squamous intraepithelial lesions and invasive carcinoma.

In the fight against cervical malignancy and its precursors, several adjuvant diagnostic methods have been proposed to increase the accuracy of cytologic and histologic diagnoses. Because chromosomal aneuploidy has been accepted as an early key event in tumorigenesis caused by genetic instability, the cytometric equivalent of chromosomal aneuploidy detected by DNA image cytometry (DNA‐ICM) may serve as a marker of neoplasia. During the last decade, the appearance of a new generation of hardware with high processing and storage capacities, together with the development of appropriate software, has facilitated the development of high‐performance DNA‐ICM systems. International consensus on the clinical application of DNA‐ICM has been reached. According to the statements of Task Force 8 of the International Consensus Conference on the Fight Against Cervical Cancer, indications for DNA‐ICM include the identification of prospectively malignant cells in squamous intraepithelial lesions (SILs) and atypical squamous cells of undetermined significance (ASCUS). The European Society of Analytical Cellular Pathology consensus reports on DNA‐ICM have provided standardized technical details on performance, terms, and algorithms for diagnostic data interpretation and quality‐assurance procedures. Increasing biologic evidence and clinical data have confirmed the utility of DNA‐ICM as an adjuvant method suitable for determining the diagnosis and prognosis of cervical intraepithelial lesions and invasive carcinoma. Patients with ASCUS and low‐grade SIL diagnoses that reveal DNA euploidy may return for normal screening intervals, whereas the detection of DNA aneuploidy indicates that these lesions should be removed. Formerly a research tool, today, standardized DNA‐ICM has become a useful and low‐cost laboratory method to establish objectively and reproducibly an early diagnosis of prospectively progressive cervical intraepithelial lesions at a high‐quality level. DNA‐ICM may further contribute to the monitoring of treatment in patients with invasive cervical malignancies. Cancer (Cancer Cytopathol) 2004;102:41–54.

9p21 Deletion in the Diagnosis of Malignant Mesothelioma in Serous Effusions Additional to Immunocytochemistry, DNA-ICM, and AgNOR Analysis

The diagnosis of malignant mesothelioma (MM) in serous effusions is difficult but may be achieved by the application of adjuvant methods.

Earliest Detection of Oral Cancer Using Non-Invasive Brush Biopsy Including DNA-Image-Cytometry: Report on Four Cases

Objective: We describe four patients presenting early oral cancers, detected cytologically on non‐invasive brush biopsies including DNA‐image cytometry as an adjunctive method before histology on scalpel biopsies confirmed the evidence of malignancy. Methods: Brush biopsies were performed and smears thereof investigated cytologically. After Feulgen restaining, DNA‐measurements were performed using a DNA‐Image‐Cytometer. Case reports: Oral squamous cell carcinomas were diagnosed cytologically in macroscopically suspicious lesions and malignancy confirmed by DNA‐cytometry. The initially performed scalpel biopsies did neither supply evidence of oral cancer nor of severe dysplasia. After at least one to 15 months the occurrence of cancer was finally proven histologically on a second scalpel biopsy each (three microinvasive and one in situ carcinoma). Conclusion: Non‐invasive brush biopsies are a suitable instrument for early cytologic detection of cancer of the mouth. DNA‐image‐cytometry, as an adjunctive method, can be used to confirm the cytologic diagnosis or suspicion of cancer in patients with doubtful lesions (dysplasias). DNA‐aneuploidy is a marker for (prospective) malignancy in smears of the oral cavity, which may detect malignancy months prior to histology. In future this method could be used as a mass screening tool in dentists practise. Colour figures can be viewed on http://www.esacp.org/acp/2003/25‐4/remmerbach.htm.

Immunocytochemical diagnosis of hepatocellular carcinoma and identification of carcinomas of unknown primary metastatic to the liver on fine‐needle aspiration cytologies

Difficulties with cytologic diagnoses on fine‐needle aspiration cytology (FNAC) of the liver can be overcome by the application of immunocytochemical panels applied on smears. The aim of the current study was to analyze the performance of a panel of monoclonal antibodies to differentiate hepatocellular carcinoma (HCC) from metastatic carcinoma (MC) or regenerative nodules, and to identify the to date unknown primary sites of carcinomas that had metastasized to the liver.