DNA Karyometry for Automated Detection of Cancer Cells.

Background: Microscopical screening of cytological samples for the presence of cancer cells at high throughput with sufficient diagnostic accuracy requires highly specialized personnel which is not available in most countries. Methods: Using commercially available automated microscope-based screeners (MotiCyte and EasyScan), software was developed which is able to classify Feulgen-stained nuclei into eight diagnostically relevant types, using supervised machine learning. the nuclei belonging to normal cells were used for internal calibration of the nuclear DNA content while nuclei belonging to those suspicious of being malignant were specifically identified. The percentage of morphologically abnormal nuclei was used to identify samples suspected of malignancy, and the proof of DNA-aneuploidy was used to definitely determine the state malignancy. A blinded study was performed using oral smears from 92 patients with Fanconi anemia, revealing oral leukoplakias or erythroplakias. In an earlier study, we compared diagnostic accuracies on 121 serous effusion specimens. In addition, using a blinded study employing 80 patients with prostate cancer who were under active surveillance, we aimed to identify those whose cancers would not advance within 4 years. Results: Applying a threshold of the presence of >4% of morphologically abnormal nuclei from oral squamous cells and DNA single-cell or stemline aneuploidy to identify samples suspected of malignancy, an overall diagnostic accuracy of 91.3% was found as compared with 75.0% accuracy determined by conventional subjective cytological assessment using the same slides. Accuracy of automated screening effusions was 84.3% as compared to 95.9% of conventional cytology. No prostate cancer patients under active surveillance, revealing DNA-grade 1, showed progress of their disease within 4.1 years. Conclusions: An automated microscope-based screener was developed which is able to identify malignant cells in different types of human specimens with a diagnostic accuracy comparable with subjective cytological assessment. Early prostate cancers which do not progress despite applying any therapy could be identified using this automated approach.

Diagnostic accuracy of brush biopsy-based cytology for the early detection of oral cancer and precursors in Fanconi anemia.

BACKGROUND: Individuals with Fanconi anemia (FA) have a 500-fold to 700-fold elevated risk, much earlier onset, and limited therapeutic options for oral squamous cell carcinoma (SCC) compared with the general population. The early detection of SCC, or preferably its precursors, is mandatory to retain curative therapeutic options. Due to frequent synchronic and metachronic oral lesions, tissue biopsies, as usually recommended by guidelines, often are not feasible. In the current study, an alternative strategy for early detection using oral brush biopsy-based cytology was validated regarding its diagnostic accuracy. METHODS: Over a 12-year period, the oral cavities of a large cohort of 713 individuals with FA were inspected systematically and brush biopsy-based cytology of 1233 visible oral lesions was performed. In cases of inconclusive cytology, analysis of DNA ploidy was performed whenever possible. The results were correlated to a long-term clinicopathological follow-up reference standard. RESULTS: A total of 737 lesions were suitable for statistical analysis, including 86 lesions with at least high-grade oral epithelial dysplasia in 30 patients. For cytology, the sensitivity and specificity were 97.7% and 84.5%, respectively. Additional analysis of DNA ploidy increased the sensitivity and specificity to 100% and 92.2%, respectively. CONCLUSIONS: Careful inspection of the oral cavity of individuals with FA followed by brush biopsy-based cytology appears to identify visible oral, potentially malignant and malignant lesions that warrant treatment. Approximately 63% of SCC and precursor lesions are detected at a noninvasive or early stage. Negative cytology or a lack of DNA aneuploidy can exclude high-grade oral epithelial dysplasia or SCC with high accuracy and thus reduce the need for invasive diagnostic biopsies.

Automated detection of cancer cells in effusion specimens by DNA karyometry.

BACKGROUND: The average sensitivity of conventional cytology for the identification of cancer cells in effusion specimens is only approximately 58%. DNA image cytometry (DNA-ICM), which exploits the DNA content of morphologically suspicious nuclei measured on digital images, has a sensitivity of up to 91% for the detection of cancer cells. However, when performed manually, to our knowledge to date, an expert needs approximately 60 minutes for the analysis of a single slide. METHODS: In the current study, the authors present a novel method of supervised machine learning for the automated identification of morphologically suspicious mesothelial and epithelial nuclei in Feulgen-stained effusion specimens. The authors compared this with manual DNA-ICM and a gold standard cytological diagnosis for 121 cases. Furthermore, the authors retrospectively analyzed whether the amount of morphometrically abnormal mesothelial or epithelial nuclei detected by the digital classifier could be used as an additional diagnostic marker. RESULTS: The presented semiautomated DNA karyometric solution identified more diagnostically relevant abnormal nuclei compared with manual DNA-ICM, which led to a higher sensitivity (76.4% vs 68.5%) at a specificity of 100%. The ratio between digitally abnormal and all mesothelial nuclei was found to identify cancer cell-positive slides at 100% sensitivity and 70% specificity. The time effort for an expert therefore is reduced to the verification of a few nuclei with exceeding DNA content, which to our knowledge can be accomplished within 5 minutes. CONCLUSIONS: The authors have created and validated a computer-assisted bimodal karyometric approach for which both nuclear morphology and DNA are quantified from a Feulgen-stained slide. DNA karyometry thus increases the diagnostic accuracy and reduces the workload of an expert when compared with manual DNA-ICM.

Removing defocused objects from single focal plane scans of cytological slides.

BACKGROUND: Virtual microscopy and automated processing of cytological slides are more challenging compared to histological slides. Since cytological slides exhibit a three-dimensional surface and the required microscope objectives with high resolution have a low depth of field, these cannot capture all objects of a single field of view in focus. One solution would be to scan multiple focal planes; however, the increase in processing time and storage requirements are often prohibitive for clinical routine. MATERIALS AND METHODS: In this paper, we show that it is a reasonable trade-off to scan a single focal plane and automatically reject defocused objects from the analysis. To this end, we have developed machine learning solutions for the automated identification of defocused objects. Our approach includes creating novel features, systematically optimizing their parameters, selecting adequate classifier algorithms, and identifying the correct decision boundary between focused and defocused objects. We validated our approach for computer-assisted DNA image cytometry. RESULTS AND CONCLUSIONS: We reach an overall sensitivity of 96.08% and a specificity of 99.63% for identifying defocused objects. Applied on ninety cytological slides, the developed classifiers automatically removed 2.50% of the objects acquired during scanning, which otherwise would have interfered the examination. Even if not all objects are acquired in focus, computer-assisted DNA image cytometry still identified more diagnostically or prognostically relevant objects compared to manual DNA image cytometry. At the same time, the workload for the expert is reduced dramatically.

Comparability of tumor-cytogenetics and -DNA-cytometry.

Knowledge of the karyotypes of cancer cells is the theoretical underpinning of diagnostic/prognostic DNA-cytometry. Currently the translation is hampered by different terminologies of both fields. The following letter tries to close current gaps between the two fields by harmonizing their different terminologies.

Is it useful to combine sputum cytology and low-dose spiral computed tomography for early detection of lung cancer in formerly asbestos-exposed power industry workers?

BACKGROUND: Low-dose spiral computed tomography (LDSCT) in comparison to conventional chest X-ray proved to be a highly sensitive method of diagnosing early stage lung cancer. However, centrally located early stage lung tumours remain a diagnostic challenge. We determined the practicability and efficacy of early detection of lung cancer when combining LDSCT and sputum cytology. METHODS: Of a cohort of 4446 formerly asbestos exposed power industry workers, we examined a subgroup of 187 (4.2%) high risk participants for lung cancer at least once with both LDSCT and sputum cytology. After the examination period the participants were followed-up for more than three years. RESULTS: The examinations resulted in the diagnosis of lung cancer in 12 participants (6.4%). Six were in clinical stage I. We found 10 non-small cell lung carcinomas and one small cell lung carcinoma. Sputum specimens showed suspicious pathological findings in seven cases and in 11 cases the results of LDSCT indicated malignancies. The overall sensitivity and specificity of sputum cytology was 58.0% and 98% with positive (PPV) and negative (NPV) predictive values of 70% and 97%. For LDSCT we calculated the sensitivity and specificity of 92% and 97%. The PPV and NPV were 65% and 99% respectively. CONCLUSIONS: Our results confirmed that in surveillance programmes a combination of sputum cytology and LDSCT is well feasible and accepted by the participants. Sputum examination alone is not effective enough for the detection of lung cancer, especially at early stage. Even in well- defined risk groups highly exposed to asbestos, we cannot recommend the use of combined LDSCT and sputum cytology examinations as long as no survival benefit has been proved for the combination of both methods. For ensuring low rates of false-positive and false-negative results, programme planners must closely cooperate with experienced medical practitioners and pathologists in a well-functioning interdisciplinary network.

Equivocal cytology in lung cancer diagnosis: improvement of diagnostic accuracy using adjuvant multicolor FISH, DNA-image cytometry, and quantitative promoter hypermethylation analysis.

BACKGROUND: Sometimes, cytological lung cancer diagnosis is challenging because equivocal diagnoses are common. To enhance diagnostic accuracy, fluorescent in situ hybridization (FISH), DNA-image cytometry, and quantitative promoter hypermethylation analysis have been proposed as adjuncts. METHODS: Bronchial washings and/or brushings or transbronchial fine-needle aspiration biopsies were prospectively collected from patients who were clinically suspected of having lung carcinoma. After routine cytological diagnosis, 70 consecutive specimens, each cytologically diagnosed as negative, equivocal, or positive for cancer cells, were investigated with adjuvant methods. Suspicious areas on the smears were restained with the LAVysion multicolor FISH probe set (Abbott Molecular, Des Plaines, Illinois) or according to the Feulgen Staining Method for DNA-image cytometry analysis. DNA was extracted from residual liquid material, and frequencies of aberrant methylation of APC, p16(INK4A) , and RASSF1A gene promoters were determined with quantitative methylation-specific polymerase chain reaction (QMSP) after bisulfite conversion. Clinical and histological follow-up according to a reference standard, defined in advance, were available for 198 of 210 patients. RESULTS: In the whole cohort, cytology, FISH, DNA-image cytometry, and QMSP achieved sensitivities of 83.7%, 78%, 79%, and 49.6%, respectively (specificities of 69.8%, 98.2%, 98.2%, and 98.4%, respectively). Subsequent to cytologically equivocal diagnoses, FISH, DNA-image cytometry, and QMSP definitely identified malignancy in 79%, 83%, and 49%, respectively. With QMSP, 4 of 22 cancer patients with cytologically negative diagnoses were correctly identified. CONCLUSIONS: Thus, adjuvant FISH or DNA-image cytometry in cytologically equivocal diagnoses improves diagnostic accuracy at comparable rates. Adjuvant QMSP in cytologically negative cases with persistent suspicion of lung cancer would enhance sensitivity.

Role of brush biopsy and DNA cytometry for prevention, diagnosis, therapy, and followup care of oral cancer.

Late diagnosis resulting in late treatment and locoregional failure after surgery are the main causes of death in patients with oral squamous cell carcinomas (SCCs). Actually, exfoliative cytology is increasingly used for early detection of oral cancer and has been the subject of intense research over the last five years. Significant advances have been made both in relation to screening and evaluation of precursor lesions. As this noninvasive procedure is well tolerated by patients, more lesions may be screened and thus more oral cancers may be found in early, curable stages. Moreover, the additional use of DNA image cytometry is a reasonable tool for the assessment of the resection margins of SCC. DNA image cytometry could help to find the appropriate treatment option for the patients. Finally, diagnostic DNA image cytometry is an accurate method and has internationally been standardized. In conclusion, DNA image cytometry has increasing impact on the prevention, diagnostic, and therapeutical considerations in head and neck SCC.

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.

Toward a multimodal cell analysis of brush biopsies for the early detection of oral cancer.

BACKGROUND: This report describes what to the authors' knowledge is the first clinical application of semiautomated multimodal cell analysis (MMCA), a novel technique for the early detection of cancer for cases with a limited number of suspicious cells. In this clinical study, MMCA was applied to oral cancer diagnostics on brush biopsies. The MMCA approach was based on the sequential application of multiple stainings of identical, slide-based cells and repeated relocalizations and measurements of their diagnostic features, resulting in multiparametric features of individual cells. Data integration of the variously stained cells increased diagnostic accuracy. The implementation of MMCA also enabled fully automatic, adaptive image preprocessing, including registration of multimodal images and segmentation of cell nuclei. METHODS: In a preliminary clinical trial, 47 slides from brush biopsies of suspicious oral lesions were analyzed. The final histologic diagnoses included 20 squamous cell carcinomas, 7 hyperkeratotic leukoplakias, and 20 lichen planus mucosae. RESULTS: The stepwise application of 2 additional approaches (morphology, DNA content, argyrophilic nucleolar organizer region counts) increased the specificity of conventional cytologic diagnosis from 92.6% to 100%. This feasibility study provided a proof of concept, demonstrating efficiency, robustness, and diagnostic accuracy on slide-based cytologic specimens. CONCLUSIONS: The authors concluded that MMCA may become a sensitive and highly specific, objective, and reproducible adjuvant diagnostic tool for the identification of neoplastic changes in oral smears that contain only a few abnormal cells.

Core classification of lung cancer: correlating nuclear size and mitoses with ploidy and clinicopathological parameters.

We attempted to establish a microscopy based tumour characterization providing insight into the genetics of cancer cells and in particular their DNA ploidy. The core classification defined semi-quantitative criteria for scoring the nuclear size ranging from small (core score 1) to giant nuclei (core score 4). By listing all nuclear sizes according to their relative frequencies it provided a measure for core size variability as a correlate of nuclear pleomorphism. Additionally, the mitosis size, their variability and the presence/abundance of tripolar and tetrapolar mitoses were determined. This classification was applied to 155 lung cancer samples from all major histologic types and the results were correlated with the analysis by DNA image cytometry and patient survival. The morphological assessments correlated highly significantly with the DNA ploidy parameters, e.g. small cell lung carcinomas showed the smallest values for nuclear size (mean core score of 1.18) and DNA content (DNA index mean of 2.08c) being highly significantly different from adenocarcinomas (1.95/3.10c), large cell lung carcinoma (2.00/3.26c) and squamous cell carcinoma (2.20/3.42c). In non-small cell lung carcinoma (NSCLC) in general and adenocarcinoma in particular, the core size variability correlated significantly with grading and survival. Furthermore, parameters indicative for chromosomal variability, i.e. 2c deviation index and 5c exceeding rate, were predictors of poor survival in NSCLC patients. As a complement to histologic tumour diagnosis the core classification should help to better stratify cancer subtypes.

Immunocytochemical typing of primary tumors on fine-needle aspiration cytologies of lymph nodes.

The aim of this study was to analyze the role of immunocytochemistry as an ancillary method on routine FNACs of enlarged lymph nodes, using different markers. In a validating cohort study all patients had confirmatory histological and/or clinical follow-up. 10 FNACs were analyzed for the differentiation of Non-Hodgkin Lymphoma (NHL) from metastatic carcinoma (MC), 30 cases to identify the sites of metastatic unknown primary tumors and 16 cases were checked to confirm clinical suspicion of a specific MC. Accuracy to differentiate NHL from MC was 100%, 92.3% to identify a primary tumor site of MC, and 100% to confirm a clinical suspicion of a specific MC. In 7 cases, the site of the primary tumor remained clinically unknown. Application of immunocytochemical markers on the same slide used for microscopic diagnosis is a useful tool in the routine assessment of FNACs of lymph nodes.

9p21 Deletion in the diagnosis of malignant mesothelioma in serous effusions additional to immunocytochemistry, DNA-ICM, and AgNOR analysis.

BACKGROUND: The diagnosis of malignant mesothelioma (MM) in serous effusions is difficult but may be achieved by the application of adjuvant methods. METHODS: The authors cytologically diagnosed 33 effusions as suspicious or positive for MM cells by using DNA-image cytometry (DNA-ICM), immunocytochemistry and AgNOR analysis. The authors further detected 9p21 deletions by chromosomal fluorescence in situ hybridization (FISH). In addition, 31 cases of metastatic carcinomas and 39 of tumor cell-negative effusions were investigated. All diagnoses were confirmed by histologic and/or clinical follow-up. RESULTS: DNA aneuploidy was found in 71% of MMs, 100% of metastatic carcinomas, and in none of the negative effusions. Calretinin was positive in 100% of MMs, in none of the metastatic carcinomas, and in 94.9% of negative effusions. BerEP4 showed positivity in 15.6% of MMs, 87.1% of metastatic carcinomas, and in none of the negative effusions. With AgNOR analysis, 89.3% of MMs and 96.7% of metastatic carcinomas showed >or=2.5 AgNOR dots as satellites and >or=4.5 as total AgNOR counts. 9p21 deletions were demonstrated in 90.9% of MM cases, 45.2% of metastatic carcinomas, and in none of the negative effusions. By cytology alone, 81.8% of MMs were identified unequivocally. Addition of DNA-ICM improved the prevalence of tumor cell detection to 87.9% and of AgNOR analysis to 97%. The introduction of 9p21 deletions by FISH improved this prevalence to 100%. CONCLUSIONS: Because of these results, the authors propose the sequential application of immunocytochemistry, DNA-ICM, and AgNOR analysis to establish a cytological diagnosis of malignant mesothelioma in serous effusions. In persistent doubtful diagnoses, the authors recommend fluorescence in situ hybridization to analyze the 9p21 deletion.

Early cytological diagnosis of diffuse malignant mesothelioma of the peritoneum: a case report.

Mesotheliomas are the most frequent primary malignant tumors of serosal cavities with a poor prognosis. A definitive and early diagnosis on effusion samples is important, because recent advances in therapy for patients with mesothelioma may result in an improved outcome if they are applied to stage I disease. We report a case of malignant peritoneal mesothelioma diagnosed repeatedly by cytology in ascites fluids 1.5 yr before the diagnosis was confirmed by biopsy histology. The cytological diagnoses were supported by immunocytochemistry, DNA-cytometry, and AgNOR-analysis. Routine cytology supported by three adjuvant methods enabled us to correctly establish the diagnosis. Our case suggests that a cytological diagnosis of malignant mesothelioma supported by adjuvant methods should not be rejected even if based on negative histological results.

[Minimally invasive brush-biopsy: innovative method for early diagnosis of oral squamous cell carcinoma]. / Minimalinvasive Bürstenbiopsie. Innovative Methode zur Früherkennung oraler Plattenepithelkarzinome.

The aim of this prospective and blinded study was to investigate the diagnostic accuracy of conventional cytopathology of oral brush biopsies taken from suspicious oral lesions. In addition we checked slide based DNA image cytometry as an adjuvant diagnostic tool. Our hypothesis is that DNA aneuploidy is a sensitive and specific marker for earliest detection of oral cancer using brush biopsies. Therefore the nuclear DNA contents were measured after Feulgen re-staining using a TV image analysis system. DNA aneuploidy was assumed if abnormal DNA stemlines or cells with DNA content greater 9c were observed. Sensitivity of our cytological diagnosis in oral smears for the detection of cancer cells thus was 91.3%, specificity for the detection of non-neoplastic cells was 95.1%, positive predictive value 94.4% and negative predictive value 92.3%. The adjuvant DNA image cytometry reached a sensitivity of 97.8%, the specificity and the positive predictive value were 100% and negative predictive value was 98.1%, respectively. Smears from oral brush biopsies of all visible oral lesions are an easily practicable, cheap, minimal invasive, painless and safe screening method for detection of oral precancerous lesions and squamous cell carcinomas in all stages. We conclude that DNA image cytometry is a very sensitive and highly specific, objective and reproducible adjuvant tool for identification of neoplastic cells in oral smears.

Additional use of DNA-image cytometry improves the assessment of resection margins.

BACKGROUND: Despite the histopathologic findings of tumor-free margins, patients with oral squamous cell carcinoma (SCC) often suffer from local tumor relapse. The purpose of this study was to determine the prognostic value of DNA-image cytometry in the assessment of resection margins. METHODS: DNA-image cytometry was performed in 40 SCC patients with histologically tumor-free resection margins. The follow-up period since the tumor resection was at least 3 years. RESULTS: Twenty patients showed a locoregional relapse of the SCC. Fourteen of these patients had aneuploid cells in DNA-image cytometry. Two patients who were relapse-free revealed aneuploid cells too. The sensitivity of the adjuvant use of DNA-image cytometry was 70% and the positive predictive value was 87.5%. CONCLUSIONS: The additional use of DNA-image cytometry is a reasonable tool for the assessment of the resection margins of SCCs. DNA-image cytometry could help to find the appropriate treatment option for the patients and thus might improve their prognosis.

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

BACKGROUND: 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. METHODS: In a validating cohort study, 108 FNACs coin lesions in the liver were routinely evaluated applying immunocytochemistry as an ancillary method. All patients had confirmatory histologic and/or clinical follow-up. A total of 23 HCCs were analyzed for the distinction from MC or regenerative nodules applying a panel of HepPar1, alpha-fetoprotein, BerEP4, CD31, CD68, and Ki-67. A total of 85 cases of unknown primary tumor metastatic to the liver were used to identify the tumor sites applying a panel of CK5/6, CK7, CK20, CA 125, thyroid transcription factor-1 (TTF-1), and Cdx2. RESULTS: Typing accuracy to differentiate HCC from MC or regenerative nodules was 100% and 90.3%, respectively, to identify the primary tumor site of MC. In 23 cases, the site of the primary tumor remained clinically unknown. CONCLUSIONS: The application of immunocytochemical panels on the same slide used for microscopic diagnosis is a useful tool in the routine assessment of FNACs of the liver to discriminate HCCs from MC or regenerative nodules and for the identification of primary sites of MC. Their performance should be confirmed in a larger series of cases.

Methylation of RAS association domain family protein 1A as a biomarker of lung cancer.

BACKGROUND: Promoter hypermethylation is an important mechanism for silencing tumor-suppressor genes in cancer and a promising tool for development of molecular biomarkers. This study aimed to determine the prevalence of RAS association domain family protein 1A (RASSF1A) promoter hypermethylation in bronchial aspirates of patients with suspected lung cancer and to test whether this type of methylation assay could be used as a diagnostic adjunct to conventional cytology. METHODS: Two hundred three bronchial aspirates from patients with suspected lung cancer were analyzed for RASSF1A hypermethylation by using a sensitive quantitative methylation-specific polymerase chain reaction (QMSP). RESULTS: RASSF1A hypermethylation was found in 88% (35 of 40), 28% (31 of 111), and 100% (6 of 6) of bronchial aspirates collected from patients diagnosed with small cell lung cancer, nonsmall cell lung cancer, and combined small cell lung cancer, respectively. No hypermethylation was detected in patients diagnosed with nonneoplastic lung disease (0 of 46). Depending on histologic subtype, up to 82% of cases presenting with a negative histology showed a positive methylation assay. CONCLUSIONS: The QMSP analysis of RASSF1A hypermethylation enabled a highly specific distinction between patients diagnosed with lung cancer and those with nonneoplastic lung disease. These results suggested that a QMSP assay is a promising molecular tool for diagnosis of primary lung cancer.

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 (p16(INK4a)), retinoic acid receptor beta2, 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, p16(INK4a), 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.

Immunocytochemical identification of carcinomas of unknown primary in serous effusions.

Metastases from carcinomas of unknown primary site (CUP) in serous effusion are a common clinical problem. Immunocytochemistry was applied as an adjunct to the cytological diagnosis of metastatic carcinomas in serous effusions. Subjects of this study were 118 pleural, 53 peritoneal, and 9 pericardial effusions from 180 patients routinely investigated in the Institute of Cytopathology. Specimens were primarily stained according to Papanicolaou (Pap). The avidin-biotin-complex method (ABC) was secondarily applied for the visualization of immunologic reactions. We have used a panel of six monoclonal antibodies (CK 5/6, CK 7, CK 20, CA 125, TTF-1, and cdx 2) so as to identify the primary tumor site of metastatic carcinoma cells in serous effusions. Applying an algorithm of immunocytochemical marker constellations, we were able to correctly diagnose primary tumor sites in 86 of 101 (85.1%) patients with CUP syndromes. The best result was achieved for the identification of metastatic carcinomas of the ovaries (94.7%) and the lungs (88.1%). We established an algorithm comprising six immunocytochemical markers that enabled a correct diagnosis of primary tumor sites in 85.1%. The panel studied could be useful in diagnostic routine for the identification of primary tumors of unknown origin metastatic to the serous membranes.