Malignancy rates in thyroid nodules: a long-term cohort study of 17,592 patients.

Objectives: Ultrasound diagnosis of thyroid nodules has greatly increased their detection rate. Their risk for malignancy is estimated between 7 and 15% in data from specialized centers which are used for guidelines recommendations. This high rate causes considerable anxiety to patients upon first diagnosis. Here, we retrospectively analyzed the malignancy rate of sonographically diagnosed nodules larger than 1 cm from a primary/secondary care center when long-term longitudinal follow-up was included. Patients/methods: In the study, 17,592 patients were diagnosed with a thyroid nodule larger than 1 cm, of whom 7776 were assessed by fine-needle aspiration cytology (FNAC) and 9816 by sonography alone. 9568 patients were initially discharged due to innocent results of FNAC and/or ultrasound. In 1904 patients, definitive histology was obtained, and 6731 cases were included in the long-term follow-up (up to 23 years, median 5 years). Results: Malignancy was histologically confirmed in 189 patients (1.1% of all) when excluding accidentally diagnosed papillary microcarcinomas. 155 were diagnosed during the first year of management, 25 in years 2-5 of follow-up, 9 in years 6-10 and nil in 1165 patients followed beyond 10 years. Conclusions: The malignancy rate of thyroid nodules from primary/secondary care was much lower than that previously reported. During follow-up for more than 5 years, their rate rapidly dropped to less than 1/1000 cases. This low malignancy rate may help to reassure patients first confronted with the diagnosis of a thyroid nodule, substantially reduce their anxiety and avoid unwarranted diagnostic and therapeutic procedures.

[Fine-needle aspiration cytology of thyroid nodules: molecular diagnostics in a routine diagnostic setting]. / Feinnadelzytologie von Schilddrüsenknoten: Molekulare Diagnostik in der klinischen Routine.

BACKGROUND AND OBJECTIVE: Results for the detection of point mutations and rearrangements have thus far been obtained by fresh material of fine needle aspiration cytology (FNAC). After a first retrospective study we report on the diagnostic detection in routinely obtained, consecutive air-dried FNAC smears. METHODS: RNA and DNA was extracted from 154 consecutive routine air-dried FNAC smears: 80 with microfollicular proliferation (MFP), 45 with follicular neoplasia (FN), 26 with the cytological diagnosis of papillary carcinomas (PTC) and 3 which were suspicious for malignancy. PAX8/PPARG and RET/PTC3 rearrangements were detected by qPCR, while BRAF and RAS point mutations were detected by pyrosequencing. RESULTS: Only 0.7 % and 5.3 % of the routine air-dried FNAC samples did not allow analysis of a point mutation or rearrangements, respectively. NRAS mutations could be detected in 7 MFP smears, and in one of FN and PTC samples, respectively. HRAS mutations were detected in one MPF and one FN sample. A KRAS mutation was only detected in one FN sample, whereas BRAF mutations were detected in 20 samples with PTC (but in no other sample). PAX8/PPARG was detected in 2 MFP samples, while RET/PTC was detected in only one MFP sample. In total, 13.8 % MFP-FNAC, 6.7 % FN-FNAC, and 80.8 % PTC-FNAC samples harbored a mutation. CONCLUSION: These results demonstrate that rearrangements and point mutations can be detected in routinely obtained air-dried FNAC samples.

p16 alterations and retinoblastoma protein expression in squamous cell carcinoma and neighboring dysplasia from the upper aerodigestive tract.

The progression potential of preinvasive epithelial lesions is usually evaluated by assessing the degree of histologic dysplasia. We examined p16, retinoblastoma protein (pRb), and proliferating cell nuclear antigen (PCNA) immunophenotypes in 57 cases of previously untreated squamous cell carcinoma (SCC) of the upper digestive tract and in the neighboring normal and dysplastic epithelia. Tissue samples were examined for homozygous deletion of exon 2 of the p16 gene using polymerase chain reaction (PCR) analysis. The PCNA index increased with increasing grade of dysplasia. The pRb protein was expressed in 89% of the samples of SCCs and in the neighboring dysplasias and carcinoma in situ (CIS). In cases with a lack of pRb expression, corresponding preinvasive lesions were also negative. Lack of p16 expression was found in 82% of SCCs. The prevalence of p16 expression decreased with increasing grade of dysplasia. Molecular analysis of the p16 gene showed homozygous deletion in 37% of SCCs, 33% of CIS, and 15% of the samples of normal epithelia. Our data indicate that inactivation of p16 may play an important role in early head and neck carcinogenesis, whereas the mutation of Rb may be an infrequent event. The p16 immunophenotype might be a biomarker for an increased risk of progression in squamous dysplasia.

Relation between DNA ploidy status and the expression of the DNA-mismatch repair genes MLH1 and MSH2 in cytological specimens of melanoma lymph node and liver metastases.

DNA-mismatch repair is essential for preventing genetic instability, and its important protective role has been demonstrated in several tumors. The main aim of this study was to investigate the expression of MLH1 and MSH2 (on the RNA level) in melanoma liver and lymph node metastases, and to define the relation between DNA ploidy status and mismatch repair gene expression. MLH1 was found in 29/33 melanoma lymph node and in 5/17 melanoma liver metastases. MSH2 was present in 26/33 lymph node and 5/17 liver metastases. A comparison of MLH1 and MSH2 positive and negative melanoma metastases showed that there were highly significant differences in the percentages of diploid cells, aneuploid cells between 4c and 8c, octaploid cells, and 5c exceeding rate. This fact confirms the strong relation between the loss of DNA-mismatch repair gene expression and advanced DNA aneuploidy status in melanoma metastases.

Downregulation of the retinoblastoma gene expression in the progression of malignant melanoma.

The retinoblastoma gene is a cell cycle regulator preventing cells from entering into S-phase. An altered expression of the retinoblastoma gene has been reported in the majority of human malignancies. The main aim of this study was to investigate retinoblastoma gene expression in the full spectrum of melanoma progression from naevus to melanoma metastases by applying immunohistochemistry and RT-PCR. All naevi with and without dysplasia showed high expression of the retinoblastoma gene. In primary melanomas, Rb-positive cells were found in 82 out of 106. Loss of expression correlated with an increase in Clark level and shorter survival rates. An independent prognostic role of the retinoblastoma gene was confirmed by Cox multivariate analyses (p < 0.01). In melanoma metastases, retinoblastoma gene expression (at the RNA level) was found in 18 out of 26 melanoma lymphatic metastases, and in 2 out of 5 liver metastases. Our results indicate a downregulation of the retinoblastoma gene in the progression of melanocytic tumours.

p53 and MDM2 protein expression in squamous cell carcinoma and neighboring dysplasia from the upper aerodigestive tract.

BACKGROUND: The identification of genetic changes in preinvasive epithelial lesions may provide markers for a better assessment of the progression potential. MATERIALS AND METHODS: p53, MDM2 and PCNA immunophenotypes were examined in 57 cases of squamous cell carcinoma (SCC) of the upper aerodigestive tract and the adjacent normal and dysplastic epithelia. RESULTS: PCNA index increased with increasing grade of dysplasia. p53 protein was expressed in 35% and MDM2 protein in 33% of SCCs. The p53 protein was expressed in 89% of mild and moderate and in 93% of severe dysplasia and carcinoma in situ adjacent to p53-positive SCCs. The MDM2 protein was expressed in 30% of mild and moderate and in 54% of severe dysplasia and carcinoma in situ adjacent to MDM2-positive SCCs. Preinvasive lesions adjacent to negative SCCs stained negative. CONCLUSIONS: p53 protein was detected more frequently in preinvasive lesions than MDM2 protein and seems to be of greater value as a biomarker.

Prognostic significance of newly defined ploidy related parameters in melanoma.

5c exceeding rate is the parameter, most frequently showing prognostic impact. The CAS200 image analyzer makes possible the measurement of additional parameters defining single subfractions of cells, as for example the ratios of diploid, aneuploid, tetraploid, octaploid and 16-ploid cells. The main objective of this study was to define the prognostic significance of these new parameters in 106 primary melanomas with known survival time. 29 out of 106 melanomas were euploid. 77 out of 106 showed an aneuploid histogram. Multivariate analysis with Cox regression demonstrated that the percentage of aneuploid cells between 2c and 4c and the percentage of aneuploid cells between 4c and 8c, but not 5c exceeding rate, were able to influence survival time.

Relationship between GAGE-1/-2 expression, EBV infection and interferon-gamma expression in undifferentiated carcinoma of nasopharyngeal type.

GAGE-1/-2 proteins are novel tumour markers, functionally related to tumour rejection. The objective of the present study was to identify the existence of a relationship between GAGE-1/-2 expression, Epstein Barr Virus (EBV) infection and viral infection-induced cytokine expression in cultivated tumour cells and archival specimens of undifferentiated carcinoma of nasopharyngeal type (UCNT). PCR and in situ hybridization techniques were employed. In cultivated UCNT cells, interferon-gamma (IFN-gamma) induced synthesis of GAGE-1/-2 mRNA. In archival tumour specimens (n = 10) however, GAGE-1/-2 gene expression was detected in only 3/8 cases with coincident EBV infection and IFN-gamma expression. In conclusion, EBV infection appears to induce IFN-gamma gene expression in most tumors, but GAGE-1/-2 expression in only some tumours. The role of IFN-gamma and other factors in triggering GAGE-1/-2 gene activation must be elucidated further. The relevance of GAGE-1/-2 gene expression and its detection by PCR for future immunotherapy is discussed.

Proliferative activity in the progression of pigmented skin lesions, diagnostic and prognostic significance.

Proliferative compartments of a tumour can be determined cytophotometrically, by in situ hybridisation or by immunohistochemical detection of Ki67 antigen. The main objective of this study was to analyse the proliferative activity during the progression of pigmented skin lesions with respect to differential diagnostic and prognostic applications. The material investigated consisted of 209 pigmented skin lesions (31 naevi, 30 dysplastic naevi, 106 primary melanomas, 20 lymphatic and 22 organ melanoma metastases). Comparison of the ratios of cells in the S-phase gained by two different methods (cytometry, in situ hybridisation) did not show any significant differences. The correlations between Ki67 and S-phase indices in every diagnostic group were highly significant. The results of forward and backward Cox regression were identical and only Ki67 showed an independent prognostic influence (p < 0.001, coefficient in regression 0.02) with change in risk 2% and confidence limit ranging between 1.1% and 2.9%.

Comparison of the DNA content in liver cell adenoma, hepatocellular carcinoma and regenerative nodules.

With regard to neoplasms of hepatocytes, diagnostic pitfalls have been reported for differentiation of liver cell adenoma (LCA) from well differentiated hepatocellular carcinoma (HCC). Since cytophotometric analysis of the DNA content with the help of image analysis has proven to be of diagnostic value in various neoplasms, we examined its ability to discriminate between LCA and HCC as well as regenerative liver nodules. The material investigated consisted of 54 cases of HCC, 10 benign liver tumours and 10 cases suspicious for HCC. All the benign liver tumours demonstrated an euploid histogram. 9 out of 10 borderline tumours were euploid while 1 out of 10 was suspiciously aneuploid. Among HCC, 21 out of 54 were euploid, 18 out of 54 suspiciously and 15 out of 54 clearly aneuploid. 5c exceeding rate differed significantly between benign liver changes and borderline lesions (p = 0.0474) as well as between borderline lesions and malignant tumours (p = 0.0108). We conclude that the use of image cytometry is helpful as an additional criterion for more diagnostic accuracy in morphologically difficult cases.

Radiation induced early nuclear changes in cytologic brush specimens from the upper aerodigestive tract.

OBJECTIVE: To evaluate the feasibility of DNA image cytometry to assess radiation induced early nuclear changes in cytologic smears from the upper aerodigestive tract. METHODS: 100 cytologic brush specimens from 50 postoperatively irradiated patients with upper aerodigestive tract carcinoma were examined Smears were taken from clinically inconspicuous mucosa from the former tumor site and from a control region (contralateral side). Cellular DNA was quantified using the CAS 200 image analysis system on Feulgen stained slides. DNA histograms were evaluated using the coefficient of variation (CV), the DNA index (DI), the 2c deviation index (2cDI) and the 2.5c exceeding rate (2.5cER). RESULTS: All specimens from the control region were diploid. Forty-six smears from the former tumor region were diploid and in four cases non-diploid cells were found. Single cells with a DNA content exceeding 5c ('5c exceeding events'), were identified in three non-diploid and six diploid smears from the former tumor region. CONCLUSION: DNA analysis is a useful tool in detecting early nuclear changes preceding morphologic changes.

MAGE-1, GAGE-1/-2 gene expression in FNAB of classic variant of papillary thyroid carcinoma and papillary hyperplasia in nodular goiter.

Differentiation of the papillary variant of papillary thyroid carcinoma (PTC) from papillary hyperplasia in nodular goiter may be difficult in fine-needle aspiration biopsy (FNAB) by means of morphology alone. To improve cytodiagnostic accuracy the occurrence of MAGE-1, GAGE-1/-2 gene expression was analyzed by means of RT-PCR. The genes investigated are recognized by autologous T lymphocytes and are expressed in carcinomas of various sites e.g. lung, ovary, colon but not in non-neoplastic tissue except testis. Routinely obtained smears with cytologic diagnosis of PTC confirmed by histology (n=20) and diagnosis of nodular goiter (n=10) were investigated. The MAGE-1, GAGE-1/-2 PCR products were found in 6/20 of the carcinomas but in none of the benign lesions. To identify PCR products automatic gene-sequencing in all positive cases was performed. The data indicate that MAGE-1, GAGE-1/-2 gene expression may give additional information to delineate PTC from papillary hyperplasia in FNAB.

Expression of DNA mismatch repair genes in naevi.

The significant difference of DNA mismatch repair genes expression between naevi and melanomas was demonstrated by our research group in the previous study. The main aim of this study was to compare the expression of MLH1, MSH2, PMS1 and PMS2 in 31 naevus cell naevi, 12 fibromatous naevi and 30 dysplastic naevi. The expression of DNA mismatch repair proteins was found in all naevi investigated. However, the expression (percentage of positively stained cells) was significantly higher in naevus cell naevi than in dysplastic and fibromatous ones.

Bcl2 and Bax expression in naevi and melanomas and their relation to ploidy status and proliferation.

Twenty-five naevi, 53 primary melanomas, 36 melanoma metastases were stained immunohistochemically for the presence of Bcl2, Bax and Ki-67 antigen in order to define the relation between apoptosis regulators and proliferative activity. Additionally, ploidy status and S-phase were measured. Bax demonstrated a tendency to increase and Bcl2 was decreasing along with melanoma progression. In the group of euploid cases Bcl2 showed a strong significant correlation with Bax expression (p = 0.0001) and both Bcl2 and Bax correlated with Ki-67 expression and S-phase. In the group of aneuploid cases only the correlation Bcl2-Ki-67 was preserved. Others did not reach the level of statistical significance.

Different gene expression of MDM2, GAGE-1, -2 and FHIT in hepatocellular carcinoma and focal nodular hyperplasia.

Overexpression and/or mutations of oncogenes, tumour suppressor genes and tumour rejection genes have been observed in several human malignancies. Their analyses might be of diagnostic importance. Therefore, malignant hepatocytes derived from hepatocellular carcinoma (HCC) tissue as well as non-malignant hepatocytes derived from focal nodular hyperplasia (FNH) were studied. Samples containing normal human hepatocytes (HC) served as controls. Cellular material was obtained by fine-needle aspiration biopsy guided by ultrasound. Cells were analysed for expression and mutation of the oncogene MDM2, the genes GAGE-1, -2 coding for tumour-associated antigens and the candidate tumour suppressor gene FHIT. Different patterns of non-mutant FHIT transcripts including precise deletion of exons were found in 7/10 HCC, 2/10 FNH and 2/10 HC. However, expression of non-mutant GAGE-1, -2 RNA was demonstrated exclusively in 6/10 HCC samples. Further genetic features specific of HCC were point mutations in a zinc-finger motif of MDM2 (3/10 HCC samples). Neither GAGE-1, -2 expression nor MDM2 mutations were observed in the FNH samples, or in normal hepatocytes. Our findings suggest that occurrence of variable FHIT transcripts is not restricted to hepatic malignant tumours. In contrast, MDM2 mutations and GAGE-1, -2 expression were associated with HCC specimens. Therefore, the RT-PCR assays for GAGE-1, -2 and MDM2 might be useful adjuncts in cytodiagnosis of liver neoplasms.

How can one explain aneuploidy status in fibromatous and lipomatous naevus cell naevus?

34 lightly fibromatous, 23 heavily fibromatous, 5 lipomatous and 10 naevus cell naevi were stained with Feulgen kit in order to evaluate their ploidy status with CAS 200 image analyzer. 26/34 lightly fibromatous, 18/23 heavily fibromatous, and 5/5 lipomatous naevi were either suspicious for aneuploidy (Auer III) or clearly aneuploid (Auer IV). In contrast all 10/10 naevus cell naevi were euploid. Proliferation (S-phase) was not increased in naevi fibromatously and lipomatously changed. The mechanisms leading to aneuploidy are discussed.

Loss of DNA-mismatch repair gene expression in oral melanomas.

The defect of DNA mismatch repair is postulated to be responsible for malignant transformation in many types of tumours. The main aim of this study was to evaluate the expression of DNA mismatch repair proteins in 29 cases of oral melanomas and to relate this to the ploidy status of the lesions. MLH1 expression was found in 4/29, MSH2 in 6/29, PMS2 in 2/29 and PMS1 in 0/29 cases investigated. The range of positively stained cells did not exceed 50% with MSH2, and PMS2, or 5% with MLH1. Loss of the DNA mismatch repair gene expression correlated with high aneuploidy ratio, observed in totally negative cases.

Myoepithelial sialadenitis versus low-grade non-Hodgkin's lymphoma of the salivary gland in FNAB: is discrimination by means of an image processing system possible?

The diagnosis of myoepithelial sialadenitis (MESA) in fine needle aspiration biopsy may be difficult. There is a dense lymphocytic infiltration in the gland and discrimination between a hyperimmune reaction and a low grade non-Hodgkin's lymphoma (NHL) of B-cell origin may be impossible. To get additional diagnostically helpful criteria, texture feature analysis on routinely obtained FNAB's of the salivary gland was applied. In the data set 36/36 cases of low grade B-NHL confirmed by histology and 10/13 histologically confirmed cases of MESA could be classified correctly by means of an image processing system. The chromatin structure of each nucleus was classified by texture features (n = 6), which were determined according to the method of Harms et al. For statistical analysis of the cell types a classification tree based on the commercial program CART was applied. The data set of 49 cases was proved by the crossvalidation test 10 fold. The calculated diagnosis for each case suggests that this method may be helpful in the cytologically doubtful cases.

Comparison of the DNA content in the different steps of cutaneous carcinoma progression.

The main aim of this study was to check the application of ploidy related parameters for differential diagnosis between solar keratosis, Bowen's disease and squamous cell carcinoma of the skin. 10 specimens of normal epidermis, 68 solar keratoses, 50 cases of Bowen's disease and 10 squamous cell carcinomas (SCC) were stained with Feulgen and their ploidy status was evaluated with a CAS200 image analyzer. Significant and highly significant differences between groups investigated not only for 5c exceeding rate, but also for the ratios of diploid, aneuploid, tetraploid, octaploid and 16-ploid cells are demonstrated. With respect to ploidy associated parameters solar keratosis is closer related to SCC of the skin than Bowen's disease. These findings are in accordance with a higher risk of progression to SCC inspite of a generally lower degree of atypia.