The in situ polymerase chain reaction for detection of chlamydia trachomatis.

The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features.

Gene expression analysis of the catalytic subunit of human telomerase (hEST2) in the differential diagnosis of serous effusions.

Diagnostic accuracy in effusion cytology based on morphologic examination is not always satisfactory. Therefore, various diagnostic adjuncts such as immunocytochemistry or deoxyribonucleic acid cytometry are employed in this diagnostic field. Recently, demonstration of telomerase activity has been proposed as a possible marker for malignancy. In this study a seminested reverse transcription-polymerase chain reaction (RT-PCR) strategy for expression analysis of the catalytic subunit of human telomerase (hEST2) was used in 58 serous effusions. RT-PCR results correlated with cytologic diagnoses in 14 of 17 malignant effusions. In eight effusions cytologically suspicious for malignancy, PCR results were in accordance with the clinical follow-up. However, hEST2 RT-PCR was also positive in six of 15 cytologically benign effusions that consisted predominantly of inflammatory and mesothelial cells. Using the telomeric repeat amplification protocol, it could be demonstrated that cultured, proliferating benign mesothelial cells may present a weak telomerase activity, as is known in other benign cells including activated lymphocytes. In conclusion, the simple and rapid method of hEST2 RT-PCR serves to support the cytologic diagnosis of malignancy, but false-positive PCR results resulting from activated lymphocytes and proliferating mesothelial cells must be considered.

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.

Comparative study of the expression of DNA mismatch repair genes, the adenomatous polyposis coli gene and growth arrest DNA damage genes in melanoma recurrences and metastases.

The main goal of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2), the adenomatous polyposis coli (APC) gene and growth arrest DNA damage inducible (GADD) genes (GADD34, GADD45 and GADD153) in the different stages of melanoma recurrences and metastases, and to identify any mutual consistencies in their expression pattern. All the cases of primary melanoma examined showed a reduced expression of DNA repair genes. These results demonstrate that disturbances of DNA repair begin in the early stages of melanoma. No significant differences were found in the expression of these markers between cutaneous melanomas and their recurrences and metastases (P> 0.05). Eighteen significant correlations between markers were found in the primary melanomas, and 10 significant correlations were observed in the first recurrences of melanoma. In contrast, 27 statistically significant relationships were demonstrated in metastatic lymph nodes. The different correlations found in primary and metastatic tumours confirmed the hypothetical difference in marker interaction in the diagnostic groups investigated. Our results suggest that DNA repair genes may play an important role in the recurrence and metastasis of melanomas.

The value of PCR technique in fine needle aspiration biopsy of salivary gland for diagnosis of low-grade B-cell lymphoma.

In fine needle aspiration biopsy (FNAB) of salivary gland delineation of low-grade B-cell lymphoma from benign lymphoid lesions of myoepithelial sialadenitis (MESA) may be very difficult by means of cytomorphological criteria alone. To improve cytodiagnosis PCR technique was applied on routinely stained smears to determine clonal status by amplifying the third complementarity-determining region (CDR3) of the hypervariable domain of the immunoglobulin heavy chain. Twelve cases diagnosed cytologically as suspicious of low-grade B-NHL with following histology of B-NHL (n = 5) or MESA (n = 7) were analyzed. The CDR3-IgH PCR produced distinct bands in 10/12 cases. The PCR products were analyzed with Genescan software on the DNA sequencer, which demonstrated monoclonal bands in all NHLs and in one case of MESA. The results indicate that PCR technique may be helpful in improving cytodiagnostic accuracy for recognition of low-grade B-NHL of salivary gland.

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.

Comparison of ploidy status of melanoma metastases in different locations.

The main aim of this study was to evaluate the DNA content and ploidy status of 29 melanoma metastases in lymph nodes, as well as 10 liver, 12 brain, 13 lung and 2 gastrointestinal metastases. All cases investigated were either suspicious to be aneuploid or clearly aneuploid. This study demonstrates differences of ploidy related parameters between melanoma metastases of different locations. The 5c exceeding rate values were the lowest in lymph node metastases and the highest in melanoma cells in the brain (p<0.05). The rate of cells in S-phase ranged between 12% in gastrointestinal metastases and 23% in liver metastases. The melanoma cells, which formed liver metastases had smaller area, lower mass and bigger shape factors in comparison with melanoma cells in lymph nodes.

Analysis of the DNA content in Bowen's disease.

The aim of this study was to evaluate the DNA content in Bowen's disease in comparison to healthy epidermis applying image cytophotometry. The material investigated was derived from 50 patients with Bowen's disease and 10 patients with healthy skin. For comparison of both groups the Kruskal-Wallis test was applied. Slides were stained with Feulgen and were evaluated with CAS-200 image analyzer. Only 7/50 morbus Bowen cases represented euploid histogram. The others 43/50 were either conspicious to be aneuploid (29/50) or clearly aneuploid (14/50). In contrast, all normal epidermis (10/10) were clearly euploid. Morbus Bowen cases demonstrated significantly higher 5c exceeding rate (p=0.0012) and significantly more cells in the S-phase (p=0.017). High aneuploidy rate and increased proliferative activity in morbus Bowen cases support the classification of these lesion as carcinoma in situ.

Comparison of the DNA content in low and high grade non-Hodgkin lymphomas.

In the fine needle aspiration biopsy (FNAB) of lymph nodes, the deliniation of low grade non-Hodgkin lymphoma (NHL) from high grade NHL by means of morphology may be difficult. To gain additional diagnostically helpful criteria, cytophotometric analysis of the DNA content was applied. 117 specimens of NHL were examined, including 51 cases of low grade NHL (14 centroblastic-centrocytic, 9 centrocytic, 14 lymphoplasmocytoid immnocytomas and 14 chronic Iymphocytic leukemias) and 66 cases of high grade NHL (15 centroblastic, 18 immunoblastic, 7 Burkitt and 26 lymphoblastic). The smears were stained with Feulgen and were evaluated by image analysis (CAS 200, Becton- Dickinson). The 5 c exceeding rate differed significantly (p = 0.0081) between low- and hig-grade NHL. In the group of low grade NHL, clearly aneuploid cases were not found (Auer type I 32%, type II 62%, type III 6% of cases). The high grade NHL's showed histograms Auer type II (10% of cases), type III (62% of cases) as well as clearly aneuploid type IV (28% of cases). In summary, image cytophotometry can be used along side morphology to differentiate between low and high grade lymphomas in morphologically difficult cases.

Comparative cytophotometric analysis of reactive lymphoid hyperplasia and low malignant non-Hodgkin lymphoma.

In fine needle aspiration biopsy (FNAB) of lymph nodes, differential diagnosis between reactive lymphoid hyperplasia (RLH) and low grade non-Hodgkin lymphoma (NHL) may be sometimes difficult. Cytophotometric analysis of DNA content using image analysis has found practical application in the determination of malignancy in different tumours. In this study, image cytometry was applied to compare the ploidy status of the above mentioned lesions to obtain diagnostically helpful information. Thirty smears of histologically proven low grade NHL (10 follicular lymphomas of centroblastic-centrocytic type, 10 lympho-plasmocytoid immunocytomas, 10 chronic lymphocytic leukemias) and 15 smears of RLH were stained with Feulgen and examined by image analysis (CAS 200, Becton Dickinson). The 5c exceeding rate was determined according to the Boecking's definition of aneuploidy. Concerning the histogram types the Auer classification was used. The aneuploidy was found in 4/10 follicular lymphomas and 4/10 lymphoplasmocytoid immunocytomas. All chronic lymphocytic leukemias were diploid. In none RLH a 5c exceeding rate was found. The results verify the value of image cytophotometry as a helpful method in some diagnostically difficult cases.

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.

Analysis of the DNA content in the progression of recurrent and metastatic melanomas.

Ploidy status and ploidy related parameters of 18 primary melanomas, 32 recurrences and 18 lymphatic metastases were investigated applying CAS200 image analyzer. All the tumours investigated were either suspicious for aneuploidy (Auer III) or clearly aneuploid (Auer IV). Primary melanomas differed from recurrent tumours concerning the percentage of aneuploid cells between 4c and 8c and 5c ER. Comparison of cutaneous tumours with lymphatic metastases showed a significant difference concerning the percentage of aneuploid cells between 2c and 4c. An already high aneuploidy rate in primary tumours suggests that recurrent and metastatic clones of cells are present in early stages and that aneuploidy status in primary melanomas could be regarded as one of the risk factors of recurrences and metastases.

Analysis of the DNA mismatch repair proteins expression in malignant melanomas.

The importance of properly functioning DNA mismatch repair has been shown in several tumour types both hereditary and sporadic, but not yet in malignant melanomas. The aim of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2) in primary melanomas and to define their possible prognostic impact in 106 primary melanomas. MLH1 was found in 64 and MSH2 in 61 out of 106 melanomas. PMS1 and PMS2 proteins were present in 69 and 67 tumours, respectively. Loss of the expression of DNA mismatch repair proteins correlated with the increase of Clark levels. Cox regression analysis demonstrated some prognostic significance for PMS1 (forward p = 0.0018 and backward selections p = 0.0277), MLH1 (only forward selection p = 0.0081) and MSH2 (only backward selection p = 0.0115).

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.

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.

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%.

Immunocytochemical determination of the tumor-associated glycoproteins MCA, CA 125 and BW 495/36-P in urine of patients with epithelial tumors of the kidney and urinary bladder.

Pre-operative and, in some cases, post-operative urine samples from 29 patients with renal cell or urinary bladder carcinoma were compared to samples from 24 healthy persons and 10 patients with nephrolithiasis and 9 patients with other benign disorders of the efferent urinary tract. The specimens were examined for the presence of MCA, CA 125 and BW 495/36-P expressing epithelial cells. The urine concentrations of the soluble antigens MCA and CA 125 were determined simultaneously in urine samples from 35 patients with renal cell or urinary bladder carcinoma, 10 patients with cystitis and 30 healthy individuals. MCA and BW 495/36-P expressing epithelial cells were significantly increased in all pre-operative urine samples of the tumor patients compared to the group of healthy persons. This increase was also seen with CA 125-positive cells in patients with bladder carcinoma, not however in patients with renal cell carcinoma. BW 495/36-P positive cells were also found in both groups of tumor patients in greater numbers than in the patients with nephrolithiasis or other benign urinary tract disorders. Based on a specificity of 97% when compared to the control urine samples, the cytological determination of the antigens MCA, CA 125 and BW 495/36-P in urinary tract cells of all tumor patients revealed a sensitivity of 48%, 33% and 79% as well as a positive predictive value of 92%, 89% and 95%, respectively. The sensitivity of CA 125 increased to 67% upon isolated analysis of patients with bladder carcinoma. The majority of labelled cells were not identifiable as tumor cells morphologically and appeared as normal transitional epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the tumor-associated glycoproteins MCA, CA 125 and BW 495/36-P in epithelial tumors of the kidney and the urinary bladder.

The differential expression of the tumor-associated glycoproteins MCA, CA 125 and BW 495/36-P was investigated in 11 renal cell carcinomas and 11 urinary bladder carcinomas and compared with their expression in non-neoplastic tissue preparations from the kidney (n = 9) and urinary bladder (n = 12). The glycoproteins were demonstrated immunohistologically in frozen sections and additionally, in some cases, in paraffin sections. MCA and BW 495/36-P positive cells were present in all preparations except for a grade I transitional cell carcinoma of the bladder, in which no MCA-expression could be detected. In the non-neoplastic renal tissue mainly the cells of the distal tubuli were stained by the antibodies against these two glycoproteins. Carcinoma cells of the kidney and of the urinary bladder showed an increased expression of both epitopes. CA 125, in comparison, was strongly expressed in 3 of the 11 urinary bladder carcinomas investigated but could only be shown in a few cells of a single renal cell carcinoma. Normal renal tissue showed no and the urinary bladder only very isolated CA 125 positive epithelial cells. Apart from this distribution, strong staining of the connective tissue fibers with CA 125 antibody was seen in all paraffin sections, but not in the frozen sections. This leads to the supposition that in these structures there is a CA 125 cryptantigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between DNA ploidy-related parameters and the deletions in mismatch repair genes MLH1 and MSH2 in lentigo maligna and malignant melanomas.

Defects in DNA mismatch repair genes MLH1 and MSH2, first described in hereditary nonpolyposis colon cancer (HNPCC), have been postulated to be responsible for malignant transformation in several tumours. To date there are no data on cutaneous tumours. Using a PCR assay it was possible to identify deletions in MSH2 (exonic regions 12 and 13) in 16 of 47 lentigos maligna and in 10 of 36 malignant melanomas. Deletions in MLH1 (exonic regions 15 and 16) were found in 11 of 47 lentigos and in 15 of 36 melanomas. Comparison of DNA ploidy-related parameters between lentigos with and without exonic deletions in MSH2 and MLH1 did not show any significant differences. In contrast, melanomas positive and negative for exons 12 and 13 (MSH2) (26/36 and 10/36, respectively) differed significantly with respect to the percentages of diploid cells (P = 0.0179) and tetraploid cells (P = 0.0042). Comparison of melanomas positive and negative for exons 15 and 16 (MLH1) (21/36 and 15/36, respectively) showed significant differences in the percentage of aneuploid cells between 2c and 4c (P = 0.0141) and tetraploid cells (P = 0.0404). In summary, deletions in DNA mismatch repair proteins MSH2 and MLH1 were present both in lentigo maligna and in melanomas and correlated with DNA ploidy-related parameters in malignant melanomas.