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.

Specific autologous anti-melanoma T cell response in vitro using monocyte-derived dendritic cells.

Dendritic cells (DC) are antigen-presenting cells initiating primary and secondary immune responses. Since malignant tumors are able to escape immunologic control, DC might be prime candidates to activate the immune system against tumor cells. In an autologous system, a dynamic interaction among monocyte-derived DC (MoDC), T lymphocytes, and tumor cells obtained from melanoma patients could be noted. MoDC were generated from blood monocytes in the presence of GM-CSF, IL-4, and IFN-gamma. T cells were isolated either from peripheral blood or from lymph nodes. Melanoma cells were harvested from surgically removed tumor metastases. They were then gamma-irradiated and co-cultured with autologous MoDC and T lymphocytes. After 5 days, the lymphocytes showed a high proliferative activity and the majority of them were CD8-positive. In five cases tested, they revealed a high cytotoxic activity resulting in apoptosis of tumor cells. These findings suggest that MoDC are capable of initiating an effective specific anti-tumor response in a strictly autologous mixed lymphocyte tumor culture (MLTC), even though tumor-specific antigens had not been individually defined. Therefore (I) whole melanoma cells can serve as a source of antigen, (II) monocyte-derived dendritic cells may process and present melanoma-specific antigens resulting in a strong lymphocyte proliferation, (III) the majority of responding T lymphocytes are CD8-positive, and (IV) an acquired cytotoxic response eventually leads to apoptosis of the melanoma cells. The reaction demonstrated here permits to in vitro and quantitatively monitoring the effect of T cell directed immunotherapies such as the adoptive immunotherapy of tumors.

GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis.

The main role of growth arrest and DNA damage-inducible (GADD) genes is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The goal of this study was to examine the expression of GADD genes in primary melanomas with respect to prognosis. GADD34 was found in 73% of the primary melanomas investigated. GADD45 and GADD153 were positive in 60% and 80% of primary melanomas, respectively. Cox regression demonstrated that only GADD153 had any independent prognostic impact. We therefore decided to establish a PCR assay for detection of GADD153 in paraffin-embedded tissue. GADD153 deletion was found in 3/26 melanomas. None of the 3 cases with GADD153 deletion showed any expression of GADD153. Sequencing analysis detected polymorphism T-C at amino acid position 10 in 6/23 melanomas. In 6 cases with GADD153 polymorphism, GADD153 expression was found in 2 melanomas with a maximum GADD153 index of 10%. We postulate that the GADD gene family plays an important role in melanoma progression.

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.

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.

Identification of an endoflagellar associated protein in Borrelia burgdorferi.

DNA of Borrelia burgdorferi was cleaved by the endonuclease EcoRI and ligated with the bacteriophage expression vector lambda gt11. After infection of the Escherichia coli strain Y1089, the plaques of recombinant phages were screened with a B. burgdorferi antiserum (human) for fusion proteins containing borrelia antigen.s A positive clone produced a hybrid protein (p200) of c. 200 Kda. The corresponding native borrelia protein (p97) was identified as having an Mr of 97 Kda. To localise protein p97 in the B. burgdorferi cell, immunoelectronmicroscopy and a Western blot of isolated flagella were used. Antibodies directed against proteins p200 and p97 recognised epitopes associated with the flagella.

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.

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.

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.

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.

A MboII polymorphism in exon 11 of the human MDM2 gene occuring in normal blood donors and in soft tissue sarcoma patients: an indication for an increased cancer susceptibility?

The human MDM2 oncogene, well known as the tumor suppressor gene p53's partner, plays an important role in tumorigenesis whether it is dependent on or independent of TP53. In this study, we investigated in a PCR-sequencing analysis the exon 11 of the human MDM2 gene for gene alterations. A MboII polymorphism occurs in 8% of normal blood donors (8 out of 100 probands) and in 13% of the soft tissue sarcoma patients (11 out of 82 patients). Of note was that two STS patients carried the gene alteration only in the tumor specimens heterozygously but not in normal tissue. In a Kaplan-Meier analysis, patients without the polymorphism, indicated a median survival rate of 57 months, whereas, patients with the polymorphism survived on average only 38 months. We suggest that this polymorphism might be associated with an increased cancer susceptibility.

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.

Reverse transcriptase polymerase chain reaction for detecting Ewing's sarcoma in archival fine needle aspiration biopsies.

OBJECTIVE: Previous studies on pediatric soft tissue sarcomas have demonstrated a chromosomal 11;22 (q24;q12) translocation in Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumors that appears to be a unique oncogenic marker. To investigate the usefulness of reverse transcriptase polymerase chain reaction (RT-PCR) as an ancillary method for cytodiagnosis, we tested archival aspirates derived from ES patients to establish whether any beta-actin RNA expression or tumor-specific EWS/FLI-1 gene translocation had occurred. STUDY DESIGN: Sixteen skeletal specimens aspirated 10-15 years earlier from patients with cytologic and histologic diagnoses of ES were prepared for PCR. The amplification products were sequenced. RESULTS: Amplifiable RNA was detected in smears from 12 patients by beta-actin RT-PCR. Seven aspirates from beta-actin-positive patients showed ES-specific genomic translocation (58%). Sequence analysis of the resulting PCR fragments revealed EWS/FLI-1 fusion transcriptions of varying length. CONCLUSION: When RNA was retrievable, RT-PCR applied to routinely stained aspirate smears was a highly specific method in differential diagnosis of ES.

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.

Autoantibodies to human manganese superoxide dismutase (MnSOD) in children with facial palsy due to neuroborreliosis.

AIM: Acute peripheral facial palsy due to neuroborreliosis is associated with a distal neuritis. In patients with Lyme disease the activity of antioxidant enzymes is decreased. With respect to the pathogenesis of neuroborreliosis, sera of children with acute peripheral facial palsy were investigated for autoantibodies against human manganese superoxide dismutase (MnSOD), which were suspected of raising the oxidative injury of infected tissues. METHODS: Sera of 20 children with acute peripheral palsy with neuroborreliosis, sera of 20 children with facial palsy without reference to Lyme disease and sera of 14 blood donors were tested for antibodies against human MnSOD using an ELISA. RESULTS: The concentrations of IgM autoantibodies to MnSOD of the children with neuroborreliosis were significantly increased, compared with the two control groups. CONCLUSIONS: We propose that the antibodies detected block the protective effects of MnSOD resulting in an increased oxidative inflammation.

Expression of gastrin releasing Peptide receptor in renal cell carcinomas: a potential function for the regulation of neoangiogenesis and microvascular perfusion.

PURPOSE: Gastrin releasing peptide (GRP) is a growth factor for renal cell carcinoma (RCC) and it has vasoactive properties. Blockade of GRP receptor inhibits the growth of GRP receptor positive and negative tumors in nude mice, suggesting GRP effects other than those related to tumor epithelium. Therefore, in this study we analyzed the effects of GRP receptor blockade on neoangiogenesis in RCC. MATERIALS AND METHODS: GRP receptor expression was determined in human RCC and corresponding normal tissue by real-time reverse transcriptase-polymerase chain reaction, immunohistochemistry and confocal laser scanning microscopy. Multicellular spheroids of the A498 RCC line were implanted into dorsal skin fold chambers of athymic nude mice. Neoangiogenesis was measured by intravital microscopy after blockade of GRP receptors by the GRP antagonist RC-3095. The influence of GRP on vascular endothelial growth factor secretion in A498 cells was studied in vitro. RESULTS: GRP receptor expression was immunolocalized in tumor cells and microvessels. Implanted tumor cell spheroids and spheroid microvessels of the chamber also expressed GRP receptors. Spheroid neoangiogenesis was significantly inhibited by RC-3095 when given immediately after spheroid implantation. Vascular endothelial growth factor secretion of A498 cells was not affected by GRP. CONCLUSIONS: RCC angiogenesis is sensitive to GRP receptor blockade. Therefore, GRP receptors may not only stimulate tumor cell proliferation, but also affect tumor microcirculation.

Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma.

AIMS: To determine the expression of a protein termed augmenter of liver regeneration (ALR), recently found to have a specific and beneficial effect on the process of liver regeneration in normal and diseased human liver. METHODS AND RESULTS: ALR expression in normal and cirrhotic human livers with various underlying diseases as well as in tissue samples of hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) was analysed by immunohistochemistry and quantitative reverse transciptase-polymerase chain reaction (RT-PCR). Expression analysis of ALR in total liver protein extracts by Western blotting showed mainly dimeric ALR protein. Immunohistochemically, cytosolic and perinuclear immunosignals were found in hepatocytes and cholangiocytes in normal, cirrhotic or cancerous liver tissue and only weak signals in some endothelial cells in normal livers. Quantitative mRNA analysis revealed significantly increased ALR expression in cirrhosis compared with normal liver tissue. In HCC and CCC ALR mRNA expression was also significantly enhanced compared with normal liver tissue, but expression levels did not differ from the matching non-neoplastic tissue in the same patient. CONCLUSIONS: The findings suggest an important role for ALR in hepatocellular regeneration in liver cirrhosis as well as in hepatocarcinogenesis and therefore its potential value in the clinical diagnosis of hepatic cirrhosis and cancer.

The 39-kilodalton protein of Borrelia burgdorferi: a target for bactericidal human monoclonal antibodies.

Three human monoclonal immunoglobulin M antibodies against Borrelia burgdorferi, obtained from in vitro-stimulated peripheral blood lymphocytes, reacted in Western blots (immunoblots) with a prominent 39-kDa peptide and a faint band of approximately 66 kDa. Two of these antibodies showed bactericidal activity without addition of complement. All three antibodies were reactive in an enzyme immunoassay with cloned P39 (W.J. Simpson, M.E. Schrumpf, and T.G. Schwan, J. Clin. Microbiol. 28:1329-1337, 1990), suggesting that the target molecule of these antibodies is identical to the P39 protein. In addition, the majority of supernatants from human lymphocytes stimulated in vitro with crude B. burgdorferi antigen reacted in this assay, demonstrating that P39, although a minor component of B. burgdorferi, is an immunodominant antigen in these spirochetes. A fourth monoclonal antibody, reacting with OspA, also exhibited bactericidal activity.

Analysis of the tumour suppressor genes, FHIT and WT-1, and the tumour rejection genes, BAGE, GAGE-1/2, HAGE, MAGE-1, and MAGE-3, in benign and malignant neoplasms of the salivary glands.

AIMS: Molecular genetic changes involved in tumorigenesis and malignant transformation of human tumours are novel targets of cancer diagnosis and treatment. This study aimed to analyse the expression of putative tumour suppressor genes, FHIT and WT-1, and tumour rejection genes, BAGE, GAGE-1/2, MAGE-1, MAGE-3, and HAGE (which are reported to be important in human cancers), in salivary gland neoplasms. METHODS: Gene expression was analysed by reverse transcription polymerase chain reaction (RT-PCR) in normal salivary gland tissue and 44 benign and malignant salivary gland tumours. RESULTS: Aberrant FHIT transcripts were found in one of 38 normal salivary glands, three of 28 adenomas, and two of 16 carcinomas. WT-1 mRNA was detectable in two adenomas and five carcinomas. Immunoblotting showed that WT-1 mRNA expression was associated with raised WT-1 protein concentrations. RT-PCR for detection of BAGE, GAGE, and MAGE gene expression was positive in two adenomas and nine carcinomas, but negative in normal salivary gland tissue. HAGE mRNA was found in two normal salivary glands, 11 benign, and eight malignant tumours. CONCLUSIONS: FHIT mRNA splicing does not appear to be involved in the genesis of salivary gland neoplasms. The upregulation of WT-1 mRNA in tumours of epithelial/myoepithelial phenotype may imply a potential role of WT-1 in the genesis and/or cellular differentiation of these salivary gland tumours. The tumour rejection genes were more frequently, but not exclusively, expressed in malignant salivary gland tumours than in benign neoplasms, although none was suitable as a diagnostic marker of malignancy in salivary gland neoplasms.

Genetic analysis of the human oncoprotein MDM2 in benign and malignant tumors of the salivary gland.

INTRODUCTION: Genetic alterations of oncogene MDM2 promote malignant transformation of several human tumors. In tumors of the salivary gland, however, the genetic status of MDM2 has not been evaluated so far. METHODS AND RESULTS: Benign and malignant tumors of the salivary gland (6 pleomorphic adenomas, 3 Warthin's tumors, 1 adenocarcinoma, 1 basal cell adenocarcinoma, 1 mucoepidermoid carcinoma, 3 acinic cell carcinomas, 2 adenoid cystic carcinoma, 1 squamous cell carcinoma) were analyzed by fluorescence-based PCR techniques and immunochemistry for MDM2 gene amplification, MDM2 gene expression, MDM2 gene mutation, MDM2 RNA splicing and MDM2 accumulation. Data show that all samples contained nonamplified MDM2 genes with nonmutant zinc finger regions. However, in two benign and two malignant samples, novel MDM2 mRNA splicing variant types 1 and 2 were detected. Furthermore, three malignant tumors revealed significant nuclear MDM2 accumulation. Correlation between levels of MDM2 mRNA and MDM2 protein could not be detected in the specimens. CONCLUSION: The present study suggests that MDM2 gene mutation and gene amplification do not contribute to MDM2 accumulation detected in malignant tumors of the salivary gland. However, the role of novel MDM2 splicing variants in MDM2 expression and malignant transformation must be elucidated further.