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.

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.

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.

Acute peripheral facial palsy in Lyme disease -- a distal neuritis at the infection site.

AIM: Children with acute peripheral facial palsy have often suffered tick bites and/or erythema migrans in the head/neck region on the same side. With respect to the pathogenesis of neuroborreliosis this topographical association was investigated in an animal model. METHODS: A Borrelia garinii strain, isolated from the CSF of a child with acute facial palsy, was injected in 9 rats intracutaneously in the right subauricular region. Infected rats were examined for clinical symptoms of Lyme disease, the spread of the spirochetes was investigated by PCR of necropsies (facial nerves, trigeminus nerves, heart, brain, skin) up to 47 days after infection. The nerve tissues were investigated by histology, immunohistochemistry and electron microscopy. RESULTS: None of the rats developed a facial palsy or other symptoms of Lyme disease. Borrelia DNA was found in the heart after 5 days and in the brain after 7 days of infection up to the end of investigation (47 days), as well as in the ipsilateral peripheral nerves after 7 to 33 days. Borrelia was detected by electron microscopy near endoneural vessels of the facial nerve. Peri-, epi-, and endoneural infiltrations of macrophages, plasma cells and B cells characterized an inflammation of the facial and trigeminus nerves ipsilateral to the infection site. CONCLUSION: An infection with Borrelia garinii in the subauricular region induces an ipsilateral neuritis of peripheral nerves. The particular vulnerability of the human facial nerve may be a result of its long intraosseus course. Thus, an inflammatory edema may injure the nerve in the canalis facialis.

Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation.

Testicular germ cell tumours (TGCT) represent the most common malignancies in young males. Whereas in 1970s, the survival rate in patients with metastatic testicular tumours was only 5%, these days, 80% of the patients treated by modern chemotherapy will survive their disease. The drug that revolutionised the cure rate for patients with metastatic testicular tumours was cisdiamminedichloroplatinum (cisplatin, CDDP). In vitro experiments on neoplastic germ cell lines showed that their exquisite sensitivity to CDDP could be attributed to p53-dependent and -independent pathways. Applying cDNA macroarray, semiquantitative RT-PCR and Western blot analyses, blocking experiments, caspase activity assays, and morphological methods, we sought here to define the p53-independent pathway(s) involved in the CDDP-induced apoptosis. For this purpose, we used the human TGCT cell line NCCIT, the mutated p53 of which is known to remain inactive during the course of CDDP-induced apoptosis. Our experiments showed that within hours of CDDP application, two prototype members of the 'mitogen-activated protein kinase' (MAPK) family, designated 'MAPK ERK kinase' (MEK) and 'extracellular signal-regulated kinase' (ERK), were dually phosphorylated and caspase-3 became active. Functional assays using MEK inhibitors demonstrated that the phosphorylation of MEK and ERK was required for the activation of caspase-3 as the executing caspase. Interestingly, experiments with the human malignant germ cell line NTERA, which is known to possess wild-type p53, revealed the same results. Thus, our data suggest that CDDP mediates its p53-independent apoptosis-inducing effect on the malignant human testicular germ cells--at least partially--through activation of the MEK-ERK signalling pathway. July 2004

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.

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.

Cirrhotic livers reveal genetic changes in the MDM2-P14ARF system of cell cycle regulators.

The genesis of hepatocellular carcinoma is promoted by changes in the regulatory MDM2-P14ARF system. The incidence of such changes has to date not been analysed in non-tumourous livers showing regenerative proliferation. In the present study, 24 cirrhotic livers of alcohol-, autoimmue disorder- or HCV-caused genesis were screened for MDM2-P14ARF alterations at the level of protein, DNA and mRNA. Using confocal laser scanning microscopy, the absence of MDM2 and P14ARF expression was detected in all samples except three HCV-infected livers (four livers) which contained hepatocytes overexpressing MDM2 (P14ARF) protein. In two of the samples lacking P14ARF expression, laser microdissection and PCR demonstrated deletion of the P14ARF gene. The P14ARF gene amplified from other specimens did not carry mutations. MDM2 splicing variants were present in tissues from alcohol- and autoimmune disorder-induced cirrhoses. Sequencing of full-size mRNA revealed a MDM2 mis-sense mutation in an alcohol-induced cirrhosis. One sample contained regenerative nodules with genetic instability occurring at MDM2 locus D12S83 according to the data of automatic PCR fragment analysis. In summary, this study gives first evidence for different types of MDM2 and P14ARF alterations in cirrhotic livers. We suggest that the changes impair the regulatory MDM2-P14ARF system, thus possibly favouring regenerative proliferation and transformation.

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.

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas.

Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70 +/- 9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63+/-8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32+/-6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.

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.

Fragments of human oncoprotein MDM2 reveal variable distribution within and on cultivated human hepatoma cells.

Human oncoprotein MDM2 reveals a MHC class I binding motif HMDM441 characterizing MDM2 as a potential tumor antigen. To analyze the distribution of MDM2 proteins containing this motif in liver cancer cells we produced rabbit anti-HMDM441 serum. The novel antibodies bound to an MDM2 fragment of approximately 55 kDa which lacked the N-terminal region and was present in lysate and supernatant of a human hepatoma cell line overexpressing normal 90-kDa MDM2. The 55-kDa fragment was detected in the cytoplasm and nucleoli and at the nuclear envelope of hepatoma cells, whereas normal hepatocytes were negative. Double-fluorescence labeling indicated that the MDM2 fragments and MHC class I molecules were coexpressed on the surface of the hepatoma cells. Further studies must clarify whether MDM2 fragments containing motif HMDM441 are novel targets of immunotherapy and immunochemical tumor diagnosis.

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.

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.

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.

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.

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.

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.