[Show abstract][Hide abstract] ABSTRACT: We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.
Immunologic Research 12/2014; 61(1-2). DOI:10.1007/s12026-014-8600-6 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Hereditary Non-Polyposis Colorectal Cancer is an inherited disease with deleterious germline mutations in the DNA mismatch repair genes causing the development of colon cancer and other malignancies. This is the first study in Hungary screening the population of our colorectal cancer patients in order to identify the prevalence of the disease.
In families who met the Modified Amsterdam and Bethesda Criteria the removed tumor tissue was first examined by immunohistochemistry and microsatellite instability analysis. Those cases which showed high microsatellite instability underwent DNA sequencing and multiple ligation dependent probe amplification.
Of the 1576 patients with colorectal cancer underwent screening for the modified Amsterdam and Bethesda criteria, 69 (4.4%) and 166 (10.5%) fulfilled the criteria respectively. 15 patients (31 %) of the Amsterdam positive group and 19 patients from the Bethesda positive (18.1%) were MSI-H. There were 8 pathogenic mutations identified in 9 families (60%) in the Amsterdam positive group. 5 mutations were found in 5 families (26%) in the Bethesda positive group. 12 pathogenic mutations were identified, two of these are newly identified, and being published first in this work. These two new mutations were located on MLH1 (g.31276_35231del) and MSH2 (c.969_970delTC) genes.
The prevalence of the mutations in the MLH1 and MSH2 genes was almost equal in our Hungarian colorectal cancer patients. One mutation in the MLH1 gene (c.143A>C; p.Q48P) was identified in three different families. Whether this mutation is the most frequent in the Hungarian population is still unidentified and warrant further investigation.
European Journal of Surgical Oncology 11/2014; 40(11). DOI:10.1016/j.ejso.2014.07.032 · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lynch syndrome (Hereditary nonpolyposis colorectal cancer, HNPCC) is an inherited disease with variable phenotype causing the development of colon cancer and other malignancies. The basis of the disease is believed to be the mismatch repair gene mutations. Genetic screening has been performed among the patients who have undergone surgery for colon cancer at the University of Debrecen, Department of Surgery. Tumor samples of the screened patients were submitted to immunohistochemistry on hMLH1, hMSH2 and hMSH6 genes, microsatellite instability testing, followed by sequencing and multiple ligation dependent probe amplification. Three families were identified with the missense mutation c.143A>C (p.Q48P) of hMLH1 gene. In one of the families a segregation analysis of this particular variant was also accomplished. The segregation analysis revealed a clear correlation between the tumor cases and the occurrence of this mutation. However, none of the analyzed 100 healthy controls demonstrated the same aberration. There is only one published evidence in the literature about the presence of this rare variant in any population. The Gln to Pro switch in the ATPase domain, a conservative region of the hMLH1 gene, creates significant changes in the protein structure. These results indicate that this mutation is the abnormality responsible for the patients' phenotype and it is feasible that this particular aberration occurs more frequently among Hungarian Lynch syndrome patients.
Familial Cancer 03/2012; 11(3). DOI:10.1007/s10689-012-9515-9 · 1.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The somatostatin structural derivative, TT-232, has a special 5-residue ring structure (D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2) and very different characteristics from the known growth hormone (GH) active somatostatin analogs. This somatostatin structural derivative has no GH release inhibitory or antisecretory activity and does not bind to rat pituitary or the cortex, where all the known somatostatin receptor subtypes are expressed. TT-232 had previously been shown to inhibit the proliferation of a large number of cancer cell lines in vitro and reduce the size of different tumors in animal models in vivo.
The therapeutic efficacy of TT-232 was evaluated in different long-term administration routes: the traditional injection (i.p. or s.c.) versus infusion treatment via s.c.- or i.v.-inserted Alzet osmotic minipump, and on different types of transplantable rodent (S-180 sarcoma, P-388sc lymphoid leukemia, Colon-26 adenocarcinoma, MXT breast carcinoma, B-16 melanoma) and human tumor models (HT-18 lymphoid melanoma, T-47/D breast carcinoma, A-431 epidermoid carcinoma). On the basis of our previous experiments the optimum injected dose of TT-232 was found to be 15 microg/kg twice a day. This dose is equivalent to 0.6 dg/day by infusion therapy.
In our experiments, the best results were achieved when TT-232 was applied as an infused treatment. In the S-180 sarcoma and P-388sc lymphoid leukemia rodent tumor models the infusion treatment with TT-232 resulted in 61%-100% tumor growth inhibition and in 20%-60% of the mice being long-term and tumor-free survivors. In the aggressive Colon-26 adenocarcinoma and MXT breast carcinoma models, the infusion treatment resulted in 52%-75% tumor growth inhibition. In the B-16 melanoma model, the infusion treatments resulted in 47% -63% growth inhibition. The tumor growth inhibitory effect of infusion treatment with TT-232 on HT-18 human lymphoid melanoma tumor proved to be significant, resulting in 69%-79% decreases in tumor volume. In the T-47/D human breast carcinoma, the infusion treatment resulted in 48%-53% tumor growth inhibition. The tumor growth inhibitory effect of infusion treatment on A-431 human epidermoid carcinoma tumor resulted in 70%-74% decreases in tumor volume.
The antitumor efficacy of TT-232 was seen in almost all the tumors investigated. In our study, the route of infusion was shown to increase drug efficacy relative to conventional delivery methods (injection). The results obtained from this study suggest that TT-232 is a promising new antitumor agent in cancer chemotherapy and a good candidate for delivery by continuous (infusion) therapy.
Anticancer research 09/2008; 28(5A):2769-74. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hereditary Non-polyposis Colorectal Carcinoma is the most frequent genetic disease leading to colon and other malignancies. Recognizing the condition requires extensive family history going back several generations focusing particularly on the types of tumors occurring in the family at different age groups.
In families who met the Amsterdam and Bethesda Criteria, the removed tumor tissue was first examined by immunohistochemistry and microsatellite instability analysis. Subsequently DNA sequencing was performed to detect an underlying Mismatch Repair Gene mutation and multiple ligation dependent probe amplification was applied for recognizing large deletions in Mismatch Repair Genes.
In the investigated families 3 pathogen mutations, 1 large deletion and 2 cases of polymorphism were found. There is considerable difference between the families in terms of the types of malignancies and the age in which those appeared.
Recognizing families with Hereditary Non-polyposis Colorectal Carcinoma presents great difficulties because of the variety of phenotypes in presentation. Special attention should be paid to small families and those who present with cancer of other than colon origin. Practicing physicians should be made aware of the fact that this disease may have atypical presentations. Follow up of families who have already been screened may be difficult for social, economical or religious reasons.
European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 03/2008; 34(12):1322-7. DOI:10.1016/j.ejso.2008.01.006 · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of germline inactivation of the adenomatosis polyposis coli (APC) gene in hereditary colorectal cancer is well known, being the most important cause of familial adenomatosus polyposis (FAP) syndrome. Hereditary cases with germline mutations, however, account only for 5-10% of colorectal cancers. The somatic inactivation of this gene has also been observed in sporadic cases. In order to examine the inactivation mechanisms of the APC gene we screened 70 sporadic colorectal cancer cases (27 rectal, 43 intestinal) of different stages for promoter hypermethylation, allelic imbalance (AI) and somatic mutations. The presence of promoter hypermethylation was observed in 21 cases (30%). Fifteen of the examined tumors (21%) showed AI, and also 15 tumors (21%) carried at least one somatic mutation. Thirteen of the detected alterations were novel variations: seven frameshifts, four missense mutations and two polymorphisms. Biallelic inactivation was found in 15 patients (21%). These results suggest that the inactivation of the APC gene is very common in sporadic colorectal cancer, and the main inactivation mechanism of the APC gene is promoter hypermethylation. Allelic imbalance has the same frequency as mutations, and mutations in the APC gene are more common in the early stages and in tumors located in the rectum.
[Show abstract][Hide abstract] ABSTRACT: Retinoids as important growth and differentiation regulating agents have a potential role in the chemoprevention of head and neck squamous cell carcinoma (HNSCC). Despite the promising preclinical and early clinical findings, limitations of application are raised by intrinsic resistance acquired during carcinogenesis. Retinoic acid receptor beta2 (RAR beta2) is one of the proximate mediators of retinoid signalling and its expression is often diminished in early stages of head and neck carcinogenesis. One form of retinoid resistance has been associated with the methylation-induced silencing of the RAR beta gene. We studied primary HNSCC samples of different anatomical sites in respect of methylation, expression and allelic loss of RAR beta gene. A strong correlation (p<0.01) was found between hyper-methylation and reduced expression of RAR beta2, however the allelic loss at 3p24, the locus of RAR beta, did not considerably influence its mRNA level. Hypopharynx tumors showed significantly lower hypermethylation (p<0.05) and higher mRNA expression levels of RAR beta2 compared to the tumors located at other sites of the head and neck. We could also provide evidence that poorly differentiated grade 3 tumors had significantly higher RAR beta2 expression and lower methylation levels (p<0.05) than better differentiated grade 1 and grade 2 tumors. In addition, we found a good correlation between the methylation degree of the RAR beta2 promoter and the ages of patients. Collectively, our results suggest that evaluation of several factors such as tumor location, age, histology and methylation state of the RAR beta gene might contribute to the selection of patients for retinoid-based chemoprevention.
[Show abstract][Hide abstract] ABSTRACT: Direct antitumor activity of sea lamprey (Petromyzon marinus) gonadotropin-releasing hormone III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2); lGnRH-III) was described on several tumor cells. To improve the selectivity of antitumor effects without increasing the hormone releasing activity and to enhance the enzymatic stability, lGnRH-III dimers were prepared via disulfide bond formation. Our results demonstrate that the lGnRH-III dimer derivatives exhibited higher antiproliferative effect and enzymatic stability in comparison with the native lGnRH-III, while lower LH-releasing potency was determined. In order to find a correlation between the biological and structural features of these compounds, the conformation of lGnRH-III and its dimer derivatives was determined by ECD, VCD, FT-IR and (1)H NMR.
[Show abstract][Hide abstract] ABSTRACT: The phenotype of HNPCC shows great diversity. Investigation of the disease needs the application of both the Amsterdam and Bethesda Guidelines. The clinical diagnosis of HNPCC can be established by means of thorough family history containing more generations. The immunohistochemistry and MSI investigation of the tumorous tissue as well as the detection of mutations based on DNA sequencing could reinforce the existence of the possible hereditary tendency.
Two pedigrees were selected based on the above-mentioned protocol at the Surgical Institute of the University of Debrecen, Medical and Health Science Center. Amongst first-degree relatives of the 31-year old male patient suffering from colorectal carcinoma (1st patient), three other colorectal, one gastric, one breast and one lung tumors have been found. Two genetic alterations of hMSH2 gene were detected in this family, which were also detectable in other family members. The mutation of exon 7 was not at that time available in international databases, so it was detected by us for the first time. We were able to find alterations of both hMLH1 and hMSH2 genes in the case of the 25-year old patient with synchronous colorectal carcinomas (2nd patient). These alterations could be detected in other family members as well. The whole pedigree contains only one other case of colorectal carcinoma besides the index person.
Several HNPCC families would be missed in case of considering the Amsterdam Criteria alone. The application of the Bethesda Guidelines is absolutely necessary for the detection of families with poor history at the first screening. The association of a polymorphism and a pathogen mutation in one person could lead to early onset of colorectal carcinoma.
Magyar Sebészet (Hungarian Journal of Surgery) 01/2007; 59(6):411-20.
[Show abstract][Hide abstract] ABSTRACT: The Bethesda guidelines may offer more useful criteria in patients' selection for germline mismatch repair gene mutation analysis than guidelines merely based on family background. An early onset double primary colorectal cancer patient with poor family history with MSI-H status was investigated for MLH1 promoter methylation, expression of the MLH1 and MSH2 gene by immunohistochemistry and mutations in the MLH1 and MSH2 genes. The index patient carried two germline alterations, the p.Val716Met in MLH1 and the c.2210+1G>C in MSH2 genes, and both tumors failed to express MLH1 and MSH2 proteins. After subsequent analysis of the whole family of the index patient, the p.Val716Met variant can be defined as a rare polymorphism with the possible contribution of pathogenicity to tumor formation and c.2210+1G>C as a true pathogenic mutation causing an out-of-frame deletion of exon 13.
[Show abstract][Hide abstract] ABSTRACT: The tumor growth inhibitory efficacy of the somatostatin structural derivative TT-232 was studied using different routes of administration and treatment schedules in various human tumor models. TT-232, containing a five-residue ring structure, has a strong antitumor activity both in vitro and in vivo. The antineoplastic activity of TT-232 has been found to be associated with the induction of programmed cell death in tumor cells, resulting in highly-selective elimination of the neoplastic tissue. The study compared the antitumor efficacy of TT-232 in various long-term administration routes; the intermittent (injection) versus continuous (infusion) treatment via subcutaneously-inserted Alzet osmotic minipumps in different human tumor models: T-47/D human breast carcinoma and A-431 human epidermoid carcinoma. Treatment with TT-232 started after disease development. The antitumor activity of TT-232 was evaluated on the basis of the tumor growth inhibition. In the case of T-47/D human breast carcinoma, the intermittent treatment resulted in 23%-26% and the infusion treatment resulted in 48%-53% tumor growth inhibition. The tumor growth inhibitory effect of TT-232 on A-431 human epidermoid carcinoma tumor resulted in 35%-43% (intermittent treatment) and 70%-74% (continuous treatment) decreases in tumor volume. This antitumor efficacy of TT-232 was observed in the two human tumors investigated. In this study, the route of infusion was shown to increase drug efficacy relative to conventional delivery methods. The results suggest that TT-232 is an effective and promising antitumor agent.
Anticancer research 07/2006; 26(4B):3011-5. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To screen a suspected Hungarian HNPCC family to find specific mutations and to evaluate their effect on the presentation of the disease.
The family was identified by applying the Amsterdam and Bethesda Criteria. Immunohistochemistry was performed, and DNA samples isolated from tumor tissue were evaluated for microsatellite instability. The identification of possible mutations was carried out by sequencing the hMLH1 and hMSH2 genes.
Two different mutations were observed in the index patient and in his family members. The first mutation was located in exon 7, codon 422 of hMSH2, and caused a change from Glu to STOP codon. No other report of such a mutation has been published, as far as we could find in the international databases. The second mutation was found in exon 3 codon 127 of the hMSH2 gene, resulting in Asp-->Ser substitution. The second mutation was already published, as a non-pathogenic allelic variation.
The pedigree analysis suggested that the newly detected nonsense mutation in exon 7 of the hMSH2 gene might be responsible for the development of colon cancers. In family members where the exon 7 mutation is not coupled with this missense mutation, colon cancer appears after the age of 40. The association of these two mutations seems to decrease the age of manifestation of the disease into the early thirties.
World Journal of Gastroenterology 03/2006; 12(8):1192-7. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prognostic significance of spontaneous regression of primary melanoma is a controversial issue. Studies on sentinel lymph node status and circulating tumour cells may represent a step towards a better understanding. The clinical details of 269 melanoma patients who underwent sentinel lymph node biopsy were analysed. Correlation was sought between the parameters of the primary tumour, particularly tumours showing a partial intermediate level of regression, and sentinel lymph node status. The presence of circulating tumour cells was studied by reverse transcription-polymerase chain reaction for tyrosinase messenger RNA preoperatively in 94 patients. Of the examined tumours, 27.8% showed histological features of a partial intermediate level of regression. Regressive tumours were localized predominantly on the trunk (P=0.006), were significantly thinner (P<0.0000) and were less frequently ulcerated (P=0.003) than tumours without regression. Moreover, the majority of regressive melanomas were of the superficial spreading type (P<0.0000) and their sentinel node status was more favourable (P=0.026). We demonstrated the presence of circulating tumour cells in five of 26 (19.2%) regressive and 19 of 68 (29.4%) non-regressive tumours. The difference was not significant (P=0.32). By multivariate analysis, however, the Breslow thickness and ulceration of the primary tumour were predictors of the sentinel lymph node status, in agreement with literature data. A partial intermediate level of regression of the primary tumour did not affect unfavourably the sentinel lymph node status in our study. We failed to demonstrate a significant relationship between the presence of circulating tumour cells and either primary tumour regression or the sentinel lymph node status.
Melanoma Research 12/2005; 15(6):509-13. DOI:10.1097/00008390-200512000-00005 · 2.10 Impact Factor