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Mammaglobin A, in contrast to other factors, is a breast specific member of uteroglobin gene family. Expression is restricted to normal and neoplastic breast epithelium. A highly homologous mammaglobin B is not specific to breast tissue. In this pilot feasibility study we examined expression of both markers for minimal residual disease in the bone marrow of patients with breast cancer. We obtained bone marrow aspirates of 34 patients with stage I (41%), II (56%) and III (3%) breast cancer who underwent either immediate complete resection of the tumor or neoadjuvant therapy with subsequent curative surgery. mRNA was isolated using QIAamp RNA blood mini kit (Qiagen). Subsequently two-step nested RT-PCR for the expression of mammaglobin A and mammaglobin B was performed. Mammaglobin A was detected in samples from 4 (12%) out of 34 patients. None of the specimens was positive for mammaglobin B. With a median follow-up of 21 month we observed only 2 recurrences, one in patient with mammaglobin A positive bone marrow.RT-PCR assay for mammaglobin A may be a useful tool for detection of occult breast cancer cells in the bone marrow. Clinical and prognostic relevance of minimal residual disease should be further investigated.

Heterozygous carriers of germ-line mutations in the BRCA1 gene are at high risk for the development of breast and ovarian cancer. Inactivating mutations have been identified in the whole coding region of the gene, however, repeatedly occuring mutations can explain a large proportion of gene alterations detected in certain ethnic groups. In Czech patients, the 5382insC and 185delAG mutations may account for approximately 50% of all BRCA1 abnormalities (unpublished data). In the present study, a rapid and simple method to identify these short insertions and deletions that alter the size of the polymerase chain reaction (PCR) product is described. The analysis involves the separation of fragments amplified with primers that flank altered sites in the BRCA1 gene on non-denaturing polyacrylamide gels containing Spreadex Polymer NAB. The increased resolving power of Spreadex gels enables full separation of two DNA fragments that differ by 1-bp on gels that are 5 cm long. The method gave interpretable results with the genetic material obtained from all tested mutation carriers and control persons. Defective alleles were also detected in DNA samples from carriers of the 1135delA mutation in BRCA1 and the 4206ins4 mutation in BRCA2. These results suggest that electrophoresis on Spreadex gels can be used universally for detection of the most frequent frameshift mutations in BRCA genes. The method is suitable even for rapid screening of frequent germ-line mutations in BRCA genes in breast and ovarian cancer patients not selected for family history of cancer or age at diagnosis.

Molecular methods tend to belong to the standard armamentarium of modern pathology. In some instances, these methods are able to identify nosological entities with better accuracy than conventional technique. These methods give useful complementary information to choose appropriate therapeutic strategy. C-erbB-2 overexpression in pancreatic cancer vary widely between 17 to 82%. C-erbB-2 gene is perspective target of anticancer therapies. 57 histologically confirmed tumors (51 pancreatic adenocarcinoma, 5 pancreatic neuroendocrine tumors and 1 carcinoma of Vater's ampullae) were analyzed for the presence of c-erbB-2 expression by immunohistochemistry. Correlation with time from initial symptoms until diagnosis, tumor size and TNM stage at diagnosis, tumor grade, type of operation and overall survival were investigated. C-erbB-2 overexpression was detected in 19.6% samples of pancreatic adenocarcinoma and in one case of Vater's ampullae carcinoma. C-erbB-2 overexpression was found in two of four insulinomas. Univariate statistical correlation stage between c-erbB-2 overexpression and time from initial symptoms until diagnosis, tumor size and TNM at diagnosis, tumor grade, type of operation and overall survival did not reach statistical significans in any parameter studied. C-erbB-2 oncogene was not found to be prognostic factor in pancreatic cancer. Its value to predict therapeutical response remains to be determined in prospective clinical trials.

Biochemical specifity of malignant melanoma is represented in part by the formation of specific cytoplasmatic particles of the pigment cell--melanosomes--in which the synthesis of pigment eumelanin and pheomelanin takes place and in part by the presence of a specific enzyme--tyrosinase--which catalyzes the formation of pigment eumelanin and pheomelanin and even the formation of specific metabolites (so called melanogens) which are excreted in increased amounts in the course of the disease. Tyrosinase and melanogens are specific for pigment cell and therefore can be used for monitoring of melanogenesis in malignant melanoma. When comparing our results and the results of other authors we can conclude that following of specific markers of melanogenesis in malignant melanoma should serve for the evaluation of prognosis of the disease. The study of melanoma markers is by far not finished. We do hope that nearly future will be able to give a sufficient answer to the question, whether melanogenuria is actually an expression of expected different biochemical or metabolic types of malignant melanoma on the one hand and/or biochemically or immunologically conditioned responses of the host organism on the other.

9672 Background: Early hematogenous spread of occult tumor cells is a crucial event in tumor progression and formation of distant metastases. Several markers mainly cytokeratins have been validated to date using immunohistochemistry (IHC) or more sensitive reverse transcriptase polymerase chain reaction (RT-PCR). The role of Cytokeratin 19 (CK-19) remains controversial regarding it's sensitivity and specificity. In contrast Mammaglobin was reported as a breast specific marker with very high specificity. In this feasibility study we validated expression of CK19, Mammaglobin A and B in the bone marrow samples of patients with breast cancer. Methods: We obtained bone marrow aspirates of 48 patients with stage I (38%), II (58%) and III (4%) breast cancer who underwent either immediate complete resection of the tumor or neoadjuvant therapy with subsequent curative surgery. mRNA was isolated using QIAamp RNA blood mini kit (Qiagen ®). Subsequently two-step nested RT-PCR for the expression of CK-19, Mammaglobin ...

The clinical results from postoperative active specific immunotherapy using autologous polymerized tumor material in six patients suffering from metastasized melanoma is reported. Correction of an alleged systemic deficiency leading to malignant cell transformation was attempted by administrating certain essential trace elements, amino acids, vitamins, and a diet containing lipids, extracted from the mammalian central nervous system, after heating. Vaccinations against influenza were also given as a precaution against certain viral infections sometimes seen to precede melanoma recurrence. The clinical results with this postoperative adjuvant therapy are so encouraging that we suggest that sterile tumor tissue should be saved at operation and treated to produce insoluble particles as an option for postoperative treatment of patients suffering from metastasized melanoma. Prospective randomized studies are indicated.

Activities of alkaline phosphatase (ALP), gamma-glutamyl transferase (GMT), lactate dehydrogenase (LD), beta-glucuronidase (GLU) and cathepsin B-like (CB-like) were determined in blind-coded sera from 50 patients with primary liver carcinoma, liver cirrhosis and acute hepatitis, and from 40 control subjects of comparable age range. CB-like activity averaged 700% (p less than 0.01), 1590% (p less than 0.01) and 1600% (p less than 0.01) of control subjects in liver cirrhosis (n = 30), acute hepatitis (n = 5) and primary liver carcinoma (n = 15), respectively. In acute hepatitis group we have found significant correlation between CB-like and GLU activities (r greater than 0.95). This correlation, however, was not observed in primary liver carcinoma suggesting that alteration in CB-like activity is not due to generalized increases in lysosomal membrane instability. The primary liver carcinoma group exhibited also the modest increments in serum ALP, GMT and LD activities (p less than 0.01). This increment, however, was not detected in any of acute hepatitis or liver cirrhosis patients. For the first time the alkaline-stable form of CB-like in human serum is described. This form representing 40% of overall CB-like activity was present in all primary liver carcinoma patients. This form, however, was not present in sera of any of control subjects or in sera of patients with acute hepatitis and liver cirrhosis with the exception of two men, in whom we have probably dealing with an early stage of primary liver carcinoma. Although the nature of the increment in CB-like activity in cancer remains to be determined, such analyses may help to the early detection of malignant hepatoma (primary liver carcinoma).

The excretion of Thormählen positive melanogens (TPM), zincuria, serum dopaoxidase activity of tyrosinase and serum sialic acid were determined in 60 patients with primary and/or metastatic cutaneous melanoma and in 20 healthy persons. On the basis of our results we can recommend the following of TPM urinary excretion and serum dopaoxidase activity in the course of malignant melanoma as specific markers of the tumor growth. The following of serum sialic acid in the course of malignant melanoma is valuable from the standpoint of prognosis of the disease. The following of zincuria in the course of malignant melanoma is not recommended because of its low value for monitoring melanoma patients.

The authors point out that melanogenuria, i.e. the excretion of specific phenolic and, particulary, indole metabolites in melanoma, is undoubtedly due to elevated tyrosinase activity in this disease. Other enzymes, in particular glucuronyltransferase, catechol-O-methyltransferase and possibly hydroxyindole-O-methyltransferase, also evidently participate in the further biochemical conversion of the resultant metabolites, however.

Cathepsin B-like activity (CB-like) was estimated in sera of 30 male patients with colorectal cancer (n = 20), benign polyps of the rectum (n = 10) and in sera of control subjects (n = 50). Both total and residual activities of the enzyme in colorectal cancer patients showed a significant elevation in comparison with control subjects and patients with benign polyps. In the course of antitumor therapy a decline in catheptic activity was observed when a reduction in the volume of tumor tissue was present. On the other hand, an increment in CB-like activity was observed when antitumor therapy had no effects or tumor relapse was present.

The Thormählen positive melanogen previously determined as "A" has been isolated from melanotic urine by means of column chromatography on Amberlite IR 45, DEAE cellulose and Dowex 50. According to its behavior during a study with 1H--NMR spectra it has been found that the isolatd compound is 6-methoxy-5-indolyglucosiduronate, the metabolite of 5,6-dihydroxyindole. The possible formation and biotransformation of this compound is discussed.

The isomeric metabolites, 5-hydroxy-6-methoxy-2-indolylcarboxylic and 6-hydroxy-5-methoxy-2-indolylcarboxylic acids have been identified in the urine of hamsters bearing transplantable melanotic melanoma. Their identification was carried out by spectral and chromatographic comparison of the two isomers isolated from urine with the same compounds achieved synthetically.

Urinary melanogens are indolic and phenolic compounds which are excreted in an elevated amount in the urine of melanoma patients. Urinary melanogens can be divided into two principal groups according to their behavior during Thormählen test--Thormählen positive melanogens (TPM) and Thormählen negative melanogens (TNM). When comparing TPM and TNM (represented by homovanillic and vanillactic acids) urinary excretion in stage III of melanoma patients we have found two significantly different relations for melanogens excretion, i. e. predominating TPM urinary excretion in melanotic forms of melanomas and predominating homovanillic acid and vanillactic acid urinary excretion in amelanotic forms of melanomas. We assumed that it could be an indirect evidence of the different catalytic activity of either cresolase or catecholase activities of tyrosinase.

Ascitic fluid of patients with primary liver cancer was shown to contain a latent thiol proteinase which can be activated by pepsin treatment or by autolysis at acidic environment. This enzyme resembles cathepsin B (EC 3.4.22.1) in many physical-chemical properties including substrate specificity, requirement for thiol activators and inhibition both by thiol blocking reagents and by peptidyl diazomethyl ketones, but has a higher molecular size even after activation. Pepsin treatment and autolysis reduce its Mr from 41 800 to 33 400 and 27 700, respectively, but all these forms are larger than human liver cathepsin B. The latent enzyme may be, therefore, an enzyme--inhibitor complex or an inactive precursor of cathepsin B due to an altered processing in Golgi endoplasmatic reticulum-lysosome compartment.

The article presents a review of basic information on incidence and detection of BRCA 1 and BRCA 2 genes mutations. Results of investigation in a group of women with ovarian and breast cancer are presented.Retrospective clinical-laboratory study and review.Department of Gynaecology and Obstetrics, 1st Medical Faculty of Charles University, Prague, Apolinárská 18, Czech Republic.Investigated group consisted of 16 persons--12 patients with ovarian or breast cancer and 4 healthy relatives of a woman--breast cancer patient and a carrier of BRCA 1 gene mutation. Protein truncation test (PTT) was performed in order to detect BRCA 1 gene mutation. This test detects mutations leading to premature termination of protein synthesis. Truncated proteins are easily discriminated from full size.Three BRCA 1 gene alterations were identified in the investigated group of women suffering from ovarian or breast cancer. One asymptomatic person--carrier of BRCA 1 gene mutation--was identified in this study. She was daughter of a woman, a carrier of BRCA 1 gene mutation, with early onset of breast cancer and positive family history.BRCA 1 and BRCA 2 gene mutations are of particular importance in the increasing risk of ovarian cancer and early onset of breast cancer as well as some other malignancies. Genetic testing and counselling including investigation of some other genetic and environmental factors, related to cancer risk, may be of clinical significance in patients with increased risk of certain malignancies.

9638 Background: Carcinoembryonic antigen (CEA) is widely used as a serum tumor marker. Several systems for the CEA-RT-PCR approach have been reported to date. In this study, we have evaluated quan...