Molecular detection of disseminated breast cancer cells in the bone marrow of early breast cancer patients using quantitative RT PCR for CEA.

Carcinoembryonic antigen (CEA) is widely used as a serum tumor marker in various types of cancer. Several systems for the CEA-RT-PCR approach have been reported to date. In this study, we have evaluated the quantitative CEA-RT-PCR as a diagnostic tool for detection of isolated tumor cells in bone marrow of early breast cancer patients prior to the administration of any adjuvant systemic therapy. We obtained bone marrow aspirates of 70 patients with stage I (37%), II (60%), 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 quantitative RT-PCR for the expression of CEA has been performed. CEA transcripts were detected in samples from 29 (41%) out of 70 patients. With a median follow-up of 22 months we observed 8 disease free survival (DFS) events including 4 systemic recurrences, 1 ductal in-situ carcinoma (DCIS), 1 local recurrence, and 2 deaths without tumour. Four DFS events (2 systemic recurrences, 2 deaths without tumor) occurred in patients with CEA transcripts in the bone marrow and 4 (2 systemic recurrences, 1 DCIS, 1 locoregional recurrence) in patients without CEA in the bone marrow. There was a trend to shorter DFS in the group with CEA in the bone marrow (p=0.05548). Overall survival was not assessed because only 2 deaths (both in patients without tumor) have been reported to date. Quantitative RT-PCR assay for CEA may be a useful tool for detection of occult breast cancer cells in the bone marrow. Clinical and prognostic relevance of minimal residual disease using this technique remains unproven. Our results should be interpreted with caution with regard to 2 deaths in CEA positive group with no relationship to disease recurrence.

Mammaglobin A, a novel marker of minimal residual disease in early stages breast cancer.

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.

[Hereditary predisposition for the development of breast and ovarian carcinoma]. / Hereditární dispozice ke vzniku karcinomu prsu a ovaria.

Part of breast and ovarian cancer cases develops on the hereditary predisposition, i.e. mutation in one of predisposing genes. Although this proportion is relatively small, 5-10% of all breast and ovarian carcinomas, it represents a group with clearly defined etiologic factor. Predictive analysis of unaffected family members allows to identify individuals at high risk of cancer and to include them into the programme of primary and secondary cancer prevention. Following article presents basic review of the hereditary predisposition to breast and ovarian cancer focusing especially on BRCA1 and BRCA2 genes, which are responsible for almost three-quarters of those hereditary tumours.

Detection of the most frequent mutations in BRCA1 gene on polyacrylamide gels containing Spreadex Polymer NAB.

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.

[Detection and occurrence of BRCA 1 gene mutation in patients with carcinoma of the breast and ovary]. / Detekce a výskyt mutací genu BRCA 1 u pacientek s karcinomem prsu a ovaria.

OBJECTIVE: 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. DESIGN: Retrospective clinical-laboratory study and review. SETTING: Department of Gynaecology and Obstetrics, 1st Medical Faculty of Charles University, Prague, Apolinárská 18, Czech Republic. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Apoptosis and its importance in the development and therapy of tumors (review)]. / Apoptóza a její význam z hlediska vzniku a lécby nádorových onemocnení (prehledový clánek).

This review focuses on apoptosis and its regulation as a tool of principal control mechanism of tissue homeostasis. Defects in regulation of apoptosis contribute to a various pathological processes, including tumor development, chronic inflammatory diseases, immunological disorders and many others. Apoptosis influences sensitivity to radiotherapy and chemotherapy of tumours. Microscopically the apoptosis is characterized by morphological changes, which result in the formation of apoptotic bodies. Apoptosis is an active process, which require synthesis and activation of a set of the specific cellular proteins. Among them, the key role belongs to the family of cystein proteases--caspases activated either through the death receptors or via activation steps starting with the release of a mitochondrial cytochrome c. Activation of caspases promotes the activation of downstream effectors leading to the cleavage of target cellular proteins and genomic DNA. The members of Bcl-2 family and p53 are- the others important proteins influencing the regulation of apoptosis. Enhancing of our knowledge about apoptosis and its mechanisms highly improves the rationale for diagnostics and therapy in oncology.

[Minimal residual disease in breast carcinoma]. / Minimální reziduální choroba u karcinomu prsu.

The incidence of breast cancer continuously increases in developed countries. The introduction of screening methods such as mammography or ultrasound lead to higher proportion of early diagnosed tumors. However, even in early stage tumors occult neoplastic cells can spread to the organism. Such tumor cells are very likely precursors of distant metastases. Using several monoclonal antibodies against epithelial mucins or cytokeratins on the cell surface could be detected one tumor cell among 10(5) or 10(6) of normal bone marrow cells. These cells are not detectable by routine histopathologic exam. More sensitive but also more costly and technically demanding are PCR assays. The sensitivity might reach almost 1:10(7). Prospective clinical trials using immunocytochemistry have shown that the presence of stained cells in bone marrow is clearly associated with shorter disease free survival and overall survival. In the near future we may use the bone marrow examination for the presence of occult tumor cells in order to improve current staging system or as a surrogate marker in the decision-making in regard to adjuvant systemic therapy or in the assessment of efficacy of adjuvant treatment. The review summarizes contemporary knowledge assembled in preclinical and clinical studies.

Prognostic significance of c-erbB-2 gene expression in pancreatic cancer patients.

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.

[The importance of iron in humans]. / Význam zeleza v lidském organismu.

The authors submitted a very brief review of some biological properties of iron in particular protein-bound iron and on the immense amount of iron which surrounds us (it is the fourth most frequent element). In the human organism there is no physiological system for iron elimination. Therefore its absorption is regulated and restricted. Some substances promote its absorption, other inhibit it. An important factor in the regulation of iron absorption are the iron reserves of the organism, the amount of dietary iron and the enhanced erythropoiesis (incl. the ineffective one), while reduced erythropoiesis does not affect iron absorption. Ascorbic acid forms chelates with iron and thus remains soluble and can be absorbed despite the alkaline pH in the duodenum. Unbound trivalent iron remains insoluble. An important asset to rational iron therapy are Lauberger's discovery of ferritin (1936), assessment of the iron plasma level (Heilmeyer and Plötner, 1937), Laurell's discovery of transferrin (1947), and Addison's RIA method for assessment of the serum ferritin level (1974). Sideropenia remains the most widespread deficiency in human pathology, sideropenic anaemia the most readily diagnosed anaemia which is, however, most wrongly treated.

Markers of melanogenesis in malignant melanoma.

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.

[Synthesis of eumelanin pigment precursors. II. 6-Hydroxy-5-methoxy- and 5-hydroxy-6-methoxyindol]. / Syntéza prekurzoru pigmentu eumelaninu. II. 6-Hydroxy-5-methoxy-a 5-hydroxy-6-methoxyindol.

Following up of specific melanogenesis metabolite excretion in the course of malignant melanoma disease is useful for the disease prognosis assessment. The authors elaborated a modified synthesis technique of 6-hydroxy-5-methoxy and 5-hydroxy-6-methoxyindole, eumelanin pigment precursors. The elaborated synthesis is economically and temporally reasonable and the synthesized compounds accord, as proved by means of elementary analysis, thin layer (TLC) and high performance liquid chromatography (HPLC) and last but not least by nuclear magnetic resonance (NMR), with demands for reference compounds needed for isomer hydroxymethoxyindole quantification in urine.

[Synthesis of eumelanin pigment precursors. II. 6-hydroxy-5-methoxy- and 5-hydroxy-6- methoxyindole]. / Syntéza prekurzoru pigmentu eumelaninu. II. 6-hydroxy-5-methoxy- a 5-hydroxy-6-methoxyindol.

Following up of specific melanogenesis metabolite excretion in the course of malignant melanoma disease is useful for the disease prognosis assessment. The authors elaborated a modified synthesis technique of 6-hydroxy-5-methoxy and 5-hydroxy-6-methoxyindole, eumelanin pigment precursors. The elaborated synthesis is economically and temporally reasonable and the synthesized compounds accord, as proved by means of elementary analysis, thin layer (TLC) and high performance liquid chromatography (HPLC) and last but not least by nuclear magnetic resonance (NMR), with demands for reference compounds needed for isomer hydroxymethoxyindole quantification in urine.

Tyrosinase of hamster melanoma: its purification and estimation by radioimmunoassay.

1. Tyrosinase was purified from melanosomal fraction of hamster melanoma. 2. A radioimmunoassay was developed to quantitate the tyrosinase protein in hamster serum and hamster melanoma tissue using polyclonal anti-tyrosinase antibodies and 125I-labeled enzyme. 3. The serum tyrosinase levels were found to be about 0.24 micrograms and 1.14-4.48 micrograms/ml in normal hamsters and melanoma-bearing hamsters, respectively. 4. Tyrosinase protein in serum correlated significantly with the enzyme activity in hamsters with melanoma (r = 0.733). 5. In the cytosol fraction of hamster melanoma, a level of 2.2 micrograms of tyrosinase/mg protein was determined. 6. The usefulness and possible applications of the tyrosinase radioimmunoassay are discussed.

Biochemical markers of malignant melanoma.

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.

Postoperative active specific immunotherapy with supportive measures in patients suffering from recurrent metastasized melanoma: case reports of six patients.

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 administering 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.

Specific inhibition by antityrosinase antibodies of tyrosinase-mediated melanogenesis.

Polyclonal antibodies to hamster melanoma tyrosinase were raised in rabbits, and series of immunoinhibition experiments with a purified enzyme and specific immunoglobulins were carried out. Tyrosinase activity was determined by a set of radiochemical and spectrophotometric methods utilizing tyrosine, dopa, dopamine, or dihydroxyindole (DHI) as substrates. The quantitative data obtained indicated that the complexing of tyrosinase with its specific antibody inhibited melanogenesis in a specific manner: dopachrome formation from dopa and dopamine conversion to melanin were not affected and all other enzyme activities comprising the DHI oxidation step were inhibited to various degrees. Additionally, tyrosine hydroxylation was also slightly inhibited. The data obtained implied that melanogenesis was restricted at the point of DHI oxidation. From observations on the immunoinhibition of a DHI oxidation at varying dopa-cofactor concentrations, we propose that dopa-cofactor may be bound at separate site than DHI and thus may act as a positive allosteric effector for DHI oxidation by tyrosinase. Study of tyrosinase immunoinhibition by the antibodies against the enzyme thus seems to provide a valuable system for investigating the tyrosinase-mediated melanogenesis.

A spectrophotometric assay for mammalian tyrosinase utilizing the formation of melanochrome from L-dopa.

A simple spectrophotometric method for a rapid determination of tyrosinase (EC 1.14.18.1) is described. The basis of the assay is the incubation of the enzyme with L-dopa in the presence of an optimal concentration of Zn2+ ions and the measurement of the formation of melanochrome, as indicated by the rise in absorbance at 540 nm. Final absorbance change reflects probably two activities of tyrosinase: the oxidation of dopa to dopaquinone and the conversion of 5,6-dihydroxyindole to melanochrome. Using a purified preparation from hamster melanoma, the assay was found to be more sensitive than the commonly used dopachrome assay. Comparison with some other currently available methods for assaying tyrosinase is presented and potential applications of the assay are discussed.

Changes in serum cathepsin B-like activity in patients with colorectal cancer.

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.