Metabolomic fingerprinting employing DART-TOFMS for authentication of tomatoes and peppers from organic and conventional farming.

The rapidly growing demand for organic food requires the availability of analytical tools enabling their authentication. Recently, metabolomic fingerprinting/profiling has been demonstrated as a challenging option for a comprehensive characterisation of small molecules occurring in plants, since their pattern may reflect the impact of various external factors. In a two-year pilot study, concerned with the classification of organic versus conventional crops, ambient mass spectrometry consisting of a direct analysis in real time (DART) ion source and a time-of-flight mass spectrometer (TOFMS) was employed. This novel methodology was tested on 40 tomato and 24 pepper samples grown under specified conditions. To calculate statistical models, the obtained data (mass spectra) were processed by the principal component analysis (PCA) followed by linear discriminant analysis (LDA). The results from the positive ionisation mode enabled better differentiation between organic and conventional samples than the results from the negative mode. In this case, the recognition ability obtained by LDA was 97.5% for tomato and 100% for pepper samples and the prediction abilities were above 80% for both sample sets. The results suggest that the year of production had stronger influence on the metabolomic fingerprints compared with the type of farming (organic versus conventional). In any case, DART-TOFMS is a promising tool for rapid screening of samples. Establishing comprehensive (multi-sample) long-term databases may further help to improve the quality of statistical classification models.

[The preparation of anticancer vaccine for patients with multiple myeloma on the base of monoclonal immunoglobulin loaded dendritic cells]. / Príprava protinádorové vakcíny pro pacienty s mnohocetným myelomem na bázi dendritických bunek nalozených monoklonálním imunoglobulinem.

BACKGROUND: On June 2006, phase II clinical trial focused on anticancer vaccination of multiple myeloma patients, was started. On September 2007, the immune and clinical response evaluation of first four patients was finished.The anticancer vaccine contained dendritic cells loaded with monoclonal immunoglobulin produced by myeloma cells. METHODS AND PATIENTS: Within the frame of phase II clinical trial were vaccinated four myeloma patients with stable disease. It was administered six vaccines for each patient, monthly. The dendritic cells were cultured from the patient's peripheral blood mononuclear cells and loaded with autologous monoclonal immunoglobulin under the good manufacturing practice conditions. After the safety and quality control, the satisfactory vaccine was administered to the patient. The functional characteristic of dendritic cells was evaluated using flow cytometry, the immune response was evaluated using ELISpot. The clinical response was monitored using monoclonal immunoglobulin concentration in patient's sera. RESULTS AND CONCLUSION: The immune response detected using ELISpot was observed in 3/4 patients. The monoclonal immunoglobulin concentration was changeable for all twelve months, but never exceeded the range of 25% for minimal clinical response achievement. During the vaccination, no significant toxicities or negative side-effects were observed. The clinical trial is going on with vaccination other patients with multiple myeloma.

Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis.

BACKGROUND: Matrix metalloproteinases are notable contributors to neuroinflammation and blood-brain barrier disruption in multiple sclerosis (MS). OBJECTIVE: The goal of this study was to determine the serum levels of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-2 (MMP-2), and their tissue inhibitors (TIMP-1) and (TIMP-2), and to investigate their possible relations to type, disability, and severity of MS. MATERIALS AND METHODS: Eighty-seven patients with definite MS according to the McDonald criteria and 50 healthy controls were enrolled in the study. Their clinical status was evaluated with the Expanded Disability Status Scale. Serum levels were analyzed by enzyme-linked immunoassay. RESULTS: A significant elevation in MMP-9 serum levels and in the MMP-9/TIMP-1 ratio was found in the whole MS group (P<0.001), in the relapsing-remitting MS (RRMS) (P<0.001), and secondary-progressive MS (SPMS) (P<0.001) groups when compared with the controls. A significant elevation in MMP-2 serum levels and in the MMP-2/TIMP-2 ratio was observed in the primary progressive (P<0.001) and the SPMS (P<0.002) groups when compared with the RRMS group, and this increase was also associated with the disability (P<0.001) and severity (P<0.05) of the disease. CONCLUSION: We confirmed that metalloproteinases are useful biological markers in MS, providing information about the clinical type, disability, and severity of the disease.

Tumor marker M2-pyruvate-kinase in differential diagnosis of chronic pancreatitis and pancreatic cancer.

BACKGROUND/AIMS: This study addresses the possibility of very difficult differential diagnosis of pancreatic cancer and chronic pancreatitis, especially in cases where pancreatic cancer appears in the course of chronic pancreatitis. A combination of graphical methods and pancreatic biopsy targeted with endosonography seems to be the most precise diagnostic technique. Even negative findings for this procedure cannot exclude the risk of existing tumor and is additionally an invasive technique. Therefore there are different conditions that have to be fulfilled to allow use of both endoscopic and bioptic instruments (biopsy with biopsy needle). Tumor markers of blood serum are used in the practice, but this use seems to be limited by the sensitivity, which is on the level of 60-70% in average. METHODOLOGY: The authors examined M2-pyruvate-kinase concentration in their group, which included patients with chronic pancreatitis, different grading of pancreatic cancer as well as patients with pancreatic cancer which appeared in the course of chronic pancreatitis. M2-pyruvate-kinase was used as a marker of tumor hyperplasia as it is present in higher concentration in tumors of gastrointestinal tract. RESULTS: The authors observed important growth in advanced forms of pancreatic tumor compared to patients with chronic pancreatitis. M2-PK was increased in a similar way in patients with pancreatic cancer in the course of chronic pancreatitis. The results led to the conclusion that evaluation of M2-PK concentration helps differentiating between pancreatic cancer and chronic pancreatitis, especially in cases where morphological changes of the gland have focal character and are imitating pancreatic cancer.

International Staging System required standardization of biochemical laboratory testing in multiple myeloma.

The standardization of biochemical measurement procedures in multiple myeloma is necessary for reliable prognostic stratification of patients in multicentric trials. The new prognostic index International Staging System for multiple myeloma uses only two laboratory markers, albumin and beta-2 microglobulin. Our study compared results of albumin, beta-2 microglobulin and monoclonal immunoglobulin measurements from six centers which provide treatment for multiple myeloma in the Czech Republic and attempted to standardize the analytic procedures. We have found that the measurement of albumin is well standardized and the results from all laboratories were comparable. The measurement of beta-2 microglobulin achieved comparability only after a partial unification of analytical methods. The determination of monoclonal immunoglobulin concentration provided comparable results for concentrations higher than 20 g/l with higher variability for lower values.

Low mutational rate of K-ras codon 12 in singular bronchoscopy specimens in suspected lung cancer.

Mutations of the K-ras gene are found in a subset of non-small- cell lung carcinomas (NSCLC). The aim of our study was to determine the K-ras codon 12 mutation in the first, singular bronchoscopy specimen in parallel with the cytological examination for the diagnosis of lung cancer. Samples were obtained by diagnostic bronchoscopy in 140 patients with suspected lung tumors. The analysis of K-ras mutations was carried out by a sensitive two-step mutation- enriched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. This method has been confirmed earlier to be positive for mutated tumor cells and negative for normal lung parenchyma and bronchus. Of the 140 patients with suspected cancer, 93 were diagnosed as NSCLC by cytology or histology in either the same specimen used for the detection of K-ras mutation or in later biopsies. However, only four K-ras codon 12 mutations were detected in the first bronchoscopic material: one in adenocarcinoma, two in squamous cell tumors, and one mutation was found in a patient with dysplasia which was diagnosed later as a squamous cell carcinoma. Our findings indicate that although the K-ras (codon 12) mutation is a gene lesion infrequently detectable in a singular specimen taken at the first bronchoscopy examination in cases of clinically suspected lung cancer, the detection of this mutation can help to confirm the cytological diagnosis of NSCLC or may be even diagnostic in cytologically negative cases.

[Comparison of diagnostic validity of carbohydrate-deficient transferrin and other laboratory markers in chronic ethanol abuse]. / Srovnání diagnostické validity karbohydrát deficientního transferinu a dalsích laboratorních markeru chronického abúzu etanolu.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) has been reported to be the best laboratory marker of the chronic alcohol abuse, but there are conflicting data on its accuracy and sensitivity ranging from 19% to 96% in various studies. The aim of this study was to compare the diagnostic efficiency of CDT with the other markers of alcohol abuse used in clinical practice with respect to possible sex differences. METHODS AND RESULTS: The serum CDT (using the method of anion-exchange chromatography and TIA), mean corpuscular volume (MCV), gamma-glutamyl-transferase (GMT) values and platelet count were evaluated in 50 alcohol-dependent patients admitted to the Center of Detoxification and in the reference group of 85 healthy teetotallers. The cut-off values for %CDT where established in the level of 2.2% and 2.5% for men and women respectively. In men we proved a comparatively high diagnostic efficiency of CDT (AUC 0.94, sensitivity 82.6%, specificity 96.7%) and GMT, MCV seem to be less accurate marker of chronic alcohol abuse. In contrast there was a lower diagnostic validity of CDT in women in comparison with common markers (AUC 0.83, sensitivity 60%, specificity 88%). CONCLUSIONS: The laboratory diagnosis of chronic alcohol consumption can be improved by using a combination of several markers. The specificity and also the cumulative sensitivity of such a battery of laboratory markers can be elevated by CDT evaluation. In a part of patients, CDT can be the only detectable abnormality.

[Diagnostic value of carbohydrate-deficient transferrin, gamma-glutamyltransferase and mean erythrocyte volume as laboratory markers of chronic alcohol abuse]. / Diagnostická výteznost karbohydrát deficitního transferinu, gama-glutamyltransferázy a stredního objemu erytrocytu jako laboratorních markeru chronického abúzu alkoholu.

INTRODUCTION: Our research aimed at finding out values of carbohydrate-deficient transferin (CDT), gamma-glutamyltransferase (GGT) and mean corpuscular volume (MCV) for the purposes of future etiological diagnostics of alcohol neuropathy in thin fibres. METHODS: We examined the serum of 80 control subjects (50 women and 30 men), and the serum of 33 alcoholics (20 men and 13 women) with the daily consumption of more than 60 g alcohol in the course of the last four weeks. CDT was determined with the use of microcolumn separation after iron saturation followed by turbidimetric immunoassay (ChronoAlcoI. D., Sangui Biotech, Inc.) on Cobas-Mira analyser. CDT is expressed as a percentage of the total transferin. Senzitivity, specificity, positive likelihood ration (+LR), ROC and the area under the ROC curve were determined using statistical program MedCalc. RESULTS: The senzitivity, specificity and positive likelihood ratio (+LR) for CDT-%, respectively, were 82.6, 96.7 and 24.8 for men (cut off 2.2%), and 60.0, 88.0 and 5.0 for women (cut off 2.5%). The respective values for GMT were 95.7, 90.0 and 9.6 for men (cut off 0.64 mu kat/l), and 90.0, 80.0 and 4.5 for women (cut off 0.38 mu kat/l); for MCV 82.6, 96.7 and 24.8 for men (cut off 95.0 fL), and 80.0, 100.0 and 20.0 for women (cut off 97.2 fL). The area under the ROC curve for CDT-%, GMT and MCV, respectively, were 0.940, 0.964 and 0.896 for men, and 0.829, 0.917 and 0.906 for women. CONCLUSION: In men, CDT-% and MCV showed the same values of the statistical parameters studied. GGT was more sensitive and less specific. In women, all the parameters studied presented a lesser diagnostic value, except for MCV with 100% specificity and +LR 20.0.

Age-related extracellular to total body water volume ratio (Ecv/TBW)--can it be used for "dry weight" determination in dialysis patients? Application of multifrequency bioimpedance measurement.

The article suggests a novel method for quantitative determination of optimal dry weight in dialysis patient based on their extracellular volume (ECV) to total body water (TBW) ratio and its relation to age. Values of ECV and TBW are evaluated by means of whole body multifrequency bioimpedometry. In an effort to find a suitable marker of hydration status in an individual from bioimpedance data, significant correlation has been found between ECV/TBW ratio and age in health. Assuming that all excess fluid in dialysis patients is stored exclusively in ECV and that distribution of their TBW at the state of optimal dry weight corresponds to that of a healthy person of the same age, the pre-dialysis ECV/TBW could be used for quantitative determination of optimal dry weight and/or of the ultrafiltration to reach this weight. Practical bioimpedance measurement of ECV/TBW in a group of dialysis patients both pre- and post-dialysis confirmed both above assumptions, i.e. nearly exclusively extracellular origin of ultrafiltration as well as normalisation of the ECV/TBW ratio towards the end of dialysis. Supporting evidence of increasing ECV/TBW value with age was also found in literature. Although the suggested method needs detailed analysis of possible disturbing factors (ethnic "specificity" of the reference ECV/TBW vs. age characteristics in health, possible difference in "biological" and "physical" age of dialysis patient and others), the article is published at this early stage to enable wider testing of the proposed novel method by different investigators.

Genomic organization and chromosomal mapping of SPARC-like 1, a gene down regulated in cancers.

Human SPARC-like 1 (SPARCL1), also known as MAST9 or hevin, is a member of the SPARC protein family. Originally we identified SPARCL1 as one of the genes down regulated in human non-small cell lung cancer (NSCLC). Recent reports indicate that the down regulation of SPARCL1 also occurs in prostate and colon carcinomas, suggesting that SPARCL1 inactivation is a common event not only in NSCLCs but also in other tumors of epithelial origin. In the present work we report the cloning and mapping of the genomic locus of human SPARCL1. Using fluorescence in situ hybridization analysis, SPARCL1 was localized to chromosome 4q22-25, a region often deleted in human cancers. Furthermore, we show that the intron/exon organization of the human SPARCL1 gene is similar to its murine homologue SC1. SPARCL1 contains 11 exons and 10 introns which span approximately 47 kb of the genome. We also sequenced the 5'-flanking region of the human SPARCL1 gene containing 2.4 kb of the putative promoter region. The data presented herein are a prerequisite for deletion/mutation analysis of the SPARCL1 gene in tumors. In addition, knowledge of the SPARCL1 promoter sequence allows to investigate the regulation of SPARCL1 expression on the transcriptional level. Taken together our results will help to clarify the function of SPARCL1 in tumor formation.

[Cystatin C in estimates of glomerular filtration in patients with renal disease--initial experience]. / Cystatin C v odhadu glomerulární filtrace u pacientu s nemocemi ledvin, první zkusenosti.

Cystatin is reported in the literature with increasing frequency as a new reliable parameter for estimates of glomerular filtration. The authors examined cystatin C by the PET method of DAKO Co. in 151 patients from the nephrological out-patient clinic. 91 patients had normal renal functions, 60 suffered from renal insufficiency of different severity. Between cystatin C and glomerular filtration assessed by creatinine clearance was a close correlation, R = -0.787 according to Pearson. The authors evaluated separately a group of 36 patients with glomerulonephritis, 34 diabetic patients with diabetic nephropathy, 38 patients with tubulointerstitial nephritis and 43 subjects with other kidney diseases. The groups did not differ significantly when compared with the whole group nor mutually (p = n.s.). The authors of the study confirmed that a good correlation of cystatin with creatinine filtration is not influenced by the type of basic nephrological disease. The effect of administration of insulin, antihypertensive drugs, cyclosporin A and glucocorticoids was not proved in the investigated group. The published method is accurate, not demanding from the technical aspect. The examined subject is not restricted by conditions ensuring the accuracy of assessment and seems thus useful and perspective for nephrological patients.

Transcriptional repression of the microphthalmia gene in melanoma cells correlates with the unresponsiveness of target genes to ectopic microphthalmia-associated transcription factor.

In the melanocyte, expression of genes required for pigment formation is mediated by the microphthalmia transcription factor, which is also critical for the development and survival of normal melanocytes during embryogenesis. Here we show that the expression of the melanocyte-specific isoform of microphthalmia transcription factor is lost in a subset of human melanoma cell lines, accompanied by the repression of tyrosinase and tyrosinase-related proteins 1 and 2, the three transcriptional target genes for microphthalmia. After the forced expression of microphthalmia transcription factor in melanoma cells where the expression of endogenous microphthalmia gene was found to be extinguished, no restoration of the melanogenic phenotype occurred and the transcription of the three microphthalmia transcription factor target genes remained silent. The transcription activation domain of microphthalmia transcription factor, tested as a GAL-MITF fusion protein, remained fully functional in these cells, however, and ectopic microphthalmia transcription factor localized normally to the nucleus and bound to the tyrosinase initiator E-box in gel retardation assays. Thus, the block of differentiation in microphthalmia-transcription-factor-negative melanomas extended the transcriptional repression of the microphthalmia transcription factor gene alone, and endogenous promoters in these melanoma cells became no longer responsive to microphthalmia transcription factor when this was substituted exogenously. The data presented suggest that a specific nuclear context is required for the transcriptional activation of the melanocyte markers by the microphthalmia transcription factor in malignant melanocytes and this specificity is lost concomitantly with the transcriptional repression of microphthalmia transcription factor.

Expression of genes for microphthalmia isoforms, Pax3 and MSG1, in human melanomas.

Microphthalmia (MITF) gene product, a transcription factor of the basic-helix-loop-helix type, is thought to play a role in the regulation of genes encoding the enzymes necessary for melanogenesis. These include tyrosinase, TRP-1 and TRP-2. Melanocyte-specific isoform of microphthalmia, MITF-M, is expressed in normal and malignant melanocytes. The presence of two other isoforms of microphthalmia, MITF-A and MITF-H, which differ from MITF-M in the amino-terminus, was demonstrated also in some non-melanocytic lineages. Here we have analyzed the presence of all three known isoforms of MITF mRNA in a panel of 17 human melanoma cell lines by a reverse transcriptase-polymerase chain reaction using isoform-specific primers. While, as expected, the predominant form in melanoma cell lines was MITF-M, low amounts of MITF-A mRNA was found in almost all melanomas, as well as in most of 20 tumor cell lines of the non-melanocyte origin (lung and colon carcinomas, osteosarcomas and neuroblastomas). The expression of MITF-H was not detected, with a few exceptions, in the tested cell lines. Pax3 transcription factor was reported earlier to regulate positively the melanocyte-specific promoter of the MITF gene. We found here that the Pax 3 mRNA was expressed in all melanoma cell lines, even in those that had repressed the MITF-M and were amelanotic. This suggests that additional factors, besides Pax3, are required for the MITF expression. The MSG1 (melanocyte-specific gene 1), a gene originally isolated from melanocytes and containing a strong transcription activation domain, was also found expressed in all melanomas and most non-melanocyte tumor cell lines. Together, these data indicate that the MITF-M isoform is the major type of MITF mRNA present in human melanoma cell lines and show that the expression of the isoform MITF-A and the MSG1 is not restricted to malignant melanocytes and occurs in a wide range of tumor cell lines.

Expression of the aromatic L-amino acid decarboxylase mRNA in human tumour cell lines of neuroendocrine and neuroectodermal origin.

Neuroendocrine differentiation of lung tumours is characterised by the expression of several neuroendocrine markers and is confined mostly to specific histological subtypes, i.e. small cell carcinomas and carcinoids. One of the markers seen in neuroendocrine tumours, high activity of the aromatic L-amino acid decarboxylase (AADC), is helpful in distinguishing the classic and variant small cell lung tumour subtypes. Here, we have analysed the expression and quantified the level of mRNA coding for AADC in human tumour cell lines by use of the reverse transcription and polymerase chain reaction (RT-PCR). High amounts of mRNA were detected in classic small cell lung carcinomas and a neuroblastoma cell line. Other cell lines (melanomas, non-small cell lung carcinomas and osteosarcoma) also showed AADC expression, but the levels were 2-3 orders lower. Also, the tissue-specific (neuronal versus liver-specific) mRNA type has been estimated. Small cell lung carcinomas, neuroblastoma and melanoma expressed messenger RNA specific for neuronal tissues. Importantly, the non-small cell lung carcinoma cell lines expressed either liver-specific (non-neuronal) mRNA (cell line A549) or predominantly the neuronal (cell line NCI-H520) AADC message. These data indicate that a range of tumour cell lines transcribe the AADC gene and that two distinct types of AADC mRNA which reflect the embryonal (neuronal or non-neuronal) origin of the tumour may be produced in non-small cell lung cancer cells.

Fluorescence in situ hybridization for detecting erbB-2 amplification in breast tumor fine needle aspiration biopsies.

OBJECTIVE: To evaluate the feasibility of erbB-2 amplification analysis of fine needle aspiration (FNA) biopsies. STUDY DESIGN: FNA smears and dissociated nuclei from 58 breast cancer samples were examined by dual-labeling fluorescence in situ hybridization (FISH) with probes for centromere 17 and the erbB-2 gene. The results were compared with the outcome of erbB-2 immunohistochemistry. RESULTS: Tumors were categorized according to the erbB-2/centromere 17 signal ratio. There were 23 tumors with high-level amplification, four cases with a low-level erbB-2 gain and 27 tumors with normal erbB-2 content. Four tumors showed an erbB-2 deletion, all in patients < or = 42 years of age. ErbB-2 amplification was strongly associated with positive erbB-2 immunostaining (P < .0001). Comparison of FISH analysis on dissociated cells and on FNA biopsies showed high correspondence (P < .0001). CONCLUSION: FISH allows reliable detection of erbB-2 gene amplification on FNA biopsies.

Patterns of p53, erbB-2, and EGF-r expression in premalignant lesions of the urinary bladder.

Frequent recurrences and multicentricity of bladder cancer suggest that alterations of the urothelium distant from the tumor may be relevant to prognosis. In this study immunohistochemistry and fluorescence in situ hybridization (FISH) were used to examine expression of p53, erbB-2, and epidermal growth factor receptor (EGF-r), genomic aberrations, and tumor cell proliferation (Ki67 LI) in normal and dysplastic urothelium. Biopsy specimens examined included normal urothelium (n = 40), mild dysplasia (n = 34), moderate dysplasia (n = 18) and carcinoma in situ (CIS; n = 20). Several different oncogene expression patterns were found, only some of which were associated with dysplasia. EGF-r expression was equally frequent in normal and dysplastic urothelium and showed a strong association with Ki67 LI (P < .0001). A purely superficial erbB-2 positivity was present in both normal and dysplastic biopsies. However, diffuse erbB-2 positivity and p53 overexpression were both associated with advanced dysplasia (P < .0001 each). FISH analysis showed erbB-2 gene amplification and p53 deletions in selected CIS, as well as a marked chromosome 17 copy number heterogeneity in all six CIS examined. These findings indicate a considerable genomic instability in bladder CIS. They show that both erbB-2 and p53 are altered during malignant transformation. Detectable oncogene expression alone, however, is not diagnostic of malignancy in bladder urothelium.

[Pleural effusion in plasmacytic leukemia]. / Pohrudnicní výpotek pri plazmacelulární leukémii.

Multiple myeloma affects predominantly osseous spaces and the close vicinity of bones. A plasmacellular exudate is rare. The authors describe the course of plasmacellular leukaemia in a 68-year-old female patient where the first symptom of the disease was anaemia and a dextrolateral pleural exudate with a cytological finding of plasma cells. The exudate disappeared after the first cycle of chemotherapy and intrapleural administration of cytostatic. After the third cycle of chemotherapy remission of the disease was recorded which was, however, short. After three months' remission (six months after establishment of the diagnosis) the disease exacerbated violently, the dextrolateral exudate reappeared and gradually the insufficiency of the infiltrated bone marrow increased. The patient died one month after the relapse of the disease. The finding of a plasmacellular exudate must be considered the sign of a very poor prognosis and in that case very aggressive treatment is indicated.

Y chromosome loss detected by FISH in bladder cancer.

To examine the significance of Y chromosome losses in bladder cancer, fluorescence in situ hybridization (FISH) was used to determine its prevalence and associations with known parameters of malignancy. Cells were dissociated from formalin-fixed paraffin-embedded bladder tumors from 68 male patients and from 11 post-mortem bladder washes of male patients with a negative bladder cancer history, and were examined by FISH using centromeric probes for chromosomes X, Y, 7, 9, and 17. Nullisomy for chromosome Y was seen in 23 of 68 tumors (34%), monosomy in 28 of 68 tumors (41%), and polysomy in 17 of 68 tumors (25%). There was no association between chromosome Y loss and tumor grade, stage, tumor growth fraction (Ki67 LI), p53 immunostaining, and presence of p53 deletions. Patient age was higher for tumors with a Y loss (73.5 +/- 12.0 years) than for tumors without Y loss (66.6 +/- 10.8 years; p = 0.0207). In one normal bladder wash, a distinct subpopulation (38% of cells) with Y nullisomy was seen. These data suggest that Y loss is a frequent event that can occur early in bladder cancer, although there is no evidence for a role of Y loss in tumor progression.

Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung.

Mutations of the K-ras gene have been implicated in the pathogenesis of human lung adenocarcinomas. In most studies published so far, squamous cell lung cancers harbored ras mutations only exceptionally or no mutations were detected at all. We have examined 141 lung tumor DNA samples for mutations in codons 12, 13, and 61 of K-ras and H-ras oncogenes. A large panel of 118 squamous cell carcinomas was included in the study. For K-ras codon 12, we used a sensitive two-step PCR-restriction fragment length polymorphism method which detects <1% of mutated DNA in the sample. K-ras mutations were found in 17 tumors (12%; 14 in codon 12 and 3 in codon 13). Among 19 adenocarcinomas, mutation was revealed in 7 samples (37%). Of these, one sample harbored two point mutations in codon 12. Nine mutational events were found in squamous cell carcinomas (8%, one adenosquamous carcinoma included, all in codon 12). Of four large cell carcinomas, one contained a mutation. Mutant-enriched PCR products harboring mutations were directly sequenced. Fifteen mutational events were G-->T transversions or G-->A transitions, one was a G-->C transition, and one sample revealed a frameshift deletion of one G from codon 12. Similar mutational spectrum was found in both squamous cell carcinomas and adenocarcinomas, suggesting similar carcinogenic pathways in both histological types of the tumor. The presence of mutations did not correlate with the stage of the disease. Moreover, we analyzed all samples for mutations in codons 12, 13, and 61 of the H-ras gene. We found only one mutation in codon 12. Thus, H-ras mutations apparently play an inferior role in lung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not only lung adenocarcinomas but also of a subset (about 8%) of squamous cell carcinomas.

[Difficulties in the therapy of primary amyloidosis]. / Obtízná lécba primární amyloidózy.

The therapy of primary amyloidosis is still unsatisfactory. The response rate after the cytostatics, dimethylsulphoxide, colchicin and vitamin E is usually low. None of these treatment modalities prolongs significantly the survival in majority of treated patients. The success of interferon alpha in the maintenance therapy of follicular non-Hodgkin's lymphoma and in the remission of multiple myeloma, as well as successful treatment of primary cryoglobulinaemia, brought us to the idea to test interferon alpha in the therapy of primary amyloidosis. Interferon alpha-2b was administered to the patient with three years history of primary amyloidosis. Interferon alpha was used in the dose of 3 x 10(6) daily for the treatment period of 10 weeks. The evaluation of the response was based on the weekly assessment of the light chain lambda concentration in the morning spot of urine. No significant decrease of the light chain concentration during the course of the therapy was observed. The administration of interferon alpha-2b was interrupted in the 10th week of the therapy because of manic psychosis. The question is, whether a higher dose than 3 x 10(6) IU daily would be able to decrease the light chain production, or if this disease is resistant to interferon alpha therapy. Because of the low incidence of primary amyloidosis, the experiences will be collected on the base of small groups of case reports.