Circulating tumor DNA as a biomarker in metastatic colorectal carcinoma case report.

INTRODUCTION: Colorectal cancer (CRC) is the third most common malignant disease worldwide. The stage of the disease at the time of diagnosis and the capture of an early recurrence have a direct impact on long-term survival. Existing control screening methods often do not reflect real-time metastatic disease. In patients with detectable circulating tumor DNA (ctDNA), liquid biopsy can be an effective monitoring tool. CASE REPORT: In 2012, we performed sigmoid resection in a 57 years old patient for advanced CRC. The follow-up assessments included: blood samples for CA 19-9 and CEA, endoscopy and imaging methods. We also sampled peripheral blood to determine the level of ctDNA. Its value corresponded to the development of the disease throughout the period. Twice it outperformed imaging methods. CEA showed some degree of unreliability, especially after prolonged illness. CA 19-9 was in the normal range at all times. CONCLUSION: Circulating tumor DNA is an effective tool in the diagnosis of recurrent metastatic CRC. In patients with detectable ctDNA, its level correlates with the tumoral mass in real time. It has a predictive value in monitoring the treatment response. Its implementation in the follow-up of patients with CRC may have an impact on the choice of treatment strategy and consequently on patient survival.

IDH1/2 Mutation and MGMT Promoter Methylation - the Relevant Survival Predictors in Czech Patients with Brain Gliomas.

Gliomas are a heterogeneous group of tumours varying in prognosis, treatment approach, and overall survival. Recently, novel markers have been identified which are linked to patient prognosis and therapeutic response. Especially the mutation of the enzyme isocitrate dehydrogenase 1 or 2 (IDH1/2) gene and the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status seem to be the most important predictors of survival. From 2012 to 2015, 94 Czech patients with primary brain tumours were enrolled into the study. The IDH1/2 mutation was detected by denaturing capillary electrophores.The methylation status of the MGMT gene and other 46 genes was revealed by MS-MLPA. In all 94 patients, the clinical data were correlated with molecular markers by Kaplan-Meier analyses and Cox regression model. The MGMT promoter methylation status was established and compared to clinical data. In our study eight different probes were used to elucidate the MGMT methylation status; hypermethylation was proclaimed if four and more probes were positive. This 3 : 5 ratio was tested and confirmed by Kaplan-Meier and Cox analyses. The study confirmed the importance of the IDH1/2 mutation and hypermethylation of the MGMT gene promoter being present in tumour tissue. Both markers are independent positive survival predictors; in the Cox model the IDH hazard ratio was 0.10 and in the case of MGMT methylation it reached 0.32. The methylation analysis of the panel of additional 46 genes did not reveal any other significant epigenetic markers; none of the candidate genes have been confirmed in the Cox regression analyses as an independent prognostic factor.

Serum albumin is a strong predictor of survival in patients with advanced-stage non-small cell lung cancer treated with erlotinib.

Molecular targeted therapy based on tyrosine kinase inhibitors (TKI), directed at epidermal growth factor receptor (EGFR) is one of the novel effective agents in management of advanced-stage of Non Small Cell Lung cancer (NSCLC). However several candidate predictors have been extensively studied, apart from activating EGFR gene mutations, no reliable biochemical or molecular predictors of response to erlotinib have been validated. The aim of our retrospective study was to evaluate the association of baseline serum albumin with outcomes in a large cohort of patients with advanced-stage NSCLC treated with erlotinib. Clinical data of 457 patients with locally-advanced (III B) or metastatic stage (IV) NSCLC treated with erlotinib were analysed. Serum samples were collected and the measurement was performed one day before the initiation of erlotinib treatment. Before the treatment initiation, low albumin was (<35 g/l) measured in 37 (8.1%) patients and normal albumin (≥ 35 g/l) was measured in 420 (91.9%). The median PFS and OS for patients with low serum albumin was 0.9 and 1.9 months compared to 1.9 and 11.4 months for patients with normal serum albumin (p=0.001 and p<0.001). The multivariate Cox proportional hazards model revealed that EGFR mutation status (HR=2.50; CI: 1.59-3.92; p<0.001) and pretreatment serum albumin (HR=1.73; CI: 1.21-2.47; p=0.003) were significant independent predictive factors for PFS, whereas EGFR mutation status (HR=3.14; CI: 1.70-5.81; p<0.001), stage (HR=1.48; CI: 1.09-2.02; p=0.013), ECOG PS (HR=1.77; CI: 1.37-2.29; p<0.001) and pretreatment serum albumin (HR=4.60; CI: 2.98-7.10; p<0.001) were significant independent predictive factors for OS. In conclusion, the results of present retrospective study indicate that pretreatment hypoalbuminemia is associated with poor outcome of NSCLC patients treated with erlotinib. Based on these results, measuement of serum albumin is an objective laboratory method feasible for estimation of prognosis of patients with advanced-stage NSCLC.

Genetic testing in the management of relatives of patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is the most common genetic cardiac disease with vast genetic heterogeneity. First-degree relatives of patients with HCM are at 50% risk of inheriting the disease-causing mutation. Genetic testing is helpful in identifying the relatives harbouring the mutations. When genetic testing is not available, relatives need to be examined regularly. We tested a cohort of 99 unrelated patients with HCM for mutations in MYH7, MYBPC3, TNNI3 and TNNT2 genes. In families with identified pathogenic mutation, we performed genetic and clinical examination in relatives to study the influence of genetic testing on the management of the relatives and to study the usefulness of echocardiographic criteria for distinguishing relatives with positive and negative genotype. We identified 38 genetic variants in 47 patients (47 %). Fifteen of these variants in 21 patients (21 %) were pathogenic mutations. We performed genetic testing in 52 relatives (18 of them (35 %) yielding positive results). Genetic testing of one HCM patient allowed us to omit 2.45-5.15 future cardiologic examinations of the relatives. None of the studied echocardiographic criteria were significantly different between the relatives with positive and negative genotypes, with the exception of a combined echocardiographic score (genotype positive vs. genotype negative, 3.316 vs. -0.489, P = 0.01). As a conclusion, our study of HCM patients and their relatives confirmed the role of genetic testing in the management of the relatives and found only limited benefit of the proposed echocardiographic parameters in identifying disease-causing mutation carriers.

Erlotinib in the treatment of advanced squamous cell NSCLC.

Erlotinib is an epidermal growth factor receptor tyrosine-kinase inhibitor. Clinical trials have shown its efficacy in advanced non-small cell lung cancer (NSCLC). We conducted a large retrospective study based on clinical experience aiming to prove erlotinib's efficacy and safety in patients with advanced-stage squamous cell NSCLC. Totally 375 patients with advanced-stage (IIIB, IV) squamous cell NSCLC were treated with erlotinib. Erlotinib was continued until disease progression or intolerable toxicity. 1 (0.3%) complete response (CR), 28 (7.5%) partial responses (PR) and 198 (52.8%) stable diseases (SD) were achieved. Overall response rate (ORR) and disease control rate (DCR) were 7.8% and 60.5%, respectively. Median progression-free survival (PFS) was 3.0 months and median overall survival (OS) was 7.6 months. PFS of patients with CR/PR, SD and PD were 7.6, 3.9 and 1.0 months, respectively (P<0.001). OS of patients with CR/PR, SD and PD were 13.3, 10.9 and 3.8 months, respectively (P<0.001).The most common adverse effects were rash and diarrhoea. In conclusion ertlotinib is effective and well-tolerated in patients with advanced-stage squamous cell NSCLC.

Comparison of EGFR-TKI and chemotherapy in the first-line treatment of advanced EGFR mutation-positive NSCLC.

Molecular targeted therapy based on EGFR tyrosine kinase inhibitors (EGFR-TKI) is currently astate of the art option for management of advanced stage NSCLC. Activating EGFR mutations are preferable for a good treatment response to EGFR-TKI. The presented retrospective study evaluated a clinical observation of EGFR-TKI aiming at its efficacy and safety in comparison to a standard chemotherapy in the first-line treatment of advanced stage NSCLC. Total number of patients with advanced stage (IIIB, IV) EGFR mutation-positive NSCLC was 54 of which 23 were treated with EGFR-TKI and 31 patients with various chemotherapy regimens in the first line. The treatment efficacy was characterized in terms of disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). The comparison of DCR was performed using Fisher's exact test and the differences in survival were tested using log-rank test. DCR for EGFR-TKI treatment was 95.6% vs. 70.9% for chemotherapy (p=0.032). Median of PFS in patients treated with EGFR-TKI was 7.2 months vs. 2.5 months in patients treated with chemotherapy (p<0.001). Median of OS was 14.5 months vs. 21.4 months (p=0.729). EGFR-TKI was associated with higher incidence of skin rash and diarrhoea; chemotherapy was associated with higher incidence of haematologic adverse events and nausea or vomiting. The analysis results showed a favourable DCR and PFS in patients treated with EGFR-TKI in the first line. The non-significant difference in OS could be attributed to a cross-over during the patient follow-up as well as the differences in performance status and age between both groups. EGFR-TKI is the optimal choice for the first-line treatment of EGFR mutation-positive NSCLC.

Second line treatment in advanced non-small cell lung cancer (NSCLC): comparison of efficacy of erlotinib and chemotherapy.

Molecular targeted therapy based on tyrosine kinase inhibitors, directed at the epidermal growth factor receptor (EGFR) is one of novel options for management of NSCLC. Erlotinib is EGFR tyrosine kinase inhibitor used for treatment of the advanced NSCLC. This presented study is focused on comparison of erlotinib and chemotherapy efficacy in the second line treatment of the advanced NSCLC. DCR and PFS became the primary endpoints.Total number of patients was 290. A group treated with chemotherapy in the second line consisted of 150 patients and a group treated with erlotinib in the second line consisted of 140 patients. Comparison of DCR was performed using Fisher's exact test, visualization of PFS was performed using Kaplan-Meier survival curves and differences were tested using the log-rank test. Genetic testing was performed using PCR direct sequencing. In the group treated with chemotherapy 2 CR, 23 PR and 51 SD were achieved vs. 5 CR, 10 PR and 55 SD in the group treated with erlotinib in the second line. DCR in patients treated with chemotherapy was 54.0% vs. 51.3% in patients without EGFR mutation treated with erlotinib (p=0.707); in patients harboring EGFR mutation, treated with erlotinib (n=9) outstanding results were achieved: 4 CR, 2 PR and 3 SD (not tested). Median of PFS in patients treated with chemotherapy was 2.1 months vs. 1.9 months in patients without EGFR mutation (p=0.879) vs. 8.4 months in patients harboring EGFR mutation treated with erlotinib (p=0.017). Results of analysis show that even patients without EGFR mutation are able to benefit from erlotinib treatment in the second line. The efficacy (DCR, PFS) of erlotinib in patients without EGFR mutation was comparable with chemotherapy. The treatment efficacy in a subgroup of patients harbouring EGFR mutation treated with erlotinib was significantly better than in patients without EGFR mutation.

Skin rash as useful marker of erlotinib efficacy in NSCLC and its impact on clinical practice.

Erlotinib is an epidermal growth factor receptor tyrosine kinase inhibitor used in treatment of advanced NSCLC. Patients harboring EGFR or KRAS mutations represent minority of all patients in caucasian population and there is no available predictor for a predominant group of patients harboring the wild-type EGFR and wild-type KRAS genes. Skin rash is the most frequent manifestation of cutaneous toxicity of erlotinib. Rash is associated with a good therapeutic response. We aimed at the evaluation of rash as a predictor of therapeutic effect of erlotinib in patients harboring the wild-type EGFR and KRAS wild-type genes and to assess its possible usage in a clinical practice.Totally 184 patients with advanced stage NSCLC (IIIB, IV) harboring the wild-type EGFR and wild-type KRAS genes were analysed. Comparison of ORR, PFS and OS according to the occurrence of rash was performed. In order to assess the impact of rash in clinical practice it was conducted landmark analysis of the group of patients whose rash was observed during first month of treatment (n=124). Patients in whom progression was observed during the first month of treatment were excluded from the landmark analysis. The comparison of ORR was performed using Fisher's exact test, visualization of survival was performed using Kaplan-Meier survival curves and the differences in survival were tested using the log-rank test. Median PFS in patients who were observed with rash during the treatment was 3.0 vs. 1.2 months in patients with no rash (p<0.001), median of OS in patients who were observed with rash during the treatment was 13.9 vs. 5.8 months in patients with no rash (p<0.001). ORR in patients who were observed with rash during the treatment was 17.4% vs. 3.3% in patients with no rash (p=0.001). Median of PFS after 1 month of treatment in patients who were observed with rash during the first month was 2.9 vs. 1.1 months in patients with no rash (p=0.027). Median of OS after 1 month of treatment in patients who were observed with rash during the first month was 13.8 vs. 9.9 months in patients with no rash (p=0.082). Rash is strongly associated with better survival and ORR in patients harboring wild-type EGFR and wild-type KRAS genes. Occurrence of rash during the first month of treatment is a useful predictor of better effect of erlotinib after one month of treatment. Patients who were not observed with rash during the first month of treatment are in high risk of progression. Optimization of the treatment of these patients can contribute restaging after two months of treatment, assessment of plasma levels of erlotinib and eventually attempt to dose escalation.

Mutation-based detection and monitoring of cell-free tumor DNA in peripheral blood of cancer patients.

Prognosis of solid cancers is generally more favorable if the disease is treated early and efficiently. A key to long cancer survival is in radical surgical therapy directed at the primary tumor followed by early detection of possible progression, with swift application of subsequent therapeutic intervention reducing the risk of disease generalization. The conventional follow-up care is based on regular observation of tumor markers in combination with computed tomography/endoscopic ultrasound/magnetic resonance/positron emission tomography imaging to monitor potential tumor progression. A recent development in methodologies allowing screening for a presence of cell-free DNA (cfDNA) brings a new viable tool in early detection and management of major cancers. It is believed that cfDNA is released from tumors primarily due to necrotization, whereas the origin of nontumorous cfDNA is mostly apoptotic. The process of cfDNA detection starts with proper collection and treatment of blood and isolation and storage of blood plasma. The next important steps include cfDNA extraction from plasma and its detection and/or quantification. To distinguish tumor cfDNA from nontumorous cfDNA, specific somatic DNA mutations, previously localized in the primary tumor tissue, are identified in the extracted cfDNA. Apart from conventional mutation detection approaches, several dedicated techniques have been presented to detect low levels of cfDNA in an excess of nontumorous (nonmutated) DNA, including real-time polymerase chain reaction (PCR), "BEAMing" (beads, emulsion, amplification, and magnetics), and denaturing capillary electrophoresis. Techniques to facilitate the mutant detection, such as mutant-enriched PCR and COLD-PCR (coamplification at lower denaturation temperature PCR), are also applicable. Finally, a number of newly developed miniaturized approaches, such as single-molecule sequencing, are promising for the future.

[EGFR mutations in patients with advanced NSCLC]. / Mutace genu EGFR u pacientu s pokrocilým NSCLC.

BACKGROUND: Molecular targeted therapy based on tyrosine kinase inhibitors, directed at the epidermal growth factor receptor (EGFR) is one of novel options for management of NSCLC. EGFR gene mutations, exon 19 deletions and exon 21 point mutations (L858R) are good predictors of response to EGFR-TKI treatment. The aim of this study was to assess the incidence of EGFR mutations in a large cohort of Europeans with advanced NSCLC and subsequently to evaluate their impact on the effect of EGFR-TKI treatment. PATIENTS AND METHODS: In total, 613 patients with advanced stage NSCLC (IIIB, IV) were genetically tested. The effect of treatment was evaluated in 410 patients treated with EGFR-TKI. Survival was evaluated using Kaplan-Meier method, and statistical comparison was performed using log-rank test. RESULTS: EGFR mutations were detected in 73 (11.9%) patients. Exon 19 deletions were detected in 49 patients, exon 21 point mutations (L858R) were detected in 22 patients, and both mutation types were detected in 2 patients. An increased incidence of EGFR mutations among patients with adenocarcinoma (14.9% vs 7.8%, p = 0.008), women (20.2% vs 7.1%, p < 0.001) and nonsmokers (29.9% vs 7.0%, p < 0.001) was demonstrated. Sixty patients with EGFR mutation and 350 patients with wild-type EGFR were treated with EGFR-TKI. Median PFS in patients harboring EGFR mutation was 7.2 vs 2.0 months in patients harboring wild-type EGFR (p < 0.001), median OS in patients harboring EGFR mutation was 14.5 vs 7.5 months in patients harboring wild-type EGFR (p = 0.019). CONCLUSION: The incidence of EGFR mutations in the studied population, their increased incidence among patients with adenocarcinoma, women and non-smokers correlated with data previously published. Results of survival analysis in patients treated with EGFR-TKI confirmed high potential of EGFR mutations to predict good effect of the EGFR-TKI treatment. Genetic testing in patients with NSCLC should be a standard part of diagnostic procedures

Extraordinary response to erlotinib therapy in a patient with lung adenocarcinoma exhibiting KRAS mutation and EGFR amplification.

Case reports on the co-incidence of Kirsten rat sarcoma (KRAS) mutation and epidermal growth factor receptor (EGFR) amplification in patients with NSCLC are very rare. This combination is usually considered a negative prognostic factor, despite EGFR amplification alone having positive predictive value. The whole course of treatment of a patient with both EGFR amplification and KRAS mutation present is decribed. The patient in question was a smoker for whom both first- and second-line chemotherapy had been unsuccessful. In stage IV disease biological therapy was administered and proved highly beneficial. Today, 38 months since commencing the treatment, the patient still has no signs of progression and the therapy is still in progress.

Impact of urban drainage on metal distribution in sediments of urban streams.

The concentrations of total and extractable heavy metals (Cu, Zn, Pb, Cd, Cr and Ni) and other sediment properties were determined in sediments of three urban streams in Prague. The mean sediment concentrations (in mg kg(-1) (dry weight)) ranged within 0.2-3.2 (Cd), 20.2-61.7 (Cr), 16.3-135.2 (Cu), 17.8-42.5 (Ni), 20.2-114.8 (Pb) and 79.4-446.3 (Zn). The chemical distribution of metals, determined in four chemical sediment fractions of the sediment, indicates an increase of the distribution of Cu and Zn in more easily available fractions in sediments affected by combined sewer overflows. The highest percentages of Cd and Zn are in the most labile acid-soluble fraction (38-64% and 15-43% respectively), whereas Pb is bound mainly to reducible fraction.

Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species.

In the present paper a new extraction technique based on the combination of solid-phase/supercritical-fluid extraction (SPE/SFE) with subsequent reversed-phase HPLC is described. The SPE/SFE extractor was originally constructed from SPE-cartridge incorporated into the SFE extraction cell. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes (4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives (o-coumaric, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acid) were extracted. Cyclic addition of binary extraction solvent system based on methanol:water (1:1, v/v) and methanol/ammonia aqueous solution was used for extraction at 40MPa and 80 degrees C. The p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic and chlorogenic acid; 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were identified by HPLC-electrospray mass spectrometry in SPE/SFE extracts of acid hydrolyzates of microalga (Spongiochloris spongiosa) and cyanobacterial strains (Spirulina platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). For the identification and quantification of the compounds the quasi-molecular ions [M-H](-) and specific fragments were analysed by quadrupole mass spectrometry analyzer. Our analysis showed that the microalgae and cyanobacteria usually contained phenolic acids or aldehydes at microg levels per gram of lyophilized sample. The proposed SPE/SFE extraction method would be useful for the analysis of different plant species containing trace amount of polar fraction of phenols.

Ultrahigh-pressure liquid chromatography of isoflavones and phenolic acids on different stationary phases.

Complete separation of aglycones and glucosides of selected isoflavones (genistin, genistein, daidzin, daidzein, glycitin, glycitein, ononin, sissotrin, formononetin, and biochanin A) was possible in 1.5 min using an ultrahigh-pressure liquid chromatography (U-HPLC) on a different particular chemically modified stationary phases with a particle size under 2 microm. In addition, selected separation conditions for simultaneous determination of isoflavones together with a group of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acid) allowed separation of all 19 compounds in 1.9 min. Separations were conducted on a non-polar reversed phase (C(18)) and also on more polar phases with cyanopropyl or phenyl groups using a gradient elution with a mobile phase consisting of 0.3% aqueous acetic acid and methanol. Chromatographic peaks were characterised using parameters such as resolution, symmetry, selectivity, etc. Individual substances were identified and quantified using UV-vis diode array detector at wavelength 270 nm. Limits of detection (3S/N) were in the range 200-400 pg ml(-1). Proposed U-HPLC technique was used for separation of isoflavones and phenolic acids in samples of plant materials (Trifolium pratense, Glycine max, Pisum sativum and Ononis spinosa) after acid hydrolysis of the samples and modified Soxhlet extraction.

Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts.

A rapid-resolution HPLC/UV-VIS DAD separation method (which takes <1 min) for the determination and identification of genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A in fmol quantities in submicroliter sample volumes was optimized. A linear gradient elution (0 min 22% B, 1.0 min 80% B, 1.4 min 100% B, 1.8 min 22% B) using a mobile phase containing 0.2 % (v/v) acetic acid (solvent A) and methanol (solvent B) was applied on a Zorbax SB C18 column (1.8 microm particle size) at 80 degrees C. The method was verified using samples of bits of soy and methanolic extracts from Trifolium pratense, Iresine herbstii and Ononis spinosa plants. Pseudobaptigenin glucoside, irilone, prunetin, texasin, tlatlancuayin and other isoflavones, in addition to aglycones of isoflavones and their beta-glucosides and malonyl and acetyl derivatives, were identified by UV-VIS DAD and electrospray mass spectrometric (ESI-MS) detection in the extracts.

Acquired resistance of pulmonary adenocarcinoma to initially successful targeted therapy due to EGFR mutation T790M.

According to previous reports in the literature, the T790M mutation indicates an acquired resistance to tyrosine kinase inhibitors. The initial positive effect of combination chemotherapy with erlotinib as the first line of treatment correlates with several positive predictors including the type of carcinoma, non-smoking status, occurrence of rash and the presence of exon 19 EGFR gene mutation. The case of a 32-year-old, non-smoker with non-contributory history patient, who was diagnosed with adenocarcinoma in the left lung T4N0M0 stage IIIB is reported. The patient underwent 6 cycles of chemotherapy with erlotinib, gemcitabine and cisplatin, followed by complete remission. Fifteen months after commencing therapy, disease recurred over subsequent therapy with erlotinib and then gefitinib. During that time, bone and cerebral metastases with pericardial effusion were detected. The patient died 7 months later. Genetic examination of tumour tissue collected at the beginning of therapy revealed activating exon 19 mutation in the EGFR gene. Later, during the relapse, the same mutation was still present and, in addition, a T790M mutation in exon 20 of EGFR was found. The subsequently acquired resistance against both erlotinib, as well as gefitinib was most likely a result of tumor cells acquiring the T790M mutations and escaping the drug effect. The authors recommend testing for T790M mutation presence in selected patients prior to targeted therapy with tyrosine kinase inhibitors.

Evaluation of clinical relevance of examining K-ras, p16 and p53 mutations along with allelic losses at 9p and 18q in EUS-guided fine needle aspiration samples of patients with chronic pancreatitis and pancreatic cancer.

AIM: To establish an optimum combination of molecular markers resulting in best overall diagnostic sensitivity and specificity for evaluation of suspicious pancreatic mass. METHODS: Endoscopic ultrasound (EUS)-guided fine needle aspiration cytology (FNA) was performed on 101 consecutive patients (63 males, 38 females, 60 +/- 12 years; 81 with subsequently diagnosed pancreatic cancer, 20 with chronic pancreatitis) with focal pancreatic mass. Samples were evaluated on-site by an experienced cytopathologist. DNA was extracted from Giemsa stained cells selected by laser microdissection and the presence of K-ras, p53 and p16 somatic mutations was tested by cycling-gradient capillary electrophoresis (CGCE) and single-strand conformation polymorphism (SSCP) techniques. In addition, allelic losses of tumor suppressor genes p16 (INK4, CDKN2A) and DPC4 (MADH4, SMAD4) were detected by monitoring the loss of heterozygosity (LOH) at 9p and 18q, respectively. RESULTS: Sensitivity and specificity of EUS-guided FNA were 75% and 85%, positive and negative predictive value reached 100%. The remaining 26% samples were assigned as inconclusive. Testing of molecular markers revealed sensitivity and specificity of 70% and 100% for K-ras mutations (P < 0.001), 24% and 90% for p53 mutations (NS), 13% and 100% for p16 mutations (NS), 85% and 64% for allelic losses at 9p (P < 0.001) and 78% and 57% for allelic losses at 18q (P < 0.05). When tests for different molecular markers were combined, the best results were obtained with K-ras + LOH at 9p (92% and 64%, P < 0.001), K-ras + LOH at 18q (92% and 57%, P < 0.001), and K-ras + LOH 9q + LOH 18q (96% and 43%, P < 0.001). When the molecular markers were used as complements to FNA cytology to evaluate inconclusive samples only, the overall sensitivity of cancer detection was 100% in all patients enrolled in the study. CONCLUSION: EUS-guided FNA cytology combined with screening of K-ras mutations and allelic losses of tumor suppressors p16 and DPC4 represents a very sensitive approach in screening for pancreatic malignancy. Molecular markers may find its use particularly in cases where FNA cytology has been inconclusive.

[Detection of DNA specific for Mycobacterium tuberculosis in archeological material using the polymerase chain reaction]. / Detekce DNA specifické pro Mycobacterium tuberculosis v archeologických materiálech metodou polymerázové retezové reakce.

The paleopathological diagnosis of bone tuberculosis in archeological findings may be confirmed by the polymerase chain reaction (PCR). If the M. tuberculosis-specific DNA fragment is amplified, then the presence of this microorganism in the sample is demonstrated. The pilot study presented investigated whether our molecular biology laboratory can collaborate with anthropologists in paleopathological analyses and to verify the use of the commercial diagnostic kit Cleanmix (Talent, Italy), for DNA isolation from archeological samples. The results were compared with the conclusions of anthropologists. Successful amplification of specific DNA fragments was achieved in a specimen from the period of the 13th to 15th century. The specimen consists of four thoracic vertebrae modified by osseous tuberculosis (gibbus). The PCR result was also positive in a five-year-old femur sample of a patient with chronic pulmonary tuberculosis. All other specimens of various ages but without macroscopic symptoms of osseous tuberculosis, were PCR negative. These results suggest that it is possible to detect former infections with pathogenic microorganisms in archeological bones find.

A note on the preparation of particulate material for an electron microscopic study.

Modifications of two rapid and satisfactory procedures of purification of minute biological particulate material for electron microscopic observations were described. These modifications eliminate the alterations of the investigated particles and the possibility of their artificial disintegration. In addition, these procedures preserve the grouping of the particles as compared with the original suspensions. The dialysis is made directly on the grids coated with the supporting formvar membrane. In the second procedure the filtration of the soluble components of the material into the agar is combined with the transfer of the particles on the membrane covered grids.