Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique

Purpose. RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. Materials. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. Results. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Conclusion. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation (AU)

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Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

PURPOSE: RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. MATERIALS: We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. RESULTS: 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. CONCLUSION: Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

Type of monocyte immunomagnetic separation affects the morphology of monocyte-derived dendritic cells, as investigated by scanning electron microscopy.

Dendritic cells (DCs) are increasingly being used for multiple applications and are useful tools for many immunotherapeutic strategies. The understanding of the possible impact of the DCs-generation methods on the biological capacities of these cells is therefore essential. Although the immunomagnetic separation is regarded as a fast and accurate method yielding cells with the high purity and efficiency, still little is known about its impact on the properties of the generated DCs. The aim of this study was to compare the morphology of the monocyte derived dendritic cells (MoDCs), generated from monocytes selected with anti-CD14 mAbs (positive separation) and treated with anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123, glycophorin A (negative separation), using laser scanning microscopy. We found that the type of the immunomagnetic separation method used strongly influences the shape and cell dimension of the MoDCs. We observed that the height of both immature and LPS-matured DCs generated from monocytes isolated by negative separation was significantly higher compared to the cells obtained by positive separation.

High intensity interval and moderate continuous cycle training in a physical education programme improves health-related fitness in young females.

The aim of the study was to investigate the effects of eight weeks of regular physical education classes supplemented with high intensity interval cycle exercise (HIIE) or continuous cycle exercises of moderate intensity (CME). Forty-eight collegiate females exercising in two regular physical education classes per week were randomly assigned to two programmes (HIIE; n = 24 or CME; n = 24) of additional (one session of 63 minutes per week) physical activity for 8 weeks. Participants performed HIIE comprising 2 series of 6x10 s sprinting with maximal pedalling cadence and active recovery pedalling with intensity 65%-75% HRmax or performed CME corresponding to 65%-75% HRmax. Before and after the 8-week programmes, anthropometric data and aero- and anaerobic capacity were measured. Two-way ANOVA revealed a significant time main effect for VO2max (p < 0.001), similar improvements being found in both groups (+12% in HIIE and +11% in CME), despite body mass not changing significantly (p = 0.59; +0.4% in HIIE and -0.1% in CME). A significant main time effect was found for relative fat mass (FM) and fat-free mass (FFM) (p < 0.001 and p < 0.001, respectively). A group x time interaction effect was found for relative FM and FFM (p = 0.018 and p = 0.018); a greater reduction in FM and greater increase in FFM were noted in the CME than the HIIE group. Improvements in anaerobic power were observed in both groups (p < 0.001), but it was greater in the HIIE group (interaction effect, p = 0.022). Weight loss is not mandatory for exercise-induced effects on improving aerobic and anaerobic capacity in collegiate females. Eight weeks of regular physical education classes supplemented with CME sessions are more effective in improving body composition than physical education classes supplemented with HIIE sessions. In contrast to earlier, smaller trials, similar improvements in aerobic capacity were observed following physical activity with additional HIIE or CME sessions.

Evaluation of 11 single-locus and seven multilocus DNA barcodes in Lamium L. (Lamiaceae).

The aim of this work was to evaluate the suitability of selected DNA regions in the barcoding of plants, based on the species belonging to the genus Lamium (Lamiaceae). For this purpose, nine chloroplast barcodes, that is, accD, matK, rbcL, rpoA, rpoB, rpoC1, rpoC2, trnH-psbA, trnL-trnF, as well as ITS nuclear region, and intron of mitochondrial nad5 gene were tested. Among the single-locus barcodes, most effective in the identification of Lamium species was the trnH-psbA spacer and matK gene. The high level of variability and resolving power was also observed in the case of rpoA and rpoC2 genes. Despite the high interspecies variability of ITS region, it turned out to be inapplicable in Lamium identification. An important disadvantage of ITS as a barcode is a limitation of its use in polyploid plants, samples contaminated with fungal material or samples with partially degraded DNA. We have also evaluated five-two-locus and two-three-locus barcode regions created from a combination of most effective single loci. The best-performing barcode combinations were matK + trnH-psbA and matK + rpoA. Both of them had equally high discriminative power to identify Lamium species.

Central nervous system involvement in mantle cell lymphoma: clinical features, prognostic factors and outcomes from the European Mantle Cell Lymphoma Network.

BACKGROUND: Central nervous system (CNS) involvement in mantle cell lymphoma (MCL) is uncommon, and the manifestations and natural history are not well described. PATIENTS AND METHODS: We present the data on 57 patients with MCL who developed CNS involvement, from a database of 1396 consecutively treated patients at 14 institutions. RESULTS: The crude incidence of CNS involvement was 4.1%, with 0.9% having CNS involvement at diagnosis. Blastoid histology, B-symptoms, elevated lactate dehydrogenase, Eastern Cooperative Group performance status ≥2 and a high Mantle Cell Lymphoma International Prognostic Index score were enriched in the cohort with CNS involvement, and the presence of ≥1 of these features defined a high-risk subset (an actuarial risk of CNS involvement 15% at 5 years) in a single-institution subset. The median time to CNS relapse was 15.2 months, and the median survival from time of CNS diagnosis was 3.7 months. The white blood cell count at diagnosis <10.9 × 109/l, treatment of CNS involvement with high-dose anti-metabolites, consolidation with stem cell transplant and achievement of complete response were all associated with improved survival. CONCLUSIONS: In MCL, CNS involvement is uncommon, although some features may predict risk. Once manifest outlook is poor; however, some patients who receive intensive therapy survive longer than 12 months.

First Report of 'Candidatus Phytoplasma asteris' Associated with Oilseed Rape Phyllody in Poland.

Winter oilseed rape (Brassica napus L.) is widely grown in Poland to produce vegetable oil for industrial processing of human and animal feed. In recent years, according to European Union directives on the use of biofuels (Directive 2003/30/EC), the area under oilseed rape cultivation in Poland has dramatically increased to 810,000 ha in 2009 and is still increasing. Morphological deformations of winter oilseed rape indicative of phytoplasma infection have been observed sporadically in Poland since 2000 (3). Plants exhibiting floral virescence, phyllody, as well as auxiliary bud proliferation, reduced leaves, and malformation of siliques were identified during surveys of research fields in Wielkopolska during May and June of 2009 and 2010. To confirm phytoplasma infection of these plants, inflorescence and leaf tissues were collected from nine diseased and three symptomless plants from three different field locations with 1 to 16% disease incidence. Total DNA was extracted from each plant tissue sample with a modified cetyltrimethylammoniumbromide method (2). Samples were analyzed for phytoplasma DNA with a nested PCR assay employing phytoplasma universal rRNA operon primer pair P1/P7 followed by R16F2n/R16R2, using previously described conditions (1). PCR products of 1.8 and 1.2 kb were obtained from all diseased plants only following PCRs with P1/P7 and nested primer pair R16F2n/R16R2, respectively. PCR products were not obtained from symptomless plants. Eight 1.2-kb amplicons were sequenced (GenBank Accession Nos. JN193475 to JN193482). Comparative analysis of the R16F2n/R16R2 rDNA sequences confirmed the phytoplasma origin of the rDNA sequences that shared 100 to 99% identity with Maize bushy stunt phytoplasma (GenBank Accession No. HQ530152), Alfalfa stunt phytoplasma (GenBank Accession No. GU289675), Primula green yellows phytoplasma (GenBank Accession No. HM590623), and other aster yellows group phytoplasmas. A 1.8-kb amplicon of isolate designated RzW14 was sequenced (GenBank Accession No. HM561990) and had 99% identity with Aster yellow group phytoplasmas from Lithuania (GenBank Accession Nos.GU223208 and AY744071). A virtual restriction fragment length polymorphism analysis of the 16S rDNA sequences from the R16F2n/R16R2 amplicons was performed with iPhyClassifier (4). Restriction profile comparisons identified all aster yellows group phytoplasmas as subgroup 16SrI-B strains. To our knowledge, this is the first report of a 'Candidatus Phytoplasma asteris'-related strain infecting oilseed rape in Poland. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) A. C. Padovan et al. Aust. J. Grape Wine Res. 1:25, 1995. (3) M. Starzycki and E. Starzycka. Oilseed Crops 21:399, 2000. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Adipohormones as prognostric markers in patients with nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) represents one of the most common liver diseases. It is strongly associated with obesity and insulin resistance and is thought to be part of the metabolic syndrome. NASH can progress to cirrhosis and liver failure. Adipohormones, synthesized in adipose tissue, are involved in the pathophysiology of many acute and chronic liver diseases. The aim of this study was to evaluate the plasma concentrations of adiponectin, resistin, leptin, TNF-alpha and Il-6 in patients with NASH, as well as their correlation with the pathologic parameters. Serum concentration of leptin, adiponectin, resistin, insulin, TNF-alpha, IL-6 were measured with ELISA method. Liver biopsies were obtained from 18 (age 42.55+/-21 years) patients. NASH has been classified according to Dixon score. The control group was represented by 16 non-obese subjects. Mean serum concentration of adiponectin in patients with NASH was significantly lower than in healthy subjects (4.87+/-1.96 vs. 8.33+/-4.56 ng/ml; p<0.05). Mean serum levels of TNF-alpha in patients with NASH were significantly higher than in controls (34.2+/-19.7 vs. 20.7+/-15.5 ng/ml; p<0.05). In patients with more advanced inflammation (grade 2-3) and fibrosis (stage 2) in pathology, serum concentration of leptin was significantly higher than in patients with steatosis and less advanced inflammation (grade 1) and fibrosis (stage 1) (median 8.94 vs. 16.2 ng/ml; p<0.05). No significant differences of serum concentration of others adipohormones between these two groups of patients were stated. Moreover, we observed the correlation in serum levels (examined group vs controls) between: resistin and TNF-alpha (r = 0.62; p<0.05), adiponectin and IL-6 (r = -0.60; p<0.05) and leptin and insulin (r = -0.51; p<0.05). In conclusion, based on our study we speculate that changes of adipohormones levels may be markers of NASH and the serum level of leptin can be associated with more advanced form of NASH.

First Report of a Phytoplasma Affecting Tomato in Poland.

During 2006, two tomato plants exhibiting dwarfing, twisting of shoots and leaves, and virescence and phyllody of flowers were observed in a greenhouse in western Poland. Total genomic DNA was extracted from approximately 3.5 g of leaf midribs and petioles using the modified cetyltrimethylammoniumbromide (CTAB) buffer method (3). Direct PCR was done with universal phytoplasma primers P1/P7 for amplification of ribosomal 16S rDNA. The PCR product (1.8 kb) was diluted 1:30 with sterile distilled water and used as DNA template for nested PCR with primers R16F2n/R16R2 (1). The final product was an expected 1.2-kb rDNA fragment amplified from infected tomato tissues. The DNA extracted from a Vinca sp. infected with phytoplasma of 16SrI-B subgroup and from a healthy tomato plant were used as positive and negative assay controls, respectively. Restriction fragment length polymorphism analysis of the final PCR product (1.2 kb) using enzymes MseI, KpnI, AluI, HhaI, HpaII, RsaI, TaqI, and TruI (1) indicated that phytoplasma from the tomato belonged to the subgroup 16SrI-C (1,2). The sequence obtained from the final PCR product (746 bp) was deposited in the GenBank database under accession number EF164961. Multiple sequence alignments with sequences of phytoplasma available from GenBank were performed using ClustalW software. The analysis revealed that the Polish isolate was co-identical (100%) to two phytoplasma sequences (GenBank Accession Nos. AY839617 and DQ078304) that belong to 16SrI-C subgroup. To our knowledge, this is the first report of a phytoplasma affecting tomato plant in Poland. References: (1) I.-M. Lee et al. Inst. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Cell-wall free bacteria. Page 283 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et a.l, eds. The American Phytopathology Society, 2001. (3) A. C. Padovan et al. Austral. J. Grape Wine Res. 1:25, 1995.

Dysphagia training programmes: 'fixes that fail' or effective inter-disciplinary working?

A number of factors influencing the development of dysphagia training programmes are identified, some of which relate to cultural issues and to the way systems operate. Lessons can be learned from current theories of relevance to effecting change in professionals' working practice. These lessons can be applied beyond dysphagia to other areas in which speech and language therapists are involved.

A multicenter randomized phase II study of oral vs. intravenous vinorelbine in advanced non-small-cell lung cancer patients.

PURPOSE: A randomized phase II trial of oral vs. intravenous (i.v.) vinorelbine was designed to determine the efficacy and safety of oral vinorelbine with an intrapatient dose escalation in previously untreated patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Between December 1997 and April 1999, 115 patients with stage IIIB or IV NSCLC were randomized (2 to 1) to receive either oral vinorelbine at a dose of 60 mg/m2/week for the first three administrations and then increased to 80 mg/m2/week in the absence of severe neutropenia, or i.v. vinorelbine at 30 mg/m2/week. RESULTS: One hundred and fourteen patients (76 in the oral arm and 38 in the i.v. arm) were treated. Ninety-eight patients (86%) were eligible and assessable. The two treatment arms were well-balanced for demographic and prognostic features. After external panel review, the response rates in evaluable patients were 14%, in the oral arm and 12% in the i.v. arm. The median progression-free survival with oral and i.v. vinorelbine was 3.2 months and 2.1 months, respectively, and the median survival 9.3 and 7.9 months, respectively. The most common hematological toxicity was neutropenia, which was severe (grade 3-4) in 46% of patients and for 7% of administrations in the oral arm, and in 62% of patients and for 25% of administrations in the i.v. arm. Non-hematological toxicities including nausea, vomiting, anorexia, weight loss, diarrhea .and constipation were generally mild to moderate. CONCLUSION: The activity of oral and i.v. vinorelbine in advanced NSCLC appears to be comparable. The safety profiles of both formulations look qualitatively similar. Oral vinorelbine can therefore be considered a good alternative to i.v. administration.

[Early detection of COPD by high risk population spirometric screening]. / Wczesne wykrywanie POChP badaniem spirometrycznym grup wysokiego ryzyka.

UNLABELLED: COPD is the most frequent chronic lung disease in Poland. The disease is however under-diagnosed, especially at the early stages. The aim of the study was to assess the efficacy of spirometric screening for COPD in middle aged smokers. Informations on causes and symptoms of COPD were disseminated in mass media in 14 large cities. Subject aged over 39 and with smoking history of > 10 packyears were invited for a free spirometry in local chest clinic. However, everyone attending had the spirometry performed. Spirometry was performed according to ATS recommendations. Airway obstruction (AO) was diagnosed when FEV1/FVC < 85% of N and categorised as mild (FEV1 > 70% of N), moderate (FEV1 50-69% of N) or severe (FEV1 < 50% of N). Spirometry was accompanied by an antismoking advice. RESULTS: 12.781 subjects were screened (mean age 52 +/- 12 years, 57% males). In 8.269 subjects who complied with inclusion criteria AO was diagnosed in 29.8% (mild in 10.9%, moderate in 12% and severe in 6.9%). In smokers < 40 years of age and a history of < 10 packyears AO was found in 8.8% (mild in 6.0%, moderate in 1.8% and severe in 1.0%). CONCLUSION: Mass spirometry is an effective and easy method for early detection of COPD.

[Incidence of chronic obstructive pulmonary disease among chronic smokers inhabiting Krakow and Proszowice]. / Czestosc wystepowania przewleklej obturacyjnej choroby pluc wsród przewlekle palacych tyton mieszkanców Krakowa i Proszowic.

COPD is the most common chronic lung condition whose identification is delayed. This paper undertakes to identify the present COPD occurrence frequency in the urban area of Krakow and the rural area of Proszowice. It makes use of free of charge spirometry tests in > 40 year-old volunteers, with the smoking history of at least 1 pack of cigarettes per day for 10 years. One in four persons examined in Krakow and one in three persons examined in Proszowice manifested ventilation impairment mainly of the obturation type. Severe obturation was diagnosed in one in three persons with ventilation dysfunction. In the conclusion the vital influence of COPD on public health is stressed, which should prompt a serious programme of early COPD identification and treatment, on regional or national scale.

Does chemotherapy-induced leukopenia predict a response in small-cell lung cancer?

The correlation between chemotherapy-induced toxicity and treatment outcome in cancer patients has not been studied thoroughly. Our aim was to evaluate whether there is any relationship between chemotherapy-induced leukopenia and response to treatment in small-cell lung cancer (SCLC). Data derived from records of 228 patients treated within two prospective multicentre phase II studies were analysed. In the first study (101 patients) chemotherapy included vincristine, epirubicin and cyclophosphamide and, in the second (127 patients), cyclophosphamide, etoposide and epirubicin; both regimens were given every 3 weeks. In the present analysis, the correlation between treatment outcome (response rate and survival) and highest scores of leukopenia within the first two and up to the fourth chemotherapy cycle, respectively, was evaluated. The objective response rate for the entire group was 66%; 53% in patients whose white blood cells remained normal and 85% in those who developed leukopenia within the first two cycles (P = 0.000). In multifactorial analysis, also including other treatment- and patient-related factors, independent correlation with response to chemotherapy was found for leukopenia (P = 0.001), chemotherapy regimen (P = 0.002) and the combined relative dose intensity (P = 0.018), but not for patient sex, age, performance status, pre-study weight loss, extent of disease and initial white blood cell count. Leukopenia within the first two cycles of chemotherapy was not correlated with survival, whereas such correlation for leukopenia occurring up to the fourth cycle was at the borderline level (P = 0.06). These findings suggest a relationship between chemotherapy-induced leukopenia and tumour response in SCLC.

[Foreign body imitating bronchial neoplasm]. / Cialo obce imitujace raka oskrzela.

A case of 53 year-old male admitted to the Clinic with fever and persisting cough with a muco-purulent secretion, varying in intensity for about 4 months, was presented. Prior to the admission to the Clinic the patient was treated with antibiotics, which only slightly relieved the symptoms mentioned above. The patient was admitted to the Clinic with diagnosis of chronic bronchitis, right sided bronchopneumonia with a suspicion of lung cancer. Performed chest X-ray seemed to confirm this diagnosis. Later on, tomography scans and bronchoscopy demonstrated a foreign body in the bronchus. Bronchoscopy not only allowed to exclude neoplastic change but also enabled us to "treat" the patient by extracting his molar tooth remained in the main right bronchus, which caused purulent inflammatory changes in this bronchus. After subsequent antibiotic therapy patient's general condition improved and radiological chest image returned to normal. Persistent cough and recurrent fever are often the symptoms of the lung cancer. It should be emphasised that in the observed case longterm treatment of these changes lasted for 4 months without chest radiological examination.

[Neurotic disturbances, depression and anxiety disorders in the population living in the vicinity of overhead high-voltage transmission line 400 kV. Epidemiological pilot study]. / Zaburzenia nerwicowe oraz poziom depresyjnosci i leku wsród osób zamieszkujacych w poblizu linii przesylowej 400 kV. Wstepne wyniki badan epidemiologicznych.

Numerous reports suggest a relationship between the increased incidence of depressive and neurotic symptoms in humans and the exposure to extremely low frequency electromagnetic field (EMF) at the place of residence. Our study was conducted in one of the Cracow suburbs with low socio-economic status, adjacent to an easement containing two 400 kV high-voltage transmission lines (distance up to 50 m from the line). The population investigated comprised 70 persons (35 males and 35 females). This group was exposed to EMF from 1986. A control group (n = 37), non-exposed to EMF, was also tested. The EMF intensity was measured by special devices. The following assessments instruments were used: Anamnestic Data Questionnaire, Neurotic Symptoms Questionnaire "O" Spielberger Self-Rating Questionnaire, and Beck-Self-Rating Scale. The measurements of the electric field levels taken at the front walls of investigated buildings averaged much higher than normal values of safety exposure. Our investigation showed the increased psychopathological values in all clinical tests. The difference between the group exposed to EMF and the control population was statistically significant. The results of our study did not support a possible cause-and-effect relationship between EMF and psychopathology observed. Some other factors (noise) can be responsible for the data obtained. The hypothesis that EMF produced by external electric power lines may enhance the occurrence of neurotic symptoms and depression, requires further clinical and experimental investigations.

[Analysis of prognostic factors for long term survival in patients with small cell lung cancer]. / Analiza czynników rokowniczych warunkujacych dlugotrwale przezycia chorych na drobnokomórkowego raka pluca.

Prognostic factors for long term survival were analyzed in a group of 719 patients with small cell lung carcinoma treated within 4 consecutive prospective multicenter trials between 1981 and 1990. 74 patients (10.3%) survived more than 2 years; 30 of them (4.2%) with no evidence of disease. The most significant determinator of prolonged survival was extent of disease: 13.9% (59/424) of patients with limited disease vs. 5.1% (15/295) with extensive disease survived more than 2 years (p < 0.001). Of 138 female patients 24 (17.4%) were long term survivors, compared to 8.6% (50/581) of males (p < 0.01). Initial good performance status and no weight loss were also found to be correlated with long term survival. Of the group of 2-year survivors 51 patients subsequently died (median survival duration 31 months), 10 are alive with cancer or are lost to follow up and 13 are in complete remission with median follow up of 64 months. 20 patients (3.8%) survived more than 5 years. This study confirms the possibility of cure in SCLC, especially in patients with favorable prognostic factors.