Clinical significance of TP53 alterations in advanced NSCLC patients treated with EGFR, ALK and ROS1 tyrosine kinase inhibitors: An update.

 The development of targeted therapies for non-small cell lung cancer (NSCLC), such as the epidermal growth factor receptor (EGFR), anaplastic lymphoma receptor tyrosine kinase (ALK), and ROS proto-oncogene 1 (ROS1), has improved patients' prognosis and significantly extended progression-free survival. However, it remains unclear why some patients do not benefit from the treatment as much or have a rapid disease progression. It is considered that, apart from the oncogenic driver gene, molecular alterations in a number of caretaker and gatekeeper genes significantly impact the efficacy of targeted therapies. The tumor protein 53 (TP53) gene is one of the most frequently mutated genes in NSCLC. To date, numerous studies have investigated the influence of various TP53 alterations on patient prognosis and responsiveness to therapies targeting EGFR, ALK, or ROS1. This review focuses on the latest data concerning the role of TP53 alterations as prognostic and/or predictive biomarkers for EGFR, ALK, and ROS1 tyrosine kinase inhibitors (TKIs) in advanced NSCLC patients. Since the presence of TP53 mutations in NSCLC has been linked to its decreased responsiveness to EGFR, ALK, and ROS1 targeted therapy in most of the referenced studies, the review also discusses the impact of TP53 mutations on treatment resistance. It seems plausible that assessing the TP53 mutation status could aid in patient stratification for optimal clinical decision-making. However, drawing meaningful conclusions about the clinical value of the TP53 co-mutations in EGFR-, ALK- or ROS1-positive NSCLC is hampered mainly by an insufficient knowledge regarding the functional consequences of the TP53 alterations. The integration of next-generation sequencing into the routine molecular diagnostics of cancer patients will facilitate the detection and identification of targetable genetic alterations along with co-occurring TP53 variants. This advancement holds the potential to accelerate understanding of the biological and clinical role of p53 in targeted therapies for NSCLC.

The expression of circulating miR-504 in plasma is associated with EGFR mutation status in non-small-cell lung carcinoma patients.

MicroRNAs (miRNAs), key regulators of gene expression at the post-transcriptional level, are grossly misregulated in some human cancers, including non-small-cell lung carcinoma (NSCLC). The aberrant expression of specific miRNAs results in the abnormal regulation of key components of signalling pathways in tumour cells. MiRNA levels and the activity of the gene targets, including oncogenes and tumour suppressors, produce feedback that changes miRNA expression levels and indicates the cell's genetic activity. In this study, we measured the expression of five circulating miRNAs (miR-195, miR-504, miR-122, miR-10b and miR-21) and evaluated their association with EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) mutation status in 66 NSCLC patients. Moreover, we examined the discriminative power of circulating miRNAs for EGFR mutant-positive and -negative NSCLC patients using two different data normalisation approaches. We extracted total RNA from the plasma of 66 non-squamous NSCLC patients (31 of whom had tumours with EGFR mutations) and measured circulating miRNA levels using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The miRNA expression levels were normalised using two endogenous controls: miR-191 and miR-16. We found significant associations between the expression of circulating miR-504 and EGFR-activating mutations in NSCLC patients regardless of the normalisation approach used (p = 0.0072 and 0.0236 for miR-16 and miR-191 normalisation, respectively). The greatest discriminative power of circulating miR-504 was observed in patients with EGFR exon 19 deletions versus wild-type EGFR normalised to miR-191 (area under the curve (AUC) = 0.81, p < 0.0001). Interestingly, circulating miR-504 levels were significantly reduced in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated subgroup compared to EGFR-mutated patients (p < 0.0030) and those with EGFR/KRAS wild-type tumours (p < 0.0359). Our study demonstrated the feasibility and potential diagnostic value of plasma miR-504 expression analysis to distinguish between EGFR-mutated and wild-type NSCLC patients. However, quality control and normalisation strategies are very important and have a major impact on the outcomes of circulating miRNA analyses.

[Current methods to detect EGFR gene mutations as predictive factor for targeted therapies in non-small cell lung cancer - is there a "golden standard" in diagnostics?]. / Wspólczesne metody wykrywania mutacji genu EGFR jako czynnika predykcyjnego dla terapii ukierunkowanej molekularnie chorych na niedrobnokomórkowego raka pluca - czy istnieje "zloty standard" diagnostyczny?

According to current Polish and international recommendations, detection of EGFR gene somatic mutations is the essential part of routine diagnostic algorithm in advanced NSCLC patients considered for tyrosine kinase inhibitor therapy. Molecular heterogeneity of tumor tissue and cytology materials used for molecular diagnostics is challenging for classic methods of genetic analysis, such as Sanger sequencing, driving the development and implementation of specialized, highly sensitive techniques for mutations detection. Constant, dynamic progress in molecular biology techniques, particularly development of next-generation sequencing, should enable clinical implementation of simultaneous multiple therapeutic biomarkers analysis as well as non-invasive EGFR mutations diagnostic based on free-circulating DNA isolated from blood of non-small cell lung cancer patients.

Reliable detection of rare mutations in EGFR gene codon L858 by PNA-LNA PCR clamp in non-small cell lung cancer.

PNA-LNA PCR clamp real-time PCR method represents allele-specific approach to mutation analysis of EGFR gene in NSCLC. Due to its unique design, it is characterized by exceptionally high specificity and sensitivity but also allows detection of rare or not specifically-targeted EGFR mutations within examined exons, otherwise undetectable by other mutation-specific fluorescent probes. We herein present two cases of rare mutations revealed by PNA-LNA PCR clamping of NSCLC samples referred for routine EGFR gene molecular diagnostics. In one, the EGFR gene L858 codon mutation was detected by standard PNA-LNA PCR clamping, subsequently reconfirmed and characterized by direct sequencing of allele specific amplification products as the missense mutation c.2572C>A (p.L858M) paired with L861Q mutation on the same allele (in cis). In the second sample, low quality FFPE material from pleural biopsy, c.2573C>T missense mutation (p.L858P) was revealed. Still, repeated DNA analysis by PNA-LNA PCR clamp and direct sequencing demonstrated low level of mutant allele existing in a total allele pool suggesting rather artifactual c.2572C>T transition, a phenomenon quite frequent in low-volume FFPE samples upon fixation procedures. In conclusion, superior sensitivity and unique design of PNA-LNA PCR clamping are crucial for its excellent diagnostic effectiveness. As we demonstrated, the method allows detecting rare EGFR mutations, although it increases the risk of detection of a very low signal, e.g., generated by a small pool of mutated allele. Therefore, applicability of PNA-LNA PCR clamp product for the direct sequencing reevaluation is of key importance enabling reliable validation of results.

[Higher incidence of alveolar lymphocytes (AL) apoptosis in smokers depends on BCL-2 expression and specific response to tumor necrosis factor alpha (TNFalpha). Bronchoalveolar lavage (BAL) material analysis from selected interstitial lung diseases (ILD) and healthy controls]. / Czestsza apoptoza limfocytów pecherzykowych (alveolar lymphocytes, AL) u palaczy papierosów zalezy od ekspresji BCL-2 i swoistej odpowiedzi na czynnik martwicy nowotworów alpha (tumor necrosis factor alpha, TNFalpha). Analiza materialu z plukania oskrzelowo-pecherzykowego (bronchoalveolar lavage, BAL) chorych z wybranymi chorobami sródmiazszowymi pluc i osób zdrowych.

BACKGROUND: We have previously described the increased apoptosis rate in smokers alveolar lymphocytes (AL) that was independent from the FASL/ FAS system activation. Consequently, the role of intrinsic apoptosis pathway and other ligand/death receptor pairs as TNFalpha/TNFR1 and TRAIL/DR4 important for apoptosis regulation should be considered in this phenomenon. The purpose of the study was to evaluate the impact of tobacco consumption on expression of selected BCL-2 family members and ligand/receptors pairs in bronchoalveolar lavage (BAL) harvested from patients with pulmonary sarcoidosis (PS), idiopathic pulmonary fibrosis (IPF) and healthy volunteers. The results were analyzed in the context of AL apoptosis rate. METHODS: AL apoptosis from PS (n=36, incl. 22 smokers), IPF (11, incl. 5 smokers) and controls (n=17, incl. 9 smokers) was evaluated by flow cytometry (sub-G1 of cell cycle). AL were stained for BCL-2, BCL-xL, BAK, TNFR1 (CD120A) TNFR2 (CD120B) and DR4. ELISA assay was used to evaluate the BAL supernatant levels of TNFalpha and TRAIL. RESULTS: According to previous observations, AL apoptosis rate was significantly higher in smoker subgroups as compared to nonsmoking counterparts. Decreased AL BCI-2+ relative number was observed in smoking PS (80.5 +/- 6.2 vs 91 +/- 9.8% in nonsmokers) and controls (59 +/- 14.1% vs 75 +/- 16.1%, p<0.05). TNFalpha concentration in BAL supernatant was significantly higher only in healthy smokers (2.32 +/- 0.77 vs 0.42 +/- 0.27 pg/ml, p<0.05), whereas TRAIL levels were remarkably enhanced in IPF smokers (44.8 +/- 12.8 vs 13.5 +/- 5.0 pg/ml, p<0.05) only. However, TUNEL. detected AL apoptosis positively correlated with TNFalpha. in smokers (p<0.05) and negatively with AL CD120B:CD120A expression ratio. Paradoxically, TNFalpha levels were positively correlated with AL BCL-2 expression in nonsmokers (Rs +0.58, p<0.01), but not in smokers. No differences were observed in all subgroups in respect to AL expression of DR4, BCL-xL or BAK. CONCLUSIONS: 1. AL were not sufficiently protected against apoptosis in smokers. 2. The most likely mechanisms involve down-regulation of BCL-2 expression and altered AL susceptibility to TNFalpha, mediated by imbalance between AL membrane expression of TNF receptor type 1 (death receptor) and type 2 (survival mediator). 3. Mechanisms regulating the increased AL apoptosis in smokers seem to be different in each tested group.

Targeted Next-Generation Sequencing of Thymic Epithelial Tumours Revealed Pathogenic Variants in KIT, ERBB2, KRAS, and TP53 in 30% of Thymic Carcinomas.

A better understanding of the molecular pathogenesis of thymic epithelial tumours (TETs) could revolutionise their treatment. We evaluated thymomas and thymic carcinomas by next-generation sequencing (NGS) of somatic or germline single nucleotide variants (SNVs) in genes commonly mutated in solid tumours. In total, 19 thymomas and 34 thymic carcinomas were analysed for nonsynonymous SNVs in 15 genes by targeted NGS (reference genome: hg19/GRCh37). Ten SNVs in TP53 (G154V, R158P, L194H, R267fs, R273C, R306 *, Q317 *), ERBB2 (V773M), KIT (L576P), and KRAS (Q61L) considered somatic and pathogenic/likely pathogenic were detected in 10 of 34 (29.4%) thymic carcinomas. No somatic SNVs confirmed as pathogenic/likely pathogenic were found in thymomas. Rare SNVs of uncertain or unknown functional and clinical significance, to our knowledge not reported previously in TETs, were found in ERBB2 (S703R), KIT (I690V), and FOXL2 (P157S) in 3 of 19 (16%) thymomas. The most frequent germline SNVs were TP53 P72R (94% TETs), ERBB2 I655V (40% TETs), and KIT M541L (9% TETs). No significant difference in median disease-free survival (DFS) was found between thymic carcinoma patients with and without pathogenic SNVs (p = 0.190); however, a trend toward a longer DFS was observed in the latter (16.0 vs. 30.0 months, respectively). In summary, NGS analysis of TETs revealed several SNVs in genes related to the p53, AKT, MAPK, and K-Ras signalling pathways. Thymic carcinomas showed greater genetic dysregulation than thymomas. The germline and rare SNVs of uncertain clinical significance reported in this study add to the number of known genetic alterations in TETs, thus extending our molecular understanding of these neoplasms. Druggable KIT alterations in thymic carcinomas have potential as therapeutic targets.

Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world evaluation of two popular diagnostic systems.

PURPOSE: The detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations. METHODS: We analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I-IV) before treatment (n â€‹= â€‹60) and after clinical progression on EGFR tyrosine kinase inhibitors (n â€‹= â€‹30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.) and therascreen EGFR Plasma RGQ PCR kit (Qiagen GmbH). RESULTS: There was higher concordance between plasma cfDNA and matched tumor tissue EGFR mutations with cobas (66.67%) compared with therascreen (55.93%). The concordance rate increased to 90.00% with cobas (Cohen's kappa coefficient, κ â€‹= â€‹0.80; p â€‹< â€‹0.0001) and 73.33% with therascreen (κ â€‹= â€‹0.49; p â€‹= â€‹0.0009) in advanced NSCLC patients. In treatment-naïve patients, cobas was superior to therascreen (sensitivity: 82.35% vs. 52.94%; specificity: 100% vs. 100%). In patients with clinical progression on EGFR tyrosine kinase inhibitors, EGFR exon 20 p.T790M was detected in 30% and 23% of cfDNA samples by cobas and therascreen, respectively. CONCLUSIONS: Cobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.

miR-625-3p and lncRNA GAS5 in Liquid Biopsies for Predicting the Outcome of Malignant Pleural Mesothelioma Patients Treated with Neo-Adjuvant Chemotherapy and Surgery.

Combining neo-adjuvant chemotherapy and surgery is part of multimodality treatment of malignant pleural mesothelioma (MPM), but not all patients benefit from this approach. In this exploratory analysis, we investigated the prognostic value of circulating miR-625-3p and lncRNA GAS5 after neo-adjuvant chemotherapy. 36 MPM patients from the SAKK 17/04 trial (NCT00334594), whose blood was available before and after chemotherapy were investigated. RNA was isolated from plasma and reverse transcribed into cDNA. miR-16-5p and ß-actin were used as a reference gene for miR-625-3p and GAS5, respectively. After exclusion of samples due to hemolysis or RNA degradation, paired plasma samples from 32 patients before and after chemotherapy were further analyzed. Quantification of miR-625-3p levels in all 64 samples revealed a bimodal distribution and cloning and sequencing of miR-625-3p qPCR product revealed the presence of miR-625-3p isomiRs. Relative change of the circulating miR-625-3p and GAS5 levels after chemotherapy showed that increased circulating miR-625-3p and decreased GAS5 was significantly associated with disease progression (Fisher's test, p = 0.0393). In addition, decreased levels of circulating GAS5 were significantly associated with shorter overall and progression-free survival. Our exploratory analysis revealed a potential value of circulating non-coding RNA for selection of patients likely to benefit from surgery after platinum-based adjuvant chemotherapy.

[Molecular diagnostics of alpha-1-antitrypsin deficiency in clinical practice]. / Diagnostyka molekularna niedoboru alfa-1-antytrypsyny w praktyce klinicznej.

The deficiency of serine protease inhibitor, alpha-1-antitrypsin (AATD), is genetically determined defect that increases the risk of lung and liver disease development. The results of recent epidemiological studies indicate the overwhelming majority of individuals with alpha-1-antitrypsin deficiency still remain undiagnosed. The complete laboratory diagnosis of AATD is based on combination of quantitative and qualitative methods. The measurement of plasma/serum AAT concentration is always the initial test performed in clinically suspected individuals. Nevertheless, only the AAT phenotype or genotype identification allows the full medical verification of the diagnosis. Among the various techniques of either AAT variant phenotyping or genotyping accepted by reference medical centers worldwide, the isoelectric focusing, real-time-PCR and restriction fragment-length polymorphism PCR (RFLP-PCR) are "considered the most effective" performed the most commonly. The AAT diagnostics in Poland still awaits for introduction into clinical routine. The aim of present review is to outline the major methods of AATD diagnosis and discuss with the special issuing of their potential benefits and disadvantages.

miRNAs as Biomarkers and Therapeutic Targets in Non-Small Cell Lung Cancer: Current Perspectives.

Lung cancer is the most common cancer worldwide. Up to 85% of lung cancer cases are diagnosed as non-small cell lung cancer (NSCLC). The effectiveness of NSCLC treatment is expected to be improved through the implementation of robust and specific biomarkers. MicroRNAs (miRNAs) are small, non-coding molecules that play a key role in the regulation of basic cellular processes, including differentiation, proliferation and apoptosis, by controlling gene expression at the post-transcriptional level. Deregulation of miRNA activity results in the loss of homeostasis and the development of a number of pathologies, including lung cancer. During lung carcinogenesis, miRNAs exhibit dual regulatory function: they act as oncogenes to promote cancer development or as tumour suppressors. Unique miRNA sequences have been detected in malignant tissues and corresponding healthy tissues. Furthermore, stable forms of tumour-related miRNAs are detectable in the peripheral blood of patients with NSCLC. The potential benefits of using extracellular miRNAs present in body fluids as part of the diagnostic evaluation of cancer include low invasiveness (compared with tumour cell/tissue sampling), and the repeatability and ease of obtaining the specimens. Apart from the diagnostic applications of altered miRNA expression profiles, the dual regulatory role of miRNA in cancer might drive the further development of personalised therapies in NSCLC. The clinical usefulness of miRNA expression analysis to predict the efficacy of various treatment strategies including surgery, radio- and chemotherapy, and targeted therapies has been evaluated in NSCLC. Also, the capacity of a single miRNA to regulate the expression of multiple genes simultaneously presents an opportunity to use these small molecules in personalised therapy as individualised therapeutic tools.

Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD). Assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine.

Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n = 13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for (1) IGF-I, BCL-2, Fas and Fas Ligand expression and (2) cell cycle (incl. sub-G1 peak of late apoptosis) with propidium iodide (PI). Flow cytometry (FC) and immunocytochemistry were used. AL early apoptosis was detected by Annexin V FITC/PI staining. IGF-I was present in AL of all tested groups. The number of IGF-I positive AL was significantly higher in IPF (52 +/- 6.7%) and in later (II and III) stages of sarcoidosis (39 +/- 7.8 vs 16 +/- 4.0% in controls, p < 0.05). Increased BCL-2 expression in AL was detected in IPF and sarcoidosis. In all tested groups, AL were almost exclusively Fas+ T cells. Generally, a low number of AL entered apoptosis; no significant differences were found between patient groups, except decreased apoptosis rate in sarcoidosis (0.60 +/- 0.17 vs 1.15 +/- 0.33% in controls, p < 0.05). Proportion of AL positive for IGF-I was significantly correlated with parameters reflecting AL and AM cell proliferation and BCL-2 expression (e.g. AL IGF-I+ vs AM in S phase of cell cycle: r(S) = +0.50, p = 0.001), but not with apoptosis. The results show that human alveolar lymphocytes express IGF-I in normal conditions, as well as in ILD. The proportion of IGF-I+ lymphocytes was significantly increased in IPF and at later stages of sarcoidosis. In our material there was no evidence for profibrogenic or antiapoptotic activity of IGF-I. We suggest that IGF-I originating from AL may be locally active as a mitogen for alveolar macrophages and lymphocytes in ILD.

[Aptamers in clinical diagnostics]. / Aptamery w diagnostyce klinicznej.

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro from combinatorial libraries in a process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Aptamers play a role of artificial nucleic acid ligands that can recognize and bind to various organic or inorganic target molecules with high specificity and affinity. They can discriminate even between closely related targets and can be easily chemically modified for radioactive, fluorescent and enzymatic labeling or biostability improvement. Aptamers can thus be considered as universal receptors that rival antibodies in diagnostics as a tool of molecular recognition. To date aptamers have been successively used instead of monoclonal antibodies in flow cytometry, immunochemical sandwich assays and in vivo imaging as well to detect wide range of small or large biomolecules.

Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules.

Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. The analysis of cell-free DNA (cfDNA) in blood may greatly aid the early detection of lung cancer by evaluating cancer-related alterations. The plasma cfDNA levels and integrity were analysed in 65 non-small cell lung cancer (NSCLC) patients, 28 subjects with benign lung tumours, and 16 healthy controls using real-time PCR. The NSCLC patients demonstrated significantly higher mean plasma cfDNA levels compared with those with benign tumours (P = 0.0009) and healthy controls (P < 0.0001). The plasma cfDNA integrity in healthy individuals was significantly different than that found in patients with NSCLC or benign lung tumours (P < 0.0003). In ROC curve analysis, plasma cfDNA levels >2.8 ng/ml provided 86.4% sensitivity and 61.4% specificity in discriminating NSCLC from benign lung pathologies and healthy controls. cfDNA integrity showed better discriminatory power (91% sensitivity, 68.2% specificity). These data demonstrate that plasma cfDNA concentration and integrity analyses can significantly differentiate between NSCLC and benign lung tumours. The diagnostic capacity of the quantitative cfDNA assay is comparable to the values presented by conventional imaging modalities used in clinical practice.

[Studies on insulin-like growth factor-I expression in human lower airways]. / Badania nad ekspresja insulinopodobnego czynnika wzrostu I w dolnych drogach oddechowych u ludzi.

UNLABELLED: Antisense techniques that inhibit intracellular expression of insulin-like growth factor-I (IGF-I) were efficient in gene therapy of some tumor diseases. IGF-I in the airways is considered to induce lung fibrosis in interstitial lung diseases, inhibit apoptosis of epithelial cells and participate in local carcinogenesis. AIM OF THE STUDY: The aim of the study was--by examining the IGF-I expression in the lower airways--to evaluate preliminary the efficacy of anti-IGF-I antisense technicques in the treatment of airways diseases. MATERIAL AND METHODS: The IGF-I expression was examined in the reverse transcriptase--polimerase chain reaction (RT-PCR) applied to A549 human cell line, that is representative for lower airway epithelium and exemplifies the model of bronchioloalveolar adenocarcinoma. IGF-I expression in non-neoplastic lower airways cells was assessed by immunocytochemical staining (anti-IGF-I) in cytological materials originating from bronchoalveolar lavage (BAL) of sarcoidosis and asbestosis patients and in individuals free of lung pathology. RESULTS: The IGF-I expression was detected in A549 cells with use of RT-PCR method (3 independent probes). In BAL cytological specimens the appearance of IGF-I was found, mainly in alveolar macrophages (62 +/- 6, 5 in sarcoidosis vs. 36 +/- 6 in controls, p=0,09), as well as in BAL lymphocytes. CONCLUSIONS: Summing up, the antisense technicques, blocking the intracellular IGF-I expression may be potentially useful in treatment of selected lower airways, both tumor (non-small cell lung carcinoma) and non-tumor (ILD complicated with lung fibrosis) conditions.

Coexistence of Borrelia burgdorferi s.l. genospecies within Ixodes ricinus ticks from central and eastern Poland.

The purpose of the study was to assess the prevalence and coinfection rates of Borrelia burgdorferi sensu lato genotypes in Ixodes ricinus (L.) ticks sampled from diverse localities in central and eastern regions of Poland. In years 2009-2011, questing nymphs and adults of I. ricinus were collected using a flagging method at 18 localities representing distinct ecosystem types: urban green areas, suburban forests and rural woodlands. Molecular detection of B. burgdorferi s.l. genospecies was based on amplification of a fla gene using nested PCR technique, subsequent PCR-RFLP analysis and bidirectional sequencing. It was revealed that 45 samples (2.1%) harboured two different B. burgdorferi s.l. genospecies, whereas triple infections with various spirochetes was found in 11 (0.5%) individuals. Generally, the highest average coinfection rates were evidenced in arachnids gathered at rural woodlands, intermediate at suburban forests, while the lowest were recorded at urban green areas. Overall, single spirochete infections were noted in 16.3% (n = 352/2,153) ticks. Importantly, it is the first report evidencing the occurrence of Borrelia miyamotoi (0.3%, n = 7/2153) in I. ricinus populations within central Poland. Circumstantial variability of B. burgdorferi s.l. genospecies in the common tick individuals sampled at various habitat types in central and eastern Poland was displayed. The coexistence of two or three different spirochete genospecies in single adult ticks, as well as the presence of B. miyamotoi were demonstrated. Therefore, further studies uncovering the co-circulation of the tested bacteria and other human pathogens in I. ricinus ticks are required.

Studies of hepatocyte growth factor in bronchoalveolar lavage fluid in chronic interstitial lung diseases.

INTRODUCTION:  Previous studies have suggested that hepatocyte growth factor (HGF) inhibits lung fibrosis as an antagonist of transforming growth factor ß (TGF­ß). OBJECTIVES:  We assessed HGF expression levels in the lower airways of patients with selected interstitial lung diseases. PATIENTS AND METHODS:  HGF levels were examined by an enzyme­linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid supernatants from patients with pulmonary sarcoidosis (PS, n = 52), idiopathic pulmonary fibrosis (IPF, n = 23), nonspecific interstitial pneumonia (NSIP, n = 14), extrinsic allergic alveolitis (EAA, n = 6), bronchiolitis obliterans organizing pneumonia (BOOP, n = 8), chronic eosinophilic pneumonia (EP, n = 6), and in control subjects (n = 13). Intracellular HGF expression in BAL cells was evaluated by flow cytometry. RESULTS:  HGF concentrations were elevated in BAL fluid from nonsmokers with IPF (261 ±204 pg/ml, P <0.02), smokers with IPF (220 ±13 pg/ml, P <0.001), and smokers with PS (172 ±33 pg/ml, P <0.02), as compared with controls (148 ±17 pg/ml for nonsmokers; 137 ±9 pg/ml for smokers). HGF levels were positively correlated with TGF­ß concentrations in BAL fluid (r = 0.3; P = 0.02) and negatively-with vital capacity (r = -0.2; P = 0.02). BAL neutrophils, and, for the first time, BAL lymphocytes, were identified as intracellular HGF­positive cells. CONCLUSIONS:  Our results do not support evidence for strong antifibrotic HGF activity. The highest HGF concentrations were observed in BAL fluid from patients with IPF, and they were also positively correlated with TGF­ß levels. Thus, although the local protective mechanisms such as the HGF expression are upregulated in chronic interstitial lung diseases, they are not enough to prevent lung fibrosis.

Quantitative analysis of free-circulating DNA in plasma of patients with resectable NSCLC.

OBJECTIVE: Minute amounts of free-circulating DNA are present in plasma of healthy individuals, whereas its increased concentration was observed in patients with malignant tumors including non-small cell lung cancer (NSCLC). This study aimed at demonstrating the potential usefulness of plasma DNA concentration monitoring in NSCLC patients for therapy effectiveness assessment throughout the treatment and follow-up period. METHODS: Plasma DNA concentration was assessed in 50 NSCLC patients (stage I - IIIA) prior and following the radical treatment using real-time quantitative PCR method. 10 orthopedic patient undergoing hip joint surgery and 40 healthy volunteers comprised control groups. RESULTS: NSCLC patients (8.02 ng/ml) demonstrated significantly higher mean plasma DNA concentration with respect to healthy controls (2.27 ng/ml; p < 0.0000). Drastic increase in plasma DNA levels up to mean 68.74 ng/ml was detected a week after primary tumor resection. Still, similar phenomenon was observed in patients subjected to orthopedic surgical treatment (from 3.00 to 28.38 ng/ml, p < 0.0015). Most resected NSCLC patients with no disease recurrence during 3- to 6-month follow-up demonstrated reduced plasma DNA levels (mean 2.77 ng/ml) with respect to their presurgical values, whereas in relapsed subjects plasma DNA levels were significant higher. CONCLUSION: Free-circulating DNA concentration in plasma was significantly higher in NSCLC patients versus healthy controls. Its drastic increase following radical NSCLC treatment was most likely due to the surgical trauma. Importantly, the kinetics of plasma free-circulating DNA seems to be a promising marker of long-term effects of radical surgery in NSCLC.

Molecular evidence of Anaplasma phagocytophilum and Babesia microti co-infections in Ixodes ricinus ticks in central-eastern region of Poland.

The aim of the study was to elucidate the distribution of Anaplasma phagocytophilum and Babesia microti co-infection in Ixodes ricinus populations within the central-eastern region of Poland. The prevalence of analysed tick-borne human pathogens in single and polymicrobial infections in I. ricinus ticks were analysed using the conventional and nested PCR techniques. A total number of 1,123 questing tick individuals (291 females, 267 males and 565 nymphs) were collected at different ecosystems (municipal parks, suburban forests, and woodlands). In the presented study, 95 samples of ticks (8.5%) were infected with A.phagocytophilum, 3.1% (n=35) with B. microti, whereas the co-existence status of these human pathogens was detected in 1.8% (n=20) of all tested samples. It has been demonstrated that the prevalence of co-infection status was the highest among females of I. ricinus (11 samples, 3.8%), whereas the lowest within tested nymphs (5 samples, 0.9%). Ticks collected at city parks in Warsaw and suburban areas of this town characterized the highest prevalence of co-infections (3.3 and 4.8%, respectively). Furthermore, it was established that co-infection rates of ticks inhabiting woodlands within Kampinos National Park and Nadbuzanski Landscape Park were similar and reached the levels of 1.4% (n=5) and 1.1% (n=4), respectively.

Simple flow cytometric protocol of CD4+/CD8+ lymphocyte ratio assessment in bronchoalveolar lavage fluids from patients with interstitial lung diseases.

OBJECTIVE: To validate the fast and accurate flow cytometric (FCM) protocol using blood-standardized antibodies for alveolar lymphocyte subtyping with respect to standard immunocytochemistry (IC). STUDY DESIGN: FCM and IC were applied to immunophenotype T cell subsets in bronchoalveolar lavage (BAL) fluids from patients with interstitial lung diseases. Diagnostic BAL specimens from 50 patients with suspected sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis were evaluated by both IC and FCM. In FCM, CD4+ and CD8+ T cells were identified by light scatter gating with CD3 selection using basic tricolor cytometer. RESULTS: Relative amounts of CD4+, CD8+ T cells, and CD4+/CD8+ ratios demonstrated by the FCM showed excellent, significant correlations with IC results. FCM values did not differ significantly from IC results. However, the sensitivity and specificity of conventional IC staining were not sufficient to assess CD4+/ CD8+ ratio in most idiopathic pulmonary fibrosis cases. Additionally, performing IC immunophenotyping in BAL samples with low lymphocyte content introduced a remarkable error into CD4+/CD8+ ratio assessment. CONCLUSION: FCM allowed reliable, precise, and fast T-cell subset measurement in all BAL samples, overcoming the IC disadvantages. Our validated FCM protocol provides diagnostically relevant CD4+/CD8+ ratio determination by simple light scatter gating strategy with CD3 selection.