Marek Sawicki

A comparative analysis of phenotypic and genotypic methods for the determination of the biofilm-forming abilities of Staphylococcus epidermidis

The collection of 146 Staphylococcus epidermidis strains isolated from the nasopharynx of lung cancer patients has been studied for the ability of slime secretion and biofilm formation using the Congo red agar (CRA) test and the microtiter plate (MtP) method, respectively. The prevalence of the icaAD and the aap genes was also analyzed. Some isolates (35.6%) were biofilm positive by the MtP method, while 58.9% of isolates exhibited a slime-positive phenotype by the CRA test. The sensitivities of the CRA test evaluated using the MtP method as a gold standard of biofilm production were 73.1%, 97.3% and 13.3% for all the strains screened, ica-positive and ica-negative strains, respectively. The genotype ica(+)aap(+) was correlated with a strong biofilm-producer phenotype. Interestingly, some of the ica(-)aap(-) isolates could also form a biofilm. The correlation between the presence of icaAD genes and the biofilm-positive phenotype by the MtP method as well as slime production by the CRA test was statistically significant (P<0.0001). However, some S. epidermidis strains possess the potential ability of ica-independent biofilm formation; thus, further studies are needed to determine reliable, short-time criteria for an in vitro assessment of biofilm production by staphylococci.

Anaerobic bacteria colonizing the lower airways in lung cancer patients.

Anaerobes comprise most of the endogenous oropharyngeal microflora, and can cause infections of airways in lung cancer patients who are at high risk for respiratory tract infections. The aim of this study was to determine the frequency and species diversity of anaerobes in specimens from the lower airways of lung cancer patients. Sensitivity of the isolates to conventional antimicrobial agents used in anaerobe therapy was assessed. Respiratory secretions obtained by bronchoscopy from 30 lung cancer patients were cultured onto Wilkins-Chalgren agar in anaerobic conditions at 37°C for 72-96 hours. The isolates were identified using microtest Api 20A. The minimal inhibitory concentrations for penicillin G, amoxicillin/clavulanate, piperacillin/tazobactam, cefoxitin, imipenem, clindamycin, and metronidazole were determined by E-test. A total of 47 isolates of anaerobic bacteria were detected in 22 (73.3%) specimens. More than one species of anaerobe was found in 16 (53.3%) samples. The most frequently isolated were Actinomyces spp. and Peptostreptococcus spp., followed by Eubacterium lentum, Veillonella parvula, Prevotella spp., Bacteroides spp., Lactobacillus jensenii. Among antibiotics used in the study amoxicillin/clavulanate and imipenem were the most active in vitro (0% and 2% resistant strains, respectively). The highest resistance rate was found for penicillin G and metronidazole (36% and 38% resistant strains, respectively). The results obtained confirm the need to conduct analyses of anaerobic microflora colonizing the lower respiratory tract in patients with lung cancer to monitor potential etiologic factors of airways infections, as well as to propose efficient, empirical therapy.

Molecularly targeted therapies in head and neck cancers.

Head and neck cancers (HNC) are 6th most common malignancies according to the incidence rate. Over 85% of tumors of this region are epithelial tumors, especially squamous cell carcinomas (head and neck squamous cell carcinomas - HNSCC). Surgery, chemotherapy and radiotherapy are still the standard for the treatment of HNC. Despite the great development of the various methods of treatment, survival of patients have not improved significantly over the last 30 years, with the overall, 5-year survival not exceeding 50%. Progress in understanding the biology of cancer leads to personalization of therapy and introduction of drugs with molecular mechanism of action to everyday practice. At present, the effectiveness of monoclonal antibodies against EGFR in the treatment of HNSCC has already been proven. Cetuximab in combination with radiotherapy was found to be effective in patients with advanced and locally advanced HNSCC. There are also some promising results of phase III trials with zalutumumab and panitumumab. Initial efficacy of sorafenib (an inhibitor of the intracellular domain of VEGFR, PDGFR and c-Kit) and afatinib (an irreversible inhibitor of pan-HER tyrosine kinase) have been demonstrated. Great hopes for the future are linked with the potential use of STAT3, EGFRvIII, abnormal proteins K-ras, H-ras and PTEN as well as proteasome as a target for therapy.

Intraspinal K-wire migration after humeral fracture fixation

1010-7940/

Sensitive methods for the detection of an insertion in exon 20 of the HER2 gene in the metastasis of non‑small cell lung cancer to the central nervous system

— see front matter # 2010 European Association for Cardio-Thoracic doi:10.1016/j.ejcts.2010.09.014 A 54-year-old man presented 1 month after surgical osteosynthesis of right humeral fracture. Chest X-ray and computed tomography (CT) revealed transpleural, intraspinal K-wire migration to the level of Th3/Th4 (Figs. 1 and 2). The rod was removed by right thoracotomy, with ligation of meningeal laceration. There were no postoperative neurological dysfunctions.

Screening for ALK abnormalities in central nervous system metastases of non-small-cell lung cancer: ALK abnormalities in CNS metastases of NSCLC.

The HER2 (ErbB2/neu) protein is a member of the HER (ErbB) receptor family (EGFR, HER2, HER3 and HER4) that expresses tyrosine kinase activity in the intracellular domain. EGFR and HER2 overexpression is observed in numerous types of cancer, nevertheless, the susceptibility of patients with non-small cell lung cancer (NSCLC) to therapy with EGFR and HER2 tyrosine kinase inhibitors (TKIs) depends on mutations present in the respective coding genes (driver mutations). In the present study, PCR and amplified DNA fragment length analysis (FLA) were used along with the multi-temperature single-strand conformation polymorphism (MSSCP) technique in order to identify the 12 base pair insertion in exon 20 of the HER2 gene in 143 patients with NSCLC metastasis to the central nervous system. The prevalence of the HER2 gene mutation was correlated with mutations in the EGFR and BRAF genes. The insertion in exon 20 of the HER2 gene was observed in a single 77-year-old, non-smoking male, with poorly-differentiated adenocarcinoma of the lung (1.5% of adenocarcinoma patients). No other genetic abnormalities were identified in this patient. In the therapy of NSCLC patients with HER2 gene mutations, drugs that inhibit the EGFR and HER2 receptors, for example afatinib, may be effective. The identification of other driving mutations in NSCLC cells appears to be key to the appropriate qualification of molecular targeted therapies.

Screening for gene mutations in central nervous system metastases of non-small-cell lung cancer: Letter to the Editor

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%–7% of primary non‐small‐cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non‐smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ2 = 16.783) in former‐smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.

Management of myasthenic crisis in a child.

Non-small-cell lung cancer (NSCLC) is characterized by aggressive clinical course including frequent occurrence of distant metastases. Central nervous system (CNS) metastases are diagnosed in 20%– 40% of NSCLC patients and they are considered as a pharmacological sanctuary lesions for most cytotoxic agents. Molecularly targeted therapies have shown relatively high activity in CNS metastases in patients harboring “drugable” abnormalities in EGFR or ALK genes. Especially promising activity of the secondgeneration ALK inhibitors (alectinib, ceritinib) and sequential or concurrent application of EGFR/ALK TKIs and WBRT/stereotactic radiotherapy has been postulated (4, 6, 8). To date the knowledge on the effectiveness of molecularly targeted therapies in NSCLC patients with CNS metastases is relatively scarce. The patients with untreated CNS metastases are excluded from recent clinical trials investigating new therapies in lung cancer (4, 8). Therefore, we retrospectively assessed the spectrum of “drugable” abnormalities in 10 genes, in CNS metastases of NSCLC (145 FFPE tissue samples—45 females and 100 males; median age 60 6 8.8 years; PS 5 0 or 1; all patients chemotherapy and TKI na€ıve), and determined the relationship between molecular status and clinical characteristics of our patients. The studied group was heterogeneous in terms of histopathology (80 adenocarcinoma, 29 squamous cell carcinoma, 22 large cell cancer and 14 nototherwise-specified NSCLC patients) and smoking status (73 current-smokers, 21 former smokers, 36 non-smokers). The molecular profile of selected mutations was assessed using different molecular methods. Mutations in EGFR (exons 18–21), KRAS (codons: 12; 13; 61), NRAS (codons: 12; 61), BRAF (codon: 600), PTEN (codon: 233) and AKT1 (codon: 17) genes were analyzed with commercially available kits certified for in vitro diagnostic (Entrogen, Woodland Hills, California, USA) or TaqMan probes for research use only (Applied Biosystem, Carlsbad, California, USA). To analysis mutation in, PIK3CA (codons: 542; 545; 1047), MEK1 (codons: 56; 57; 67), HER2 (exon 20) and DDR2 (codon: 768) genes we used originally designed methods which based on allele-specific PCR (ASP-PCR) and highresolution melting PCR (HRM-PCR). Moreover, direct sequencing and multi-temperature single strand conformation polymorphism (MSSCP) techniques were used to confirm results obtained by originally designed methods. ALK abnormal protein was determined using automated immunohistochemistry with Positive Rabbit Monoclonal Antibody D5F3 (Ventana, Tucson, Arizona, USA) according to manufacturer instructions (9). To confirm ALK gene rearrangement we used FISH technique with Vysis ALK Break Apart FISH Probe Kit (Abbot Molecular, Des Plaines, Illinois, USA). The criteria of FISH analysis were in accordance with FDA guidelines (10). In 30 patients, the material was simultaneously available from primary and metastatic NSCLC tumors. We identified at least one abnormality in 59 cases (41%): KRAS—21.4% (31/145), EGFR—6.2% (9/145), ALK—4.8% (7/145), DDR2—2.1% (3/145), PIK3CA—2.1% (3/145), NRAS— 1.4% (2/145) and HER2, AKT1, PTEN, MEK1 respectively in 0.7% of patients (1/145) (Figure 1A). Coexistence of two mutations (KRAS and DDR2) was found in one patient. Mutations were significantly more frequently observed in adenocarcinoma compared to other histologic types of NSCLC (P 5 0.001; v 5 15.9; Figure 1B,C) and in non-smokers compared to former/current smokers (P 5 0.034; v 5 11.39, Figure 1D,E). The mOS of patients with mutations was insignificantly longer than in patients with wild-type of analyzed genes (16 vs. 11.7 months; P 5 0.084; HR 5 1.36). Cox multivariate logistic regression demonstrated that the factors significantly prolonging patients’ survival were younger age (<60 years) and mutations’ presence (overall model: P 5 0.0459; v 5 6.161). In 30 matched primary NSCLC tumors, 23% had EGFR and 7% had KRAS mutations. Heterogeneity between primary tumor and CNS metastases concerned only KRAS mutations. In five cases KRAS gene mutations were identified in both specimens, in one case only in primary tumor and in one case only in CNS metastases. Most of our observations are in concordance with large epidemiological data from studies focusing on primary tumors of NSCLC. Barlesi et al indicated the presence of driver mutations in six examined genes in 50% out of 17 664 European and Caucasian patients. Prevalence of analyzed mutations was as follows: KRAS mutations in 29% (4894/17 001) of cases, EGFR mutations in 11% (1947/17 706) of cases, ALK rearrangement in 5% (388/8134) of cases, BRAF mutations in 2% (262/13 906) of cases, PIK3CA mutations in 2% (252/ 10 678) of cases and HER2 mutations in 1% of patients (98/11 723). The presence of a genetic alterations was significantly associated with longer duration of response to both firstand second-line of treatment, as well as with longer first-line PFS and with longer mOS. Cox multivariate analysis confirmed that the presence of ALK rearrangements and EGFR and HER2 genes mutations had a favorable effect on prognosis (1). Kris et al reported the driver mutations in 64% (466/733) of American patients with adenocarcinoma. The analysis included ten genes and spectrum of particular disorders was fallowing: KRAS mutations in 25% (182/733) of cases, EGFR activation mutations in 17% (122/733) of cases, ALK rearrangements in 8% (57/733) of cases, HER2 mutations in 3% (19/733) of cases, BRAF mutations in 2% (16/733) of cases; PIK3CA mutations in <1% (6/733) cases; MET amplification in <1% (5/733) of cases; NRAS mutations in <1% (5/733) of cases; MEK1 mutation in <1% (1/733) of cases. The authors have not identified a mutation in AKT1 gene. Moreover, they noted the co-existence of two or more mutations in 3% (24/733) of cases. PIK3CA gene mutations commonly overlapped with others genetic abnormalities. Kris et al observed significantly longer mOS in patients with molecular abnormalities who received targeted therapies

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

Myasthenia gravis is an autoimmune disorder of peripheral nervous system, leading to fluctuating muscle weakness. It is caused by circulating antibodies that block acetylcholine nicotinic postsynaptic receptors at the postsynaptic neuromuscular junction. Myasthenic crisis is a life-threatening complication, which is defined as weakness from acquired myasthenia gravis. In this paper we described a 15-year-old boy who was admitted to the Paediatric Intensive Care Unit due to myasthenic crisis. He had suffered not only from myasthenia gravis but also hypothyroidism, cerebral palsy and epilepsy. The patient required mechanical ventilation and was successfully treated with both plasmapheresis and intravenous immunoglobulins. He recovered from the crisis and then thymectomy was performed. Perioperative period and anaesthesia passed uncomplicated. Discharged home from the hospital after 2.5 month-treatment, for the last 4 years, he has only come on scheduled outpatient medical appointments. This case reveals that myasthenic crisis, albeit rare, may occur in male adolescents. In such cases multidisciplinary care followed by surgery becomes a procedure of choice. Concomitant medical problems, if well controlled, do not affect the results of outcome of the underlying disease.

Prevalence of NRAS, PTEN and AKT1 gene mutations in the central nervous system metastases of non-small cell lung cancer

PurposeRT-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.MaterialsWe 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.Results21% 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.ConclusionUtility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.