Resistance to osimertinib in advanced EGFR-mutated NSCLC: a prospective study of molecular genotyping on tissue and liquid biopsies.

BACKGROUND: Resistance to osimertinib in advanced EGFR-mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent therapeutic implications. METHODS: This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR-mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed. RESULTS: Sixty-five advanced EGFR-mutated NSCLC patients treated with osimertinib in first- (n = 56) or in second-line (n = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification (n = 8), EGFR C797S (n = 3), and SCLC transformation (n = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively. CONCLUSIONS: Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.

An undiagnosed pleural effusion with surprising consequences.

We present the case of a 43-year-old Italian woman with a left undiagnosed pleural effusion, which in subsequent months presented a clinically unexpected evolution with the appearance at first of a right wrist tenosynovitis and subsequently a bilateral lung involvement caused by M. Tuberculosis. With this case report, we would like to underline the importance of making a correct diagnosis of any pleural effusion as soon as possible by at least a thoracocentesis. If untreated, tuberculosis may easily disseminate to other organs. Some considerations and suggestions for antibiotic treatment of pleural effusion will also be given, since many antibiotics have some anti-tuberculosis effect and may delay the diagnosis of this infectious disease.

Mechanical evaluation of new injection needles for dental anesthesia.

AIM: In this study Authors want to evaluate the static and dynamic flexural behaviour of innovative injection needles for dental anaesthesia comparing to the traditional needles. METHODS: Four kinds of innovative injection needles (Carpule Free Flow-Heraeus Kulzer), different for length and external diameter, have been evaluated in comparison with the traditional ones (Carpule-Heraeus Kulzer), dimensions being equal except for the internal diameter. Static stiffness tests (10 for each kind of needles, resulting in a totality of 80 tests) and fatigue bending test (10 for each kind of needles, each tested at 3 different bending angles, resulting in a totality of 240 tests) have been conducted following the ISO 9626 norms. The final comparison among the obtained data have been conducted using a one way statistical analysis of variance (ANOVA). RESULTS: After stiffness tests the Carpule Free Flow injection needles and the traditional ones showed the same static behaviour, resulting in statistical comparable values of mean deflection at the maximum load allowed by ISO norms tests, whereas the 0.4 mm external diameter showed a statistically significant difference because the Carpule Free Flow needles achieved lower deflection values. After dynamic bending tests at 20 degrees both needle types showed the same behaviour, whereas at 30 degrees bending the Carpule Free Flow injection needles showed a higher number of breaks comparing to normal type, up to a maximum value of breaks (10 out of 10) for the 0.4 mm external diameter needles. After 50 degrees fatigue bending tests the Carpule Free Flow needles came to the break point after lower number of cycles than the Carpule needles. CONCLUSION: This study pointed out that, among anaesthesia injection needles, the Carpule Free Flow needles not only showed better clinical characteristics but also a mechanical behaviour, both static and dynamic, statistically similar to traditional ones, according to international standards. After fatigue bending higher than 20 degrees, exclusively made in this research in order to simulate critical or extreme conditions, all analysed Carpule Free Flow needles have been broken after a lower number of cycles. It is concluded that it is recommended not to repeatedly bend this kind of needles at more than 90 degrees. At the end of this study it may be suggested that this kind of needles cannot be repeatedly bended at angles more than 90 degrees.

Effects of Nd:YAG laser on dental enamel.

Caries prevention should be an important goal of the dentistry practitioner. In addition to traditional preventive techniques, laser beam has been a recent suggestion. This study aimed to evaluate Nd:YAG laser treatment effects on dental enamel surfaces, irradiated at different energy levels, by scanning electron microscopy (SEM) observations and surface micro-hardness tests to evaluate if caries prevention can be achieved by laser treatment. Forty human teeth were divided in four groups of 10 specimens each: in three groups the enamel surface was treated with three different laser beam energy levels, 60, 120 and 160 mJ, in one group the enamel was not lased. Five samples from each group were subjected to the Vickers microhardness test and five samples underwent SEM investigation. Results of the microhardness test demonstrated no significant differences between treated and non-treated dental enamel samples. SEM observation demonstrated a rougher enamel surface in all treated groups: the 60 mJ treated group showed tooth surfaces with vertical scratches, the 120 mJ treated group showed the enamel surface covered by craters and cracks, and the 160 mJ treated group showed a completely changed enamel structure with columns separated by voids and with a glass-like surface. These investigations demonstrated that dental enamel laser treatment at low energy levels (not >60 mJ) produces a protective glass-like surface, without the loss of its integrity and could be an advisable technique to prevent caries. Higher energy laser treatment leads to modifications of the enamel morphology such as craters and cracks, even if it does not cause any change in enamel hardness characteristics, which could be more useful in conservative dentistry.

Bronchoalveolar lavage in intensive care units.

Pneumonia is common in those patients placed in intensive care units, especially in mechanically ventilated patients. The high mortality rate of ventilator-associated pneumonia requires a rapid initiation of the appropriate antibiotic treatment. Patients who do not respond to initial antibiotic regimens could have the additional benefit of the use of invasive techniques such as bronchoalveolar lavage. Moreover, BAL is of clinical use to identify several non-infectious pulmonary conditions that may mimic pneumonia in these patients. Such conditions include pulmonary haemorrhages, acute eosinophilic pneumonia, malignancy, drug-induced toxicity, adult respiratory distress syndrome and cardiogenic pulmonary oedema. It is important to distinguish these conditions from pneumonia because the management and prognosis of these entities is quite different.

Mechanical and ultrastructural evaluation of quartz post-endodontic reconstructions.

Aesthetics is a very important element in dentistry, but requires the support of good mechanical performance. Quartz fiber used in post-endodontic reconstruction is an aesthetic material, although there is little research concerning its mechanical properties. This study evaluated the retentive property of post-endodontic reconstruction, composed of a quartz fiber post. Different thermal stresses were applied in vitro to post-endodontic reconstructions, in order to simulate oral thermal action on post-system dental structure linkage. We chose 30 human extracted teeth, endodontically treated and restored, and then divided them into three groups of 10 teeth. A different treatment was applied to each group before mechanical testing: in the 1st group no treatment was done (controls); in the 2nd group teeth were subjected, in a climatic chamber, to 10 thermo-cycles between 4 degrees C and 58 degrees C; in the 3rd group teeth were stored in a saline solution at 37 degrees C for 48 hr. The teeth then underwent tensile shear stress tests at break point using a computerized electronic dynamometer. After mechanical testing, two teeth from each group were longitudinally half-sectioned, sputter-coated in gold and observed by scanning electron microscopy (SEM). The extracted quartz fiber post of each tooth also underwent SEM observation. Mechanical test results demonstrated that thermal cyclic variations could affect bond stability between dental structures and posts in quartz fiber reconstructions, whereas their bond strength seemed unaffected by humidity increases. Quartz fiber post SEM observation demonstrated a homogeneous structure and a regular fiber disposition. Dental root canal morphology SEM images always showed a different thickness in the cement layer. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 156-61).

Seasonal variations of T-cell cytokine pattern in peripheral blood from atopic subjects.

Previous studies on the effect of seasonal exposure to the sensitizing antigen on T-cell cytokine pattern from atopic subjects evaluated T-cell cytokine production by titration in the serum or culture supernatants. The purpose of this study was to determine the seasonal variations of T-cell cytokine pattern from atopic subjects at the single-cell level. We examined the interleukin-4 (IL-4) and interferon-gamma expression in peripheral blood CD4 + and CD8 + T cells from 11 subjects with grass-pollen-sensitive allergy before and during the 1999 grass pollen season using a flow cytometric method of intracellular cytokine detection. Eight healthy volunteers served as the control group. Flow cytometric analysis of peripheral blood lymphocytes showed no seasonal variations of IL-4- and interferon-gamma-producing T cells in atopic subjects. However, there was a decreased percentage of IL-4-producing cells among peripheral blood CD4+ and CD8+ T cells from the atopic subjects both during and outside the pollen season in comparison to the controLs. We did not find seasonal variations of T-cell cytokine pattern in peripheral blood from atopic subjects. However, we observed a decreased percentage of IL-4-producing T cells in peripheral blood from these subjects in comparison to healthy controls. These data add to the view of a continuous migration of T helper 2 (TH2) cells from the blood to the tissues of primary allergen exposure.

Long-term treatment with low doses of interleukin-2 and interferon-alpha: immunological effects in advanced renal cell cancer.

PURPOSE: We aimed to determine the immunological effects of low doses of recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha (rIFN-alpha) in patients bearing advanced renal cell carcinoma. METHODS: Twenty-seven patients received therapeutic cycles consisting of subcutaneous rIL-2 for 5 days per week and intramuscular rIFN-alpha twice weekly, for 4 consecutive weeks. The cycle was repeated indefinitely at regular 4-month intervals, for all patients. rIL-2 (1 x 10(6) IU/m2) was administered every 12 h on days 1 and 2 and once a day on days 3-5 of each week; rIFN-alpha (1.8 x 10(6) IU/m2) was given on days 3 and 5. In the enrolled patients, total and differential white blood cell counts, phenotypic analysis of some lymphocyte subsets, and soluble IL-2 receptor (sIL-2R), were investigated before and after each of the first six cycles of therapy (about 24 months of follow-up). RESULTS: The cycles of immunotherapy induced a significant increase of total lymphocytes (37%, P < 0.001), eosinophils (222%, P < 0.001), CD25+ cells (27%, P=0.004), sIL-2R (174%, P < 0.001) and natural killer (NK) cells (CD3-CD56+) (61%, P < 0.001); the subset that expresses CD56 with high density (CD56+ bright) expanded more (233%, P < 0.001) than the subset expressing the same marker with low density (CD56+ dimmer) (15%, P = 0.043). Unlike the previous subsets, the treatment decreased significantly T-lymphocytes with NK cell marker (CD3+ CD56+) (28%, P = 0.011). No significant differences of effectiveness were found among the subsequent treatment cycles, except for CD25+ cells and sIL-2R (P = 0.036 and P = 0.005, respectively): the increase induced by immunotherapy was maximum after the first cycle and decreased progressively thereafter. CONCLUSIONS: Long-term repeated cycles of low-dose immunotherapy induced repeated and significant expansion of one of the most important lymphocyte subsets for the non-MHC-restricted immune response to the tumour mass: CD3-CD56+ cells.

Effects of glucocorticoids on endogenous and transcellular metabolism of eicosanoids in asthma.

BACKGROUND: Human blood polymorphonuclear cells, which biosynthesize eicosanoids from the 5-lipoxygenase (5-LO) pathway, are likely to be involved in asthma, in which glucocorticoids represent the first line of therapy. Their effects on leukotriene release after a short course of treatment, which have been reported in several studies, are controversial. OBJECTIVE: We sought to investigate whether long-term oral glucocorticoids inhibit lipid mediators from the 5-LO pathway. METHODS: Twelve normal control subjects, 29 asthmatic subjects, and 50 glucocorticoid-dependent asthmatic subjects were included in the study. Polymorphonuclear cells were studied for endogenous and transcellular metabolism of eicosanoids. RESULTS: Total leukotriene B(4) production was significantly lower in cells from glucocorticoid-dependent asthmatic subjects (mean +/- SD, 177 +/- 26 ng/10(7) cells) than in control subjects (406 +/- 27), untreated asthmatic subjects (421 +/- 34), and asthmatic subjects treated with inhaled glucocorticoids (290 +/- 56). When incubated with arachidonic acid, these polymorphonuclear cells released very low amounts of 5(S)- and 12(S)-hydroxy-eicosatetraenoic acid (HETE), whereas endogenous 15(S)-HETE was found in substantial amounts. The transformation of exogenous 15(S)-HETE into 5(S),15(S)-diHETE and lipoxins was significantly more important in untreated asthmatic subjects than in control subjects and glucocorticoid-dependent asthmatic subjects. CONCLUSION: This study showed that long-term oral corticotherapy affects the 5-LO activity and leads to a decrease production of all metabolites in contrast to short-term or inhaled glucocorticoids. This study also questions the site of action of glucocorticoids in regulating the availability of arachidonic acid and potential eicosanoid regulation, as previously held in phospholipase A2 studies.

Inhaled corticosteroids in stable COPD patients: do they have effects on cells and molecular mediators of airway inflammation?

STUDY OBJECTIVE: To investigate possible changes in cells and molecular mediators of airway inflammation following inhaled steroid treatment of stable COPD patients. DESIGN: Six-week open preliminary prospective study. SETTING: A university respiratory disease clinic. PATIENTS: : Stable COPD patients with mild disease. INTERVENTION: Six-week treatment with inhaled beclomethasone (1.5 mg die). MEASUREMENTS: The levels of interleukin (IL)-8, myeloperoxidase, eosinophilic cationic protein and tryptase, and cell numbers in bronchial lavage specimens were determined, and the symptom score, the endoscopic bronchitis index, and functional parameters were recorded. RESULTS: After treatment there were significant reductions in the lavage levels of IL-8 ([mean +/- SEM] 1,603.4 +/- 331.2 vs 1,119.2 +/- 265.3 pg/mL, respectively; p = 0. 01) and myeloperoxidase (1,614.5 +/- 682.3 vs 511.2 +/- 144.2 microg/L, respectively; p = 0.05), in cell numbers (250.6 +/- 27.7 vs 186.3 +/- 11.5 cells x 10(3)/mL, respectively; p = 0.04), neutrophil proportion (59.7 +/- 14.3% vs 31.5 +/- 10.1%; p = 0.01), symptom score (4.5 +/- 0.6 vs 1.4 +/- 0.5; p = 0.01), and bronchitis index (8.5 +/- 0.8 vs 5.5 +/- 0.7; p = 0.007). CONCLUSIONS: In stable patients with COPD, inhaled steroid treatment may induce changes on some cellular and molecular parameters of airway inflammation.

Bronchoalveolar lavage in bronchiolitis obliterans organizing pneumonia primed by radiation therapy to the breast.

BACKGROUND: Growing evidence indicates that unilateral lung irradiation for breast cancer may "prime" the development of migratory lung infiltrates with histologic features of bronchiolitis obliterans organizing pneumonia. OBJECTIVE: Our purpose was to evaluate the cytologic and immunocytologic features of bronchoalveolar lavage in this condition. METHODS: We analyzed the profile bronchoalveolar lavage cell differentials and lymphocyte subpopulations of 11 women with bronchiolitis obliterans organizing pneumonia syndrome after radiation therapy for breast cancer in comparison to 9 healthy women. RESULTS: The bronchoalveolar lavage analysis demonstrated a significant increase in the percentage of lymphocytes (36.7% +/- 5.4% vs 8.6% +/- 1.1%, P =.0002), neutrophils (3.8% +/- 1.2% vs 0.6% +/- 0.2%, P =.005), eosinophils (2.4% +/- 1% vs 0.3% +/- 0.1%, P =.01), and mast cells (1.4% +/- 0.6% vs 0.1% +/- 0.02%, P =.05) with a significant decrease in the percentage of macrophages (56.1% +/- 6% vs 90.3% +/- 1.4%, P =.0002) in patients with bronchiolitis obliterans organizing pneumonia compared with the control subjects. The percentage of CD3(+) cells was significantly increased in patients with bronchiolitis obliterans organizing pneumonia (93.7% +/- 1.3% vs 70.9% +/- 4%, P =.0004), with a significant decrease in CD4(+) cells (32.7% +/- 4.7% vs 55.4% +/- 2. 6%, P =.002) and a significant increase in CD8(+) cells (61.2% +/- 4. 8% vs 37.5% +/- 2.9%, P =.003) in comparison to control subjects. The CD4/CD8 ratio was significantly reduced in patients with bronchiolitis obliterans organizing pneumonia compared with control subjects (0.6% +/- 0.1% vs 1.5% +/- 0.1%, P =.001). CONCLUSION: These data add to the view that unilateral lung irradiation for breast cancer may "prime" the development of a syndrome quite similar to idiopathic bronchiolitis obliterans organizing pneumonia.

T-cell cytokine pattern at three time points during specific immunotherapy for mite-sensitive asthma.

BACKGROUND: Several lines of evidence indicate that specific immunotherapy may act by modifying the patterns of cytokines produced by helper T cells. However, different protocols have been used and different results obtained. OBJECTIVES: To quantify the effect of specific immunotherapy on the TH1/TH2 T-cell cytokine pattern at the single cell level. METHODS: We examined the interferon-gamma/interleukin-4 ratio in peripheral blood CD4+ and CD8+ T cells from 12 subjects with house dust mite-sensitive asthma using a flow cytometric method of intracellular cytokine detection. Cytokine production was determined following stimulation with phorbol myristate acetate/ionomycin, a policlonal activator. Subjects were examined at three occasions: before specific immunotherapy, after the 3-months dose increase phase and after 1 year of treatment. During the treatment year patients kept a diary in which they recorded: (a) symptoms of asthma according to a 0-3 grading (0 = absent, 1 = mild, 2 = moderate, 3 = severe); (b) number of puffs (100 microg) per day of salbutamol required to control symptoms; and (c) peak expiratory flow. RESULTS: Specific immunotherapy improved clinical indices of disease activity including symptom scores and medication use during the treatment year, and had a marked effect in increasing the interferon-gamma/interleukin-4 ratio in peripheral blood CD4+ T cells already after the dose increase phase (5.47 +/- 1.5 vs 4.07 +/- 1.49%, P = 0.03) with and a further rise after 1 year's treatment (16.12 +/- 2.8 vs 4.07 +/- 1.49 and 16.12 +/- 2.8 vs 5.47 +/- 1.5%, P = 0.001 and P = 0.002, respectively). There were no significant changes in the interferon-gamma/interleukin-4 ratio in peripheral blood CD8+ T cells at the three times of the study. CONCLUSIONS: These data add to view that the efficacy of specific immunotherapy may be attributed to a modified cytokine secretion of CD4+ T cells.

Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease.

BACKGROUND: Nitric oxide (NO) plays an important role as an inflammatory mediator in the airways. Since chronic obstructive pulmonary disease (COPD) is characterised by airway inflammation, a study was undertaken to determine NO levels in the exhaled air of patients with COPD. METHODS: Two groups of patients with clinically stable COPD were studied, 10 current smokers and 10 ex-smokers. Two control groups of healthy subjects consisting of 10 current smokers and 20 non-smokers were also studied. Exhaled NO levels were measured by the collection bag technique and NO chemiluminescence analyser. RESULTS: Mean (SE) levels of exhaled NO in ex-smokers and current smokers with COPD (25.7 (3.0) ppb and 10.2 (1.4) ppb, respectively) were significantly higher than in non-smoker and current smoker control subjects (9.4 (0.8) ppb and 4.6 (0.4) ppb, respectively). In current smokers with COPD exhaled levels of NO were significantly lower than in ex-smokers. In this latter group of patients there was a significant negative correlation between smoking history (pack years) and levels of exhaled NO (r = -0.8, p = 0.002). A positive correlation was seen between forced expiratory volume in one second (FEV1) and levels of exhaled NO (r = 0.65, p = 0.001) in patients with COPD. CONCLUSIONS: These data show that exhaled NO is increased in patients with stable COPD, both current and ex-smokers, compared with healthy control subjects.

Predominant TH1 cytokine pattern in peripheral blood from subjects with chronic obstructive pulmonary disease.

BACKGROUND: Previous studies have provided evidence for an inflammatory process in the large airways of subjects with chronic obstructive pulmonary disease (COPD), consisting predominantly of activated T cells. No data are available on the TH1 /TH2 T-cell cytokine pattern in this disease. OBJECTIVE: We sought to characterize the TH1 /TH2 T-cell cytokine pattern in subjects with COPD. METHODS: We examined the IFN-gamma and IL-4 expression in peripheral blood CD4+ and CD8+ T cells from 20 patients with COPD and 25 control subjects by using a flow cytometric method of intracellular cytokine detection. We also examined the expression of 2 surface activation markers (CD25 and HLA-DR) on peripheral blood CD4+ and CD8+ T cells. RESULTS: There was an increased percentage of IFN-gamma-producing cells (30.3% [range, 12.9% to 60.4%] vs 19.1% [range, 4% to 31.2%], P =.003) and a decreased percentage of IL-4-producing cells (4.55% [range, 0.6% to 11.3%] vs 9.5% [2.1% to 21.3%], P =.0008) among peripheral blood CD4+ T cells from the patients with COPD compared with control subjects. There was no significant difference between the 2 groups in the percentage of peripheral blood CD8+ T cells producing IFN-gamma or IL-4 or in the percentage of peripheral blood CD4+ and CD8+ T cells expressing CD25 and HLA-DR. CONCLUSION: These findings provide evidence for a TH1 -like immune response of peripheral blood CD4+ T cells in subjects with COPD.

Neutrophils infiltrating bronchial epithelium in chronic obstructive pulmonary disease.

In order to characterize neutrophil and eosinophil presence in the airways of patients with chronic obstructive pulmonary disease (COPD), bronchoscopy with bronchial washings and bronchial biopsies was performed in 12 smoking stable COPD subjects and 18 normal non-smoking control subjects. Bronchial biopsies were examined by light microscopy using plastic embedding and histochemical techniques to identify different cell types. Bronchial washing fluid of COPD patients was characterized by a predominance of neutrophils (P = 0.001), and a slight, but significant (P = 0.03), increase of eosinophil fraction. Subjects with COPD had higher number of neutrophils in the epithelium (P = 0.01), and eosinophils in the lamina propria (P = 0.01) than did control subjects. The thickness of reticular basement membrane was increased for COPD patients in comparison to control subjects (P = 0.01). The present study provides evidence of neutrophil infiltration both in bronchial washing and bronchial epithelium of patients with COPD, suggesting that the source of neutrophils in airway lumen may be the bronchial mucosa. Although less common than in asthma, airways of COPD subjects reveal eosinophil presence and airway remodelling.

Influence of atmospheric nitric oxide concentration on the measurement of nitric oxide in exhaled air.

BACKGROUND: Measurement of nitric oxide (NO) in exhaled air shows promise as a non-invasive method of detecting lung inflammation. However, variable concentrations of NO are measured in environmental air. The aim of this study was to verify a possible relationship between exhaled NO and atmospheric NO values during high atmospheric NO days. METHOD: Exhaled air from 78 healthy non-smokers of mean age 35.3 years was examined for the presence of NO using a chemiluminescence NO analyser and NO levels were expressed as part per billion (ppb). The exhaled air from all the subjects was collected into a single bag and into two sequential bags. Before each test atmospheric NO was measured. RESULTS: The mean (SE) concentration of exhaled NO collected into the single bag was 17.1 (0.6) ppb while the mean values of exhaled NO in bags 1 and 2 were 16.7 (1.3) ppb and 13.8 (1.2) ppb, respectively. The atmospheric NO concentrations registered before each test varied from 0.4 to 71 ppb. There was a significant correlation between exhaled NO in the single bag and atmospheric NO (r = 0.38, p = 0.001). The atmospheric NO concentration also correlated with exhaled NO both in bag 1 (r = 0.44, p = 0.0001) and in bag 2 (r = 0.42, p = 0.0001). These correlations disappeared with atmospheric NO concentrations lower than 35 ppb. CONCLUSIONS: These results indicate a relationship between atmospheric NO and NO levels measured in exhaled air, therefore exhaled NO should not be measured on very high atmospheric NO days.