José Ramírez

EML4-ALK Rearrangement in Non-Small Cell Lung Cancer and Non-Tumor Lung Tissues

A fusion gene, echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK), with transforming activity has recently been identified in a subset of non-small cell lung cancer (NSCLC), but its pathogenetic, diagnostic, and therapeutic roles remain unclear. Both frequency and type of EML4-ALK transcripts were investigated by reverse transcription PCR in 120 frozen NSCLC specimens from Italy and Spain; non-neoplastic lung tissues taken far from the tumor were used as controls. In cases carrying the fusion transcript, we determined EML4-ALK gene and protein levels using fluorescence in situ hybridization, Western blotting, and immunoprecipitation. We also analyzed ALK protein levels in paraffin samples from 662 NSCLC specimens, including the 120 cases investigated in the molecular studies. EML4-ALK transcripts (variants 1 and 3) were detected in 9 of 120 NSCLC samples but were not specific for NSCLC since they were also found in non-cancerous lung tissues taken far from the tumor. Notably, no transcripts were detected in matching tumor samples from these patients. Fluorescence in situ hybridization analysis of cases expressing EML4-ALK transcripts showed that only a minority of cells harbored the EML4-ALK gene. None of these cases was found to express the EML4-ALK protein as examined by immunohistochemistry, Western blotting, and immunoprecipitation. The EML4-ALK transcript cannot be regarded as a specific diagnostic tool for NSCLC. Our results show therefore that the causal role and value of EML4-ALK as a therapeutic target remain to be defined.

Development of aortic aneurysm/dilatation during the followup of patients with giant cell arteritis: A cross‐sectional screening of fifty‐four prospectively followed patients

OBJECTIVEnGiant cell arteritis (GCA) may involve the aorta. Retrospective studies have demonstrated a higher prevalence of aortic aneurysm among patients with GCA compared with the general population. We investigated the prevalence of aortic aneurysm in a cohort of patients with biopsy-proven GCA using a defined protocol and assessed whether persisting low-grade disease activity is associated with higher risk of developing aortic aneurysm.nnnMETHODSnFifty-four patients with GCA (14 men and 40 women) were cross-sectionally evaluated after a median followup of 5.4 years (range 4.0-10.5 years). The screening protocol included a chest radiograph, abdominal ultrasonography scan, and computed tomography scan when aortic aneurysm was suspected or changes with respect to the baseline chest radiograph were observed. Clinical and laboratory data, corticosteroid requirements, and relapses were prospectively recorded.nnnRESULTSnTwelve patients (22.2%) had significant aortic structural damage (aneurysm/dilatation), 5 of them candidates for surgical repair. Aortic aneurysm/dilatation was more frequent among men (50%) than women (12.5%; relative risk 3.5, 95% confidence interval 1.53-8.01, P = 0.007). At the time of screening, patients with aneurysm/dilatation had lower serum acute-phase reactants, lower relapse rate, and needed shorter periods to withdraw prednisone than patients without aortic structural damage.nnnCONCLUSIONnThere is a substantial risk of developing aortic aneurysm/dilatation among patients with GCA. Our data do not support that aneurysm formation mainly results from persistent detectable disease activity. Additional factors including characteristics of the initial injury or the target tissue may also determine susceptibility to aortic aneurysm/dilatation.

Usefulness of Serum Tumor Markers, Including Progastrin-Releasing Peptide, in Patients with Lung Cancer: Correlation with Histology

Background: Tumor markers have been extensively studied in lung cancer, reporting some relationship to the histology, but their clinical utility is not clear. Methods: ProGRP, CEA, SCC, CA 125, CYFRA 21-1 and NSE were studied prospectively in 155 patients with unconfirmed suspicion of lung cancer and in 647 patients with lung cancer: 182 squamous, 205 adenocarcinomas, 19 large-cell lung cancer (LCLC), 175 small-cell lung cancer (SCLC) and 66 unspecific non-small-cell lung cancer (NSCLC). Results: Abnormal tumor marker serum levels were found in less than 5.3% of the patients with benign diseases, excluding CA 125 (21.3%). Tumor markers were related to histological type and tumor extension with significantly higher CEA (p <0.01) and CA 125 (p <0.007) serum levels in adenocarcinomas, SCC (p <0.0001) and CYFRA 21-1 (p <0.008) in squamous tumors and ProGRP (p <0.0001) and NSE (p <0.0001) in SCLC. Tumor markers may be useful in the histological differentiation of NSCLC and SCLC. Patients with SCC serum levels >2 ng/ml were always NSCLC, while those with SCC <2 ng/ml and ProGRP >100 pg/ml and NSE >35 ng/ml were all SCLC patients. The sensitivity was 76.7 and 79.5%, specificity was 97.2 and 99.6%, with a positive predictive value of 98.6 and 98.6% and a negative predictive value of 60.7 and 92.9% in the differentiation of NSCLC and SCLC, respectively. Conclusions: Tumor marker determination in patients with suspicious signs of lung cancer suggests, in a few hours, the histological diagnosis in the majority of lung cancer patients.

A Dual Role for KRT81: A miR-SNP Associated with Recurrence in Non-Small-Cell Lung Cancer and a Novel Marker of Squamous Cell Lung Carcinoma

MicroRNAs (miRNAs) play an important role in carcinogenesis through the regulation of their target genes. miRNA-related single nucleotide polymorphisms (miR-SNPs) can affect miRNA biogenesis and target sites and can alter microRNA expression and functions. We examined 11 miR-SNPs, including 5 in microRNA genes, 3 in microRNA binding sites and 3 in microRNA-processing machinery components, and evaluated time to recurrence (TTR) according to miR-SNP genotypes in 175 surgically resected non-small-cell lung cancer (NSCLC) patients. Significant differences in TTR were found according to KRT81 rs3660 (median TTR: 20.3 months for the CC genotype versus 86.8 months for the CG or GG genotype; Pu200a=u200a0.003) and XPO5 rs11077 (median TTR: 24.7 months for the AA genotype versus 73.1 months for the AC or CC genotypes; Pu200a=u200a0.029). Moreover, when patients were divided according to stage, these differences were maintained for stage I patients (Pu200a=u200a0.002 for KRT81 rs3660; P<0.001 for XPO5 rs11077). When patients were divided into sub-groups according to histology, the effect of the KRT81 rs3660 genotype on TTR was significant in patients with squamous cell carcinoma (Pu200a=u200a0.004) but not in those with adenocarcinoma. In the multivariate analyses, the KRT81 rs3660 CC genotype (ORu200a=u200a1.8; Pu200a=u200a0.023) and the XPO5 rs11077 AA genotype (ORu200a=u200a1.77; Pu200a=u200a0.026) emerged as independent variables influencing TTR. Immunohistochemical analyses in 80 lung specimens showed that 95% of squamous cell carcinomas were positive for KRT81, compared to only 19% of adenocarcinomas (P<0.0001). In conclusion, miR-SNPs are a novel class of SNPs that can add useful prognostic information on the clinical outcome of resected NSCLC patients and may be a potential key tool for selecting high-risk stage I patients. Moreover, KRT81 has emerged as a promising immunohistochemical marker for the identification of squamous cell lung carcinoma.

Extracorporeal lung membrane provides better lung protection than conventional treatment for severe postpneumonectomy noncardiogenic acute respiratory distress syndrome

OBJECTIVEnWe compared conventional treatment with pumpless extracorporeal lung membrane (Interventional Lung Assist [iLA] Novalung; Novalung GmbH, Hechingen, Germany) support in a pig model of postpneumonectomy severe acute respiratory distress syndrome.nnnMETHODSnAdult pigs underwent a left thoracotomy without (group I) or with a left extrapericardial pneumonectomy and radical lymphadenectomy (groups II to V). After stabilization, pigs belonging to group II were observed only, whereas in those belonging to groups III to V, a surfactant-depletion severe (Pao(2)/Fio(2) < 100) postpneumonectomy acute respiratory distress syndrome was induced. This was followed by observation (group III); treatment with conventional therapy including protective ventilation, steroids, and nitric oxide (group IV); or femoral arteriovenous iLA Novalung placement, near-static ventilation, steroids, and nitric oxide (group V). Each group included 5 animals. Primary outcome was extubation 12 hours postoperatively or postpneumonectomy acute respiratory distress syndrome.nnnRESULTSnA severe postpneumonectomy acute respiratory distress syndrome was obtained after 9 +/- 2 alveolar lavages over 90 +/- 20 minutes. In group V pigs, the iLA Novalung device diverted 17% +/- 4% of the cardiac output, permitted an oxygen transfer and carbon dioxide removal of 298.4 +/- 173.7 mL/min and 287.7 +/- 87.3 mL/min, respectively, and static ventilation (tidal volume, 2.2 +/- 1 mL/kg; respiratory rate, 6 +/- 2.9 breaths/min). All but 1 pig belonging to group V could be extubated compared with none in groups III and IV (P < .01), and only their lungs normalized cytokine release (P < .001) and surfactant (P < .03) and displayed fewer parenchymal lesions (P < .05).nnnCONCLUSIONSnThe pumpless extracorporeal lung membrane and near-static ventilation achieved a significantly better outcome than conventional treatment in this pig model of severe postpneumonectomy acute respiratory distress syndrome, probably because the injured lungs were not forced to work and this rest gave them more time to heal.

Guidelines for biomarker testing in advanced non-small-cell lung cancer. A national consensus of the Spanish Society of Medical Oncology (SEOM) and the Spanish Society of Pathology (SEAP)

Patients with advanced non-small-cell lung cancer (NSCLC) carrying epidermal growth factor receptor (EGFR) mutations can now have specific treatment based on the result of biomarker analysis and patients with rearrangements of the anaplastic lymphoma kinase (ALK) gene will probably soon be able to. This will give them better quality of life and progression-free survival than conventional chemotherapy. This consensus statement was conceived as a joint initiative of the Spanish Society of Medical Oncology (SEOM) and the Spanish Society of Pathology (SEAP), and makes diagnostic and treatment recommendations for advanced NSCLC patients based on the scientific evidence on biomarker use. It therefore provides an opportunity to improve healthcare efficiency and resource use, which will undoubtedly benefit these patients. Although this field is in continuous evolution, at present, with the available data, this panel of experts recommends that all patients with advanced NSCLC of non-squamous cell subtype, or non-smokers regardless of the histological subtype, should be tested for EGFR gene mutations within a maximum of 7 days from the pathological diagnosis. Involved laboratories must participate in external quality control programmes. In contrast, ALK gene rearrangements should only be tested in the context of a clinical trial, although the promising data obtained will certainly justify in the near future its routine testing in patients with no EGFR mutations. Lastly, routine testing for other molecular abnormalities is not considered necessary in the current clinical practice.

Low miR-145 and high miR-367 are associated with unfavourable prognosis in resected nonsmall cell lung cancer

The transcription factors SRY-related HMG box (SOX)2 and octamer-binding transcription factor (OCT)4 regulate the expression of the miR-302–367 cluster. miR-145 regulates SOX2 and OCT4 translation and p53 regulates miR-145 expression. We analysed the expression of the miR-302–367 cluster and miR-145 and the mutational status of p53 in resected nonsmall cell lung cancer (NSCLC) patients and correlated results with time to relapse (TTR). Tumour and paired normal tissue samples were obtained from 70 NSCLC patients. MicroRNA expression was assessed with TaqMan MicroRNA Assays. p53 exons 5 to 8 were sequenced. miR-145 was downregulated (p<0.0001) and miR-367 was upregulated (p<0.0001) in tumour compared with normal tissue. Mean TTR was 18.4 months for patients with low miR-145 levels and 28.2 months for those with high levels (p=0.015). Mean TTR was 29.1 months for patients with low miR-367 levels and 23.4 months for those with high levels (p=0.048). TTR was shorter for patients with both unfavourable variables (p=0.009). Low miR-145 expression (p=0.049), the combination of unfavourable microRNA levels (p=0.02) and the combination of low miR-145 with p53 mutations (p=0.011) were independent markers of shorter TTR. In conclusion, miR-145 and miR-367 expression could be novel markers for relapse in surgically treated NSCLC. p53 may play a role in modulating miR-145 expression in NSCLC.

Determinación en plasma del factor transformador del crecimiento β1 en la fibrosis pulmonar idiopática

Objetivo El factor transformador del crecimiento β1 (TGF-β1) es uno de los mediadores fibrogenicos con mas relevancia en la patogenia de la fibrosis pulmonar idiopatica (FPI). El objetivo del estudio ha sido investigar el valor pronostico de la determinacion en plasma del TGF-β1 en la FPI. Pacientes y metodos Se ha realizado un estudio prospective en el que se incluyo a 29 pacientes con FPI y 27 controles sanos. La determinacion del TGF-β1 se realizo mediante enzimoinmunoanalisis. Resultados La concentracion de TGF-β1 fue significativamente mayor en los pacientes con FPI que en los controles (mediaxa0±xa0desviacion estandar: 11,1xa0±xa07,5 frente a 4xa0±xa02,4xa0ng/ml; pxa0 Conclusiones Aunque la concentracion plasmatica de TGF-β1 esta elevada en los pacientes con FPI, este parametro no parece ser util como marcador del pronostico de la enfermedad ni de la respuesta terapeutica.

Pre-treatment with mesenchymal stem cells reduces ventilator-induced lung injury.

Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). This study investigated whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. 24 Sprague–Dawley rats (250–300 g) were subjected to high-volume mechanical ventilation (25 mL·kg−1). MSCs (5×106) were intravenously or intratracheally administered (n=8 each) 30 min before starting over-ventilation and eight rats were MSC-untreated. Spontaneously breathing anesthetised rats (n=8) served as controls. After 3 h of over-ventilation or control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI features. Lung oedema, histological lung injury index, concentrations of total protein, interleukin-1&bgr;, macrophage inflammatory protein-2 and number of neutrophils in BALF and vascular cell adhesion protein-1 in lung tissue significantly increased in over-ventilated rats. All these indices of VILI moved significantly towards normalisation in the rats treated with MSCs, whether intravenously or intratracheally. Both local and systemic pre-treatment with MSCs reduced VILI in a rat model.

Physician alert suggesting alpha-1 antitrypsin deficiency testing in pulmonary function test (PFT) results

Alpha-1 antitrypsin deficiency (AATD) is under-recognized by clinicians, with long diagnostic delays between patients first symptom and initial diagnosis. Recent recommendations by official societies encourage testing for AATD in all symptomatic adults with spirometric evidence of COPD, though compliance with this recommendation has been variable. For 6 months, the following physician alert was added to PFT reports of patients with airflow obstruction of GOLD Stage II or higher: “The American Thoracic Society recommends testing for AATD in all patients with FEV1 < 80% predicted and FEV1/FVC less than 0.70, if clinically indicated. Appropriate counseling suggested.” During the “Pre-alert Period,” 821 spirometry tests were performed; 178 of these 821 unique patients (22%) satisfied spirometric criteria of > GOLD Stage II and 11 (6%) were tested for AATD. In contrast, during the “Physician Alert” intervention period, 689 spirometry tests were performed on 689 unique patients, of whom 140 (20%) satisfied criteria for > GOLD II airflow obstruction; AAT testing was done more frequently (18 [13%], p = 0.04). The overall rate of misclassifying patients reports for testing by the respiratory therapists was very low (3.3%). An analysis of “number needed to test” suggests that 98–290 patients must be tested to have 95% certainty to identify a single patient with severe AATD. In conclusion, implementing a physician alert on PFT reports of patients with COPD can increase physicians testing for AATD. The incomplete rate of testing suggests the need for additional strategies to enhance clinicians detection of individuals with AATD.