Biomarkers for cystic fibrosis lung disease: Application of SELDI-TOF mass spectrometry to BAL fluid

AbstractBackgroundFor cystic fibrosis (CF) patients there is a lack of good assays of disease activity and response to new therapeutic interventions, including gene therapy. Current measures of airways inflammation severity are insensitive or non-specific.MethodsBronchoalveolar lavage fluid from 39 CF children and 38 respiratory disease controls was obtained at bronchoscopy and analysed by surface enhanced laser desorption ionisation time of flight (SELDI-TOF) mass spectrometry. Recognized proteins were assessed for CF disease specificity. Individual protein identification of specific peaks was performed.Results1277 proteins/peptides, >4 kDa, were detected using 12 different surfaces and binding conditions. 202 proteins/peptides were differentially expressed in the CF samples (p<0.001), 167 up-regulated and 35 down-regulated. The most discriminatory biomarker had a mass of 5.163 kDa. The most abundant, with a mass of 10.6 kDa, was identified as s100 A8 (calgranulin A).ConclusionsThe application of SELDI-TOF mass spectrometry allows evaluation of proteins in BAL fluid avoiding the limitations of only analysing predetermined proteins and potentially identifying proteins not previously appreciated as biomarkers. Its application to cystic fibrosis should enable appropriate evaluation of evolving illness, of gene therapy and other new therapies

Homologous recombination DNA repair defects in PALB2- associated breast cancers

Abstract: Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

CYP3A7*1C allele: linking premenopausal oestrone and progesterone levels with risk of hormone receptor-positive breast cancers

Funder: Breast Cancer Now (BCN); doi: https://doi.org/10.13039/100009794Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH)Funder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): HEALTH-F2-2009-223175, HEALTH-F2-2009-223175Funder: Genome Canada (Génome Canada); doi: https://doi.org/10.13039/100008762Funder: Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada); doi: https://doi.org/10.13039/501100000024Funder: Quebec Breast cancer Foundation Genome QuebecFunder: U.S. Department of Health & Human Services | NIH | U.S. National Library of Medicine (NLM); doi: https://doi.org/10.13039/100000092Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))Funder: European Union’s Horizon 2020Funder: Deutsche Krebshilfe (German Cancer Aid); doi: https://doi.org/10.13039/501100005972Funder: BCAST - European Union’s Horizon 2020Funder: Breast Cancer Now; doi: https://doi.org/10.13039/501100007913Abstract: Background: Epidemiological studies provide strong evidence for a role of endogenous sex hormones in the aetiology of breast cancer. The aim of this analysis was to identify genetic variants that are associated with urinary sex-hormone levels and breast cancer risk. Methods: We carried out a genome-wide association study of urinary oestrone-3-glucuronide and pregnanediol-3-glucuronide levels in 560 premenopausal women, with additional analysis of progesterone levels in 298 premenopausal women. To test for the association with breast cancer risk, we carried out follow-up genotyping in 90,916 cases and 89,893 controls from the Breast Cancer Association Consortium. All women were of European ancestry. Results: For pregnanediol-3-glucuronide, there were no genome-wide significant associations; for oestrone-3-glucuronide, we identified a single peak mapping to the CYP3A locus, annotated by rs45446698. The minor rs45446698-C allele was associated with lower oestrone-3-glucuronide (−49.2%, 95% CI −56.1% to −41.1%, P = 3.1 × 10–18); in follow-up analyses, rs45446698-C was also associated with lower progesterone (−26.7%, 95% CI −39.4% to −11.6%, P = 0.001) and reduced risk of oestrogen and progesterone receptor-positive breast cancer (OR = 0.86, 95% CI 0.82–0.91, P = 6.9 × 10–8). Conclusions: The CYP3A7*1C allele is associated with reduced risk of hormone receptor-positive breast cancer possibly mediated via an effect on the metabolism of endogenous sex hormones in premenopausal women

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Sputum Proteomics in Inflammatory and Suppurative Respiratory Diseases

Rationale: Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful

Avaliação da eficácia e segurança da associação de budesonida e formoterol em dose fixa e cápsula única no tratamento de asma não controlada: ensaio clínico randomizado, duplo-cego, multicêntrico e controlado Evaluation of the efficacy and safety of a fixed-dose, single-capsule budesonide-formoterol combination in uncontrolled asthma: a randomized, double-blind, multicenter, controlled clinical trial

OBJETIVO: Avaliar a eficácia e a segurança da associação de budesonida e formoterol em dose fixa e cápsula única, em comparação ao uso de budesonida isolada em pacientes com asma não controlada. MÉTODOS: Ensaio clínico randomizado, duplo-cego, multicêntrico, de fase III, com grupos paralelos, comparando a eficácia de curto prazo e a segurança da formulação em pó de budesonida (400 µg) e formoterol (12 µg) com a formulação em pó de budesonida (400 µg) em 181 participantes com asma não totalmente controlada. A idade dos participantes variou de 18-77 anos. Após um período de run-in de 4 semanas, durante o qual todos os participantes receberam budesonida duas vezes por dia, houve a randomização para um dos tratamentos do estudo. O tratamento foi administrado duas vezes ao dia por 12 semanas. Os principais desfechos foram VEF1, CVF e PFE matinal. Os dados foram analisados por intenção de tratar. RESULTADOS: O grupo tratado com a associação, quando comparado ao grupo budesonida isolado, teve uma melhora significativa no VEF1 (0,12 L vs. 0,02 L; p = 0.0129) e no PFE matinal (30,2 L/min vs. 6,3 L/min; p = 0,0004). Esses efeitos foram acompanhados por boa tolerabilidade e segurança, como demonstrado pela baixa frequência de eventos adversos menores. CONCLUSÕES: A associação em cápsula única de budesonida e formoterol mostrou ser eficaz e segura. Os resultados demonstram que essa formulação é uma opção terapêutica válida para a obtenção e manutenção do controle da asma.<br>OBJECTIVE: To evaluate the efficacy and safety of a fixed-dose, single-capsule budesonide-formoterol combination, in comparison with budesonide alone, in patients with uncontrolled asthma. METHODS: This was a randomized, double-blind, multicenter, phase III, parallel clinical trial, comparing the short-term efficacy and safety of the combination of budesonide (400 µg) and formoterol (12 µg), with those of budesonide alone (400 µg), both delivered via a dry powder inhaler, in 181 patients with uncontrolled asthma. The age of the patients ranged from 18 to 77 years. After a run-in period of 4 weeks, during which all of the patients received budesonide twice a day, they were randomized into one of the treatment groups. for 12 weeks. The treatment consisted of the administration of the medications twice a day for 12 weeks. The primary outcome measures were FEV1, FVC, and morning PEF. We performed an intention-to-treat analysis of the data. RESULTS: In comparison with the budesonide-only group patients, those treated with the budesonide-formoterol combination showed a significant improvement in FEV1 (0.12 L vs. 0.02 L; p = 0.0129) and morning PEF (30.2 L/min vs. 6.3 L/min; p = 0.0004). These effects were accompanied by good tolerability and safety, as demonstrated by the low frequency of adverse events, only minor adverse events having occurred. CONCLUSIONS: The single-capsule combination of budesonide and formoterol appears to be efficacious and safe. Our results indicate that this formulation is a valid therapeutic option for obtaining and maintaining asthma control. (ClinicalTrials.gov Identifier: NCT01676987 [http://www.clinicaltrials.gov/]