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A proteomics workflow reveals predictive autoantigens in idiopathic pulmonary fibrosis (cohort 1)

ABSTRACT: Rationale: The diagnosis of idiopathic pulmonary fibrosis (IPF) requires exclusion of an underlying autoimmune disease, as present in interstitial lung diseases associated with connective tissue diseases (CTD-ILD). The prevalence of autoantibodies in IPF patients is currently unknown. Objectives: An unbiased assay for de novo discovery of autoantigens can help characterizing autoreactivities in IPF patients beyond clinically established autoimmune panels. Methods: We developed the proteomic Differential Antigen Capture (DAC) assay, capturing patient antibodies from plasma, followed by affinity purification coupled to mass spectrometry (AP-MS). The DAC assay quantifies the binding capacity of patient antibodies to proteins in a pooled native extract from lung explants (ILD explants n=41; donor controls n=12). Plasma antibodies from patients with IPF (n=35), CTD-ILD (n=24) and age-matched controls (n=32) were analyzed and validated in an independent cohort (IPF: n=40; CTD-ILD: n=20). Plasma antibody binding profiles were associated with clinical meta-data including diagnosis, lung function and transplant free survival. Measurements and Main Results: We identified 586 putative autoantigens in both study cohorts with a broad heterogeneity among disease entities and cohorts. On average, in IPF a mean±SD of 16±40 autoantigens and in CTD-ILD a mean±SD of 9±15 autoantigens were identified per patient. We identified 18 IPF-specific autoantigens validated in the second cohort. Interestingly, there was also a high number of shared autoantigens in IPF and CTD-ILD patients, with 17 being present in IPF and CTD-ILD of both cohorts. Presence of antibodies to Thrombospondin 1 (THBS1) and tubulin beta-1 chain (TUBB1) was associated with a significantly reduced survival in patients with IPF (p=0.002 and p=0.019, respectively). This signature was often associated with autoreactivity against talin-1 (TLN1), latent-transforming growth factor beta-binding protein 1 (LTBP1), epididymis secretory protein Li 112 (HEL-S-112), zyxin (ZYX), the LIM and senescent cell antigen-like-containing domain protein 1 (LIMS1) and caldesmon (CALD1). Conclusions: Unbiased proteomic profiling reveals that the overall prevalence of autoantibodies is similar in IPF and CTD-ILD patients and identifies novel IPF specific autoantigens associated with patient survival.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung, Blood Serum

SUBMITTER: Christoph Mayr

LAB HEAD: Herbert Schiller

PROVIDER: PXD024113 | Pride | 2021-02-22

REPOSITORIES: Pride

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Project description:Rationale: The diagnosis of idiopathic pulmonary fibrosis (IPF) requires exclusion of an underlying autoimmune disease, as present in interstitial lung diseases associated with connective tissue diseases (CTD-ILD). The prevalence of autoantibodies in IPF patients is currently unknown. Objectives: An unbiased assay for de novo discovery of autoantigens can help characterizing autoreactivities in IPF patients beyond clinically established autoimmune panels. Methods: We developed the proteomic Differential Antigen Capture (DAC) assay, capturing patient antibodies from plasma, followed by affinity purification coupled to mass spectrometry (AP-MS). The DAC assay quantifies the binding capacity of patient antibodies to proteins in a pooled native extract from lung explants (ILD explants n=41; donor controls n=12). Plasma antibodies from patients with IPF (n=35), CTD-ILD (n=24) and age-matched controls (n=32) were analyzed and validated in an independent cohort (IPF: n=40; CTD-ILD: n=20). Plasma antibody binding profiles were associated with clinical meta-data including diagnosis, lung function and transplant free survival. Measurements and Main Results: We identified 586 putative autoantigens in both study cohorts with a broad heterogeneity among disease entities and cohorts. On average, in IPF a mean±SD of 16±40 autoantigens and in CTD-ILD a mean±SD of 9±15 autoantigens were identified per patient. We identified 18 IPF-specific autoantigens validated in the second cohort. Interestingly, there was also a high number of shared autoantigens in IPF and CTD-ILD patients, with 17 being present in IPF and CTD-ILD of both cohorts. Presence of antibodies to Thrombospondin 1 (THBS1) and tubulin beta-1 chain (TUBB1) was associated with a significantly reduced survival in patients with IPF (p=0.002 and p=0.019, respectively). This signature was often associated with autoreactivity against talin-1 (TLN1), latent-transforming growth factor beta-binding protein 1 (LTBP1), epididymis secretory protein Li 112 (HEL-S-112), zyxin (ZYX), the LIM and senescent cell antigen-like-containing domain protein 1 (LIMS1) and caldesmon (CALD1). Conclusions: Unbiased proteomic profiling reveals that the overall prevalence of autoantibodies is similar in IPF and CTD-ILD patients and identifies novel IPF specific autoantigens associated with patient survival.

Project description:Performing GWAS on multiple myeloma in relation to the development of the toxicity neuropathy. This set was used as validation set. We performed a genome-wide association study using Affymetrix HD-SNP arrays 6.0 to identify risk variants for developing bortezomib-induced peripheral neuropathy (BiPN) in 469 multiple myeloma (MM) patients who received bortezomib-dexamethasone therapy prior to autologous stem-cell transplantation and conducted validation in an independent cohort of 116 MM patients. We identified one previously unreported BiPN risk locus at 21q22.3 (rs2839629, PKNOX1; OR = 0.53, 95% CI: [0.40-0.69]). PKNOX1 is known to regulate MCP-1, a potent mediator of chemotherapy-induced peripheral neuropathy. rs2839629 is in strong linkage disequilibrium ( r2 = 0.87) with rs915854, localized 6.5kb centromeric to CBS encoding endogenous H2S-producing enzyme. CBS-H2S signalling pathway is implicated in the pathogenesis of a variety of neurodegenerative and inflammatory disorders, and specifically in neuropathy models. Our data provide conclusive evidence for genetic susceptibility to BiPN in MM and new potential targets in neuro-protective strategies of treatment. Each patient of both IFM and HOVON cohorts was genotyped using the Affymetrix SNP6.0 Human DNA chip (Affymetrix, Santa Clara, CA) according to manufacturerâs instructions (Affymetrix, Santa Clara, CA). The HOVON65 samples were genotyped using CRLMM v2 resulting in a call rate higher than 95%. Unannotated SNPs according to the hg19 na32 SNP6 Affymetrix annotations (n= 130) along with SNPs from MT and sexual chromosomes (n= 37,326) were not considered in the study. To perform GWAS in the IFM cohort, we used stringent criteria to select a total of 370,605 SNPs from the 872,166 SNPs available onto the array. A SNP was analysed if an AA or BB genotype was present in more than 5% of patients, if its genotype was determined in at least 95% of the patients and if the Hardy-Weinberg rule was not rejected. Statistical analyses were performed using SNPTEST v2.5.11. Firstly, we compared 370,605 genotypes from 155 IFM patients with neuropathy (grade >= 2) after bortezomib exposure to 314 control patients treated with bortezomib who did not develop neuropathy (grade 0-1). Secondly, we performed a validation in the HOVON 65/GMMG-HD4cohort for the highest associated SNPs as identified in the discovery cohort. We compared 41 bortezomib-treated patients with neuropathy (grade >= 2) with 75 bortezomib-treated controls who did not develop neuropathy. We applied a one-sided logistic regression with 10,000 label swapping permutations to correct for multiple testing in order to confirm BiPN association in this independent cohort. This set was used as a validation set. An independent dataset was used for discovery [GSE65777].

Project description:Introduction: Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare. Methods: We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n=45) to healthy controls (n=17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n=23) and without significant renal involvement (n=18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n=23) and longitudinal samples (88 patient visits) to test its accuracy. Results: Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B-cell activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV & X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91% accuracy. Conclusions: Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.

Project description:Background Currently, no gene-expression signature (GES) established from node-positive cohorts, able to predict breast cancer evolution after systemic adjuvant chemotherapy, exists. Methods Gene-expression profiles of 252 node-positive patients (median follow-up: 7.7 years), mostly included in a randomized clinical trial (PACS01), receiving systemic adjuvant regimen, were determined by means of cDNA custom array representing 5,776 distinct genes. Findings In the training cohort, we established a 38-GES for the purpose of predicting time to distant metastasis. The 38-GES yielded unadjusted hazard ratio of 4.86 (95% CI=2.76-8.56). Even when adjusted with the two best clinicopathological prognostic scoring: NPI and Adjuvant!, 38-GES HR were 3.30 (1.81-5.99) and 3.40 (1.85-6.24), respectively. Furthermore, 38-GES improved mostly NPI and Adjuvant! intermediate-risk classified patients. NPI intermediate-risk patients (7-year MFS=87.2%) were divided into 2/3 (7-year MFS=95.5%) close to NPI low-risk group and 1/3 (7-year MFS=69.3%) close to NPI high-risk group (HR=6.97 [2.51-19.36]). Adjuvant! intermediate-risk patients (7-year MFS=88.0%) were divided into 2/3 (7-year MFS=94.8%) close to Adjuvant! low-risk group and 1/3 (7-year MFS=71.7%) close to Adjuvant! high-risk group (HR=5.31 [5.38-11.87]). The 38-GES was validated on gene-expression datasets from three external node-positive breast cancer subcohorts (n=224) generated from different microarray platforms. The 38-GES yielded unadjusted HR=2.95 (1.74-5.01). Furthermore, 38-GES showed performance in supplementary cohorts with different lymph-node status and endpoint (1,031 new patients). Interpretation The 38-GES represents a robust tool able to type systemic adjuvant treated node-positive patients at high risk of metastatic relapse, and especially powerful to separate NPI or Adjuvant! intermediate-risk node-positive patients. Keywords: disease-state analysis 252 breast cancer patients at diagnosis examined with spotted cDNA nylon membrane. Patient details: PACS01x are 2 parts of a clinical trial : PACS01A : patients had received 6 cycles of FEC100 PACS01B : patients had received 3 cycles of FEC100 then 3 cycles of Docetaxel CCRG are patients from our local cancer center (Cancer Center René Gauducheau) included with the same criteria as PACS01A (6 cycles of FEC100).

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A proteomics workflow reveals predictive autoantigens in idiopathic pulmonary fibrosis (cohort 1)

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