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Proteomic profiling to predict therapeutic response to anti-PD1 therapy in advanced melanoma

ABSTRACT: Purpose Antibody-blockade of PD-1 shows durable responses, but so far, few biomarkers accurately predict if patients with metastatic melanoma will respond. Experimental design We analyzed baseline serum samples (n=56) and tumor cell cultures (n=8), from melanoma patients treated with anti-PD1 therapy. Two approaches concentrating low abundant proteins in serum and two different mass spectrometry (MS) methods were applied: depletion of high abundant proteins with shotgun-MS, and N-glycosite enrichment combined with SWATH-MS. Tumor cell cultures were processed as whole cell lysates with subsequent shotgun-MS. Results Focusing on the non-responder proteome, we identified pathways such as inflammatory processes, cell adhesion and migration, scavenger receptor activity, RAGE receptor binding, platelet aggregation, neutrophil degranulation and integrin signaling to be upregulated. Analyzing the tumor cell proteome revealed a distinctive immunophenotype with CD antigens highly overexpressed in non-responders. Validation of a subset of these markers, performed by immunofluorescence staining of a tissue microarray, revealed a higher mean of positive cells in the tumor microenvironment of non-responders. Validation of serum proteome markers on a second baseline cohort by using a timsTOF for ion mobility MS, revealed significant differences between responder and non-responder for the proteins ORM2, SERPINA1, EFEMP1, and VASN. TCGA survival analysis demonstrated prognostic significance for multiple proteins of our signature correlating with lower overall survival in melanoma patients. Based on this study, we suggest a protein-panel for responsiveness to PD-1 blockade consisting of 14 serum and 33 tumor cell markers that will be the basis for further research.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Serum

DISEASE(S): Melanoma

SUBMITTER: Christopher Gerner

LAB HEAD: Christopher Gerner

PROVIDER: PXD027525 | Pride | 2023-08-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	21-Prot-0818_RA1_1_4730.tdf_bin	Other
	21-Prot-0821_RA4_1_4734.tdf_bin	Other
	21-Prot-0822_RA5_1_4735.tdf_bin	Other
	21-Prot-0823_RA6_1_4736.tdf_bin	Other
	21-Prot-0825_RA8_1_4742.tdf_bin	Other

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Project description:TNF antagonists are routinely used in severe rheumatoid arthritis (RA) patients who failed conventional DMARD therapy. According to large clinical trials, the three available drugs (adalimumab, infliximab and etanercept) display similar effects in terms of efficacy, tolerability and side effects. These studies also indicate that about 25% of RA patients treated with TNF-antagonists do not display any significant clinical improvement. The aim of this study was to investigate global molecular patterns in synovial biopsies from RA patients obtained 12 weeks after initiation of adalimumab therapy. All patients had rheumatoid arthritis (RA), according to the American College of rheumatology criteria for the diagnosis of RA. They had active disease at the time of initiation of adalimumab therapy and were resistant to conventional therapy. They all had erosive changes imaged on conventional x-rays of the hands and/or feet. All patients were treated with disease-modifying antirheumatic drugs (DMARD’s), 23 with methotrexate (median dose 15 mg/week, range 7.5 – 20 mg/week), and 2 with leflunomide (20 mg/day); 18 of them were treated with low-dose steroids (prednisolone ≤ 7.5 mg/day). Six patients had been included in double-blind clinical trials before inclusion in the present study (1 in a Golimumab versus placebo trial, 3 in a MapKinase inhibitor versus placebo trial and 2 in a TACE-inhibitor versus placebo trial). These trials were stopped at least 3 months prior to initiation of TNF-blocking therapy. All drug dosages were stable from at least 3 months prior to initiation of TNF blocking therapy until completion of the study. No steroid injections were allowed during the duration of the study. Adalimumab therapy was initiated at a dosage of 40 mg subcutaneously every other week. Disease activity at baseline and 12 weeks after initiation of therapy (T12) was evaluated using DAS(28)-CRP (3- and 4-variables) scores, and response to therapy was assessed according to the EULAR response criteria that categorize patients in responders (good- or moderate-) and non- (or poor-) responders based on changes in DAS activity between T0 and T12 and absolute DAS values at T12. Synovial biopsies were obtained by needle-arthroscopy of knee of the patients at T12. The aim of the study was to compare gene expression profiles in synovial tissue of RA patients who responded versus not responded to adalimumab therapy.

Project description:Steroid-refractory acute rejection is a risk factor for inferior renal allograft outcome. We aimed to gain insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid-refractory acute rejection. Eighty-three kidney transplant recipients (1995-2005), who were treated with methylprednisolone during a first acute rejection episode, were included in this study. Gene expression patterns were investigated in a discovery cohort of 36 acute rejection biopsies, and verified in a validation cohort of 47 acute rejection biopsies. In the discovery set, expression of metallothioneins (MT) was significantly (P<0.000001) associated with decreased response to steroid treatment. Multivariate analysis resulted in a predictive model containing MT-1 as an independent covariate (AUC=0.88, P<0.0000001). In the validation set, MT-1 expression was also significantly associated with steroid resistance (P=0.029). Metallothionein expression was detected in macrophages and tubular epithelial cells. Parallel to the findings in patients, in vitro experiments of peripheral blood mononuclear cells from 11 donors showed that non-response to methylprednisolone treatment is related to highly elevated MT levels. High expression of metallothioneins in renal allografts is associated with resistance to steroid treatment. Metallothioneins regulate intracellular concentrations of zinc, through which they may diminish the zinc-requiring anti-inflammatory effect of the glucocorticoid receptor. Transcriptional profiles of 40 renal allograft biopsy samples were analyzed using Illumina HumanRef-8 v3.0 BeadChips (Illumina, San Diego, CA). Expression profiles were investigated in total RNA isolated from biopsy samples of 18 patients with steroid responsive acute rejection and 18 patients with steroid resistant acute rejection. Four biopsies, taken during acute decrease of graft function but with no histomorphologic indication of rejection, were included as controls.

Project description:Background: Although the use of TNF inhibitors has fundamentally changed the way rheumatoid arthritis (RA) is treated, not all patients respond well. It is desirable to facilitate the identification of responding and non-responding patients prior to treatment, not only to avoid unnecessary treatment but also for financial reasons. In this work we have investigated the transcriptional profile of synovial tissue sampled from RA patients before anti-TNF treatment with the aim to identify biomarkers predictive of response. Methodology/Principal Findings: Synovial tissue samples were obtained by arthroscopy from 62 RA patients before the initiation of infliximab treatment. RNA was extracted and gene expression profiling was performed using an in-house spotted long oligonucleotide array covering 17972 unique genes. Tissue sections were also analyzed by immunohistochemistry to evaluate cell infiltrates. Response to infliximab treatment was assessed according to the EULAR response criteria. The presence of lymphocyte aggregates dominated the expression profiles and a significant overrepresentation of lymphocyte aggregates in good responding patients confounded the analyses. A statistical model was set up to control for the effect of aggregates, but no differences could be identified between responders and non-responders. Subsequently, the patients were split into lymphocyte aggregate positive- and negative patients. No statistically significant differences could be identified except for 38 transcripts associated with differences between good- and non-responders in aggregate positive patients. A profile was identified in these genes that indicated a higher level of metabolism in good responding patients, which indirectly can be connected to increased inflammation. Conclusions/Significance: It is pivotal to account for the presence of lymphoid aggregates when studying gene expression patterns in rheumatoid synovial tissue. In spite of our original hypothesis, the data do not support the notion that microarray analysis of synovial biopsy specimens can be used in the context of personalized medicine to identify non-responders to anti-TNF therapy before the initiation of treatment. All patients were hybridized in a dual channel reference design where the reference was always labeled with cy3 and the samples with cy5. In total, RNA was extracted from synovial biopsies of 62 patients. 18 patients were good responding, 30 moderate responding and 14 non-responding. Universal Human Reference RNA from Stratagene was used as reference. All samples were amplified with the RiboAmp II kit before labeling and hybridization.

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Proteomic profiling to predict therapeutic response to anti-PD1 therapy in advanced melanoma

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