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Discovery and Validation of Predictive Biomarkers of Survival for Non-small Cell Lung Cancer Patients Undergoing Radical Radiotherapy: Two Proteins With Predictive Value.

Walker MJ, Zhou C, Backen A, Pernemalm M, Williamson AJ, Priest LJ, Koh P, Faivre-Finn C, Blackhall FH, Dive C, Whetton AD - EBioMedicine (2015)

Bottom Line: Identification of such markers would allow treatment options to be considered for more effective therapy.Plasma samples from patients pre- and during radiotherapy who had survived > 18 mo were compared to the same time points from patients who survived < 14 mo using an 8 channel isobaric tagging tandem mass spectrometry discovery proteomics platform.Over 650 proteins were detected and relatively quantified.

View Article: PubMed Central - PubMed

Affiliation: Stoller Biomarker Discovery Centre, Manchester Academic Health Science Centre, The University of Manchester, Wolfson Molecular Imaging Centre, Manchester M20 3LJ, UK.

ABSTRACT
Lung cancer is the most frequent cause of cancer-related death world-wide. Radiotherapy alone or in conjunction with chemotherapy is the standard treatment for locally advanced non-small cell lung cancer (NSCLC). Currently there is no predictive marker with clinical utility to guide treatment decisions in NSCLC patients undergoing radiotherapy. Identification of such markers would allow treatment options to be considered for more effective therapy. To enable the identification of appropriate protein biomarkers, plasma samples were collected from patients with non-small cell lung cancer before and during radiotherapy for longitudinal comparison following a protocol that carries sufficient power for effective discovery proteomics. Plasma samples from patients pre- and during radiotherapy who had survived > 18 mo were compared to the same time points from patients who survived < 14 mo using an 8 channel isobaric tagging tandem mass spectrometry discovery proteomics platform. Over 650 proteins were detected and relatively quantified. Proteins which showed a change during radiotherapy were selected for validation using an orthogonal antibody-based approach. Two of these proteins were verified in a separate patient cohort: values of CRP and LRG1 combined gave a highly significant indication of extended survival post one week of radiotherapy treatment.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of study design and experimental workflow. (a) Therapeutic plan, two baseline samples are collected prior to the start of RT (T1 and T2). RT is delivered over 6 weeks (green boxes) with a sample collected during week 2 (T3). After treatment patient survival was followed and patients were retrospectively assigned to either the < 14 mo or > 18 mo survival groups. (b) Experimental plan and workflow for identification and relative quantification of plasma proteins. Proteins were depleted of high abundance proteins using a MARS 14 depletion system followed by digestion with trypsin and labelling with the correct iTRAQ reagent. Each iTRAQ 8 plex was designed to contain the samples from 2 patients (one < 14 mo one > 18 mo) A portion of all samples included in the discovery cohort were collected into a pool sample which was used to assess technical variation and allow comparisons across isobaric tagging experiments. Peptides were fractionated prior to mass spectrometry by 2-dimensional reverse phase liquid chromatography, the 1st dimension at pH 10.5 and the second at pH 3. The mass spectrometry was run with IDA methods and the raw result files analysed by Protein Pilot. Protein quantification is then reconstituted from high confidence peptide spectral matches and the proteins with elevated levels postradiotherapy identified.
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f0005: Schematic representation of study design and experimental workflow. (a) Therapeutic plan, two baseline samples are collected prior to the start of RT (T1 and T2). RT is delivered over 6 weeks (green boxes) with a sample collected during week 2 (T3). After treatment patient survival was followed and patients were retrospectively assigned to either the < 14 mo or > 18 mo survival groups. (b) Experimental plan and workflow for identification and relative quantification of plasma proteins. Proteins were depleted of high abundance proteins using a MARS 14 depletion system followed by digestion with trypsin and labelling with the correct iTRAQ reagent. Each iTRAQ 8 plex was designed to contain the samples from 2 patients (one < 14 mo one > 18 mo) A portion of all samples included in the discovery cohort were collected into a pool sample which was used to assess technical variation and allow comparisons across isobaric tagging experiments. Peptides were fractionated prior to mass spectrometry by 2-dimensional reverse phase liquid chromatography, the 1st dimension at pH 10.5 and the second at pH 3. The mass spectrometry was run with IDA methods and the raw result files analysed by Protein Pilot. Protein quantification is then reconstituted from high confidence peptide spectral matches and the proteins with elevated levels postradiotherapy identified.

Mentions: Blood was collected from donors in lithium heparin coated tubes and centrifuged within 30 min of collection at 2500 ×g for 15 min at 4 °C before aliquots of the plasma layer were stored at − 80 °C. Samples were collected at the following time points for each patient; before RT, during RT (days 2, 3, 8, then weekly) and on completion after RT (months 1, 3, 6) (Fig. 1). Blood samples were taken from 29 randomised patients with lung cancer enrolled in the RADAR study at the Christie Hospital, Manchester, UK following written informed consent with ethical approval from the Central Manchester Local Research Ethics Committee. This proteomic analysis was undertaken on two samples per patient collected prior to the start of radiotherapy and a third sample on day 8 of the treatment regimen.


Discovery and Validation of Predictive Biomarkers of Survival for Non-small Cell Lung Cancer Patients Undergoing Radical Radiotherapy: Two Proteins With Predictive Value.

Walker MJ, Zhou C, Backen A, Pernemalm M, Williamson AJ, Priest LJ, Koh P, Faivre-Finn C, Blackhall FH, Dive C, Whetton AD - EBioMedicine (2015)

Schematic representation of study design and experimental workflow. (a) Therapeutic plan, two baseline samples are collected prior to the start of RT (T1 and T2). RT is delivered over 6 weeks (green boxes) with a sample collected during week 2 (T3). After treatment patient survival was followed and patients were retrospectively assigned to either the < 14 mo or > 18 mo survival groups. (b) Experimental plan and workflow for identification and relative quantification of plasma proteins. Proteins were depleted of high abundance proteins using a MARS 14 depletion system followed by digestion with trypsin and labelling with the correct iTRAQ reagent. Each iTRAQ 8 plex was designed to contain the samples from 2 patients (one < 14 mo one > 18 mo) A portion of all samples included in the discovery cohort were collected into a pool sample which was used to assess technical variation and allow comparisons across isobaric tagging experiments. Peptides were fractionated prior to mass spectrometry by 2-dimensional reverse phase liquid chromatography, the 1st dimension at pH 10.5 and the second at pH 3. The mass spectrometry was run with IDA methods and the raw result files analysed by Protein Pilot. Protein quantification is then reconstituted from high confidence peptide spectral matches and the proteins with elevated levels postradiotherapy identified.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563120&req=5

f0005: Schematic representation of study design and experimental workflow. (a) Therapeutic plan, two baseline samples are collected prior to the start of RT (T1 and T2). RT is delivered over 6 weeks (green boxes) with a sample collected during week 2 (T3). After treatment patient survival was followed and patients were retrospectively assigned to either the < 14 mo or > 18 mo survival groups. (b) Experimental plan and workflow for identification and relative quantification of plasma proteins. Proteins were depleted of high abundance proteins using a MARS 14 depletion system followed by digestion with trypsin and labelling with the correct iTRAQ reagent. Each iTRAQ 8 plex was designed to contain the samples from 2 patients (one < 14 mo one > 18 mo) A portion of all samples included in the discovery cohort were collected into a pool sample which was used to assess technical variation and allow comparisons across isobaric tagging experiments. Peptides were fractionated prior to mass spectrometry by 2-dimensional reverse phase liquid chromatography, the 1st dimension at pH 10.5 and the second at pH 3. The mass spectrometry was run with IDA methods and the raw result files analysed by Protein Pilot. Protein quantification is then reconstituted from high confidence peptide spectral matches and the proteins with elevated levels postradiotherapy identified.
Mentions: Blood was collected from donors in lithium heparin coated tubes and centrifuged within 30 min of collection at 2500 ×g for 15 min at 4 °C before aliquots of the plasma layer were stored at − 80 °C. Samples were collected at the following time points for each patient; before RT, during RT (days 2, 3, 8, then weekly) and on completion after RT (months 1, 3, 6) (Fig. 1). Blood samples were taken from 29 randomised patients with lung cancer enrolled in the RADAR study at the Christie Hospital, Manchester, UK following written informed consent with ethical approval from the Central Manchester Local Research Ethics Committee. This proteomic analysis was undertaken on two samples per patient collected prior to the start of radiotherapy and a third sample on day 8 of the treatment regimen.

Bottom Line: Identification of such markers would allow treatment options to be considered for more effective therapy.Plasma samples from patients pre- and during radiotherapy who had survived > 18 mo were compared to the same time points from patients who survived < 14 mo using an 8 channel isobaric tagging tandem mass spectrometry discovery proteomics platform.Over 650 proteins were detected and relatively quantified.

View Article: PubMed Central - PubMed

Affiliation: Stoller Biomarker Discovery Centre, Manchester Academic Health Science Centre, The University of Manchester, Wolfson Molecular Imaging Centre, Manchester M20 3LJ, UK.

ABSTRACT
Lung cancer is the most frequent cause of cancer-related death world-wide. Radiotherapy alone or in conjunction with chemotherapy is the standard treatment for locally advanced non-small cell lung cancer (NSCLC). Currently there is no predictive marker with clinical utility to guide treatment decisions in NSCLC patients undergoing radiotherapy. Identification of such markers would allow treatment options to be considered for more effective therapy. To enable the identification of appropriate protein biomarkers, plasma samples were collected from patients with non-small cell lung cancer before and during radiotherapy for longitudinal comparison following a protocol that carries sufficient power for effective discovery proteomics. Plasma samples from patients pre- and during radiotherapy who had survived > 18 mo were compared to the same time points from patients who survived < 14 mo using an 8 channel isobaric tagging tandem mass spectrometry discovery proteomics platform. Over 650 proteins were detected and relatively quantified. Proteins which showed a change during radiotherapy were selected for validation using an orthogonal antibody-based approach. Two of these proteins were verified in a separate patient cohort: values of CRP and LRG1 combined gave a highly significant indication of extended survival post one week of radiotherapy treatment.

No MeSH data available.


Related in: MedlinePlus