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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

Verification of putative biomarkers in additional cohort and with orthogonal method. (a) Scatter plots of concentration of LBP, LRG1 and CRP in plasma with small cell lung cancer prior to radiotherapy (SCLC—circle), adenocarcinoma NSCLC (AC—square) or squamous cell carcinoma NSCLC (SqCC—triangle). Levels of the proteins in plasma were assayed by commercial ELISA. (b) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients prior to radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (c) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (d) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from AC and SCLC during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). Significance was tested using a 2-tailed unpaired Mann Whitney test.
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f0015: Verification of putative biomarkers in additional cohort and with orthogonal method. (a) Scatter plots of concentration of LBP, LRG1 and CRP in plasma with small cell lung cancer prior to radiotherapy (SCLC—circle), adenocarcinoma NSCLC (AC—square) or squamous cell carcinoma NSCLC (SqCC—triangle). Levels of the proteins in plasma were assayed by commercial ELISA. (b) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients prior to radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (c) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (d) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from AC and SCLC during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). Significance was tested using a 2-tailed unpaired Mann Whitney test.

Mentions: To verify if the three shortlisted proteins are predictive of survival during radiotherapy an additional cohort of 23 additional lung cancer patients were analysed using enzyme linked immunosorbent assay (ELISA) for CRP, LBP and LRG1. In this further part of the study other lung cancer subtypes were included (Supplementary Table 3). Thus to test if the effect is only specific to squamous cell carcinoma NSCLC (SqCC), six patient samples from adenocarcinoma NSCLC (AC) and small cell lung cancer (SCLC) patients respectively were included in the verification samples set as well as eleven SqCC patients. Each patient had a single pre-treatment sample as well as an early radiotherapy treatment sample collected as for the SqCC NSCLC discovery proteomics sample set using the same standard operating procedures for sample collection and storage. The hypothesis that the putative biomarkers mentioned above had relevance in diseases other than SqCC NSCLC, like AC and SCLC, was tested by examining the level of each protein in patients' plasma prior to and after radiotherapy (Fig. 3a). CRP showed no significant difference between any patient groups prior to therapy. LRG1 levels in the plasma of SqCC patients (127 ± 11 mg/L mean ± SD N = 11) was significantly different to AC (78 ± 18 mg/L, mean ± SD N = 6 p < 0.047) and SCLC (62 ± 8 mg/L mean ± SD N = 6, p < 0.0003) prior to therapy. LBP levels in the plasma of SqCC patients (33 ± 11 mg/L mean ± SD N = 11) was significantly different to AC (13 ± 7 mg/L mean ± SD N = 6, p < 0.0048) and SCLC (3 ± 1 mg/L mean ± SD N = 6, p < 0.0031). Due to the differences seen in protein levels between the different patient sets the effect of radiotherapy on levels were only analysed between patients with the same histology. The levels of all three proteins (CRP, LBP and LRG1) prior to radiotherapy showed no significant difference in the plasma of patients with poor or good prognosis prior to radiotherapy (Fig. 3b). The level of each of the proteins was compared for their changes during radiotherapy and only SqCC patients showed any significant difference in protein levels between patients with good and poor prognosis (Fig. 3c). LBP was down regulated following radiotherapy, consistent with the MS/MS results, but it showed no significant difference between the > 18 mo survival SqCC patient group and the < 14 mo survival SqCC patient group. The level of CRP and LRG1 were both significantly different in the SqCC < 14 mo survival group (CRP 48 ± 27 mg/L, LRG1 156 ± 19 mg/L mean ± SD N = 5) compared to the SqCC > 18 mo survival group (CRP 8.5 ± 2 mg/L p = 0.0173, LRG1 94 ± 7 mg/L p = 0.0087, mean ± SD N = 6). Analysis of the levels seen before and during radiotherapy levels for LRG1, LBP and CRP in adenocarcinoma and small cell lung cancer showed no significant difference in values (Fig. 3d). A post-hoc power analysis of our two phase study (Mass spectrometry identification followed by ELISA verification) was carried out based on simulation, a power of 82.6% was achieved, indicating that our results are highly repeatable.


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)

Verification of putative biomarkers in additional cohort and with orthogonal method. (a) Scatter plots of concentration of LBP, LRG1 and CRP in plasma with small cell lung cancer prior to radiotherapy (SCLC—circle), adenocarcinoma NSCLC (AC—square) or squamous cell carcinoma NSCLC (SqCC—triangle). Levels of the proteins in plasma were assayed by commercial ELISA. (b) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients prior to radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (c) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (d) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from AC and SCLC during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). Significance was tested using a 2-tailed unpaired Mann Whitney test.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0015: Verification of putative biomarkers in additional cohort and with orthogonal method. (a) Scatter plots of concentration of LBP, LRG1 and CRP in plasma with small cell lung cancer prior to radiotherapy (SCLC—circle), adenocarcinoma NSCLC (AC—square) or squamous cell carcinoma NSCLC (SqCC—triangle). Levels of the proteins in plasma were assayed by commercial ELISA. (b) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients prior to radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (c) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from SqCC patients during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). (d) Scatter plots of the concentration of LRG1, CRP and LBP in plasma from AC and SCLC during radiotherapy comparing survival < 14 mo (circle) and > 18 mo (square). Significance was tested using a 2-tailed unpaired Mann Whitney test.
Mentions: To verify if the three shortlisted proteins are predictive of survival during radiotherapy an additional cohort of 23 additional lung cancer patients were analysed using enzyme linked immunosorbent assay (ELISA) for CRP, LBP and LRG1. In this further part of the study other lung cancer subtypes were included (Supplementary Table 3). Thus to test if the effect is only specific to squamous cell carcinoma NSCLC (SqCC), six patient samples from adenocarcinoma NSCLC (AC) and small cell lung cancer (SCLC) patients respectively were included in the verification samples set as well as eleven SqCC patients. Each patient had a single pre-treatment sample as well as an early radiotherapy treatment sample collected as for the SqCC NSCLC discovery proteomics sample set using the same standard operating procedures for sample collection and storage. The hypothesis that the putative biomarkers mentioned above had relevance in diseases other than SqCC NSCLC, like AC and SCLC, was tested by examining the level of each protein in patients' plasma prior to and after radiotherapy (Fig. 3a). CRP showed no significant difference between any patient groups prior to therapy. LRG1 levels in the plasma of SqCC patients (127 ± 11 mg/L mean ± SD N = 11) was significantly different to AC (78 ± 18 mg/L, mean ± SD N = 6 p < 0.047) and SCLC (62 ± 8 mg/L mean ± SD N = 6, p < 0.0003) prior to therapy. LBP levels in the plasma of SqCC patients (33 ± 11 mg/L mean ± SD N = 11) was significantly different to AC (13 ± 7 mg/L mean ± SD N = 6, p < 0.0048) and SCLC (3 ± 1 mg/L mean ± SD N = 6, p < 0.0031). Due to the differences seen in protein levels between the different patient sets the effect of radiotherapy on levels were only analysed between patients with the same histology. The levels of all three proteins (CRP, LBP and LRG1) prior to radiotherapy showed no significant difference in the plasma of patients with poor or good prognosis prior to radiotherapy (Fig. 3b). The level of each of the proteins was compared for their changes during radiotherapy and only SqCC patients showed any significant difference in protein levels between patients with good and poor prognosis (Fig. 3c). LBP was down regulated following radiotherapy, consistent with the MS/MS results, but it showed no significant difference between the > 18 mo survival SqCC patient group and the < 14 mo survival SqCC patient group. The level of CRP and LRG1 were both significantly different in the SqCC < 14 mo survival group (CRP 48 ± 27 mg/L, LRG1 156 ± 19 mg/L mean ± SD N = 5) compared to the SqCC > 18 mo survival group (CRP 8.5 ± 2 mg/L p = 0.0173, LRG1 94 ± 7 mg/L p = 0.0087, mean ± SD N = 6). Analysis of the levels seen before and during radiotherapy levels for LRG1, LBP and CRP in adenocarcinoma and small cell lung cancer showed no significant difference in values (Fig. 3d). A post-hoc power analysis of our two phase study (Mass spectrometry identification followed by ELISA verification) was carried out based on simulation, a power of 82.6% was achieved, indicating that our results are highly repeatable.

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