WO2009034562A2 - Methode permettant d'evaluer le risque de cancer chez un patient - Google Patents

Methode permettant d'evaluer le risque de cancer chez un patient Download PDF

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WO2009034562A2
WO2009034562A2 PCT/IE2008/000086 IE2008000086W WO2009034562A2 WO 2009034562 A2 WO2009034562 A2 WO 2009034562A2 IE 2008000086 W IE2008000086 W IE 2008000086W WO 2009034562 A2 WO2009034562 A2 WO 2009034562A2
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abundance
protein
cancer
sequence
proteins
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PCT/IE2008/000086
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WO2009034562A3 (fr
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Paul Dowling
Martin Clynes
John Crown
Michael Moriarty
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Dublin City University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to a method of assessing cancer status in an individual.
  • the invention relates to a method of screening patients to identify patients at risk of having a lung squamous cell carcinoma.
  • Lung cancer comprises broadly of two groups, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC).
  • SCLC small cell lung carcinoma
  • NSCLC non-small cell lung carcinoma
  • a major factor in the high mortality of lung cancer patients is the presence of metastatic tumours in approximately two-thirds of patients at time of diagnosis.
  • the identification of lung cancer-specific biomarkers is critical to early detection.
  • the pathophysiology of lung squamous cell carcinoma development is complex and incompletely understood. Genetic alterations involved in the pathogenesis of lung cancer produce proteins involved in cell growth, invasion/metastasis, differentiation, cell cycle processes, apoptosis and angiogenesis.
  • expression profiling can sub-classify lung adenocarcinoma in terms of predicting length of survival. Takeuchi, T., Tomida, S., Yatabe, Y., Kosaka, T., Osada, H., Yanagisawa, K., Mitsudomi, T., Takahashi, T., J. Clin. Oncol. 2006, 24, 1679-1688.
  • the invention is based on the finding that the serum level of certain proteins is modulated in patients with lung squamous cell carcinoma (lung SCC) compared with control patients. Certain proteins have been found to be present in greater abundance in sera from lung SCC patients, and certain other proteins have been found to be present in lower abundance in sera from lung SCC patients. Thus, the expression levels of these proteins function as biomarkers of lung SCC status in an individual. In particular, the biomarkers find application in screening at-risk patients (i.e. smokers over 40 years of age) to identify patients that should undergo more investigative procedures such as biopsy and further screens/scans.
  • lung SCC lung squamous cell carcinoma
  • a method of assessing the status of a lung SCC cancer in an individual comprising a step of assessing a biological sample from the individual for the abundance of a protein selected from the group consisting of: SEQUENCE ID NO'S: 1 to 11, and correlating the expression level of the protein with lung SCC cancer status.
  • each of the protein biomarkers of the invention is provided in Table 2 below, along with a Gene Index number for each protein, and the average ratio of abundance for each biomarker between control and cancer patients.
  • the ratio of abundance of this protein in sera from lung SCC patients to the abundance of the marker in sera from the cohort of control patients is 1:2.43 (-2.43).
  • the abundance of this protein in sera from lung SCC patients is on average 7.81 times greater that the average abundance of the marker in sera from the cohort of control patients (control abundance value).
  • control abundance value the ratio of abundance of this marker in a test patient to a control abundance value is found to be 7.81 or greater, then this would indicate that the patient is at risk of having lung SCC.
  • the invention encompasses each of the biomarkers of SEQUENCE ID NO's: 1 to 11 functioning as individual variables of lung SCC status, and also combinations of these markers, including a combination of all of the biomarkers.
  • the method comprises a step of assessing a biological sample obtained from the individual for the expression level of a protein selected from the group consisting of: SEQUENCE ID NO'S: 4, 7 and 8.
  • the method comprises a step of assessing a biological sample obtained from the individual for the expression level of a protein selected from the group consisting of: SEQUENCE ID NO:'s: 7 and 8. In another embodiment, the method comprises a step of assessing a biological sample obtained from the individual for the expression level of a protein selected from the group consisting of: SEQUENCE ID NO: 8.
  • the method comprises a step of assessing a biological sample obtained from the individual for the expression level of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 proteins selected from the group consisting of: SEQUENCE ID NO:'s: 1 to 11.
  • the invention comprises a step of assessing a biological sample obtained from the individual for the expression level of each of the proteins in the group consisting of SEQUENCE ID NO:'s: 1 to 11.
  • the invention comprises a step of assessing a biological sample obtained from the individual for the expression level of each of the proteins in the group consisting of SEQUENCE ID NO:'s: 2; 3; 4; 5; 7 and 8.
  • the invention comprises a step of assessing a biological sample obtained from the individual for the expression level of each of the proteins in the group consisting of SEQUENCE ID NO.'s: 1; 6; 9; 10 and 11.
  • cancer status should primarily be taken to mean an assessment of cancer risk in which a positive identification of one of the diagnostic variables disclosed herein would indicate that the person is at risk of having cancer.
  • positive identification of a diagnostic variable according to the invention would indicate that the person is at a sufficient risk of having lung SCC that they should undergo further investigation.
  • the term should also be taken to encompass early cancer diagnosis, invasive/metastases potential of a cancer, assessment of likely patient outcome due to the cancer, and assessment of effectiveness of a treatment for a cancer.
  • cancer should be taken to mean any cancer, including a cancer selected from the group consisting of: fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcom; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma; lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumor; leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma; bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma; chori
  • biological sample should primarily be taken to mean serum, however the assay of other biological samples, especially fluid samples such as, for example, blood, serum, saliva, urine, and cerebrospinal fluid, is also envisaged.
  • the individual will be a person suspected of having cancer (i.e. symptomatic), or pre-disposed to developing cancer as determined by other phenotypic, genotypic or hereditary traits.
  • the individual may be a person known to have cancer, and who is undergoing a therapeutic treatment regime, in which case the method of the invention may be employed to monitor the effectiveness of the treatment, or may be a post-operative patient being monitored for re-occurrence of the disease.
  • the method is a method for the early detection of a cancer, in which over-abundance or under-abundance of a protein selected from the group consisting of SEQUENCE ID NO's: 1 to 11 is correlated with early detection of the cancer.
  • the cancer is lung SCC.
  • over-abundance of a protein selected from the group of SEQUENCE ID NO's: 2, 3, 4, 5, 7 and 8 correlates with early detection of cancer.
  • over-abundance of each protein in the group of SEQUENCE ID NO's: 4, 7 and 8 correlates with early detection of cancer.
  • under-abundance of a protein selected from the group of SEQUENCE ID NO's: 1, 6, 9, 10 and 11 correlates with early detection of cancer.
  • the method is a method of monitoring the effectiveness of a treatment for a cancer, especially a treatment for preventing metastases of the cancer, in which changes in the abundance of a protein selected from the group consisting of SEQUENCE ID NO's: 1 to 11 is correlated with effectiveness of the treatment.
  • the cancer is lung SCC.
  • the protein being assayed is a protein selected from the group of SEQUENCE ID NO's: 2, 3, 4, 5, 6, 7 and 8, then a decrease in abundance of the protein is generally indicative of effectiveness of the treatment.
  • the method will involve an initial assay to determine the starting abundance level of the or each biomarker, and then further periodic measurements of the abundance level of the or biomarker during and/or after the course of the treatment to monitor abundance levels of the or each marker.
  • the protein being assayed is a protein selected from the group of SEQUENCE ID NO's: 1, 6, 9, 10 and 11 then a decrease in abundance of the protein is' generally indicative of effectiveness of the treatment.
  • the method will involve an initial assay to determine the starting abundance level of the or each biomarker, and then further periodic measurements of the abundance level of the or biomarker during .and/or after the course of, the treatment to monitor abundance levels of the or each marker'.'
  • over-abundance as applied to a specific protein in Table 2 should be taken to mean an abundance of the protein (relative to a control abundance for that protein) which is equal to or greater than the abundance ratio provided in Table 2 for that protein.
  • the term "under-abundance" as applied to a specific protein in Table 2 should be taken to mean an abundance of the protein (relative to a control abundance for that protein) which is equal to or less than the abundance ratio provided in Table 2 for that protein.
  • the invention relates to a method of screening an individual for risk of lung squamous cell carcinoma, the method involving assaying a serum sample from the individual for the abundance of one or more biomarkers relative to a control abundance level for the or each biomarker, and correlating the relative abundance for the or each biomarker with Table 2 to identify individuals at risk for lung SCC.
  • more than one biomarker is employed.
  • all biomarkers of Table 2 are employed.
  • the identification in the sera of an individual of any biomarker of Table 2 having a relative abundance equal to or exceeding the average limits of Table 2 is indicative that the patients is at risk of having lung SCC, and therefore should undergo further investigations.
  • the individual undergoing this assay would be a person who would be considered to be in an at-risk category, such as for example being over 40, a smoker, and/or having a family history of lung cancer.
  • the diagnostic methods of the invention involve relative quantification or relative abundance of the biomarkers of Table 2; in other words, the methods involve a determination of the abundance of one or more of the biomarkers of Table 2 relative to a control abundance value for each biomarker.
  • the method will be carried out using a control sample, or on an apparatus in which the control abundance values have been pre-set.
  • the control abundance value for each biomarker is determined from a control sample.
  • the control sample should be a pooled sample of sera from at least eight patients having no documented history of cancer. Ideally, each sample of sera in the control cohort is age and sex matched to the subject. Age matching should be taken to mean that the age of each person in the control cohort is within +/- 10 years of the age of the subject. Ideally, the sera employed in the control sample will be obtained from individuals who are ethnically matched to the subjects of the diagnosis. Thus, if the diagnosis is to be performed on patients of Western European ethnicity, then the control sample should be obtained from Western European individuals.
  • the diagnostic methods of the invention may also involve absolute quantification of the or each biomarker of Table 2, and comparison with known control abundance values for the or each biomarker, to determine if the ratio of control and sample abundance levels correlates with risk of cancer.
  • the literature in the field provides (as indicated below) absolute values of serum concentration for many of the biomarkers on Table 2, and these values may be employed in the method of diagnosis involving determining absolute abundance values for the serum biomarkers from a test patient, and comparing the absolute abundance value obtained with known control abundance value to obtain an abundance ratio which, when compared with the average ratio of Table 2 may be correlated with cancer risk.
  • the concentration of Alpha-2-HS- glycoprotein in human serum is approximately 361 ⁇ 55 ug/ml; P ⁇ 0.05 (Oikawa et al., 2007).
  • Methods of determining absolute levels of circulating proteins will be well known to those skilled in the art such as, for example, ELISA or direct mass spectrometer analysis.
  • the methods of the invention involve gel electrophoresis, especially 2-DIGE, separation of proteins in a sample, followed by relative quantification of the separated proteins by gel imaging.
  • the proteins in the control sample and patient sample are differentially labelled.
  • the samples are immunodepleted to remove high- abundance proteins prior to separation of the proteins.
  • the samples are serum samples.
  • the control sample is used to "zero" the mass spectrometer in respect of abundance values for the biomarkers, wherein abundance values obtained for biomarkers present in the test sample are provided in a the form of a relative value (relative to a control).
  • Lung cancer is the leading cause of cancer-related mortality in both men and women.
  • the prevalence of lung cancer is second only to that of prostate cancer in men and breast cancer in women. Early detection of lung cancer would greatly improve the survival rates.
  • Serum biomarker have great potential to facilitate the early detection and for monitoring of treatments/recurrence in patients with lung cancer in a non-invasive manner.
  • the analysis of the serum proteome in patients with squamous cell carcinoma of the lung could identify potential biomarkers and provide a greater understanding of the pathophysiology.
  • the concentration of Haptoglobin in the sera of patients with squamous cell carcinoma of the lung was found to be greater than that of the disease free control group.
  • Haptoglobin was elevated beyond the normal capacity of the immunodepletion column and thus appeared in the flow-through fraction, with an average ratio of 3.9 fold greater concentration in the patients with squamous cell carcinoma of the lung. Due to the Haptoglobin levels being greater that the capacity of the column which might have distorted the result, western blot analysis was performed on raw serum from both the normal and cancer samples. This analysis confirmed a significant elevation in the abundance level of Haptoglobin in the cancer samples compared to normal samples. The full range of biological functions of Haptoglobin in cancer is not well understood at present Dobryszycka, W., Eur. J. Clin. Chem. Clin. Biochem. 1997, 35, 647-654.
  • Apolipoprotein A-IV precursor Chain F
  • Human Complement Component C3c Serum amyloid A protein precursor
  • Ras-related protein Rab-7b Ras-related protein Rab-7b.
  • Apolipoprotein is a carrier of lipids and regulates many cellular functions.
  • Complement components are important mediators of inflammation and contribute to the regulation of the immune response. It was found that Chain F; Human Complement Component C3c to be 1.53-fold increased in squamous cell carcinoma sera. Two recent reports have shown that elevated levels of C3a were found in the ascitic fluids of ovarian cancer patients and in patients with chronic hepatitis C/HCV -related HCC. Bjorge, L., Hakulinen, J., Vintermyr, O.K., Jarva, H., Jensen, T.S., Iversen, O.E., Meri, S., Br. J.
  • the serum amyloid A family comprises a number of differentially expressed apolipoproteins including acute-phase SAAl and SAA2.
  • the serum level of this apolipoprotein increases in a wide range of different disease conditions.
  • Rab7 a member of the Ras Oncogene family, is a small Rab GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway.
  • Northern blot analysis shows that Rab7b mRNA is expressed in lung cells. Yang, M., Chen, T., Han, C, Li, N., Wan, T., Cao, X., Biochem. Biophys. Res. Commun. 2004, 318, 792- 799.
  • Alpha-2-HS-glycoprotein is a glycoprotein present in the serum, is synthesized by hepatocytes and is important in blocking transforming growth factor-betal signal transduction, which is associated with tumor progression.
  • Alpha2HS-glycoprotein has been observed to be depleted in certain tumors compared with normal tissue. Barcellos-Hoff, M.H., Ewan, K.B., Breast Cancer Res.
  • Kawakami et al. reported that alpha2-HS glycoprotein was differentially expressed in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment.
  • HCC hepatocellular carcinoma
  • Hemopexin is a serum glycoprotein that binds heme and transports it to the liver for breakdown and iron recovery and has been reported to be over-expressed in nipple aspirate fluid (NAF) from patients with early-stage breast cancer.
  • NAF nipple aspirate fluid
  • Proapolipoprotein which is hydrolyzed by the signal peptidase and propeptidase, through which apolipoprotein is generated, was recently reported to be increased in breast cancer serum patients using 2 -D differential gel electrophoresis.
  • Antithrombin III is a plasma protein synthesised in the liver. Normal plasma levels are 115 to 160mg/L.
  • SP-40,40 serum protein 40 kD,40 kD
  • Clusterin is an 80 kD disulfide-linked, heterodimeric glycoprotein.
  • Rodriguez-Pineiro et al. have recently shown that serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.
  • Zhang reported that a loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma.
  • FIG. 1 shows a Colloidal Coomassie Blue stained 1-D gel of raw and immuno-depleted serum showing an increased number of visible proteins that were previously masked due to the presence of high abundance proteins and western blot analysis of raw and immuno- depleted serum using an antibody to albumin showing the effectiveness of the immuno- depletion column in removing selected high abundance proteins;
  • FIG. 2 is a D-DIGE image of Cy 2, Cy 3 and Cy 5 labelled squamous cell carcinoma and normal serum proteins. Protein differences were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-DIGE) to resolve proteins based on their isoelectric point, in this case a range of 4-7 was employed and their molecular weight was used to generate a protein expression map (PEM);
  • 2D-DIGE two-dimensional polyacrylamide gel electrophoresis
  • FIG. 3 is a 3-D image of Alpha2HS -glycoprotein. Images were generated using the BVA module of DeCyder software and visually show the abundance levels for Alpha2HS-glycoprotein are lower in the cancer samples compared to normal;
  • FIG. 4 is a statistical analysis of Alpha2HS-glycoprotein. Statistics were generated using the BVA module of DeCyder software and show an average of 2.43-fold lower abundance levels for Alpha2HS-glycoprotein in the cancer samples compared to normal; and
  • FIG. 5 is a Western blot analysis of raw serum from normal and cancer samples using antibodies to actin (loading control) and haptoglobin. The results confirm the increase in abundance levels of haptoglobin in cancer samples compared to normal. This is a representative blot for analysis on all samples performed in triplicate.
  • the patient group studied comprised of 8 individuals with NSCLC. All patients were treated at St. Vincent's University Hospital (SVUH), Dublin, between 2002/2003 and approval to conduct this study was granted by the SVUH Ethics Committee.
  • a pooled sample consisting of equal amounts of each of the sixteen experimental samples, was made and used as a pooled internal standard.
  • 10 niL blood samples were collected preoperatively in glass tubes without additive (10 mL BD Vacutainer No Additive, BD) and allowed to clot at room temperature for 120 mins. Serum was separated by centrifugation at 1500 rpm for 15 min at room temperature. 1 mL aliquots of serum were taken and stored at -80 0 C until ready for use. The time from collection to frozen storage was no more than 3 hrs.
  • Serum samples were processed using a Multiple Affinity Removal Spin Cartridge (Agilent Technologies), which selectively removes albumin, IgG, IgA, anti-trypsin, transferrin, and haptoglobin from the serum sample. Samples were processed according to manufacturer's instructions. For each sample, a low abundance fraction was collected and concentrated using 5000 Da molecular weight cut-off spin concentrators (Agilent Technologies). Samples were subsequently cleaned prior to labelling using a 2-D Cleanup Kit (Biorad). The protein pellets were resuspended in ice-cold 20 mM Tris, 7 M Urea, 2 M Thiourea, 4% CHAPS pH 8.5 buffer. Protein quantification was performed using the Quick Start Bradford Protein Assay (Biorad) absorbance at 595 nm using bovine serum albumin as a protein standard. Approximately 90% of total serum protein is removed by this method.
  • a Multiple Affinity Removal Spin Cartridge Agilent Technologies
  • Immobilized 24cm linear pH gradient (IPG) strips pH 4-7, were rehydrated in rehydration buffer (7M Urea, 2M Thiourea, 4% CHAPS 5 0.5% IPG Buffer, 50 mM DTT) overnight, according to the manufacturers guidelines.
  • Isoelectric focusing was performed using an IPGphor apparatus (GE Healthcare) for a total of 40 kV/h at 20°C, 50 mA. Strips were equilibrated for 20 mins in 50 mM Tris-HCl, pH 8.8, 6 M Urea, 30% (v/v) Glycerol, 1% (w/v) SDS containing 65 mM DTT and then for 20 mins in the same buffer containing 240 mM iodoacetamide.
  • SyproRuby stained gels were imaged using a Typhoon 9400 Variable Mode Imager (GE Healthcare) at the appropriate excitation and emission wavelengths for the stain.
  • the subsequent gel image was imported into the BVA module of DeCyder software and was matched to images generated from DIGE analysis. Spots of interest were selected and confirmed using this software for subsequent picking using an Ettan Spot Picker.
  • Gel plugs were placed into a presilconized 1.5 mL plastic tube for destaining, desalting and washing steps. The remaining liquid above the gel plugs was removed and sufficient acetonitrile was added in order to cover the gel plugs.
  • Mass spectra were recorded using the MALDI ToF instrument operating in the positive reflector mode at the following parameters: accelerating voltage 20 IcV; and pulsed extraction: on (focus mass 2500). Internal and external calibration was performed using trypsin autolysis peaks at m/z 842.50, m/z 2211.104 and Pep4 mix respectively. The mass spectra were analysed using MALDI evaluation software (GE Healthcare), and protein identification was achieved with the PMF Pro-Found search engine for peptide mass fingerprints. Results were also confirmed using MASCOT, an alternate search engine to identify proteins by peptide mass fingerprinting.
  • All buffers used for nano LC separation contained 0.1% formic acid (Fluka) as the ion pairing reagent.
  • Full scan mass spectra were recorded in profile mode and tandem mass spectra in centroid mode. The peptides were identified using the information in the tandem mass spectra by searching against the SWISS PROT database using SEQUESTTM.
  • Two-sided, Student's t-tests were used to analyze differences in protein levels between squamous cell carcinoma and normal disease free samples. A p-value of less than 0.05 was considered statistically significant.
  • Nitrocellulose replicas were subsequently twice washed for 10 min in blocking solution and then incubated with corresponding peroxidase-conjugated secondary antibody for 1 h at room temperature. Nitrocellulose membranes were washed twice for 10 min in blocking solution and twice rinsed for 10 min in PBS. Visualization of immuno- decorated ID bands was carried out using an enhanced chemiluminescence kit (GE Healthcare).
  • GE Healthcare enhanced chemiluminescence kit
  • Sera from eight male patients with squamous cell lung carcinoma were analyzed in this study.
  • the patient's age was in a range from 61-79 years.
  • the tumor ranges in size from 2.1-7 centimetres.
  • Eight healthy male disease free samples were used in the control group with an age range of 26-56 years.
  • Table 1 illustrates Clinical information for patients diagnosed with squamous cell carcinoma of the lung.
  • the serum proteome constitutes a highly complex array of circulating proteins, and is a rich source of potential diagnostic and prognostic biomarkers.
  • the immunodepletion technique involves using multiple affinity removal columns which contain affinity- purified polyclonal antibodies to rapidly remove more than 99 percent of targeted proteins (albumin, immunoglobulin G, immunoglobulin A, alpha- 1 -antitrypsin, transferrin and haptoglobin) with minimal non-specific removal of other proteins.
  • targeted proteins albumin, immunoglobulin G, immunoglobulin A, alpha- 1 -antitrypsin, transferrin and haptoglobin
  • the result of immunodepletion on a colloidal Coomassie Blue stained ID gels were removal of high abundant proteins like albumin allow evaluation of lower abundant proteins previously masked due to the overpowering presence of the immuno-depleted proteins.
  • Figure 1 depicts Colloidal Coomassie Blue stained 1-D gel of raw and immuno-depleted serum showing an increased number of visible proteins that were previously masked due to the presence of high abundance proteins and western blot analysis of raw and immuno- depleted serum using an antibody to albumin showing the effectiveness of the immunodepletion column in removing selected high abundance proteins.
  • Protein differences were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-DIGE) to resolve proteins based on their isoelectric point, in this case a range of 4-7 was employed and their molecular weight to generate a protein expression map (PEM).
  • 2D-DIGE two-dimensional polyacrylamide gel electrophoresis
  • Figure 3 shows 3-D image of Alpha2HS-glycoprotein. Images were generated using the BVA module of DeCyder software and visually show the abundance levels for Alpha2HS -glycoprotein are lower in the cancer samples compared to normal.
  • Figure 4 To show visually alterations in corresponding spot intensity proportions, selected spots are displayed as three-dimensional (3D) images.
  • Figure 4 also displays the associated graph views of standardized log abundances of the selected spots among analyzed gel replicates.
  • Figure 4 depicts statistical analysis of Alpha2HS-glycoprotein. Statistics were generated using the BVA module of DeCyder software and show an average of 2.43-fold lower abundance levels for Alpha2HS-glycoprotein in the cancer samples compared to normal.
  • the differentially expressed proteins included in the table all had a p-value of less than 0.05.
  • Mass spectra were recorded using the Ettan MALDI-ToF Pro instrument from GE Healthcare operating in the positive reflector mode at the following parameters: accelerating voltage 20 kV; and pulsed extraction: on (focus mass 2500). Internal and external calibration was performed using trypsin autolysis peaks at m/z 842.50, m/z 2211.104 and Pep4 mix respectively. The mass spectra were analysed using MALDI evaluation software (GE Healthcare), and protein identification was achieved with the PMF Pro-Found search engine for peptide mass fingerprints.
  • Table 2 shows proteins found to have higher or lower abundance levels in squamous cell carcinoma sera compared to normal sera. Listed are the protein identities obtained from MALDI-ToF/LC-MS analysis, gene index number, average ratio, theoretical mw and theoretical pi. All proteins listed in the table were found to have a statistically significant p-value of less than 0.05.
  • Raw serum from normal and cancer samples were electrophoretically separated and transferred to nitrocellulose before probing with an antibody to haptoglobin.
  • the results show an increase in abundance levels of haptoglobin in cancer samples compared to normal.
  • These results from western blot analysis were in good agreement with results from the 2D-DIGE data.
  • Figure 5 shows Western blot analysis of raw serum from normal and cancer samples using antibodies to actin (loading control) and haptoglobin. The results confirm the increase in abundance levels of haptoglobin in cancer samples compared to normal. This is a representative blot for analysis on all samples performed in triplicate.

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Abstract

L'invention concerne une méthode de dépistage permettant d'identifier des patients présentant un risque de développer un carcinome pulmonaire à cellules squameuses. Selon cette méthode, un échantillon de sérum prélevé sur un individu est analysé pour déterminer l'abondance d'une protéine sélectionnée dans le groupe constitué par SEQUENCE ID NO'S: 1 à 11, par rapport à une abondance témoin pour cette protéine, puis l'abondance relative obtenue est corrélée à un risque de carcinome pulmonaire à cellules squameuses. Ce diagnostic peut impliquer une variable diagnostique unique ou un ensemble de variables diagnostiques. Pour chaque biomarqueur, l'abondance relative peut être obtenue par séparation 2-DiGE et imagerie par gel. Les prélèvements sont généralement prétraités pour éliminer du sérum les protéines à haute abondance.
PCT/IE2008/000086 2007-09-11 2008-09-09 Methode permettant d'evaluer le risque de cancer chez un patient WO2009034562A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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US8044179B2 (en) 2005-09-13 2011-10-25 National Research Council Of Canada Methods and compositions for modulating tumor cell activity
US8802826B2 (en) 2009-11-24 2014-08-12 Alethia Biotherapeutics Inc. Anti-clusterin antibodies and antigen binding fragments and their use to reduce tumor volume
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US8426562B2 (en) 2005-09-13 2013-04-23 National Research Council Of Canada Methods and compositions for modulating tumor cell activity
US8802826B2 (en) 2009-11-24 2014-08-12 Alethia Biotherapeutics Inc. Anti-clusterin antibodies and antigen binding fragments and their use to reduce tumor volume
US9512211B2 (en) 2009-11-24 2016-12-06 Alethia Biotherapeutics Inc. Anti-clusterin antibodies and antigen binding fragments and their use to reduce tumor volume
US9822170B2 (en) 2012-02-22 2017-11-21 Alethia Biotherapeutics Inc. Co-use of a clusterin inhibitor with an EGFR inhibitor to treat cancer
WO2022234070A1 (fr) * 2021-05-07 2022-11-10 Csl Behring Ag Système d'expression pour la production d'une chaîne bêta de l'haptoglobine (hp) recombinante

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