WO2009034562A2

WO2009034562A2 - A method of assessing lung squamous cell carcinoma status in an individual

Info

Publication number: WO2009034562A2
Application number: PCT/IE2008/000086
Authority: WO
Inventors: Paul Dowling; Martin Clynes; John Crown; Michael Moriarty
Original assignee: Dublin City University
Current assignee: Dublin City University
Priority date: 2007-09-11
Filing date: 2008-09-09
Publication date: 2009-03-19
Anticipated expiration: 2010-03-11
Also published as: WO2009034562A3

Abstract

The invention relates to a method of screening patients to identify patients at risk of having a lung squamous cell carcinoma. A sample of serum from the individual is assayed for the abundance of a protein selected from the group consisting of: SEQUENCE ID NO'S: 1 to 11 relative for a control abundance for that protein, and the relative abundance obtained is correlated with risk of lung SCC cancer. The diagnosis may involve a single diagnostic variable or a panel of diagnostic variables. For each biomarker, relative abundance may be obtained by means of 2-DIGE separation and gel imaging. The samples are generally pre-treated to remove highly abundant proteins from the serum.

Description

A METHOD OF ASSESSING CANCER STATUS IN AN INDIVIDUAL

Technical Field

The invention relates to a method of assessing cancer status in an individual. In particular, the invention relates to a method of screening patients to identify patients at risk of having a lung squamous cell carcinoma.

Background of the Invention

Over 1 million people are diagnosed with lung cancer each year. Parkin, D.M., Bray, F., Ferlay, J., Pisani, P., Int. J. Cancer 2001, 94, 153-156. Most lung cancer is diagnosed too late for curative treatment to be possible. Efforts at early detection and treatment have had little success to date and hence the overall prognosis remains poor. In the majority of those diagnosed with lung cancer, the disease has already metastasised at the time of diagnosis. Lung cancer comprises broadly of two groups, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). NSCLC comprises more than 80% of lung cancers and includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma, squamous carcinoma being the most common type.

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. Ries, L.A.G., Kosary, C.L., Hankey, B.F., Miller, B.A., Clegg, L.X., Edwards, B.K., SEER Cancer Statistics Review, 1999. Detection of cancer in these patients at earlier stages would increase survival rates dramatically. 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. Discovering these mechanisms and pathways will undoubtedly lead' to new ways in dealing with prevention, early detection and therapy, for example, 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.

A number of potential biomarkers have recently been identified, but currently, no satisfactory biomarkers are available to screen for lung cancer due to low specificity and sensitivity. 1. Schneider, J., Velcovsky, H.G., Morr, H., Katz, N., Neu, K., Eigenbrodt, E., Anticancer Res. 2000, 20, 5053-5058; Schneider, J., Bitterlich, N., Velcovsky, H.G., Morr, H., Katz, N., Eigenbrodt, E., Int. J. Clin. Oncol. 2002, 7, 145-151. Lung cancer biomarkers have the potential to be involved in patient screening, monitoring of cancer progression, treatment response and as a predictive factor for recurrence. Seemann, M.D., Beinert, T., Furst, H., Fink, U., Lung Cancer 1999, 26, 149-155. No single biomarker with 100% sensitivity and 100% specificity for lung cancer is likely to be discovered.

Statements of Invention

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.

According to the invention, there is provided 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.

The identity of 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. Thus, for the biomarker of SEQUENCE ID NO: 1 (Alpha-2-HS glycoprotein), 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 (control abundance value) is 1:2.43 (-2.43). Thus, if the ratio of abundance of this marker in a test patient to a control abundance value is found to -2.43 or less, then this would indicate that the patient is at risk of having lung SCC.

Likewise, for example, for the biomarker of SEQUENCE ID NO: 8 (Ras-related protein Rab-7b), 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). Thus, for example, if 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. Thus, in one 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'S: 4, 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:'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.

In another embodiment, 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. In a particularly preferred embodiment, 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. In a particularly preferred embodiment, 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. In a particularly preferred embodiment, 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.

The term "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. Thus, in the case of lung SCC 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. However, 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.

The term "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; choriocarcinoma; seminoma; embryonal carcinoma; Wilms' tumor; cervical cancer; uterine cancer; testicular tumor; lung carcinoma; small cell lung carcinoma; bladder carcinoma; epithelial carcinoma; glioma; astrocytoma; medulloblastoma; craniopharyngioma; ependymoma; pinealoma; hemangioblastoma; acoustic neuroma; oligodendroglioma; meningioma; melanoma; retinoblastoma; and leukemias. In a preferred embodiment of the invention, the cancer is a squamous cell carcinoma, especially lung squamous cell carcinoma.

In this specification, the term "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.

In most cases, 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. In other cases, 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.

In another embodiment of the invention, 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. Typically, the cancer is lung SCC. Typically, 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. Typically, over-abundance of each protein in the group of SEQUENCE ID NO's: 4, 7 and 8 correlates with early detection of cancer. Typically, 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.

In another embodiment of the invention, 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. Typically, the cancer is lung SCC. When 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. Generally, 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. Likewise, when 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. Generally, 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'.'

In this specification, the term "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.

In this specification, 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. In one aspect, 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. Suitably, more than one biomarker is employed. Ideally, 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. Typically, 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.

Generally, 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. In this context, 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. In this regard, 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. For example, 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.

Generally, 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. Ideally, the proteins in the control sample and patient sample are differentially labelled. Typically, the samples are immunodepleted to remove high- abundance proteins prior to separation of the proteins. Suitably, the samples are serum samples. In a preferred embodiment of the invention, 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). While the methods of the invention as described herein employ 2-DIGE for relative protein quantification, it will be appreciated that other techniques are applicable for relative protein quantification such as, for example ELISA and direct spectrometer analysis. Direct mass spectrometer analysis is performed using a label or label-free approach. A labelling approach involves tagging proteins or peptides and using these specific tags to later quantify the abundance levels of the proteins being quantified.

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.

Other proteins which were found to be of higher abundance in squamous cell carcinoma sera compared to normal sera included Apolipoprotein A-IV precursor, Chain F; Human Complement Component C3c, Serum amyloid A protein precursor and 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. Cancer 2005, 92, 895-905; Lee, I.N., Chen, C.H., Sheu, J.C., Lee, H.S., Huang, G.T., Chen, D.S., Yu, C.Y., Wen, C.L., Lu, F.J., Chow, L.P., Proteomics 2006, 6, 2865-2873. 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. Howard, B. A., Wang, M.Z., Campa, M.J., Corro, C, Fitzgerald, M.C., Patz, E. F. Jr. Proteomics 2003, 3, 1720-1724,- Benson, M.D., Eyanson, S., Fineberg, N.S., Cancer 1986, 57, 1783-1787. Benson et al. reported that Serum amyloid A in carcinoma of the lung was significantly elevated.

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.

Proteins found with lower abundance levels in squamous cell carcinoma sera compared to normal sera included Alpha-2-HS-glycoprotein, hemopexin precursor, proapolipoprotein, antithrombin III and SP40; 40. Alpha2-HS glycoprotein (AHSG), 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. 2000, 2, 92-99; Swallow, C.J., Partridge, E.A., Macmillan, J.C., Tajirian, T., DiGuglielmo, G.M., Hay, K., Szweras, M., Jahnen-Dechent, W., Wrana, J.L., Redston, M., Gallinger, S., Dennis, J.W., Cancer Res. 2004, 64, 6402-6409.

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. Kawakami, T., Hoshida, Y., Kanai, F., Tanaka, Y., Tateishi, K., Ikenoue, T., Obi, S., Sato, S., Teratani, T., Shiina, S., Kawabe, T., Suzuki, T., Hatano, N., Taniguchi, H., Omata, M., Proteomics 2005, 5, 4287-4295. 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. Pawlik, T.M., Hawke, D.H., Liu, Y., Krishnamurthy, S., Fritsche, H., Hunt, K.K., Kuerer, H.M., B.M.C Cancer 2006, 6, 68. 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. Gordon, J.I., Sims, H.F., Lentz, S.R., Edelstein, C, Scanu, A.M., Strauss, A. W., J. Biol. Chem. 1983, 258, 4037-4044; Huang, H.L., Stasyk, T., Morandell, S., Dieplinger, H., Falkensammer, G., Griesmacher, A., Mogg, M., Schreiber, M., Feuerstein, L, Huck, C.W., Stecher, G., Bonn, G.K., Huber, L.A., Electrophoresis 2006, 27, 1641-1650. Antithrombin III (ATH III) is a plasma protein synthesised in the liver. Normal plasma levels are 115 to 160mg/L. Reduced serum ATH III levels together with elevated Fibrinogen, vWF, and D-dimer were found to be associated with poor survival outcome in ovarian cancer. The author suggests that fibrinogen, vWF, ATH III, and D-dimer levels be used together as prognostic markers for disease outcome especially in patients with advanced ovarian cancer within 36 months of disease. Koh, S. C, Khalil, R., Lim, F.K., Ilancheran, A., Choolani, M., Clin. Appl. Thromb. Hemost. 2006, 12, 3-8.

SP-40,40 (serum protein 40 kD,40 kD) or 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. Rodriguez-Pineiro, A.M., de Ia Cadena, M.P., Lopez-Saco, A., Rodriguez-Berrocal, F.J., MoI. Cell Proteomics 2006, 5, 1647-1657. Zhang reported that a loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma. Zhang, L.Y., Ying, W.T., Mao, Y.S., He, H.Z., Liu, Y., Wang, H.X., Liu, F., Wang, K., Zhang, D.C., Wang, Y., Wu, M., Qian, X.H., Zhao, X.H., World J. Gastroenterol. 2003, 9, 650-654.

Brief Description of the Figures The invention will be more clearly understood form the following description of some embodiment thereof, given by way of example only, with reference to the following Figures in which:

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; and

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);

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.

Detailed Description of the Invention Materials and Methods Serum Samples

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 ⁰C until ready for use. The time from collection to frozen storage was no more than 3 hrs.

Removal of High Abundance Proteins from Serum Samples

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.

DIGE Labelling

Samples were labelled with N-hydroxy succinimidyl ester-derivatives of the cyanine dyes Cy2, Cy3, and Cy5 following standard protocol Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., Pognan, F., Hawkins, E., Currie, I., Davison, M., Proteomics. 2001, 1, 377-96. Typically, 50 μg of lysate was minimally labelled with 200 pmol of either Cy3 or Cy5 for comparison on the same 2D gel. Labelling reactions were performed on ice in the dark for 30 mins and then quenched with a 50-fold molar excess of free lysine to dye for 10 mins on ice. A pool of all samples was also prepared and labelled with Cy2 to be used as a standard on all gels to aid image matching and cross-gel statistical analysis. The Cy3 and Cy5 labelling reactions (50 μg of each) from each lysate were mixed and run on the same gels with an equal amount (50 μg) of Cy2-labeled standard.

Protein Separation by 2D Gel Electrophoresis and Gel Imaging

Immobilized 24cm linear pH gradient (IPG) strips, pH 4-7, were rehydrated in rehydration buffer (7M Urea, 2M Thiourea, 4% CHAPS₅ 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. Equilibrated IPG strips were transferred onto 18x20- cm 12.5% uniform polyacrylamide gels poured between low fluorescence glass plates. Strips were overlaid with 0.5% (w/v) low melting point agarose in running buffer containing bromphenol blue. Gels were run using the Ettan DaIt 6 apparatus (GE Healthcare) at 2.5 W/gel for 30 mins then 100 W in total at 10°C until the dye front had run off the bottom of the gels. All the images were collected on a Typhoon 9400 Variable Mode Imager (GE Healthcare). Statistics and quantitation of protein expression were carried out in Decyder software (GE Healthcare).

Spot digestion and Mass Spec analysis

Excision of protein spots, trypsin digestion, and protein identification by mass spectrometric analysis using an Ettan MALDI-ToF Pro instrument from GE Healthcare was performed according to an established methodology. Preparative gels containing 300μg of protein were fixed in 30% (v/v) methanol, 7.5% (v/v) acetic acid overnight and washed in water, and total protein was detected by post-staining with SyproRuby dye (Molecular Probes) for 3 hrs at room temperature or Colloidal Coomassie (Sigma) for 2 hrs at room temperature. Excess dye was removed by appropriate destaining/washing methods. 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. Following shrinkage of the gel plugs, acetonitrile was removed and the protein-containing gel pieces were rehydrated for 5 mins with a minimal volume of 100 mM ammonium bicarbonate. An equal volume of acetonitrile was added and after 15 mins of incubation the solution was removed from the gel plugs and the samples then dried down for 30 mins using a vacuum centrifuge. Individual gel pieces were then rehydrated in digestion buffer (12.5ng trypsin per μl of 10% Acetronitrile 4OmM Ammonium Bicarbonate) to cover the gel pieces. More digestion buffer was added if all the initial volume had been absorbed by the gel pieces. Exhaustive digestion was carried out overnight at 37 ⁰C. After digestion, the samples were centrifuged at 12,000 g for 10 mins using a bench top centrifuge. The supernatant was carefully removed from each sample and placed into clean and silconized plastic tubes. Samples were stored at -7O⁰C until analysed by MS. For spectrometric analysis, mixtures of tryptic peptides from individual samples were desalted using Millipore C- 18 Zip-Tips (Millipore) and eluted onto the sample plate with the matrix solution [5 mg/mL a-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0. I % trifluoroacetic acid (v/v)]. 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.

Lower abundant proteins not identified by MALDI-ToF were digested with tryspin and analysed by one-dimensional LC-MS using the Ettan™ MDLC system (GE Healthcare) in high-throughput configuration directly connected to a Finnigan^1M LTQ™ (Thermo Electron). Samples were concentrated and desalted on RPC trap columns (Zorbax™ 300SB C18, 0.3mmx5mm, Agilent Technologies, and the peptides were separated on a nano-RPC column (Zorbax 300SB C 18, 0.075mm x 100mm, Agilent Technologies) using a linear acetonitrile gradient from 0% to 65% ACN (Sigma) over 60 minutes. 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 SEQUEST™.

Statistical Analysis

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.

1-D Gel and Immunoblot analysis

8ug of protein was loaded onto a 12% NuPAGE Bis-Tris Gels (Invitrogen) and electrophoretically separated using a MOS/SDS buffer. The samples were run according to the manufacturer's instructions and were stained using colloidal Coomassie Blue (Sigma). Electrophoretic transfer of proteins to Hybond-ECL nitrocellulose membranes (GE Healthcare) was carried out using a Bio-Rad Transblot SD cell (Bio-Rad). Proteins were transferred for 50 min at 0.34 mA. Efficiency of transfer was evaluated using Ponceau-S-Red staining of nitrocellulose membranes, followed by destaining in phosphate buffered saline (PBS; 50 mM sodium phosphate, 0.9% (w/v) NaCl, pH 7.4). Membranes were blocked for 1 h in 5% (w/v) fat-free milk powder in PBS containing 0.5% Tween-20. Membranes were then incubated overnight at 4°C with the primary antibody, serum albumin (ablO241); haptoglobin (abl4248) and actin (abl801) (Abeam). 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).

Results

Clinical Specimens

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.

Table 1

Specimen

No. Diagnosis Gender Type Size Grade Smoking Metastasis

1 56 Male Sq cell Ca 5.4 3 Yes none

2 65 Male Sq cell Ca 6.6 3 Yes none

Yes, to peribronchial

3 64 Male Sq cell Ca 3.7 3 Yes lymph nodes

4 74 Male Sq cell Ca 2.1 2 Yes none

Yes 3 mediastinal

5 62 Male Sq cell Ca 7 3 Yes lymph nodes

6 58 Male Sq cell Ca 2.4 2 Yes none

Yes, to peribronchial

7 60 Male Sq cell Ca 6 2 Yes lymph nodes

8 76 Male Sq cell Ca 2.5 3 Yes none Immunodepletion of High abundant Serum Proteins

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. 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.

DIGE Analysis of the Immunodepleted Serum Proteome

Fifty micrograms of protein from each sample was labelled with Cy2, Cy3, or Cy5. Eight individual samples of squamous cell lung carcinoma serum and eight samples of normal serum were labelled with Cy3 and Cy5 respectively. All sixteen samples employed in the experiment were used in the Cy2 labelled internal pooled standard. Samples were combined and separated by 2D gel electrophoresis. For DeCyder image analysis, the differential in-gei analysis mode of DeCyder was first used to merge the Cy2, Cy3, and Cy5 images for each gel and to detect spot boundaries for the calculation of normalized spot volumes/protein abundance. At this stage, features resulting from non-protein sources, namely dust particles and scratches, were filtered out. The analysis was used to rapidly calculate abundance differences between samples run on the same gel. The biological variation analysis mode of DeCyder was then used to match all pairwise image comparisons from difference in-gel analysis for a comparative cross-gel statistical analysis. Operator intervention was required at this point to set landmarks on gels for more accurate cross-gel image superimposition. Comparison of normalized Cy3 and Cy5 spot volumes with the corresponding Cy2 standard spot volumes within each gel gave a standardized abundance. This value was compared across atl gels for each matched spot and a statistical analysis was performed. This is depicted in Figure 2 which shows 2D- 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 to generate a protein expression map (PEM).

3D Images, Statistics and Mass Spectrometry

Examples for the evaluation by DeCyder of alterations in spot intensities using the DIGE system are displayed in Figure 3. 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.

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. Lower abundant proteins not identified by MALDI-ToF Samples were digested with tryspin and analysed by one- dimensional LC-MS using the Ettan™MDLC system (GE Healthcare) in high-throughput configuration directly connected to a Finnigan™ LTQ™ (Thermo Electron). Proteins found to have higher abundance levels in squamous cell carcinoma sera compared to normal sera included Apolipoprotein A-IV precursor, Chain F; Human Complement Component C3c, Haptoglobin, Serum amyloid A protein precursor and Ras-related protein Rab-7b while proteins found to have lower abundance levels included Alpha-2- HS-glycoprotein, hemopexin precursor, proapolipoprotein, antithrombin III and SP40. 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.

Western Blot analysis

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.

The invention is not limited to the embodiments hereinbefore described which may be varied in both construction and detail without departing from the spirit of the invention. Table 2

Spot ID SEQ ID Gene index Average Method of

Number Number Protein Identification number ratio pi MW(Da) Identification

1 1 Alρha-2-HS-glycoprotein gi\30851645\ -2.43 5.4 40110 MALDI-ToF

2 2 Apolipoprotein A-IV precursor gill 14006| 3.04 5.3 45350 MALDI-ToF

(Apo-AIV)

3 3 Chain F; Human Complement gi|78101274| 1.58 4.8 40210 MALDI-ToF

Component C3c

4 4 haptoglobin gi|1212947| 4.42 6.3 38950 MALDI-ToF

5 5 haptoglobin gi[3337390[ 3.9 6.1 38730 MALDI-ToF

6 - haptoglobin gi|3337390| 3.81 6.1 38730 MALDI-ToF

7 - haptoglobin gi|3337390| 3.58 6.1 38730 MALDI-ToF

8 6 hemopexin precursor gi|386789| -1.3 6.6 52270 MALDI-ToF

9 7 Serum amyloid A protein precursor gi| 134167| 6.35 6.2 13532 LC-MS/MS

O

10 8 Ras-related protein Rab-7b gi|50401122| 7.81 6.3 22511 LC-MS/MS

1

11 proapolipoprotein gi|178775| -1.51 5.4 28940 MALDI-ToF

12 9 proapolipoprotein gi|178775| -1.53 5.4 28940 MALDI-ToF

13 10 antithrombin gi|37682619| -1.51 6.3 53040 LC-MS/MS

14 11 'SP40; 40' gi|338305| -1.3 5.7 37000 LC-MS/MS

References

Barcellos-Hoff, M.H., Ewan, K.B., Breast Cancer Res. (2000), 2, 92-99;

Benson, M.D., Eyanson, S., Fineberg, N.S., Cancer (1986), 57, 1783-1787

Bjorge, L., Hakulinen, J., Vintermyr, O.K., Jarva, H., Jensen, T.S., Iversen, O.E., Meri, S., Br. J. Cancer (2005), 92, 895-905

Chromy, B.A., Gonzales, A.D., Perkins, J., Choi, M.W., Corzett, M.H., Chang, B.C., Corzett, C.H., McCutchen-Maloney, S.L., J. Proteome Res. (2004), 3, 1120-1127.

Dobryszycka, W., Eur. J. Clin. Chem. Clin. Biochem. (1997), 35, 647-654

Gordon, J.I., Sims, H.F., Lentz, S.R., Edelstein, C, Scanu, A.M., Strauss, A. W., J. Biol. Chem. (1983), 258, 4037-4044

Howard, B.A., Wang, M.Z., Campa, MJ., Corro, C, Fitzgerald, M.C., Patz, E. F. Jr. Proteomics (2003), 3, 1720-1724

Huang, H.L., Stasyk, T., Morandell, S., Dieplinger, H., Falkensammer, G., Griesmacher, A., Mogg, M., Schreiber, M., Feuerstein, I., Huck, C.W., Stecher, G., Bonn, G.K., Huber, L.A., Electrophoresis (2006), 27, 1641-1650

Kawakami, T., Hoshida, Y., Kanai, F., Tanaka, Y., Tateishi, K., Ikenoue, T., Obi, S., Sato, S., Teratani, T., Shiina, S., Kawabe, T., Suzuki, T., Hatano, N., Taniguchi, H., Omata, M., Proteomics (2005), 5, 4287-4295

Koh, S. C, Khalil, R., him, F.K., Ilancheran, A., Choolani, M., CHn. Appl. Thromb. Hemost. (2006), 12, 3-8

Lee, I.N., Chen, C.H., Sheu, J.C., Lee, H.S., Huang, G.T., Chen, D.S., Yu, C.Y., Wen, C.L., Lu, FJ., Chow, L.P., Proteomics (2006), 6, 2865-2873

Liau CY, Chang TM, Pan JP, Chen WL, Mao SJ. J Chromatogr B Analyt Technol Biomed Life Sci. (2003) 790, 209-16

Oikawa O, Higuchi T, Yamazaki T, Yamamoto C, Fukuda N, Matsumoto K. Clin Exp Nephrol. (2007), 11, 304-8

Parkin, D.M., Bray, F., Ferlay, J., Pisani, P., Int. J. Cancer (2001), 94, 153-156

Pawlik, T.M., Hawke, D.H., Liu, Y., Krishnamurthy, S., Fritsche, H., Hunt, K.K., Kuerer, H.M., BMC Cancer (2006), 6, 68 Ries, L.A.G., Kosary, C.L., Hankey, B.F., Miller, B.A., Clegg, L.X., Edwards, B.K., SEER Cancer Statistics Review, 1999

Rodriguez-Pineiro, A.M., de Ia Cadena, M.P., Lopez-Saco, A., Rodriguez-Berrocal, F.J., MoI. Cell Proteomics (2006), 5, 1647-1657.

Schneider, J., Velcovsky, H.G., Morr, H., Katz, N., Neu, K., Eigenbrodt, E., Anticancer Res. (2000), 20, 5053-5058;

Schneider, J., Bitterlich, N., Velcovsky, H.G., Morr, H., Katz, N., Eigenbrodt, E., Int. J. Clin. Oncol. (2002), 7, 145-151

Seemann, M.D., Beinert, T., Furst, H., Fink, U., Lung Cancer (1999), 26, 149-155

Swallow, C.J., Partridge, E.A., Macmillan, J.C., Tajirian, T., DiGuglielmo, G.M., Hay, K., Szweras, M., Jahnen-Dechent, W., Wrana, J.L., Redston, M., Gallinger, S., Dennis, J. W., Cancer Res. (2004), 64, 6402-6409

Takeuchi, T., Tomida, S., Yatabe, Y., Kosaka, T., Osada, H., Yanagisawa, K., Mitsudomi, T., Takahashi, T., J. Clin. Oncol. (2006), 24, 1679-1688.

Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., Pognan, F., Hawkins, E., Currie, I., Davison, M., Proteomics. (2001), 1, 377-96

Yang, M., Chen, T., Han, C, Li, N., Wan, T., Cao, X., Biochem. Biophys. Res. Commun. (2004), 318, 792-799.

Yu, K.H., Rustgi, A.K., Blair, I.A., J. Proteome Res. (2005), 4, 1742-1751.

Zhang, L.Y., Ying, W.T., Mao, Y.S., He, H.Z, Liu, Y., Wang, H.X., Liu, F., Wang, K., Zhang, D.C., Wang, Y., Wu, M., Qian, X.H., Zhao, X.H., World! Gastroenterol. (2003), 9, 650-654.

Claims

1. A method for screening an individual for risk of lung SCC comprising a step of assaying a serum sample from the individual for the abundance of a protein selected from the group consisting of SEQUENCE ID NO'S 1 to I l relative to a control abundance for that protein, and correlating the relative abundance thus obtained for the protein with lung SCC cancer status.

2. A method as claimed in Claim 1, in which the relative abundance of the proteins of SEQUENCE ID NO'S 7 and 8 is determined.

3. A method as claimed in Claim 1, in which the relative abundance of a protein selected from the group consisting of SEQUENCE ID NO's 4, 7 and 8 is determined.

4. A method as claimed in Claim 1, in which the relative abundance of a protein selected from the group consisting of SEQUENCE ID NO's 4,. 5, 7 and 8 is determined.

5. A method as claimed in Claim 1, in which the relative abundance of a panel of at least five proteins selected from the group consisting of the proteins of SEQUENCE ID NO'S: 1 to 11 is determined.

6. A method as claimed in Claim 1, in which the relative abundance of a panel of proteins consisting of the proteins of SEQUENCE ID NO'S: 1 to 11 is determined.

7. A method as claimed in any preceding Claim in which the individual is a person at-risk for lung cancer.

8. A method for the early detection of lung SCC cancer, comprising a step of assaying a serum sample from the individual for the relative abundance of a protein selected from the group consisting of SEQUENCE ID NO'S: 1 to 11, in which 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, and under-abundance of a protein selected from the group of SEQUENCE ID NO's: 1, 6, 9 and 10 correlates with early detection of cancer.

9. A method of monitoring the effectiveness of a treatment for a lung SCC cancer, in which changes in the relative abundance of a protein selected from the group consisting of SEQUENCE ID NO's: 1 to 11 is correlated with effectiveness of the treatment.

10. A method as claimed in Claim 9 in which, when the protein being assayed is a protein selected from the group of SEQUENCE ID NO's: 2, 3, 4, 5, 7 and 8, a decrease in abundance of the protein is indicative of effectiveness of the treatment.

11. A method as claimed in Claim 9 in which, when the protein being assayed is a protein selected from the group of SEQUENCE ID NO's: 1, 6, 9, and 10, a decrease in abundance of the protein is generally indicative of effectiveness of the treatment.

12. A method as claimed in any of Claims 9 to 11 in which the method involves an initial assay to determine the starting relative abundance level of the or each biomarker, and then further periodic measurements of the relative abundance level of the or biomarker during and/or after the course of the treatment to monitor relative abundance levels of the or each marker.

13. A method as claimed in any preceding Claim in which the relative abundance of a protein is determined using 2-DIGE and gel imaging.

14. A method as claimed in any preceding Claim in which the serum sample is pre- treated to deplete the serum of high-abundance proteins.

15. A method as claimed in Claim 14 in which the pre-treatment is immunodepletion.