title: Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease.
abstract: BACKGROUND
  Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS).
  
  
  METHODS
  Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the 'GenePattern' suite of computational tools (Broad Institute, MIT, USA). Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR) test statistic was used to identify and rank differentially expressed marker peaks. The k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, 'leave-one-out' cross-validation method.
  
  
  RESULTS
  73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour verses adjacent normal mucosa tissue (P < or = 0.01, false discovery rate < or = 0.05). Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100% sensitivity and specificity (95% confidence interval: 67.9-99.2%). Supervised prediction using 'leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours (P = < 0.001; receiver-operator characteristics - ROC - error, 0.171); disease recurrence was correctly predicted in 5/6 cases and disease-free survival (median follow-up time, 25 months) was correctly predicted in 22/23 cases (P = < 0.001; ROC error, 0.105). A similar analysis of normal mucosa spectra correctly predicted 11/14 patients with, and 15/19 patients without lymph node involvement (P = 0.001; ROC error, 0.212).
  
  
  CONCLUSIONS
  Protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value in studies aimed at improved molecular classification of this disease. Further studies, with longer follow-up times and larger patient cohorts, that would permit independent validation of supervised classification models, would be required to confirm the predictive value of tumour spectra for disease recurrence/patient survival.
authors.affiliation: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ UK.
authors.forename: Christopher C L
authors.initials: CC
authors.lastname: Liao
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ward
authors.forename: Simon
authors.initials: S
authors.lastname: Marsh
authors.forename: Tan
authors.initials: T
authors.lastname: Arulampalam
authors.forename: John D
authors.initials: JD
authors.lastname: Norton
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000465:Algorithms; D014408:Biomarkers, Tumor / Q000378:metabolism*; D016022:Case-Control Studies; D016000:Cluster Analysis; D003106:Colon / Q000378:metabolism* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000145:classification / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D040081:Protein Array Analysis; D040901:Proteomics; D012007:Rectum / Q000378:metabolism* / Q000473:pathology; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D014408:Biomarkers, Tumor