Statistics for Microarray Data Analysis with R

1. Statistics for Microarray Data Analysis with R Session 8: Discrimination Class web site: http://ludwig-sun2.unil.ch/~darlene/

2. Today’s Outline • Discrimination (classification) • Some classification rules • Performance assessment • Discrimination (classification) inR • Exercises

3. cDNA gene expression data mRNA samples Data on G genes for n samples sample1 sample2 sample3 sample4 sample5 … 1 0.46 0.30 0.80 1.51 0.90 ... 2 -0.10 0.49 0.24 0.06 0.46 ... 3 0.15 0.74 0.04 0.10 0.20 ... 4 -0.45 -1.03 -0.79 -0.56 -0.32 ... 5 -0.06 1.06 1.35 1.09 -1.09 ... Genes Gene expression level of gene i in mRNA samplej = (normalized) Log(Red intensity / Green intensity)

4. Classification • Task:assign objects to classes (groups) on the basis of measurements made on the objects • Unsupervised:classes unknown, want to discover them from the data (cluster analysis) • Supervised:classes are predefined, want to use a (training or learning) set of labeled objects to form a classifier for classification of future observations

5. Discrimination • Objects (e.g. arrays) are to be classified as belonging to one of a number of predefined classes {1, 2, …, K} • Each object associated with a class label (or response) Y {1, 2, …, K} and a feature vector (vector of predictor variables) of G measurements: X = (X1, …, XG) • Aim: predict Y from X

6. Example: Tumor Classification • Reliable and precise classification essential for successful cancer treatment • Current methods for classifying human malignancies rely on a variety of morphological, clinical and molecular variables • Uncertainties in diagnosis remain; likely that existing classes are heterogeneous • Characterize molecular variations among tumors by monitoring gene expression (microarray) • Hope: that microarrays will lead to more reliable tumor classification (and therefore more appropriate treatments and better outcomes)

7. Tumor Classification Using Gene Expression Data Three main types of statistical problems associated with tumor classification: • Identification of new/unknown tumor classes using gene expression profiles (unsupervised learning – clustering) • Classification of malignancies into known classes (supervised learning – discrimination) • Identification of “marker” genes that characterize the different tumor classes (feature or variable selection)

8. Classifiers • A predictor or classifier partitions (divides) the space of gene expression profiles into K disjoint subsets, A1, ..., AK, such that for a sample with expression profile X=(X1, ...,XG)inAk the predicted class is k • Classifiers are built from a learning set (LS) L = (X1, Y1), ..., (Xn,Yn) • Classifier C built from a learning set L: C( . ,L): X {1,2, ... ,K} • Predicted class for observation X: C(X,L) = k if X is in Ak

9. Maximum likelihood discriminant rule • A maximum likelihood estimator (MLE) chooses the parameter value that makes the chance of the observations the highest • For example, if I toss a coin 10 times and I see 7 heads, the MLE for p = probability of heads is .7 • If the histograms for the individual classes are known, the maximum likelihood (ML)discriminant rule predicts the class of an observationX to be the one with the highest bar on the histogram (density scale)

10. Fisher Linear Discriminant Analysis First applied in 1935 by M. Barnard at the suggestion of R. A. Fisher (1936), Fisher linear discriminant analysis (FLDA): • finds linear combinations of the gene expression profiles X=X1,...,XG with large ratios of between-groups to within-groups sums of squares - discriminant variables; • predicts the class of an observation X by the class whose mean vector is closest to X in terms of the discriminant variables

11. Gaussian ML Discriminant Rules • For multivariate Gaussian (normal) class densities X|Y= k ~ N(k,k), the ML classifier is C(X) = argmink {(X - k) k-1(X - k)’ + log| k |} • In general, this is a quadraticrule (Quadratic discriminant analysis, or QDA) • In practice, population mean vectors k and covariance matrices k are estimated by corresponding sample quantities

12. Gaussian ML Discriminant Rules • When all class densities have the same covariance matrix, k = the discriminant rule is linear(Linear discriminant analysis,orLDA;FLDA for k = 2) • When all class densities have the same diagonal covariance matrix =diag(12… G2), the discriminant rule is again linear(Diagonal linear discriminant analysis, orDLDA)

13. Nearest Neighbor Classification • Based on a measure of distance between observations (e.g. Euclidean distance or one minus correlation) • k-nearest neighbor rule (Fix and Hodges (1951)) classifies an observation X as follows: • find thek observations in the learning set closestto X • predict the class of X by majority vote, i.e., choose the class that is most common among those k observations. • The number of neighbors kcan be chosen by cross-validation(more on this later)

14. Classification Trees • Partition the feature space into a set of rectangles, then fit a simple model in each one • Binary tree structured classifiers are constructed by repeated splits of subsets (nodes) of the measurement space X into two descendant subsets (starting with X itself) • Each terminal subset is assigned a class label; the resulting partition of X corresponds to the classifier

15. Classification Tree

16. Three Aspects of Tree Construction • Split Selection Rule • Split-stopping Rule • Class assignment Rule Different approaches to these three issues (e.g. CART: Classification And Regression Trees, Breiman et al. (1984); C4.5 and C5.0, Quinlan (1993))

17. Three Rules (CART) • Splitting: At each node, choose split maximizing decrease in impurity(e.g.misclassification error) • Split-stopping: Grow large tree, prune to obtain a sequence of subtrees, then use cross-validation to identify the subtree with lowest misclassification rate • Class assignment: For each terminal node, choose the class minimizing the resubstitution estimate of misclassification probability, given that a case falls into this node

22. Other Classifiers Include… • Support vector machines (SVMs) • Neural networks • Bayesian regression methods

23. Features • Feature selection • Automatic with trees • For DA, NN need preliminary selection • Need to account for selection when assessing performance • Missing data • Automatic imputation with trees • Otherwise, impute (or ignore)

24. Performance assessment (I) • Resubstitution estimation: error rate on the learning set • Problem: downward bias • Test set estimation: divide cases in learning set into two sets, L1 and L2; classifier built using L1, error rate computed for L2. L1 and L2 must be iid. • Problem: reduced effective sample size

25. Performance assessment (II) • V-fold cross-validation (CV) estimation: Cases in learning set randomly divided into V subsets of (nearly) equal size (for example, 5 or 10 subsets). Build classifiers leaving one set out; test set error rates computed on left out set and averaged to get overall error • Bias-variance tradeoff: smaller V can give larger bias but smaller variance • Out-of-bag estimation: covered below • ROC curves: not covered at all, I’m still learning about this!

26. Cross-validation average error

27. Performance assessment (III) • Common to do feature selection using all of the data, then CV only for model building and classification • However, usually features are unknown and the intended inference includes feature selection. Then, CV estimates as above tend to be downward biased • Features should be selected only from the learning set used to build the model (and not the entire set)

28. How NOT to estimate error • ** DON’T DO THIS ** DON’T DO THIS ** • Use the whole data set to choose which variables (features) to use in the classifier • Divide the data into (10, say) subsets for CV • Leave out a subset and build a classifier with features chosen from the whole data set • Use the classifier to predict the left out subset • Average over left out subsets to estimate error • ** DON’T DO THIS ** DON’T DO THIS **

29. Aggregating classifiers • Breiman (1996, 1998) found that gains in accuracy could be obtained by aggregating predictors built from perturbed versions of the learning set • Many possibilities for perturbing • The multiple versions of the predictor are aggregated by (weighted) voting

30. Bagging • Bagging = Bootstrap aggregating • Nonparametric Bootstrap (standard bagging): perturbed learning sets drawn at random with replacement from the learning sets; predictors built for each perturbed dataset and aggregated by plurality voting (wb = 1) • Parametric Bootstrap: perturbed learning sets are some known distribution (e.g. multivariate Gaussian) • Convex pseudo-data (Breiman 1996)

31. Aggregation By-products: Out-of-bag estimation of error rate • Out-of-bag error rate estimate: unbiased • Expect about 1/3 of cases to be left out of each bootstrap sample • Use these left out cases from each bootstrap sample as a test set • Classify these test set cases, and compare to the true class labels to get the out-of-bag estimate of the error rate

32. Boosting • Freund and Schapire (1997), Breiman (1998) • Data resampled adaptively so that the weights in the resampling are increased for those cases most often misclassified • Predictor aggregation done by weighted voting

33. Comparison of classifiers • Dudoit, Fridlyand, Speed (JASA, 2002) • FLDA • DLDA • DQDA • NN • CART • Bagging and boosting

34. Comparison study datasets • Leukemia – Golub et al. (1999) n = 72 samples, G = 3,571 genes 3 classes (B-cell ALL, T-cell ALL, AML) • Lymphoma – Alizadeh et al. (2000) n = 81 samples, G = 4,682 genes 3 classes (B-CLL, FL, DLBCL) • NCI 60 – Ross et al. (2000) N = 64 samples, p = 5,244 genes 8 classes

35. Leukemia data, 2 classes: Test set error rates;150 LS/TS runs

36. Leukemia data, 3 classes: Test set error rates;150 LS/TS runs

37. Lymphoma data, 3 classes: Test set error rates; N=150 LS/TS runs

38. NCI 60 data :Test set error rates;150 LS/TS runs

39. Results • In the main comparison, NN and DLDA had the smallest error rates, FLDA had the highest • Aggregation improved the performance of CART classifiers, the largest gains being with boosting and bagging with convex pseudo-data • Dettling and Bühlmann, improved the performance of boosting (LogitBoost)

40. Results, cont • For the lymphoma and leukemia datasets, increasing the number of genes to G=200 didn't greatly affect the performance of the various classifiers; there was an improvement for the NCI 60 dataset. • More careful selection of a small number of genes (10) improved the performance of FLDA dramatically

41. Comparison study – Discussion (I) • “Diagonal” LDA: ignoring correlation between genes helped here • Unlike classification trees and nearest neighbors, LDA is unable to take into account gene interactions • Although nearest neighbors are simple and intuitive classifiers, their main limitation is that they give very little insight into mechanisms underlying the class distinctions

42. Comparison study – Discussion (II) • Classification trees are capable of handling and revealing interactions between variables • Useful by-product of aggregated classifiers: prediction votes, variable importance statistics • Variable selection: A crude criterion such as BSS/WSS may not identify the genes that discriminate between all the classes and may not reveal interactions between genes • With larger training sets, expect improvement in performance of aggregated classifiers

43. Acknowledgements • Sandrine Dudoit • Jane Fridlyand • Yee Hwa (Jean) Yang • Terry Speed • www.bioconductor.org

44. R: discrimination • A number of R packages (libraries) contain functions to carry out discrimination, including: • MASS: lda, qda • sma: dlda • class: knn • rpart: classification and regression trees (recursive partitioning) • ipred: bagging • e1071: svm • LogitBoost: boosting

45. Exercise: discrimination • These ideas have been hard! • The handout gives a very brief guide to using some of the classification routines in R, including a little bit of cross-validation • There are many others as well, feel free to explore...