Biomarker Uncertainty Principle: A molecular signature can be either parsimonious or predictive, but not both. — FE Harrell, 2009

We have more data than ever, more good data than ever, a lower proportion of data that are good, a lack of strategic thinking about what data are needed to answer questions of interest, sub-optimal analysis of data, and an occasional tendency to do research that should not be done. — FE Harrell, 2015

20.1 Background

High-dimensional data are of at least three major types:

Data collected on hundreds of thousands or millions of subjects with a diverse array of variables

Time series of biologic signals collected every few milliseconds

Extremely large data arrays where almost all the variables are of one type (the main topic here) The last data class includes such areas as

functional imaging

gene microarray

SNPs for genome-wide association studies

RNA seq

exome sequencing

mass spectrometry

The research yield of analysis of such data has been disappointing to date, for many reasons such as:

Biology is complex

Use of non-reproducible research methodology

Use of unproven statistical methods

Multiple comparison problems and double dipping^{1}

Failure to demonstrate value of information over and above the information provided by routine clinical variables, blood chemistry, and other standard medical tests

Inadequate sample size for the complexity of the analytic task

Overstatement of results due to searching for “winning” markers without understanding bias, regression to the mean (Section 14.8), and overfitting

^{1} Double dipping refers to using the same data to test a hypothesis that was formulated from a confession from the tortured dataset.

Regarding double dipping/multiplicity a beautiful example is the dead salmon fMRI study by Bennett et al. in which the standard method of analyzing fMRI data by voxels was shown to find brain activation regions even in the dead brain (see prefrontal.org/files/posters/Bennett-Salmon-2009.pdf).

Wired, 2009-09-18.

Unfortunately, a large number of papers in genomics and imaging research have pretended that feature selection has no randomness, and have validated predictions in the same dataset used to find the signals, without informing the bootstrap or cross-validation procedure about the data mining used to derive the model so that the resampling procedure could repeat all feature selection steps afresh for each resample (Ambroise & McLachlan (2002)). Only by re-running all data analysis steps that utilized \(Y\) for each resample can an unbiased estimate of future model performance be obtained.

20.2 Global Analytic Approaches

Let \(X_{1}, X_{2}, \ldots, X_{p}\) denote an array of candidate features (gene expressions, SNPs, brain activation, etc.) and \(Y\) the response, diagnosis, or patient outcome being predicted.

20.2.1 One-at-a-Time Feature Screening

OaaT feature screening involves choosing a measure of association such as Pearson’s \(\chi^2\) statistic^{2}, computing all \(p\) association measures against \(Y\), and choosing those \(X\) features whose association measure exceeds a threshold. This is by far the most popular approach to feature discovery (and often to prediction, unfortunately) in genomics and functional imaging. It is demonstrably the worst approach in terms of the reliability of the “winning” and “losing” feature selection and because it results in poor predictive ability. The problems are due to multiple comparison problems, bias, typically high false negative rates, and to the fact that most features “travel in packs”, i.e., act in networks rather than individually. As if OaaT could not get any worse, many practitioners create “risk scores” by simply adding together individual regression coefficients computed from individual “winning” \(X\) features without regard to the correlation structure of the \(X\)s. This gives far too much weight to the selected \(X\)s that are co-linear with each other.

^{2} Oddly, many practitioners of OaaT choose the more conservative and much slower to compute Fisher’s exact test.

There is a false belief that preventing a high false discovery rate solves the problems of OaaT. Most researchers fail to consider, among other things, the high false negative rate caused by their design and sample size.

OaaT results in highly overestimated effect sizes for winners, due to double dipping.

Multiplicity Corrections

In the belief that false positive discoveries are less desirable than missed discoveries, researchers employ multiplicity corrections to \(P\)-values arising from testing a large number of associations with \(Y\). The most conservative approach uses the addition or Bonferroni inequality to control the family-wise error risk which is the probability of getting any false positive association when the global null hypothesis that there are no true associations with \(Y\) holds. This can be carried out by testing the \(p\) associations against \(\frac{\alpha}{p}\) where \(\alpha=0.05\) for example. A less conservative approach uses the false discovery rate (FDR), better labeled the false discovery risk or false discovery probability. Here one controls the fraction of false positive associations. For example one sets the critical value for association statistics to a less severe threshold than dictated by Bonferroni’s inequality, by allowing \(\frac{1}{10}^{\mathrm th}\) of the claimed associations (those with the smallest \(P\)-values) to be false positives.

It is an irony that the attempt to control for multiplicity has led not only to missed discoveries and abandonment of whole research areas but has resulted in increased bias in the “discovered” features’ effect estimates. When using stepwise regression, the bias in regression coeffients comes not from multiplicity problems arising when experimentwise \(\alpha=0.05\) is used throughout but from using an \(\alpha\) cutoff \(< \frac{1}{2}\) for selecting variables. By selecting variables on the basis of small \(P\)-values, many of the selected variables were selected because their effects were overestimated, then regression to the mean sets in.

20.2.2 Forward Stepwise Variable Selection

Forward stepwise variable selection that does an initial screening of all the features, adds the most significant one, then attempts to add the next most significant feature, after adjusting for the first one, and so on. This approach is unreliable. It is only better than OaaT in two ways: (1) a feature that did not meet the threshold for the association with \(Y\) without adjusting for other features may become stronger after the selection of other features at earlier steps, and (2) the resulting risk score accounts for co-linearities of selected features. On the downside, co-linearities make feature selection almost randomly choose features from correlated sets of \(X\)s, and tiny changes in the dataset will result in different selected features.

20.2.3 Ranking and Selection

Feature discovery is really a ranking and selection problem. But ranking and selection methods are almost never used. An example bootstrap analysis on simulated data is presented later. This involves sampling with replacement from the combined \((X, Y)\) dataset and recomputing for each bootstrap repetition the \(p\) association measures for the \(p\) candidate \(X\)s. The \(p\) association measures are ranked. The ranking of each feature across bootstrap resamples is tracked, and a 0.95 confidence interval for the rank is derived by computing the 0.025 and 0.975 quantiles of the estimated ranks.

This approach puts OaaT in an honest context that fully admits the true difficulty of the task, including the high cost of the false negative rate (low power). Suppose that features are ranked so that a low ranking means weak estimated association and a high ranking means strong association. If one were to consider features to have “passed the screen” if their lower 0.95 confidence limit for their rank exceeds a threshold, and only dismisses features from further consideration if the upper confidence limit for their rank falls below a threshold, there would correctly be a large middle ground where features are not declared either winners or losers, and the researcher would be able to only make conclusions that are supported by the data. Readers of the classic Absence of Evidence paper Altman & Bland (1995) will recognize that this ranking approach solves the problem.

An example of bootstrap confidence intervals where multivariable modeling was used rather than OaaT association measures is in the course notes for Regression Modeling Strategies, Section 5.4. An example where the bootstrap is used in the context of OaaT is below.

20.2.4 Joint modeling of All Features Simultaneously using Shrinkage

This approach uses multivariable regression models along with penalized maximum likelihood estimation, random effects / hierarchical modeling, or skeptical Bayesian prior distributions for adjusted effects of all \(p\)\(X\) features simultaneously. Feature effects (e.g., log odds ratios) are discounted so as to prevent overfitting/over-interpretation and to allow these effects to be trusted out of context. The result is a high-dimensional regression model that is likely to provide well-calibrated absolute risk estimates and near-optimum predictive ability. Some of the penalization methods used are

lasso: a penalty on the absolute value of regression coefficient that highly favors zero as an estimate. This results in a large number of estimated coefficients being exactly zero, i.e., results in feature selection. The resulting parsimony may be illusory: bootstrap repetition may expose the list of “selected” features to be highly unstable.

ridge regression (penalty function that is quadratic in the regression coefficients): does not result in a parsimoneous model but is likely to have the highest predictive value

elastic net: a combination of lasso and quadratic penalty that has some parsimony but has better predictive ability than the lasso. The difficulty is simultaneously choosing two penalty parameters (one for absolute value of \(\beta\)s, one for their sum of squares).

20.2.5 Random Forest

This is an approach that solves some of the incredible instability and low predictive power of individual regression trees. The basic idea of random forest is that one fits a regression tree using recursive partitioning (CART) on multiple random samples of candidate features. Multiple trees are combined. The result is no longer a tree; it is an uninterpretable black box. But in a way it automatically incorporates shrinkage and is often competitive with other methods in predictive ability.

There is evidence that minimal-assumption methods such as random forests are “data hungry”, requiring as many as 200 events per candidate variable for their apparent predictive discrimination to not decline when evaluated in a new sample van der Ploeg et al. (2014).

20.2.6 Data Reduction Followed by Traditional Regression Modeling

This approach uses techniques such as principle component analysis (PCA) whereby a large number of candidate \(X\)s are reduced to a few summary scores. PCA is based on additively combining features so as to maximize the variation of the whole set of features that is explained by the summary score. A small number of PCA scores are then put into an ordinary regression model (e.g., binary logistic model) to predict \(Y\). The result is sometimes satisfactory though no easier to interpret than shrinkage methods.

20.2.7 Model Approximation

Also called pre-conditioning, this method is general-purpose and promising (Paul et al. (2008),Shen et al. (2004),Ambler et al. (2002)). One takes a well-performing black box (e.g., random forest or full penalized regression with \(p\) features) that generates predicted responses \(\hat{Y}\) and incorporates the right amount of shrinkage to make the predictions well-calibrated. Then try to find a smaller set of \(X\)s that can represent \(\hat{Y}\) with high accuracy (e.g., \(R^{2} \geq 0.9\)). Forward stepwise variable selection may be used for this purpose^[Here one is developing a mechanistic prediction where the true \(R^{2}\) is 1.0.}. This sub-model is an approximation to the “gold-standard” full black box. The ability to find a well-performing approximation is a test of whether the predictive signal is parsimoneous. If one requires 500 \(X\)s to achieve an \(R^{2} \geq 0.9\) in predicting the gold-standard predictions \(\hat{Y}\), then it is not possible to be parsimoneous and predictive.

A major advantage of model approximation is that if the original complex model was well calibrated by using appropriate shrinkage, the smaller approximate model inherits that shrinkage.

20.2.8 Incorporating Biology into High-Dimensional Regression Models

This approach is likely to result in the most trustworthy discoveries as well as the best predictive accuracy, if existing biological knowledge is adequate for specification of model structure. This is a structured shrinkage method where pathway (e.g., gene pathway) information is inserted directly in the model. One may encode multiple paths into a simultaneous regression model such that genes are “connected” to other genes in the same pathway. This allows an entire path to be emphasized or de-emphasized.

20.3 Simulated Examples

Monte Carlo simulation, when done in a realistic fashion, has the great advantage that one knows the truth, i.e., the true model and values of model parameters from which the artificial population was simulated. Then any derived model or estimates can be compared to the known truth. Also with simulation, one can easily change the sample size being simulated so that the effect of sample size can be studied and an adequate sample size that leads to reliable results can be computed. One can also easily change the dimensionality of the features.

20.3.1 Simulation To Understand Needed Sample Sizes

One of the most common association measures used in genomic studies is the odds ratio. As shown in Section 6.8 and Figure 6.1 , the odds ratio (OR) is very difficult to estimate when the outcome is rare or when a binary predictive feature has a prevalence far from \(\frac{1}{2}\). That is for the case when only a single pre-specified is estimated. When screening multiple features for interesting associations, one is effectively estimating a large number of ORs, and in order to make correct decisions about which features are promising and which aren’t, one must be able to control the margins of error of the entire set of OR estimates.

In the following simulation consider varying sample size \(n\) and number of candidate features \(p\). We simulate \(p\) binary features with known true ORs against the diagnosis or outcome \(Y\). The true unknown ORs are assumed to have a normal\((\mu=0,\sigma=0.25)\) distribution. We want to judge the ability to jointly estimate \(p\) associations and to rank order features by observed associations. The analysis that is simulated does not examine multiple \(X\)s simultaneously, so we save time by simulating just the total numbers of zeros and ones for each \(X\), given \(Y\).

Code

# For a vector of n binary outcomes y, simulates p binary features# x that have a p-vector of fixed prevalences | y=0 of prev and are connected# to y by a p-vector of true population odds ratios ors.# Estimates the p odds ratios against the simulated outcomes and# returns a data frame summarizing the information## Note: the odds ratio for x predicting y is the same as the odds ratio# for y predicting x. y is simulated first so that all features will# be analyzed against the same outcomessim <-function(y, prev, or) { n <-length(y) p <-length(prev)if(p !=length(or)) stop('prev and or must have the same length')# prev = Pr(x=1 | y=0); let the odds for this be oprev = prev / (1-prev)# or = odds(x=1 | y=1) / oprev# Pr(x=1 | y=1) = oprev / ((1 / or) + oprev) oprev <- prev / (1- prev) p1 <- oprev / ((1/ or) + oprev) n0 <-sum(y ==0) n1 <-sum(y ==1)# For n0 observations sample x so that Pr(x=0 | y=0) = prev nxy0 <-rbinom(p, size=n0, prob=prev) nxy1 <-rbinom(p, size=n1, prob=p1)# Compute p sample odds ratios sor <- (n0 - nxy0) * nxy1 / (nxy0 * (n1 - nxy1)) g <-function(x) ifelse(x >=1, x, 1/ x) r1 <-rank(sor)[which.max(or) ] / p r2 <-rank(or) [which.max(sor)] / pdata.frame(prev, or, nx=nxy0 / n0, obsprev0=(nxy0 + nxy1) / n,obsprev=nxy1 / (nxy0 + nxy1), obsor=sor, n=n,N =paste('n', n, sep=':'),Features=paste('Features', p, sep=':'),mmoe =quantile(g(sor / or), 0.90, na.rm=TRUE),obsranktrue=r1, truerankobs=r2,rho=cor(sor, or, method='spearman', use='pair'))}

Code

U <-NULLset.seed(1)for(n inc(50, 100, 250, 500, 1000, 2000)) {for(p inc(10, 50, 500, 1000, 2000)) {for(yprev inc(.1, .3)) { y <-rbinom(n, 1, yprev) prev <-runif(p, .05, .5) or <-exp(rnorm(p, 0, .25)) u <-cbind(sim(y, prev, or),Yprev=paste('Prevalence of Outcome', yprev, sep=':')) U <-rbind(U, u) } }}

In the plots below red lines show the line of identity.

The last two figures use a log scale for the \(y\)-axis (estimated odds ratios), so the errors in estimating the odds ratios are quite severe. For a sample size of \(n=50\) one cannot even estimate a single pre-specified odds ratio. To be able to accurately assess 10 ORs (10 candidate features) requires about \(n=1000\). To assess 2000 features, a sample size of \(n=2000\) seems adequate only for the very smallest and very largest true ORs.

The plot below summarizes the previous plots by computing the 0.9 quantile of the multiplicative margin of error (fold change) over the whole set of estimated odds ratios, ignoring direction.

Code

ggplot(U, aes(x=n, y=mmoe)) +geom_point() +facet_grid(Features ~ Yprev) +geom_hline(aes(yintercept=1.5, col='red')) +ylim(1, 10) +ylab('0.9 Quantile of Multiplicative Margin of Error in OR Across Features')

Horizontal red lines depict a multiplicative margin of error (MMOE) of 1.5 which may be considered the minimally acceptable error in estimating odds ratios. This was largely achieved with \(n=1000\) for a low-incidence \(Y\), and \(n=500\) for a moderate-incidence \(Y\).

Another way to summarize the results is to compute the Spearman rank correlation between estimated and true underlying odds ratios over the entire set of estimates.

Code

ggplot(U, aes(x=n, y=rho)) +geom_point() +facet_grid(Features ~ Yprev) +ylab(expression(paste('Spearman ', rho, ' Rank Correlation Between ', OR, ' and ', hat(OR), ' Across Features')))

One may desire a correlation with the truth of say 0.8 and can solve for the needed sample size.

20.3.2 Bootstrap Analysis for One Simulated Dataset

Suppose that one wanted to test \(p\) candidate features and select the most “significant” one for a validation study. How likely is the apparently “best” one to be truly the best? What is a confidence interval for the rank of this “winner”? How much bias in the OR does the selection process create? The bootstrap can be used to answer all of these questions without needing to assume anything about true population parameter values. The bootstrap can take into account many sources of uncertainty. We use the bootstrap to estimate the bias in the apparent highest and apparent lowest odds ratios—the two “winners”. The sample size of the simulated data is 600 subjects and there are 300 candidate features.

The bootstrap is based on sampling with replacement from the rows of the entire data matrix \((X, Y)\). In order to sample from the rows we need to generate raw data, not just numbers of “successes” and “failures” as in the last simulation.

Code

# Function to simulate the raw data# prev is the vector of prevalences of x when y=0 as before# yprev is the overall prevalence of y# n is the sample size# or is the vector of true odds ratiossim <-function(n, yprev, prev, or) { y <-rbinom(n, 1, yprev) p <-length(prev)if(p !=length(or)) stop('prev and or must have the same length')# prev = Pr(x=1 | y=0); let the odds for this be oprev = prev / (1-prev)# or = odds(x=1 | y=1) / oprev# Pr(x=1 | y=1) = oprev / ((1 / or) + oprev) oprev <- prev / (1- prev) p1 <- oprev / ((1/ or) + oprev) x <-matrix(NA, nrow=n, ncol=p)for(j in1: p) x[, j] <-ifelse(y ==1, rbinom(n, 1, prob = p1[j] ),rbinom(n, 1, prob = prev[j]))list(x=x, y=y)}# Function to compute the sample odds ratios given x matrix and y vectorors <-function(x, y) { p <-ncol(x) or <-numeric(p)for(j in1: p) { f <-table(x[, j], y) or[j] <- f[2, 2] * f[1, 1] / (f[1, 2] * f[2, 1]) } or}

Code

# Generate sample of size 600 with 300 features# Log odds ratios have a normal distribution with mean 0 SD 0.3# x have a random prevalence uniform [0.05, 0.5]# y has prevalence 0.3set.seed(188)n <-600; p <-300prev <-runif(p, .05, .5)or <-exp(rnorm(p, 0, .3))z <-sim(n, 0.3, prev, or)# Compute estimated ORsx <- z$x; y <- z$ysor <-ors(x, y)# Show how estimates related to true ORsggplot(data.frame(or, sor), aes(x=or, y=sor)) +geom_point() +xlab('True OR') +ylab('Estimated OR')

Code

# Print the largest estimated OR and its column number,# and corresponding true OR, and similarly for the smallest.largest <-max(sor)imax <-which.max(sor)true.imax <- or[imax]mmoe.imax <- largest / true.imaxsmallest <-min(sor)imin <-which.min(sor)true.imin <- or[imin]mmoe.imin <- smallest / true.imincat('\nLargest observed OR\n')

Next use the bootstrap to get an estimate of the MMOE for the observed largest OR, and a 0.95 confidence interval for the true unknown rank of the largest observed OR from among all the features. 1000 bootstrap resamples are drawn. In estimating the MMOE we are estimating bias in the largest log odds ratio when a new “largest” OR is found in each bootstrap resample. That estimated OR is compared to the OR evaluated in the whole sample for the same column number. This is also done for the “smallest” OR.

Code

set.seed(11)B <-1000ranksS <- ranksL <- mmoeS <- mmoeL <-numeric(B)for(k in1: B) {# Draw a sample of size n with replacement i <-sample(1: n, n, replace=TRUE)# Compute sample ORs on the new sample bor <-ors(x[i, ], y[i]) blargest <-max(bor) bmax <-which.max(bor) ranksL[k] <-sum(bor <= largest) mmoeL[k] <- blargest / sor[bmax] bsmallest <-min(bor) bmin <-which.min(bor) ranksS[k] <-sum(bor <= smallest) mmoeS[k] <- bsmallest / sor[bmin]}

The bootstrap geometric mean MMOE for the smallest odds ratio was zero due to small frequencies in some \(X\)s. The median bootstrap MMOE was used to bias-correct the observed smallest OR, while the geometric mean was used for the largest.

Largest OR
CL for rank: 294 299 Median MMOE: 1.45 Geometric mean MMOE: 1.52
True MMOE: 1.72
Bootstrap bias-corrected largest OR: 1.93 Original OR: 2.94 True OR: 1.71

Smallest OR
CL for rank: 0 5 Median MMOE: 0.34 Geometric mean MMOE: 0
True MMOE: 0.3
Bootstrap bias-corrected smallest OR: 0.62 Original OR: 0.21 True OR: 0.71

The bootstrap bias-corrected ORs took the observed extreme ORs and multiplied them by their respective bootstrap geometric mean or median MMOEs. The bias-corrected estimates are closer to the true ORs.

The data are consistent with the observed smallest OR truly being in the bottom 5 and the observed largest OR truly being in the top 7.

Here is some example wording that could be used in a statistical analysis plan: We did not evaluate the probability that non-selected genes are truly unimportant but will accompany the planned gene screening results with a bootstrap analysis to compute 0.95 confidence intervals for the rank of each gene, using a non-directional measure of association when ranking. The “winning” genes will have high ranks in competing with each other (by definition) but if the lower confidence limits include mid- to low-level ranks the uncertainty of these winners will be noted. Conversely, if the upper confidence intervals of ranks of “losers” extend well into the ranks of “winners”, the uncertainty associated with our ability to rule out “losers” for further investigation will be noted.

Altman, D. G., & Bland, J. M. (1995). Absence of evidence is not evidence of absence. BMJ, 311, 485.

Ambler, G., Brady, A. R., & Royston, P. (2002). Simplifying a prognostic model: A simulation study based on clinical data. Stat Med, 21(24), 3803–3822. https://doi.org/10.1002/sim.1422

ordinary backward stepdown worked well when there was a large fraction of truly irrelevant predictors

Ambroise, C., & McLachlan, G. J. (2002). Selection bias in gene extraction on the basis of microarray gene-expression data. PNASs, 99(10), 6562–6566. https://doi.org/10.1073/pnas.102102699

Relied on an improper accuracy score (proportion classified correct) so had to use the .632 bootstrap unnecessarily

Paul, D., Bair, E., Hastie, T., & Tibshirani, R. (2008). “Preconditioning” for feature selection and regression in high-dimensional problems. Ann Stat, 36(4), 1595–1619. https://doi.org/10.1214/009053607000000578

develop consistent Y using a latent variable structure, using for example supervised principal components. Then run stepwise regression or lasso predicting Y (lasso worked better). Can run into problems when a predictor has importance in an adjusted sense but has no marginal correlation with Y;model approximation;model simplification

Shen, X., Huang, H.-C., & Ye, J. (2004). Inference after model selection. J Am Stat Assoc, 99, 751–762.

uses optimal approximation for estimating mean and variance of complex statistics adjusting for model selection

van der Ploeg, T., Austin, P. C., & Steyerberg, E. W. (2014). Modern modelling techniques are data hungry: A simulation study for predicting dichotomous endpoints. BMC Medical Research Methodology, 14(1), 137+. https://doi.org/10.1186/1471-2288-14-137

Would be better to use proper accuracy scores in the assessment. Too much emphasis on optimism as opposed to final discrimination measure. But much good practical information. Recursive partitioning fared poorly.

Source Code

```{r setup, include=FALSE}require(Hmisc)getRs('reptools.r')hookaddcap() # make knitr call a function at the end of each chunk# to try to automatically add to list of figureoptions(prType='html')source('~/r/rmarkdown/qbookfun.r')knitr::set_alias(w ='fig.width', h ='fig.height',cap ='fig.cap', scap ='fig.scap')```# Challenges of Analyzing High-Dimensional Data {#sec-hdata}`r quoteit("**Biomarker Uncertainty Principle**:<br> A molecular signature can be either parsimonious or predictive, but not both.", "FE Harrell, 2009")``r quoteit("We have more data than ever, more good data than ever, a lower proportion of data that are good, a lack of strategic thinking about what data are needed to answer questions of interest, sub-optimal analysis of data, and an occasional tendency to do research that should not be done.", "FE Harrell, 2015")`## BackgroundHigh-dimensional data are of at least three major types:* Data collected on hundreds of thousands or millions of subjects with a diverse array of variables* Time series of biologic signals collected every few milliseconds* Extremely large data arrays where almost all the variables are of one type (the main topic here)The last data class includes such areas as* functional imaging* gene microarray* SNPs for genome-wide association studies* RNA seq* exome sequencing* mass spectrometryThe research yield of analysis of such data has been disappointing todate, for many reasons such as:* Biology is complex* Use of non-reproducible research methodology* Use of unproven statistical methods* Multiple comparison problems and double dipping^[Double dipping refers to using the same data to test a hypothesis that was formulated from a confession from the tortured dataset.]* Failure to demonstrate value of information over and above the information provided by routine clinical variables, blood chemistry, and other standard medical tests* Inadequate sample size for the complexity of the analytic task* Overstatement of results due to searching for "winning" markers without understanding bias, regression to the mean (@sec-change-rttm), and overfittingRegarding double dipping/multiplicity a beautiful example is the deadsalmon fMRI study by Bennett _et al._ in which the standardmethod of analyzing fMRI data by voxels was shown to find brainactivation regions even in the dead brain (see[prefrontal.org/files/posters/Bennett-Salmon-2009.pdf](http://prefrontal.org/files/posters/Bennett-Salmon-2009.pdf)).<imgsrc="deadsalmon.png"width="80%">_Wired_, 2009-09-18.Unfortunately, a large number of papers in genomics and imagingresearch have pretended that feature selection has no randomness, andhave validated predictions in the same dataset used to find the signals,without informing the bootstrap or cross-validation procedure aboutthe data mining used to derive the model so that the resamplingprocedure could repeat all feature selection steps afresh for eachresample (@amb02sel). Only by re-running all data analysis stepsthat utilized $Y$ for each resample can an unbiased estimate of futuremodel performance be obtained.## Global Analytic ApproachesLet $X_{1}, X_{2}, \ldots, X_{p}$ denote an array of candidate features (gene expressions, SNPs, brain activation, etc.) and $Y$ the response, diagnosis, or patient outcome being predicted.### One-at-a-Time Feature ScreeningOaaT feature screening involves choosing a measure of association such as Pearson's $\chi^2$ statistic^[Oddly, many practitioners of OaaT choose the more conservative and much slower to compute Fisher's exact test.], computing all $p$ association measures against $Y$, and choosing those $X$ features whose association measure exceeds a threshold.This is by far the most popular approach to feature discovery (and often to prediction, unfortunately) in genomics and functional imaging. It is demonstrably the worst approach in terms of the reliability of the "winning" and "losing" feature selection and because it results in poor predictive ability. The problems are due to multiple comparison problems, bias, typically high false negative rates, and to the fact that most features "travel in packs", i.e., act in networks rather than individually. As if OaaT could not get any worse, many practitioners create "risk scores" by simply adding together individual regression coefficients computed from individual "winning" $X$ features without regard to the correlation structure of the $X$s. This gives far too much weight to the selected $X$s that are co-linear with each other.There is a false belief that preventing a high false discovery rate solves the problems of OaaT. Most researchers fail to consider, among other things, the high false negative rate caused by their design and sample size.OaaT results in highly overestimated effect sizes for winners, due todouble dipping.#### Multiplicity CorrectionsIn the belief that false positive discoveries are less desirable thanmissed discoveries, researchers employ multiplicity corrections to$P$-values arising from testing a large number of associations with$Y$. The most conservative approach uses the addition or Bonferroniinequality to control the _family-wise error risk_ which is theprobability of getting _any_ false positive association when the globalnull hypothesis that there are no true associations with $Y$ holds.This can be carried out by testing the $p$ associations against$\frac{\alpha}{p}$ where $\alpha=0.05$ for example. A lessconservative approach uses the _false discovery rate_ (FDR),better labeled the _false discovery risk_ or _false discovery probability_. Here one controls the fraction of falsepositive associations. For example one sets the critical value forassociation statistics to a less severe threshold than dictated byBonferroni's inequality, by allowing $\frac{1}{10}^{\mathrm th}$ ofthe claimed associations (those with the smallest $P$-values) to befalse positives.It is an irony that the attempt to control for multiplicity has lednot only to missed discoveries and abandonment of whole research areasbut has resulted in increased bias in the "discovered" features'effect estimates. When using stepwise regression, the bias inregression coeffients comes not from multiplicity problems arisingwhen experimentwise $\alpha=0.05$ is used throughout but from using an$\alpha$ cutoff $< \frac{1}{2}$ for selecting variables. By selectingvariables on the basis of small $P$-values, many of the selectedvariables were selected because their effects were overestimated, thenregression to the mean sets in.### Forward Stepwise Variable SelectionForward stepwise variable selection that does an initialscreening of all the features, adds the most significant one, thenattempts to add the next most significant feature, after adjustingfor the first one, and so on. This approach is unreliable. It isonly better than OaaT in two ways: (1) a feature that did not meet thethreshold for the association with $Y$ without adjusting for otherfeatures may become stronger after the selection of other features atearlier steps, and (2) the resulting risk score accounts forco-linearities of selected features. On the downside, co-linearitiesmake feature selection almost randomly choose features from correlatedsets of $X$s, and tiny changes in the dataset will result in differentselected features.### Ranking and SelectionFeature discovery is really a ranking and selection problem. But ranking and selection methods are almost never used. An example bootstrap analysis on simulated data is presented later. This involves sampling with replacement from the combined $(X, Y)$ dataset and recomputing for each bootstrap repetition the $p$ association measures for the $p$ candidate $X$s. The $p$ association measures are ranked. The ranking of each feature across bootstrap resamples is tracked, and a 0.95 confidence interval for the rank is derived by computing the 0.025 and 0.975 quantiles of the estimated ranks.This approach puts OaaT in an honest context that fully admits the true difficulty of the task, including the high cost of the false negative rate (low power). Suppose that features are ranked so that a low ranking means weak estimated association and a high ranking means strong association. If one were to consider features to have "passed the screen" if their lower 0.95 confidence limit for their rank exceeds a threshold, and only dismisses features from further consideration if the upper confidence limit for their rank falls below a threshold, there would correctly be a large middle ground where features are not declared either winners or losers, and the researcher would be able to only make conclusions that are supported by the data. Readers of the classic _Absence of Evidence_ paper @alt95abs will recognize that this ranking approach solves the problem.An example of bootstrap confidence intervals where multivariable modeling was used rather than OaaT association measures is in the course notes for _Regression Modeling Strategies_, Section 5.4. An example where the bootstrap is used in the context of OaaT is below.### Joint modeling of All Features Simultaneously using ShrinkageThis approach uses multivariable regression models along with penalized maximum likelihood estimation, random effects / hierarchical modeling, or skeptical Bayesian prior distributions for adjusted effects of all $p$ $X$ features simultaneously. Feature effects (e.g., log odds ratios) are discounted so as to prevent overfitting/over-interpretation and to allow these effects to be trusted out of context. The result is a high-dimensional regression model that is likely to provide well-calibrated absolute risk estimates and near-optimum predictive ability. Some of the penalization methods used are1. lasso: a penalty on the absolute value of regression coefficient that highly favors zero as an estimate. This results in a large number of estimated coefficients being exactly zero, i.e., results in feature selection. The resulting parsimony may be illusory: bootstrap repetition may expose the list of "selected" features to be highly unstable.1. ridge regression (penalty function that is quadratic in the regression coefficients): does not result in a parsimoneous model but is likely to have the highest predictive value1. elastic net: a combination of lasso and quadratic penalty that has some parsimony but has better predictive ability than the lasso. The difficulty is simultaneously choosing two penalty parameters (one for absolute value of $\beta$s, one for their sum of squares).### Random ForestThis is an approach that solves some of the incredible instability and low predictive power of individual regression trees. The basic idea of random forest is that one fits a regression tree using recursive partitioning (CART) on multiple random samples of **candidate features**. Multiple trees are combined. The result is no longer a tree; it is an uninterpretable black box. But in a way it automatically incorporates shrinkage and is often competitive with other methods in predictive ability.There is evidence that minimal-assumption methods such as randomforests are "data hungry", requiring as many as 200 events percandidate variable for their apparent predictive discrimination to notdecline when evaluated in a new sample @plo14mod.### Data Reduction Followed by Traditional Regression ModelingThis approach uses techniques such as principle component analysis (PCA) whereby a large number of candidate $X$s are reduced to a few summary scores. PCA is based on additively combining features so as to maximize the variation of the whole set of features that is explained by the summary score. A small number of PCA scores are then put into an ordinary regression model (e.g., binary logistic model) to predict $Y$. The result is sometimes satisfactory though no easier to interpret than shrinkage methods.### Model ApproximationAlso called _pre-conditioning_, this method is general-purpose and promising (@pau08pre,@she04inf,@amb02sim). One takes a well-performing black box (e.g., random forest or full penalized regression with $p$ features) that generates predicted responses $\hat{Y}$ and incorporates the right amount of shrinkage to make the predictions well-calibrated. Then try to find a smaller set of $X$s that can represent $\hat{Y}$ with high accuracy (e.g., $R^{2} \geq 0.9$). Forward stepwise variable selection may be used for this purpose^[Here one is developing a mechanistic prediction where the true $R^{2}$ is 1.0.}. This sub-model is an approximation to the "gold-standard" full black box. The ability to find a well-performing approximation is a test of whether the predictive signal is parsimoneous. If one requires 500 $X$s to achieve an $R^{2} \geq 0.9$ in predicting the gold-standard predictions $\hat{Y}$, then it is not possible to be parsimoneous and predictive.A major advantage of model approximation is that if the original complex model was well calibrated by using appropriate shrinkage, the smaller approximate model inherits that shrinkage.### Incorporating Biology into High-Dimensional Regression ModelsThis approach is likely to result in the most trustworthy discoveries as well as the best predictive accuracy, if existing biological knowledge is adequate for specification of model structure. This is a structured shrinkage method where pathway (e.g., gene pathway) information is inserted directly in the model. One may encode multiple paths into a simultaneous regression model such that genes are "connected" to other genes in the same pathway. This allows an entire path to be emphasized or de-emphasized.## Simulated ExamplesMonte Carlo simulation, when done in a realistic fashion, has thegreat advantage that one knows the truth, i.e., the true model andvalues of model parameters from which the artificial population wassimulated. Then any derived model or estimates can be compared to theknown truth. Also with simulation, one can easily change the samplesize being simulated so that the effect of sample size can be studiedand an adequate sample size that leads to reliable results can becomputed. One can also easily change the dimensionality of the features.### Simulation To Understand Needed Sample SizesOne of the most common association measures used in genomic studies isthe odds ratio. As shown in @sec-prop-rem and@fig-prop-mmeor , the oddsratio (OR) is very difficult to estimate when the outcome is rare or when abinary predictive feature has a prevalence far from $\frac{1}{2}$.That is for the case when only a single pre-specified is estimated.When screening multiple features for interesting associations, one iseffectively estimating a large number of ORs, and in order to makecorrect decisions about which features are promising and which aren't,one must be able to control the margins of error of the entire set ofOR estimates.In the following simulation consider varying sample size $n$ andnumber of candidate features $p$. We simulate $p$ binary featureswith known true ORs against the diagnosis or outcome $Y$. Thetrue unknown ORs are assumed to have a normal$(\mu=0,\sigma=0.25)$distribution. We want to judge the ability to jointly estimate $p$associations and to rank order features by observed associations. The analysisthat is simulated does not examine multiple $X$s simultaneously, so we save timeby simulating just the total numbers of zeros and ones for each $X$, given $Y$.```{r simor-sim}# For a vector of n binary outcomes y, simulates p binary features# x that have a p-vector of fixed prevalences | y=0 of prev and are connected# to y by a p-vector of true population odds ratios ors.# Estimates the p odds ratios against the simulated outcomes and# returns a data frame summarizing the information## Note: the odds ratio for x predicting y is the same as the odds ratio# for y predicting x. y is simulated first so that all features will# be analyzed against the same outcomessim <-function(y, prev, or) { n <-length(y) p <-length(prev)if(p !=length(or)) stop('prev and or must have the same length')# prev = Pr(x=1 | y=0); let the odds for this be oprev = prev / (1-prev)# or = odds(x=1 | y=1) / oprev# Pr(x=1 | y=1) = oprev / ((1 / or) + oprev) oprev <- prev / (1- prev) p1 <- oprev / ((1/ or) + oprev) n0 <-sum(y ==0) n1 <-sum(y ==1)# For n0 observations sample x so that Pr(x=0 | y=0) = prev nxy0 <-rbinom(p, size=n0, prob=prev) nxy1 <-rbinom(p, size=n1, prob=p1)# Compute p sample odds ratios sor <- (n0 - nxy0) * nxy1 / (nxy0 * (n1 - nxy1)) g <-function(x) ifelse(x >=1, x, 1/ x) r1 <-rank(sor)[which.max(or) ] / p r2 <-rank(or) [which.max(sor)] / pdata.frame(prev, or, nx=nxy0 / n0, obsprev0=(nxy0 + nxy1) / n,obsprev=nxy1 / (nxy0 + nxy1), obsor=sor, n=n,N =paste('n', n, sep=':'),Features=paste('Features', p, sep=':'),mmoe =quantile(g(sor / or), 0.90, na.rm=TRUE),obsranktrue=r1, truerankobs=r2,rho=cor(sor, or, method='spearman', use='pair'))}``````{r simor-dosim}U <-NULLset.seed(1)for(n inc(50, 100, 250, 500, 1000, 2000)) {for(p inc(10, 50, 500, 1000, 2000)) {for(yprev inc(.1, .3)) { y <-rbinom(n, 1, yprev) prev <-runif(p, .05, .5) or <-exp(rnorm(p, 0, .25)) u <-cbind(sim(y, prev, or),Yprev=paste('Prevalence of Outcome', yprev, sep=':')) U <-rbind(U, u) } }}```In the plots below red lines show the line of identity.```{r simor-plot,w=7,h=7}require(ggplot2)pl <-function(yprev) { br <-c(.01, .1, .5, 1, 2.5, 5, 25, 100)ggplot(subset(U, Yprev=yprev),aes(x=or, y=obsor)) +geom_point() +facet_grid(Features ~ N) +ggtitle(paste('Prevalence of Outcome', yprev, sep=':')) +xlab('True ORs') +ylab('Estimated ORs') +scale_x_log10(breaks=br) +scale_y_log10(breaks=br) +theme(axis.text.x =element_text(size =rel(0.8), angle=-45,hjust=0, vjust=1)) +geom_abline(col='red')}pl(0.1)``````{r simor-plotb,w=7,h=7}pl(0.3)```The last two figures use a log scale for the $y$-axis (estimated oddsratios), so the errors in estimating the odds ratios are quitesevere. For a sample size of $n=50$ one cannot even estimate a singlepre-specified odds ratio. To be able to accurately assess 10 ORs (10candidate features) requires about $n=1000$. To assess 2000 features,a sample size of $n=2000$ seems adequate only for the very smallestand very largest true ORs.The plot below summarizes the previous plots by computing the 0.9 quantile ofthe multiplicative margin of error (fold change) over the whole set of estimatedodds ratios, ignoring direction.```{r simor-mmoe,w=5.5,h=5.5}ggplot(U, aes(x=n, y=mmoe)) +geom_point() +facet_grid(Features ~ Yprev) +geom_hline(aes(yintercept=1.5, col='red')) +ylim(1, 10) +ylab('0.9 Quantile of Multiplicative Margin of Error in OR Across Features')```Horizontal red lines depict a multiplicative margin of error (MMOE) of1.5 which may be considered the minimally acceptable error inestimating odds ratios. This was largely achieved with $n=1000$ for alow-incidence $Y$, and $n=500$ for a moderate-incidence $Y$.Another way to summarize the results is to compute the Spearman rank correlationbetween estimated and true underlying odds ratios over the entire set ofestimates.```{r simor-rho,w=5.5,h=5.5}ggplot(U, aes(x=n, y=rho)) +geom_point() +facet_grid(Features ~ Yprev) +ylab(expression(paste('Spearman ', rho, ' Rank Correlation Between ', OR, ' and ', hat(OR), ' Across Features')))```One may desire a correlation with the truth of say 0.8 and can solvefor the needed sample size.### Bootstrap Analysis for One Simulated DatasetSuppose that one wanted to test $p$ candidate features and select themost "significant" one for a validation study. How likely is theapparently "best" one to be truly the best? What is a confidenceinterval for the rank of this "winner"? How much bias in the ORdoes the selection process create? The bootstrap can be used toanswer all of these questions without needing to assume anything abouttrue population parameter values. The bootstrap can take into accountmany sources of uncertainty. We use the bootstrap to estimate thebias in the apparent highest and apparent lowest odds ratios---the two"winners". The sample size of the simulated data is 600 subjectsand there are 300 candidate features.The bootstrap is based on sampling with replacement from the rows of the entiredata matrix $(X, Y)$. In order to sample from the rows we need to generate rawdata, not just numbers of "successes" and "failures" as in the lastsimulation.```{r simbf}# Function to simulate the raw data# prev is the vector of prevalences of x when y=0 as before# yprev is the overall prevalence of y# n is the sample size# or is the vector of true odds ratiossim <-function(n, yprev, prev, or) { y <-rbinom(n, 1, yprev) p <-length(prev)if(p !=length(or)) stop('prev and or must have the same length')# prev = Pr(x=1 | y=0); let the odds for this be oprev = prev / (1-prev)# or = odds(x=1 | y=1) / oprev# Pr(x=1 | y=1) = oprev / ((1 / or) + oprev) oprev <- prev / (1- prev) p1 <- oprev / ((1/ or) + oprev) x <-matrix(NA, nrow=n, ncol=p)for(j in1: p) x[, j] <-ifelse(y ==1, rbinom(n, 1, prob = p1[j] ),rbinom(n, 1, prob = prev[j]))list(x=x, y=y)}# Function to compute the sample odds ratios given x matrix and y vectorors <-function(x, y) { p <-ncol(x) or <-numeric(p)for(j in1: p) { f <-table(x[, j], y) or[j] <- f[2, 2] * f[1, 1] / (f[1, 2] * f[2, 1]) } or}``````{r simbd}# Generate sample of size 600 with 300 features# Log odds ratios have a normal distribution with mean 0 SD 0.3# x have a random prevalence uniform [0.05, 0.5]# y has prevalence 0.3set.seed(188)n <-600; p <-300prev <-runif(p, .05, .5)or <-exp(rnorm(p, 0, .3))z <-sim(n, 0.3, prev, or)# Compute estimated ORsx <- z$x; y <- z$ysor <-ors(x, y)# Show how estimates related to true ORsggplot(data.frame(or, sor), aes(x=or, y=sor)) +geom_point() +xlab('True OR') +ylab('Estimated OR')# Print the largest estimated OR and its column number,# and corresponding true OR, and similarly for the smallest.largest <-max(sor)imax <-which.max(sor)true.imax <- or[imax]mmoe.imax <- largest / true.imaxsmallest <-min(sor)imin <-which.min(sor)true.imin <- or[imin]mmoe.imin <- smallest / true.imincat('\nLargest observed OR\n')cat('OR:', round(largest, 2), ' Feature #', imax, ' True OR:',round(true.imax, 2), ' MMOE:', round(mmoe.imax, 2), '\n')cat('Rank of winning feature among true ORs:', sum(or <= or[imax]), '\n\n')cat('Smallest observed OR\n')cat('OR:', round(smallest, 2), ' Feature #', imin, ' True OR:',round(true.imin, 2), ' MMOE:', round(mmoe.imin, 2), '\n')```Next use the bootstrap to get an estimate of the MMOE for the observedlargest OR, and a 0.95 confidenceinterval for the true unknown rank of the largest observed OR fromamong all the features. 1000 bootstrap resamples are drawn. Inestimating the MMOE we are estimating bias in the largest log oddsratio when a new "largest" OR is found in each bootstrap resample.That estimated OR is compared to the OR evaluated in the whole samplefor the same column number. This is also done for the "smallest" OR.```{r bootor,cache=TRUE}set.seed(11)B <-1000ranksS <- ranksL <- mmoeS <- mmoeL <-numeric(B)for(k in1: B) {# Draw a sample of size n with replacement i <-sample(1: n, n, replace=TRUE)# Compute sample ORs on the new sample bor <-ors(x[i, ], y[i]) blargest <-max(bor) bmax <-which.max(bor) ranksL[k] <-sum(bor <= largest) mmoeL[k] <- blargest / sor[bmax] bsmallest <-min(bor) bmin <-which.min(bor) ranksS[k] <-sum(bor <= smallest) mmoeS[k] <- bsmallest / sor[bmin]}```The bootstrap geometric mean MMOE for the smallest odds ratio was zerodue to small frequencies in some $X$s. The median bootstrap MMOE wasused to bias-correct the observed smallest OR, while the geometricmean was used for the largest.```{r summarizeb}pr <-function(which, ranks, mmoe, mmoe.true, estor, or.true) { gm <-exp(mean(log(mmoe)))cat(which, 'OR\n')cat('CL for rank:', quantile(ranks, c(0.025, 0.975)),' Median MMOE:', round(median(mmoe), 2),' Geometric mean MMOE:', round(gm, 2),'\nTrue MMOE:', round(mmoe.true, 2), '\n') bmmoe <-if(which =='Largest') gm elsemedian(mmoe)cat('Bootstrap bias-corrected', tolower(which), 'OR:',round(estor / bmmoe, 2),' Original OR:', round(estor, 2),' True OR:', round(or.true, 2),'\n\n')}pr('Largest', ranksL, mmoeL, mmoe.imax, largest, true.imax)pr('Smallest', ranksS, mmoeS, mmoe.imin, smallest, true.imin)```The bootstrap bias-corrected ORs took the observed extreme ORs andmultiplied them by their respective bootstrap geometric mean or medianMMOEs. The bias-corrected estimates are closer to the true ORs.The data are consistent with the observed smallest OR truly being in thebottom 5 and the observed largest OR truly being in the top 7.Here is some example wording that could be used in a statisticalanalysis plan: We did not evaluate the probability that non-selected genesare truly unimportant but will accompany the planned gene screeningresults with a bootstrap analysis to compute 0.95 confidence intervalsfor the rank of each gene, using a non-directional measure ofassociation when ranking. The "winning" genes will have high ranks incompeting with each other (by definition) but if the lower confidencelimits include mid- to low-level ranks the uncertainty of thesewinners will be noted. Conversely, if the upper confidence intervalsof ranks of "losers" extend well into the ranks of "winners", theuncertainty associated with our ability to rule out "losers" forfurther investigation will be noted.```{r echo=FALSE}saveCap('20')```