Using singscore to predict mutation status in acute myeloid leukemia from transcriptomic signatures

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.


Introduction
The development of microarrays and more recently the rapid uptake of RNA-sequencing technologies have provided a platform to examine the transcriptional profile (or transcriptome) of biological samples 1 . Transcriptomic analyses have traditionally focused on 'differential expression' of genes between sets of samples, however with the rapid growth of publicly available RNA data there has been increasing usage of 'relative approaches', which quantify the relative concordance of a sample or samples with a specific gene signature 1 . While sequencing of genomic mutations has been important for classifying different tumour subsets based upon the presence of mutations or fusion genes, and identifying genetic lesions which may act as drivers of cancer progression, transcriptomic profiling can provide further information on the state or phenotype of cells carrying these mutations. Cancers are a heterogeneous set of diseases with a number of clinical and pathological subtypes. In diseases such as breast cancer the primary clinical classifications relate to the expression of hormone receptors (estrogen receptor: ER; and progesterone receptor: PR) or the overexpression of Erb-B2 receptor tyrosine kinase (HER2), as these features can be directly targeted with therapeutic agents. A common example of transcriptomic or gene expression data informing clinical practice is the use of prediction analysis of microarray 50 (PAM50) signatures for distinguishing the intrinsic breast cancer subtypes [2, Cieślik and Chinnaiyan 1 ]. For many other cancers, subtype classification has largely relied upon identifying sets of recurrent mutations across large patient cohorts, with whole genome or whole exome sequencing studies helping to resolve the clinically significant subtypes [3,Papaemmanuil et al. 4 ].
Perhaps the most well-known 'relative approach' is single-sample gene set enrichment analysis (ssGSEA) 5 , often used through the GenePattern web-tool. Another common approach is gene set variation analysis (GSVA) 6 which is available as an R/Bioconductor package that also includes functionality for ssGSEA, an alternative approach known as PLAGE 7 , and a z-score based approach 8 . Both ssGSEA and GSVA use a Kolmogorov-Smirnov like random-walk statistic to convert normalised gene ranks to the resulting score, however this normalisation procedure means that the scores are not truly 'single-sample', and variations in the overall sample composition for a study (e.g. variations in the presence or relative frequency of different cancer subtypes) can lead to unexpected changes in sample scores. Furthermore, the resultant scores from these methods can vary in their range and absolute value, making them difficult to interpret without further processing. To overcome this, we have developed a single-sample gene set scoring method singscore 9 which simply uses the ranks of genes within a given set, normalised relative to the maximum and minimum theoretical scores for a gene set of a given size.
Through large scale efforts such as The Cancer Genome Atlas (TCGA), transcriptomic data are available for thousands of clinical samples, often together with corresponding genomic or epigenomic (often DNA methylation) data. These transcriptomic data can help to characterise the functional effects of corresponding mutations, and provide a window to study the heterogeneity which arises within different subtypes of cancer due to epigenetic and transcriptional regulatory programs which can also influence cell behaviour. Here, we demonstrate that the single-sample gene set scoring method singscore 9 can be used to classify TCGA AML samples using transcriptional 'gene signatures' for the NPM1c mutation, KMT2A (MLL) gene fusions, and PML-RARA gene fusions that were derived from independent studies. Without any need for parameter fitting or estimation, we show that gene set scoring with singscore can distinguish samples carrying these mutations. The case studies we present demonstrate the application of gene set scoring to examine not only the differences, but also the relative similarities between established subtypes of AML that impact clinical outcome. This workflow is available as a bioconductor workflow package from https://bioconductor.org/packages/release/workflows/html/ SingscoreAMLMutations.html.

Amendments from Version 2
The updated workflow includes additional guidance on how to interpret results from singscore, especially in an unsupervised setting where labels for data are unavailable and further context on how this relates to alternative approaches. We also demonstrate the ability of our visualisation functions to label individual samples, further assisting in result interpretation Any further responses from the reviewers can be found at the end of the article

Description of the biological problem
As with most cancers, acute myeloid leukemia (AML) is a heterogeneous disease with a number of classified subtypes. Analysis of TCGA AML genomic data identified a number of subtypes based upon the presence or absence of specific 'driver mutations'; recapitulating and expanding upon previously identified clinical subsets 3 . A more recent study which focused primarily on genomic data has further refined the clinically significant AML subtypes 4 , highlighting a number of co-occurring as well as mutually exclusive mutations. As the identification of putative driver fusions/mutations continues, work has also been directed towards how these lesions interact with each other and other features (e.g. cellular proliferation, changes due to phenotypic plasticity, or variation in post-transcriptional regulators such as microRNAs) to drive transcriptional changes as discussed in a recent review 10 .
Of note for this work, one of the most common mutations in clinical AML samples is a frameshift mutation within exon 12 of the nucleophosmin (NPM1) gene 4 . This mutation leads to aberrant localisation of nucleophosmin with cytoplasmic accumulation rather than localising to the nucleolus, and accordingly this mutation is often referred to as the NPM1c mutation 11 . As noted by Verhaak et al. 12 , the NPM1c mutation is associated with dysregulated activity of the homeobox domain (Hox) family of transcription factors which are essential for developmental patterning. The effects of this mutation in disease progression have been further demonstrated in recent work which showed that loss of NPM1c leads to differentiation of AML cells 11 .
Further recurrent genetic lesions in AML relevant for this work include lysine methyl transferase 2A (KMT2A; previously known as MLL) fusion genes, partial tandem duplications within KMT2A (KMT2A-PTD), and fusion genes between promyelocytic leukemia and retinoic acid receptor alpha (PML-RARA). Given the role of NPM1c in dysregulating the Hox gene family, it is interesting to note that AML samples with MLL fusion genes also show dysregulated expression of Hox family genes [13, Ross et al. 14 ]; however, samples with MLL-PTD appear to show a relatively distinct phenotype from MLL-fusion samples 14 . While there is good evidence demonstrating the role of NPM1c mutations and other genetic lesions in blocking AML cell differentiation, the PML-RARA fusion subset is diagnostic for a specific subset of AML known as acute promyelocytic leukemia (APL). This clinically distinct subtype of AML is associated with a specific morphology under the French-American-British (FAB) classification of AML, FAB-M3, with cells showing a distinct morphology due to a differentiation block at the promyelocyte stage 15 .
In this workflow we demonstrate the ability of the singscore method for single sample gene set scoring 9 to classify tumour 'driver mutations' from transcriptomic data. We use a previously identified gene signature for the NPM1c mutation 12 . We also use signatures for PML-RARA gene fusions and MLL-fusions that were derived using pediatric AML samples but shown to work well for classifying adult AML samples with similar lesions 14 , although we note that there is evidence of relatively large differences in the mutational profiles of adult and pediatric cancers 16 . Using these signatures, which are included within the molecular signatures database (MSigDB) 17 , we demonstrate that a bi-directional scoring approach can classify TCGA AML samples with corresponding mutations with a good precision and recall. A particularly useful feature of gene set scoring is the ability to project samples onto 2D or higher-order landscapes defined by corresponding phenotypic signatures. Accordingly, by comparing scores for both the NPM1c and KMT2A-/MLL-fusion signatures, we show that this classification likely arises through the shared downstream biological effects of Hox family dysregulation. We also compare the NPM1c mutation signature to the PML-RARA signature and show a clear separation of these subtypes reflecting their divergent phenotypes and the mutually exclusive nature of these mutations.
While we demonstrate that singscore is capable of inferring mutation status from the transcriptional profile of AML samples, we note that it is best used to supplement alternative data which can provide a more definitive resolution of these lesions. Processing of raw RNA seq data will directly identify the presence of gene fusion products or mutations within protein-coding regions, although for many large data sets the quantified transcript abundance data are much easier to obtain without access agreements. The method can also be applied to legacy microarray data sets where genome and RNA sequencing data are unavailable. As such singscore provides a useful approach to supplement established methods for the study of genetic lesions in cancer. By exploring associations between different genomic and phenotypically relevant signatures, it may also help to better characterise true driver mutations which exert consistent effects on the transcriptome of AML samples and other cancers.

Downloading and preparing the data
Data from TCGA project is made available through the Genomic Data Commons (GDC). Open access data from the project can be accessed in multiple pre-processed formats. Transcriptomic data can be downloaded either at the count level or as FPKM transformed abundance, before or after upper quantile normalisation. Other pre-processed version can be found from sources such as the cBioPortal and FireBrowse. The GDC data used STAR to perform a two-pass alignment followed by quantification using HTSeq. Data from the GDC can be downloaded using the GDC data transfer tool which allows users to select the specific files of interest using the GDC portal. These files then have to be read, merged, annotated and processed into a data structure that simplifies downstream analysis. Alternatively, all the above mentioned steps, including the download, can be performed using the R package TCGAbiolinks 18 . The package supports data download using the GDC API and the GDC client. We will use the TCGAbiolinks package to download, annotate and process the data into a SummarizedExperiment R object.
The following steps need to be performed to prepare the data: 1. Create a query to select the files to download 2. Execute the query and download the data 3. Read the data into R 4. Filter out genes with low expression 5. Normalise the data for compositional bias and transform to account for gene-length biases as outlined in the singscore manuscript 9 Querying the GDC database The first step in any analysis should be to determine and report the data version and the service used to download the data. The getGDCInfo() function returns the release date of all data on the GDC along with a version. A query then needs to be run, using the GDC to identify the specific files for download. This step is similar to generating a MANIFEST file using the GDC portal. The first parameter of the query specifies the project -available projects can be accessed using getGDCprojects() or from https://portal.gdc.cancer.gov/ projects. The TCGA acute myeloid leukemia data is part of the TCGA-LAML project. Following this, the data category, data type and workflow type need to be specified. The query formed below selects files containing the count level transcriptomic measurements. Values for different parameters of the query can be identified from "searching arguments" section of the "query" vignette: vignette("query", package = "TCGAbiolinks"). The result of this query will be a dataframe containing filenames and additional annotations related to the files.
Read count level data are selected instead of the processed FPKM data as one of the downstream pre-processing analysis results in filtering out of genes. A general recommendation is to compute FPKM values after filtering genes out so as to ensure counts are normalised by the corresponding library sizes. In cases where count-level data is not available, filtering can be performed directly on FPKM values, provided that the library size is large enough. "data_type" ## [3] "updated_datetime" "file_name" ## [5] "submitter_id" "file_id" ## [7] "file_size" "cases" ## [9] "id" "created_datetime" ## [11] "md5sum" "data_format" ## [13] "access" "state" ## [15] "version" "data_category" ## [17] "type" "experimental_strategy" ## [19] "project" "analysis_id" ## [21] "analysis_updated_datetime" "analysis_created_datetime" ## [23] "analysis_submitter_id" "analysis_state" ## [25] "analysis_workflow_link" "analysis_workflow_type" ## [27] "analysis_workflow_version" "tissue.definition" Downloading the TCGA AML RNA-seq read counts The GDCdownload function then executes the query on the GDC database and begins downloading the data using the GDC API. The download method should be changed to "client", if the size of the data is expected to be large, e.g for RNA-seq read data or methylation data. It is good practice to specify the directory for data storage -we store all the data in the "GDCdata" directory in the temporary directory. Users should store their data in a permanent storage to retain the data. The function downloads the data and organises it into the folder based on parameters specified in the query. This allows multiple different levels and types of data to be stored in the same directory structure. Files with counts are stored at TEMPDIR/GDCdata/TCGA-LAML/harmonized/Transcriptome_ Profiling/Gene_Expression_Quantification/. datapath = file.path(tempdir(), 'GDCdata') GDCdownload(query_rna, directory = datapath) #(size: 39MB) Reading count-level data into R The GDCprepare function reads and processes the downloaded data into a RangedSummarizedExperiment object from the SummarizedExperiment package which allows patient annotations, gene annotations and count data to be stored in one object. Patient annotations are downloaded upon calling this function and subsequently mapped and attached to the resulting object. A RangedSummarizedExperiment object is similar to an Expression-Set object but provides added functionality such as indexing with genomic coordinates and storing multiple data matrices with the same structure. Feature annotations used to annotate the data are stored in an RDA/RDATA file.
The object contains data for 56,925 features and 151 samples. The original data files contain 60,483 features, some of which (3,881) could not be mapped to ENSEMBL GRCh38.p12. Feature and sample annotations can be accessed using rowData(se) and colData(se), respectively, and the counts data can be accessed using assay(se). TCGA data usually contains some formalin-fixed paraffin-embedded (FFPE) samples which should be discarded from the analysis as the protocol introduces biological artefacts. This procedure is only performed on solid tumours and not leukemias, therefore, no filtering is required for this data set. Filter out genes with low counts The edgeR package contains methods that assist in the data normalisation and transformation required for filtering and subsequent steps. The methods require a DGEList object therefore we begin by creating a DGEList for the AML data from the SummarizedExperiment. library(SummarizedExperiment) library(edgeR) aml_dge = DGEList(counts = assay(aml_se), genes = rowData(aml_se)) Genes with low counts across most samples are discarded from the analysis. This is a standard step in differential expression analysis as inclusion of such genes in the analysis could skew estimates of dispersion. It is also motivated in rank-based analysis, such as with singscore, to avoid rank duplication. Rank duplication reduces the discriminant power of scores as the number of unique ranks is reduced. A commonly used filter is to select only those genes that have CPMs above a certain threshold across a proportion of samples. Filtering is performed on the CPMs rather than raw counts as the former accounts for variation in library sizes, therefore, is unbiased. For instance, a CPM of 1 would equate to read counts between 19 and 50 for samples in the AML data where library sizes vary between 18.6 and 49.7 million reads. Here, we retain genes that have a CPM > 1 across more than 50% of the samples. Other methods to filter out genes with low counts exist and may be preferable in specific applications. Chen et al. 19 and Law et al. 20 filter genes based on the experimental design whereby the proportion of samples with enough read counts are evaluated per experimental group. As the AML data have many samples, filtering is performed across all samples rather than within sub-groups. Group specific filtering would be recommended for the study of rare groups. The distribution of logCPMs is much closer to the expected log-normal distribution after filtering out genes with low counts as seen in Figure 1. Transformation to FPKM values and normalisation Singscore requires gene expression measurements to be comparable between genes within a sample, therefore, correction for gene length bias needs to be performed 21 . Transformations such as transcripts per million (TPM) and reads/fragments per kilobase per million (RPKM/FPKM), that normalise by gene length, may be used. Both-TPM and RPKM/FPKM values should produce similar results when applying singscore provided that the library size is large enough, which they are here. RPKM values are generally computed after correcting for compositional biases. The calcNormFactors function in edgeR provides three methods to do so, TMM normalisation being the default. Chen et al. 19 and Law et al. 20 discuss the implications of normalisation prior to down-stream processing such as differential expression analysis. Normlisation is generally performed for cross-sample analysis where samples need to be comparable. Singscores are invariant to data normalisation as the analysis is contained within the sample of interest. The idea extends to any transformation that preserves the relative ranks of genes within a sample such as a log transformation. Here, we use TMM normalisation solely for visualisation purposes.
Data transformation to TPM or RPKM/FPKM requires the lengths for all genes to be calculated. Gene lengths need to be computed based on the alignment and quantification parameters. The TCGA transcriptomic data has been aligned using STAR and quantified using HTSeq (details of the pipeline available at https:// docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/. ). HTSeq quantifies reads mapping to the exons of each gene, therefore, effective gene lengths can be calculated as the sum of all exons spanning the gene. The GENCODE v22 annotation file was used for quantification therefore the same file needs to be used to compute gene lengths. #download v22 of the GENCODE annotation gencode_file = 'gencode.v22.annotation.gtf.gz' gencode_link = paste( 'ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_22', gencode_file, sep = '/' ) download.file(gencode_link, gencode_file, method = 'libcurl') #(size: 39MB) The rtracklayer R package provides functions to help parse GTF files.

Map Ensembl IDs to Entrez IDs
Ensembl annotations (Ensembl IDs) have higher coverage of the genome which may be useful in applications such as variant calling and similar exploratory analysis 22 . However, RefSeq annotations (Entrez IDs) may be better suited to RNA-seq analyses which require a stable reference annotation for comparison 23 . As such, we choose to map Ensembl IDs to Entrez IDs and discard any unmapped features.
Mapping can be performed using the Ensembl Biomart service, which can be queried using the biomaRt bioconductor package. This would provide the most up to date annotations. Alternatively, mapping could be performed with the bi-annually updated org.Hs.eg.db annotation R package which provides a stable set of annotations, thereby enhancing reproducibility. Mapping is performed with the mapIds function in the AnnotationDbi R package.

Transcriptional signatures to predict mutation status
The signature by Verhaak et al. 12 is now used to predict the mutation status of the NPM1c mutation. This is done by quantifying the concordance of genes in the signature with their expression in each sample. As such, high expression of up-regulated genes and low expression of down-regulated genes would result in higher scores. This single value can then be used to predict the mutation status of individual samples if these data were unavailable.
The signatures of interest are first downloaded from the MSigDB and read into GeneSet objects from the GSEABase R package. We then use the singscore R/Bioconductor package to quantify each sample for the Verhaak signature. Some of the visualisation and diagnostic tools within the singscore package are used to interpret the signatures and scores. Finally, we use a simple logistic regression model on the scores to predict the mutation status.
Download signature and load into R The Verhaak et al. 12 signature is composed of an up-regulated and a down-regulated gene set. Many signatures are developed in such a manner to improve discrimination of samples. MSigDB stores such signatures using names with suffixes "_UP" and "_DN" representing the independent components of the signature. Here, we form the download links for the signature with the base name "VERHAAK_AML_WITH_NPM1_MUTATED".  Functions in the GSEABase package help with reading, parsing and processing the signatures. Signatures from an MSigDB XML file can be read using the getBroadSets function which results in a GeneSet object. Gene symbols, Entrez IDs and affymetrix chip IDs from the original experiment (HG-U133A in this case) are stored in the XML file. Entrez IDs are read from the file as these can be mapped directly to our data. Conversions to other identifiers can be achieved using the mapIdentifiers function from GSEABase and an annotation package that contains the mappings. The advantage of using this function instead of the mapIds function from the AnnotationDbi package is that the former retains the GeneSet object after conversion of IDs. To make data indexing easier during signature scoring, row names of the SummarisedExperiment object are changed to Entrez IDs which are already part of the row annotations.
rownames(aml_se) = rowData(aml_se)$entrezgene Score TCGA AML samples using the Verhaak signature Singscore is a rank based metric of gene set enrichment in single samples. Scores for multiple signatures make use of the same ranked expression per sample. As such, it makes sense to compute the ranks only once and re-use them for scoring different signatures. The implementation separates these two phases of the analysis to reduce the computational cost of scoring. The rankGenes function will compute ranks from expression data in the form of either a numeric matrix, numeric data frame, ExpressionSet object, DGEList object or a SummarizedExperiment object. Users also have to specify what method should be used to break ties. The default is 'min' and we recommend this be used for RNA-seq data which may have many genes with zero counts. This will reduce the effect of zeros in the scores, however, appropriate pre-filtering of genes with low counts will still be required.

library(singscore)
#apply the rankGenes method to each version of the dataset, excluding counts aml_ranked = rankGenes(assay(aml_se, 'logFPKM_TMM')) Singscores can be computed using three modes, depending on the properties of the gene signature. The first mode of operation is applied when two directed gene sets (expected up-and down-regulated gene sets) form the transcriptomic signature. Many signatures in the MSigDB, including the Verhaak et al. 12 signature come in such pairs. This mode can be invoked by passing the up-and down-regulated gene sets to the arguments upSet and downSet respectively. In some cases, only one set of genes forms the signature. If all genes in the gene set are up-regulated or all down-regulated, the second mode of operation applies and is invoked by passing the gene set to the upSet argument. For sets of down-regulated genes, the score would simply be inverted (-score if scores are centered, 1 -score otherwise). Finally, if the user is unsure of the composition of genes in the gene-set, such that, the gene set may contain both up-and down-regulated genes, the final mode of singscore applies. The gene set is passed to the upSet argument similar to the previous mode with the additional argument knownDirection set to FALSE.
By default, singscores are centered such that the range of scores is [-1, 1] and [-0.5, 0.5] for the first two modes respectively. Negative scores indicate an inverse enrichment of signatures, that is, expected up-regulated genes are in fact down-regulated and vice-versa. Scores from the last mode can not be centered and have the range [0, 1]. In this mode, high scores are obtained when ranks of genes are distant from the median and low scores obtained when ranks converge to the median rank. If scores are centered in this scenario, it would lead to the conclusion that a negative score shows inverted enrichment, which is not the case. Score centering only serves the purpose of easing interpretation for users, a simple linear transformation is applied to achieve it.
Scores for the NPM1c mutation signature are computed using the default settings, with the first mode of operation being used due to the presence of an up-and down-regulated gene set. The function returns a data frame reporting the score and dispersion of ranks for the up-regulated gene set, down-regulated gene set and the combination of both. Dispersion of the combined gene set in this mode is simply the mean of the independent dispersion estimates. If any gene names/IDs are present in the signature but missing in the expression data, a warning will be reported. It should be noted that singscores are composed of two components, an enrichment score and a dispersion estimate of ranks. The quantity of interest in gene set enrichment is the distribution of the expression or ranks of genes in the signature. In an ideal scenario, all expected up-regulated genes would have high expression therefore higher values of ranks. As such, ranks would be distributed on the higher end of the entire rank spectrum. Singscore aims to quantify this distribution of ranks, therefore, computes and reports the average and dispersion of ranks of genes in the signature relative to all other genes. The first quantity is similar to scores computed from all other single sample scoring methods. We determined a two component score to be a more appropriate and accurate representation of the distribution of ranks of signature genes. The default and recommended measure of dispersion is the median absolute deviation (MAD) due to its non-parametric property. Other appropriate measure of dispersion could be the inter-quartile range (IQR) and can be used by passing the IQR function as an argument to the dispersionFun argument. Diagnostics of the Verhaak signature The singscore package provides a set of visualisation tools that enable diagnostics of the gene signature. For instance, these tools may be used to determine the importance of each component for a bidirectional signature (up-and down-regulated gene sets) to the total score, determine the importance of each gene of a signature in discriminating between the classes of interest, and to investigate the relationship between the final score and the dispersion of signature gene ranks. Sample annotations of interest (e.g. clinical annotations) can be colour coded on each plot. Singscore supports both continuous and categorical annotations, which can either be input as a vector, or as a string specifying a column within the score data frame. We begin by investigating the relationship between the score and dispersion of ranks for the up-regulated gene signature, down-regulated gene signature and the full signature. The plotDispersion functions generates a diagnostic plot with annotations overlaid. Annotations can be discrete or continuous, and can be passed as independent variables, or as a column name when the data is appended to the score data frame. It should be noted that all plotting functions in singscore can be made interactive by setting the isInteractive argument to TRUE.
#relative size of text in the figure relSize = 0.7 Figure 2 shows that the set of down-regulated genes has more discriminant power in separating NPM1c mutated samples from those those without. Moreover, the signature is able to discriminate MLL (KMT2A) fusions and PTDs from the other samples. NPM1c mutations produce higher scores for the down-regulated gene set while MLL fusions and PTDs produce moderate scores. Similar trends are observed with the set of upregulated genes; however, despite the range of scores increasing, the discriminant power drops moderately. In fact, the signature of up-regulated genes is able to discriminate samples without the genomic changes of interest ('Other') better by producing negative scores for most of these samples. Negative scores for the expected up-regulated gene set indicate that these genes are expressed below median expression, therefore, likely down-regulated within corresponding samples. Another observation from these plots is based on the trend of the dispersion (MAD). The dispersion is generally expected to be higher for scores close to zero. Zero scores are achieved in three possible scenarios: when genes are expressed at median expression level; when genes are evenly distributed at both ends of the expression spectrum; or, the more likely scenario whereby the rank of expression of all genes are uniformly distributed. The last two scenarios would result in a high dispersion. To explain these ideas, we select 3 samples and plot the ranks of genes in both signatures. The sample with the highest total score, lowest total score and highest dispersion are chosen. #overwrite title of the plot with the description of the sample #this is possible because singscore uses ggplot2 p1 = p1 + ggtitle(paste(x, mutated_gene[aml_sample], sep = '\n')) + guides(colour = guide_legend(ncol = 1)) return(p1) }) #create a multipanel plot grid.arrange(grobs = rank_plots, nrow = 1) Figure 3 shows that both the up-and down-regulated gene sets contribute to the score of the top scoring sample as these genes are at the extremes ends of the rank spectrum. Similarly, the lowest scoring sample has the ranks of genes in each set inverted. As observed in the previous plot, the up-regulated genes improve discrimination between NPM1c mutated samples and other samples and may be a stronger indicator of wild-type NPM1c samples than the down-regulated genes. Finally, the sample with the maximum dispersion exhibits uniformly distributed downregulated gene ranks and a bimodal distribution of the up-regulated gene ranks with the modes at the extremes of the spectrum.
Combined, these plots show that both the up-and down-regulated genes play an important role in discriminating between NPM1c mutated (and MLL fusion/PTD samples), and wild-type samples. These diagnostic plots help in determining the importance of genes in signatures with respect to the samples of interest and should be used prior to application of signatures. In some cases, signatures may have been developed in specific contexts due to inherent biases in datasets and yet, described or applied in a generalised setting. These diagnostic plots may help in validating their application in specific contexts and possibly assist in characterising the contexts of all existing signatures.
Predicting NPM1c mutation status using the Verhaak signature Mutation status can be predicted from singscores using a logistic regression model with a "logit" link function.
The benefits of this model over one where each gene in the signature is used as a term in the model is the simplicity of the model. The Verhaak et al. 12 signature consists of 429 genes which would result in a regression model with 429 predictors. As there are fewer samples than the predictors, some feature selection method would be required which may result in the loss of information. Moreover, automating model development on a larger selection of gene signatures would be limited with gene-level models. Additionally, models trained using singscore would inherit its non-parametric properties to some extent. For instance, such models would be invariant to all data transformations that retain within sample ranks of genes. The main limitation of such models would be the loss of accuracy due to summarisation of the data (information on 429 genes is summarised into a two-component score).
In any case, our aim here is not to discuss the various models that can be used to predict mutation status, but to exhibit the discriminant power of singscore and transcriptomic signatures. consequently, the data used to train the model are used to evaluate the basic performance properties. We begin by combining the scores with mutation annotations.  To quantify the above observations, we fit logistic regression models with each genomic lesion as the response variable and the score as the predictor. Coefficient estimates, standard errors, z-statistics, and p-values of each model are listed below.

Evaluate prediction model performance
The above statistics give insight on the models trained but not on their performance. Precision and recall provide insight on a models predictive performance. The dcanr package provides functions to compute these metrics along with other measures of performance. Precision for predictions of all genomic changes other than NPM1c is undefined because all samples are predicted to be wild-types. Consequently, their recall is zero. Precision, recall and the F1 score of the NPM1c mutation model are high as expected. As singscores are two-components scores, performance may be further improved by including the dispersion of ranks. This is observed in Figure 2.
As observed from the model below, using both components of singscores significantly improves the predictive performance. Unsupervised classification of mutations There may be some cases where sample annotation is not available. In such scenarios, we are unable to build regression models to interpret scores. A higher singscore would provide stronger evidence for the signature but the magnitude is difficult to interpret without a reference. One approach to deal with this situation is to compare scores to those from other datasets where the mutations status is known. An alternative approach would be to compare scores within the dataset using unsupervised learning methods.
Here we demonstrate the use of three clustering methods (Gaussian mixture decomposition, k-means clustering, and hierarchical clustering) to stratify samples, and as we have done previously 24 use the adjusted Rand index (ARI) to compare classifications. As expected, supervised (GLM) classification results in the best prediction. This is followed by clustering based on the score using Gaussian mixture decomposition. Any other classification algorithm along with prior knowledge could be used to decompose scores into groups. The important characteristic of singscores is that they maintain the discriminant power of gene signatures therefore can be coupled with supervised, semi-supervised or unsupervised algorithms to perform stratification.

Signature landscapes with multiple signatures
Often, we are interested in the relationship between two dependent or independent phenotypes, for instance, the epithelial and mesenchymal phenotypes. The role of most signatures is to estimate an unobservable molecular phenotype so they may be considered as proxies of phenotypes. As such, we could investigate the relationship between two phenotypes using corresponding signatures. Foroutan et al. 25 first introduced the idea of molecular signature landscapes to investigate the relationship between signatures related to the epithelial-mesenchymal transition (EMT) and TGFβ-induced EMT. Subsequently, Cursons et al. 26 computed epithelial and mesenchymal phenotype signature singscores and demonstrated an epithelial phenotype shift following miR-200c transfection into a mesenchymal cell line using a signature landscape. Foroutan et al. 9 used signature landscapes to stratify breast cancer subtypes along the epithelial-mesenchymal axis and included it as part of the singscore package. Here, we show how such landscapes can be used beyond the current application of the epithelial-mesenchymal axis. We demonstrate how transcriptomic signatures of different mutations can be used to stratify AML samples. The gene set is a composition of both up-and down-regulated genes as genes were selected based on their ability to discriminate mutants from wild-types. We use the third mode of singscore, which does not require the direction of genes in the gene set to be known. The knownDirection parameter of the simpleScore function is set to FALSE to induce this mode. Ranks computed previously can be reused to compute scores with the new signature.

rossmll_scores = simpleScore(aml_ranked, rossmll_sig[[1]], knownDirection = FALSE)
The plotScoreLandscape function plots a hexbin plot to visualise the two-dimensional distribution of scores. Scores computed using the Verhaak et al. 12  Points are projected onto an existing landscape using the projectScoreLandscape function. This functions uses the p_mll_npm1c plot generated using the plotScoreLandscape and overlays new data points onto it. Scores for the new data must be computed with the same signatures that were used to compute the landscape. As we are using existing data, scores computed in earlier sections are re-used. The subSamples can be used to select a subset of samples to project. Here, we select samples with the mutations we are interested in annotating.  Ross PML-RARA fusion signature vs. Verhaak signature landscape It is evident from the previous analysis that PML-RARA samples differ from all other samples examined here. We repeat the analysis with the PML-RARA signature from Ross et al. 14 to verify this distinction. The signature is available on the MSigDB and is named "ROSS_AML_WITH_PML_RARA_FUSION". We download and parse the signature, score all samples against it, plot a landscape with scores from the Verhaak et al. 12 signature, and finally, project samples onto the plot. The signature was constructed in a similar manner to the MLL fusions signature, therefore, samples are scored using the same settings.  signatures. As discussed above (Description of the biological problem) the PML-RARA fusion is diagnostic of a specific subtype of AML known as acute promyelocytic leukemia, with a highly distinct cell phenotype reflecting a block on differentiation at the promyelocyte stage 15 . The distinct features of this subtype are correspondingly reflected in the L-shaped landscape for these two signatures and the different mechanisms by which these lesions drive leukemogenesis.

Summary
The singscore package provides an easy interface to apply gene set scoring methods within the R/Bioconductor environment. The TCGAbiolinks package allows relatively easy access to large clinically relevant data sets such as TCGA, together with appropriate annotation functions for interpreting biological data. Diagnostic and plotting functions included with singscore allow the user to investigate gene sets of interest to determine their power for distinguishing differences between samples. Different gene signatures can then be combined to explore how different cellular phenotypes are associated across a large cohort of cancer samples. As demonstrated, when appropriate gene set signatures are used, metrics calculated by singscore can be used for sample classification and this may be useful for further interrogation of large transcriptomic data sets where no genomic data are available

Packages used
This workflow depends on various packages from version 3.9 of the Bioconductor project, running on R version 3.6.1 (2019-07-05) or higher. The complete list of the packages used for this workflow are shown below:

Data availability
All data analyzed in the workflow is read automatically from public websites as part of the code. Mutation data for the samples in this study are available as part of the R/Bioconductor package SingscoreAMLMutations (v1.0.0).

Introduction
The article presents a new package, which was developed from a previous one ('singscore') elaborated by the same authors. The package 'singscore' is a single-sample gene set scoring method which is valuable for analysis of transcriptomes of samples collected through the long time and not sequenced in the same run, experiment, platform or laboratory. Here, the authors apply the method for classification of TCGA acute myeloid (AML) samples using transcriptional 'gene signatures' identified by other authors (Verhaak and Ross) as typical for the NPM1c mutation, KMT2A (MLL) gene fusions, and PML-RARA gene fusions.
AML is a heterogeneous and multi-clonal disease. The transcriptome picture of AML is very complex and can result from different mutations, genomic rearrangements, and aberrant regulation of gene expression at different levels (see my last review paper ). Sometimes, the gene expression profiles can overlap between samples with different genetic lesions. The examples are HOX-gene-based discriminative signatures determined by Verhaak and Ross, not limited to AML with mutated NPM1, but specific also for AML cases with 11q23 abnormalities and KMT2A (MLL) gene rearrangements. The authors are aware of this fact and underline it in the article, too. On the other side, samples with the same mutation can present 1 this fact and underline it in the article, too. On the other side, samples with the same mutation can present varying expression profiles because of additional features which also affect the transcriptome. Therefore, interpretation of the results must be done with caution. For me, the authors interpret the results a little bit too optimistically. The presented method is not sufficient to determine the mutation of interest, but can be used as a supplementary approach, a screening tool or (in future) a personalized medicine tool which could classify patients based on transcriptomic profiles, associated with specific treatment response.
Testing package I have used the package in R with TCGA data and confirm the code works, it is fast and generates the same results and plots as presented in the article. Moreover, I have tested the whole procedure on my own RNA-seq dataset (not published yet but used for supplementary expression analysis of NPM1 alternative transcripts, see the article ). My dataset contains 28 AML samples, including 8 with NPM1 mutation (verified with three independent approaches). The data load was more tricky as I started from the csv table with counts and I had to convert the DESeqDataSet object into the RangedSummarizedExperiment object. The best discrimination was achieved with the Verhaak signature, but only with the down-regulated genes. All samples with NPM1 mutation were clearly separated from others, however, 2 additional samples, without NPM1 mutation, were grouped together with NPM1c. It is possible they have KMT2A (MLL) rearrangements, but I cannot verify it now. Analyzing the plots, up-regulated genes and all genes form the signature are not as efficient, separating only 2 or 3 NPM1c samples from the rest. In the article, the authors also admit that the set of down-regulated genes has more discriminant power, but they claim up-regulated genes also contribute to the discrimination.
I have no samples with PML-RARA fusion in my dataset and the status of KMT2A (MLL) gene was unknown in my patients, so I could not compare the efficiency of signatures other than NPM1c. APL with PML-RARA is the most distinctive AML subtype which is easily separated from other AML samples based on transcriptome profile, so I would expect good results. This example shows the package works well for samples with very specific gene expression profile.

Interpretation of the results
Considering the result interpretation, I have some doubts. The singscores are composed of two components, an enrichment score and a dispersion estimate of ranks. I am informed that "high expression of up-regulated genes and low expression of down-regulated genes would result in higher scores". This is logical. I also know the maximal ranges ([−1, 1] for signatures involving up-and down-regulated genes). However, which value is really high? For example, is 0.2 enough (it looks like from the plots in the article and from my own data, too) or maybe I should expect much more, e.g. 0.7? Similarly, which value of dispersion should I expect? I suppose, the lowest, but which is low enough or which range is optimal? What is difficult to understand is that "despite the range of scores increasing, the discriminant power drops moderately" (for up-regulated genes from Verhaak signature, when compared to down-regulated genes). I see the same paradox in my data -the scores are higher for up-reg. genes, which are much less efficient in discrimination between NPM1c+ and NPM1c-samples. It looks like scores only do not reflect the trends observed from score vs dispersion plots.

Possible improvements
Although the package is generally useful and well described, the following improvements will make it more friendly for less advanced R users, e.g.
Instruction how to prepare and load the data other than those deposited in the GDC database. Annotation of samples on the plots with unique sample IDs (when a sample is misclassified, a user does not know which one it is; it will also be helpful to localize a sample on various plots, e.g. generated with a signature of up-and down-regulated genes) -it is written in the article that the 'singscore' package supports different types of annotations ("Annotations of interest can be 2 'singscore' package supports different types of annotations ("Annotations of interest can be overlayed on each plot") but I found only color code annotation whereas I would like to have color code for mutation type and additional text labels with sample IDs on the same plot.
A command which lists the samples typed as strong candidates for having particular mutation, ordered according to the calculated metrics.
Generating a threshold line between samples with and without mutation. In future, it would be also good to include other signatures, e.g. the signature of 369 genes identified by Alcalay . in 2005 , discriminating AML patients with NPMc+ from patients with NPMc-, even in cases et al with rare chromosomal abnormalities.
What I would expect the most from a package designed to identify mutations in transcriptomes, is a direct identification of mutations in RNAseq data. The results of mutation typing based on gene expression profile will be strongly supported when a particular mutation will be covered by RNAseq reads. From my own experience, I know it is possible (for NPM1 mutation detection, see ). For genes with high and middle expression level, the coverage can be even higher than that obtained from genome-or exome-level data. And in the case when DNA data are unavailable, it would be really fantastic. Because it demands completely different data processing, it may be considered for future versions of the package.

Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly
Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly No competing interests were disclosed. Competing Interests: Reviewer Expertise: Genomics, transcriptomics, biology of acute myeloid leukemia.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Introduction
: The article presents a new package, which was developed from a previous one ('singscore') LH elaborated by the same authors. The package 'singscore' is a single-sample gene set scoring method which is valuable for analysis of transcriptomes of samples collected through the long time and not sequenced in the same run, experiment, platform or laboratory. Here, the authors apply the method for classification of TCGA acute myeloid (AML) samples using transcriptional 'gene signatures' identified by other authors (Verhaak and Ross) as typical for the NPM1c mutation, KMT2A (MLL) gene fusions, and PML-RARA gene fusions.
: We note that the purpose of the initial manuscript may have been unclear, in part because it DDB is listed under the F1000 Software Tool Article section. The purpose of this article is to present a workflow demonstrating the use of singscore, and it is a R/Bioconductor workflow illustrating the use of singscore, and so is not intended as a new package or tool. https://www.bioconductor.org/packages/release/workflows/ https://www.bioconductor.org/packages/release/workflows/html/SingscoreAMLMutations.html Some of the review comments below assume that this manuscript presents a specific software package for detecting NPM1 mutations, so we have clarified the manuscript as outlined below in order to make the purpose and intention more clear (note that the title of the manuscript has changed from 'predicting mutations' to 'predicting mutation status' based upon feedback from reviewer 1). This work arose from an observation in another project that the Verhaak signature scored with singscore appeared to correlate strongly with mutation status, and we thought this would be an interesting example that might also help researchers to investigate the links between genetic lesions and corresponding transcriptional changes, an area in which the reviewer clearly has lesions and corresponding transcriptional changes, an area in which the reviewer clearly has expertise.
: AML is a heterogeneous and multi-clonal disease. The transcriptome picture of AML is very LH complex and can result from different mutations, genomic rearrangements, and aberrant regulation of gene expression at different levels (see my last review paper ). Sometimes, the gene expression profiles can overlap between samples with different genetic lesions. The examples are HOX-gene-based discriminative signatures determined by Verhaak and Ross, not limited to AML with mutated NPM1, but specific also for AML cases with 11q23 abnormalities and KMT2A (MLL) gene rearrangements. The authors are aware of this fact and underline it in the article, too. On the other side, samples with the same mutation can present varying expression profiles because of additional features which also affect the transcriptome.
: Therefore, interpretation of the results must be done with caution. For me, the authors interpret LH the results a little bit too optimistically. The presented method is not sufficient to determine the mutation of interest, but can be used as a supplementary approach, a screening tool or (in future) a personalized medicine tool which could classify patients based on transcriptomic profiles, associated with specific treatment response.

DDB:
We agree with the reviewer that gene set scoring of transcriptomic data should not be the only method used to identify patient samples carrying genetic lesions. The Bioconductor workflow we present is intended to provide an example of applying singscore to study mutation/fusion based gene sets as we believe that singscore provides a relatively flexible and intuitive approach for investigating different gene sets across large data sets. We feel that a particularly useful feature is the ability to combine different signatures/gene sets (including phenotype/cell-cycle signatures etc) to explore how these transcriptional changes are associated over different samples, demonstrated in Figures 5-7. We have modified the "Description of biological relevance" section to address the reviewer's comments around the complexity of AML genomic lesions and corresponding transcriptomic changes. The review highlighted is particularly relevant for this work and accordingly we mention the complexity of AML and direct the reader towards this resource by extending the first paragraph (new text ): underlined .. A more recent study which has focused primarily on genomic data has further refined the clinically significant AML subtypes [Papaemmanuil (2016), NEJM], highlighting a number of co-occurring as well as mutually exclusive mutations. As the identification of putative driver fusions/mutations continues, work has also been directed towards how these lesions interact with each other and other features (e.g. cellular proliferation, changes due to phenotypic plasticity, or variation in post-transcriptional regulators such as microRNAs) to drive transcriptional changes as discussed in a recent review [Hanschuch (2019), J. Oncol.].
We have also added a paragraph at the end of this section which discusses some of the limitations for our approach and provides more context in which it could be applied: While we demonstrate that singscore is capable of inferring mutation status from the transcriptional profile of AML samples, we note that it is best used to supplement alternative data which can provide a more definitive resolution of these lesions. Processing of raw RNAseq data will directly identify the presence of genefusion products or mutations within protein-coding regions, although for many large data sets the quantified transcript abundance data are much easier to obtain without access agreements. The method can also be applied to legacy microarray data sets where genome and RNA sequencing data are unavailable. As such singscore provides a useful approach to supplement established methods for the study of genetic lesions in cancer. By exploring 1 to supplement established methods for the study of genetic lesions in cancer. By exploring associations between different genomic and phenotypically relevant signatures, it may also help to better characterise true driver mutations which exert consistent effects on the transcriptome of AML samples and other cancers.

Testing package
: I have used the package in R with TCGA data and confirm the code works, it is fast and LH generates the same results and plots as presented in the article. Moreover, I have tested the whole procedure on my own RNA-seq dataset (not published yet but used for supplementary expression analysis of NPM1 alternative transcripts, see the article ). My dataset contains 28 AML samples, including 8 with NPM1 mutation (verified with three independent approaches). The data load was more tricky as I started from the csv table with counts and I had to convert the DESeqDataSet object into the RangedSummarizedExperiment object. The best discrimination was achieved with the Verhaak signature, but only with the down-regulated genes. All samples with NPM1 mutation were clearly separated from others, however, 2 additional samples, without NPM1 mutation, were grouped together with NPM1c. It is possible they have KMT2A (MLL) rearrangements, but I cannot verify it now. Analyzing the plots, up-regulated genes and all genes form the signature are not as efficient, separating only 2 or 3 NPM1c samples from the rest. In the article, the authors also admit that the set of down-regulated genes has more discriminant power, but they claim up-regulated genes also contribute to the discrimination.
: I have no samples with PML-RARA fusion in my dataset and the status of KMT2A (MLL) gene LH was unknown in my patients, so I could not compare the efficiency of signatures other than NPM1c. APL with PML-RARA is the most distinctive AML subtype which is easily separated from other AML samples based on transcriptome profile, so I would expect good results. This example shows the package works well for samples with very specific gene expression profile.

DDB:
We thank the reviewer for the exceptional effort and time invested to test our workflow on independent data -we hope results from this analysis have been informative for identifying other features within their data. Our workflow includes guidance for users who wish to import data from other sources such as those used by the reviewer.

Interpretation of the results
: Considering the result interpretation, I have some doubts. The singscores are composed of LH two components, an enrichment score and a dispersion estimate of ranks. I am informed that "high expression of up-regulated genes and low expression of down-regulated genes would result in higher scores". This is logical. I also know the maximal ranges ([−1, 1] for signatures involving upand down-regulated genes). However, which value is really high? For example, is 0.2 enough (it looks like from the plots in the article and from my own data, too) or maybe I should expect much more, e.g. 0.7? Similarly, which value of dispersion should I expect? I suppose, the lowest, but which is low enough or which range is optimal? What is difficult to understand is that "despite the range of scores increasing, the discriminant power drops moderately" (for up-regulated genes from Verhaak signature, when compared to down-regulated genes). I see the same paradox in my data -the scores are higher for up-reg. genes, which are much less efficient in discrimination between NPM1c+ and NPM1c-samples. It looks like scores only do not reflect the trends observed from score vs dispersion plots.
: Interpretation of singscores is intentionally left to be problem specific as it generally requires DDB some domain-specific knowledge of the biological system and corresponding signature genesideally the computational biologist or bioinformaticians working on each project can provide some 2 ideally the computational biologist or bioinformaticians working on each project can provide some guidance. The basic interpretation of the score is the normalised mean rank of genes within the signature relative to all other genes in the sample. At the extrema this interpretation is relatively simple -near 1, a higher value would indicate that genes in the signature are expressed at higher levels relative to other genes. For scores towards zero, however, the interpretation can be much more difficult -a score of zero (on the range [-1,1]) could indicate the signature genes are tightly clustered around the sample-wide mean abundance, or it could indicate a highly-dispersed almost uniform distribution across the entire abundance range (with a mean near the mean of all genes). By exploring the singscores together with dispersion estimates this information is summarised, assisting in estimation of effect size variability. Interpretation of scores depends on the context of the experiment and the typical behaviour of the gene set. A "high" score is best determined relative to other samples. This is achieved either by comparing scores from other samples in large datasets such as TCGA, or better, across a set of samples from a given experiment with known conditions. Other methods normalise the data before computing scores, and we note that a recent paper has applied z-score normalisation to results from singscore for comparison to ssGSEA [ : Although the package is generally useful and well described, the following improvements will LH make it more friendly for less advanced R users, e.g.
: Instruction how to prepare and load the data other than those deposited in the GDC database. LH : We have noted in text that the rank matrix can be computed using either a DDB SummarizedExperiment object, DGEList object, ExpressionSet object, numeric matrix or a numeric data frame. As such, a numeric matrix with sample names as column names and genes as row names would suffice. Scoring should be performed on length bias corrected measurements such as RPKM/FPKM or TPM and not CPM or raw counts. Transcriptional signatures to predict mutation status/Score TCGA AML samples using the Verhaak signature -extract from text: "The `rankGenes` function will compute ranks from expression data in the form of either a numeric matrix, numeric data frame, ExpressionSet object, DGEList object or a SummarizedExperiment object" : Annotation of samples on the plots with unique sample IDs (when a sample is misclassified, a LH user does not know which one it is; it will also be helpful to localize a sample on various plots, e.g. generated with a signature of up-and down-regulated genes) -it is written in the article that the 'singscore' package supports different types of annotations ("Annotations of interest can be overlayed on each plot") but I found only color code annotation whereas I would like to have color code for mutation type and additional text labels with sample IDs on the same plot.
: Sample labels can be added to landscape plots but were not supported in other DDB visualisations. We have added functionality to the latest version of the singscore package (v1.5.1) to allow labelling of samples in the score vs. dispersion plots. We have modified the text to clarify, and modified Figure 6 to label samples where classification uncertainty (NMP1c vs WT) is high to demonstrate this feature. The changes below have been made to the section: "Transcriptional signatures to predict mutation status/Diagnostics of the Verhaak signature". … . … Sample annotations of interest (e.g. clinical annotations) can be colour coded on each plot  A command which lists the samples typed as strong candidates for having particular mutation, LH ordered according to the calculated metrics.
: As discussed in an earlier comment, we recommend such analyses to be problem specific. DDB Generally, a higher score would indicate a stronger effect of genes in the signature relative to WT samples therefore samples with higher scores would be stronger candidates for mutations. Alternatively, the partitioning created using Gaussian mixture modelling could be used as a guide for separation and samples with a score much higher than the threshold would be the strongest candidates for the mutation.
: Generating a threshold line between samples with and without mutation. : What I would expect the most from a package designed to identify mutations in LH transcriptomes, is a direct identification of mutations in RNAseq data. The results of mutation typing based on gene expression profile will be strongly supported when a particular mutation will be covered by RNAseq reads. From my own experience, I know it is possible (for NPM1 mutation detection, see ). For genes with high and middle expression level, the coverage can be even higher than that obtained from genome-or exome-level data. And in the case when DNA data are unavailable, it would be really fantastic. Because it demands completely different data processing, it may be considered for future versions of the package.
: As we have outlined at the start of this review there may have been some misunderstanding DDB around the purpose of this Workflow package/paper. We agree with the reviewer that direct detection of mutations/fusions from RNA-seq data is the best approach, and we now note this in the 'Description of biological relevance section' as stated above. The other gene signatures mentioned above could be incorporated in a workflow as singscore supports analysis and comparison of multiple gene sets.
No competing interests were disclosed.

Competing Interests:
shorten the paragraph by first mentioning the issue introduced by the function, then saying edgeR is applied to the data filtered of duplicate entries. (TCGAbiolinks v2.12.2). Consequently, we will be removing the text describing the identification and removal of duplicated features. The text removed is: ------------------------------Creation of a DGEList object is initially unsuccessful due to duplicated row names in the data matrix (107 features). The raw count files for individual samples do not have any duplicated features indicating this is introduced by the `GDCprepare` function. This is further supported by the fact that the counts for a single feature are the same for duplicated entries. An example of such a feature is shown below and we have verified duplicated entries of all other features to be the same.  Figure 3. The legend below each graph is identical. I suggest to only use one legend for all three plots.

DDB: The issue of duplicated features with the TCGAbiolinks package has been fixed in a recent update
DDB: We appreciate this point, however each plot in this figure is generated by a function in the package which includes the legend. For a normal use case, users would be generating a single plot to diagnose a single sample, which is why we include a legend for the plot. In this workflow, we simply combine the three plots into a single figure. Removing legends from the default plots would overcomplicate the code of this diagnostic procedure in the workflow which is why we decided to leave the legends as they are. If users were interested in manipulating figures in their application, they could add extra options to the ggplot2 object (as we show with adding a new title and modifying the legend to be a single column).
5. The authors comment on several instances about the similarities between scores for signatures of two subgroups of AML samples, NPM1c and KMT2A-/MLL-fusion, and that's due to the presence of Hox genes. I suggest to have the lists of the two gene signatures directly in the article for immediate lookup. This is not essential, as the authors already refer to the articles they take the