Tumor characterization and stratification by integrated molecular profiles reveals essential pan-cancer features
© Liu and Zhang. 2015
Received: 2 January 2015
Accepted: 5 June 2015
Published: 7 July 2015
Identification of tumor heterogeneity and genomic similarities across different cancer types is essential to the design of effective stratified treatments and for the discovery of treatments that can be extended to different types of tumors. However, systematic investigations on comprehensive molecular profiles have not been fully explored to achieve this goal.
Here, we performed a network-based integrative pan-cancer genomic analysis on >3000 samples from 12 cancer types to uncover novel stratifications among tumors. Our study not only revealed recurrently reported cross-cancer similarities, but also identified novel ones. The macro-scale stratification demonstrates strong clinical relevance and reveals consistent risk tendency among cancer types. The micro-scale stratification shows essential pan-cancer heterogeneity with subgroup-specific gene network characteristics and biological functions.
In summary, our comprehensive network-based pan-cancer stratification provides valuable information about inter- and intra- cancer stratification for patient clinical assessments and therapeutic strategies.
Cancer largely results from various molecular aberrations comprising somatic mutational events such as single nucleotide mutations, copy number changes and DNA methylations [1–3]. In addition, cancer is viewed as a wildly heterogeneous disease, consisting of different subtypes with diverse molecular implementations of oncogenesis and therapeutic responses. Many organ-specific cancers have established definitions of molecular subtypes on the basis of genomic, transcriptomic, and epigenomic characterizations [1–3], indicating diverse molecular oncogenic processes and clinical outcomes. The molecular-defined intrinsic breast cancer subtypes (luminal A, luminal B, HER2-enriched, basal-like, and normal-like) are typical examples, since they have been reported to be associated with distinct phenotype outcomes and have different chemotherapy responses and respective stratified therapy [4–8]. Similarly, endometrial cancers have also been classified into four categories (POLE ultramutated, microsatellite instability hypermutated, copy-number low, and serous-like) through a comprehensive, multiplatform analysis , and glioblastoma multiformae was stratified into four distinct molecular subtypes (proneural, neural, classical, and mesenchymal) based on the CpG island methylation phenotype . Different tumor subtypes of the same organ reflect diverse molecular oncogenic processes and various clinical outcomes, which imply that they should be treated as different cancers for treatment design in some sense .
Key genomic similarities shared by subgroups of patients across cancer types would present an opportunity to design tumor treatment strategies among tumors regardless of tissue or organ of origin and enable the extension of effective treatments from one cancer type to another . For example, the molecular commonalities between basal-like breast tumors with high-grade serous ovarian tumors indicate a related etiology and similar therapeutic opportunities . However, the current tumor heterogeneity is mostly defined for tumors of the same organ without considering the potential cross-cancer benefits. Thus, deciphering tumor heterogeneity for all cancers based on their genomic characteristics is an urgent issue.
In the past, insufficiency of high quality genomic datasets of a large number of patients across different tumor types has impeded such investigations. With great advancement in high-throughput sequencing technologies and comprehensive efforts of systematic cancer genomics projects (e.g., the Cancer Genome Atlas pan-cancer project ), studies on molecular aberrations of cancer patients have increased unprecedentedly in scale and accessibility, enabling large-scale integrative cross-cancer analysis . Very recently, Hoadley et al. conducted a comprehensive integrative analysis using data from six independent omics platforms on 3,527 specimens from 12 cancer types and reported a unified classification into 11 major subtypes (originally, there were 13 classes and 2 classes only had 3 samples and 6 samples respectively) .
Cancer has long been considered as a disease of combinations of functionally related alterations at the network level. In recent years, the molecular network as a simple but efficient presentation of complex interactions and regulatory relationships between molecules has been adopted comprehensively for understanding system-level properties of complex disease. However, Hoadley et al. only adopted very limited information on pathways and failed to employ a large-scale molecular interaction network . In contrast, we believe that aggregating genomic characterizations of patients using gene networks would contribute to identifying subgroups of patients with similar molecular-network patterns affected by diverse genetic alterations.
In this study, we adopted a network-based stratification (NBS) approach  to integrate key genetic and epigenetic features of 3299 tumor samples from 12 cancer types  to uncover novel pan-cancer heterogeneity. We found that our pan-cancer stratification is predictive of clinical outcomes, and different cancer patients falling into the same subgroup show consistent survival tendency or grade/stage severity. We identified subgroup-specific genomic alterations and networks that are responsible for distinguishing each subgroup. These subgroup networks demonstrate specific genomic characteristics and biological functions. In summary, our cross-cancer stratifications not only revealed most recurrently reported cross-cancer similarities, but also novel patient groupings, implying valuable messages for patient clinical assessments and therapeutic strategies.
Overview of the pan-cancer stratification analysis
We can observe clear consistency between every successive two classifications (e.g., k = 6 versus k = 7) of the samples (Fig. 1b). In particular, two patient subgroups were consistently identified across all 3 ~ 15 classes (samples denoted by light blue and green in Fig. 1b). One subgroup was dominated by KIRC tumors. KIRC has been reported to have a high frequency of Von Hippel-Lindau (VHL) mutation and show distinct exclusivity from other 11 cancer types . The other subgroup consists of subsets of GBM, BLCA, LUSC, and HNSC tumors. The similarity of these tumors has been implicated in the mutation or amplification of ERBB2-HER2 . The remaining patients are progressively subdivided into new subgroups as the number of classes gets larger. We will further explore those representative subgroups in terms of macro-scale (with k = 3) and micro-scale (with k = 9 and Fig. 1c) classes in the following subsections.
Macro-scale pan-cancer subgroups reveal clinical relevance
Lastly, we found that a large fraction of KIRC tumors and a subset of UCEC tumors were significantly enriched in subgroup 2 (Fig. 2a). Those KIRC tumors and UCEC tumors in subgroup 2 tend to be patients at early tumor stage and low grade (Fig. 2g, h). More than half of the KIRC tumors in subgroup 2 are at Stage I, and no UCEC tumor in subgroup 2 is at Stage IV and high grade. All these observations demonstrate that our pan-cancer macro-scale stratification reveals strong clinical relevance and shows consistent clinical tendency in some cancer types, implying distinct pan-cancer heterogeneity as well as oncogenic mechanisms.
Hoadley et al.  reported patient overall survival of their 11 identified subtypes, which is very similar to the overall survival observed in the original cancer types, indicating limited contribution to the prognosis evaluation and stratified therapy of patients. However, we note that the comparison of patient survival among cancer tissue types is limited to some extent. For example, GBM or LAML patients are often associated with poor prognosis compared to relatively moderate BRCA or UCEC patients. Seen from this angle, our pan-cancer macro-scale stratification divides almost all cancers into subgroups with consistent good or poor survival rates, revealing underlying pan-cancer similarities among cancer types and providing valuable information for patient clinical assessments and stratified therapeutic strategies.
Micro-scale pan-cancer subgroups reveal abundant cross-cancer similarities
We first found that 94.4 % of the tumors in subgroup-5 were KIRC types, making this subgroup highly exclusive to a single cancer type, and more than half of tumors (56.8 %) in subgroup-4 were BRCA types. In contrast to these two subgroups dominated by individual cancer type, other subgroups consist of multiple cancer types. For example, subgroup-7 is significantly enriched with a large fraction of GBM (60.6 %), HNSC, LUSC, and BLCA tumors. In subgroup-6, 59.1 % of LAML tumors and three molecule-defined COADREAD subtypes were clustered together, indicating potential commonalities between solid and liquid tumors (Fig. 3a).
We next explored the network modules consisting of significant differentially influenced genes for each subgroup (see Methods). We can see that the overlap of these gene sets is very limited, indicating that these gene sets are highly specific to a subgroup (Fig. 3b). Moreover, the biological functional annotations of these 9 gene sets are also very specific to individual subgroups (Fig. 3c, d).
Micro-scale pan-cancer subgroups demonstrate distinct subgroup-specific patterns
Multiple cancer types or subtypes including COADREAD-ultra and UCEC as well as BRCA-luminal A tumors are significantly enriched in pan-cancer subgroup-1 (Fig. 3a). This subgroup was marked by mutations of multiple genes that exhibit a mutually exclusive pattern in this cohort (Fig. 4 and in Additional file 1: Figure S4). Both PTEN and PIK3CA alterations were reported to have strong relationships with UCEC and COADREAD, and the loss of PTEN expression is also observed to be associated with PIK3CA mutations in metastatic colorectal cancer [21–25]. Altered PTEN expression was viewed as a diagnostic marker for early detection of UCEC , and is associated with favorable clinical and pathologic characteristics . In addition, PIK3CA mutations were reported to be present in approximately 25 % of breast cancers, particularly the estrogen receptor–positive subtypes, while they are absent in the basal-type breast cancer . This is consistent with the fact that luminal A breast tumors are significantly enriched in this subgroup. The mutation of PTEN and PIK3CA together with other alterations of genes affects a common biological network, which reflects the major similarities among subgroup-1 tumors (Fig. 5b in Additional file 1). Moreover, high methylation frequency of MLH1 was observed exclusively in the UCEC-MSI cohort of subgroup-1 (in Additional file 1: Figure S4), confirming that MLH1 promoter methylation is the primary cause of microsatellite instability in sporadic endometrial cancers . Finally, many subgroup-1-specific altered genes including PIK3CA show significant differential expression in subgroup-1 compared to all other patients (Fig. 5b), indicating the potential associations with downstream expression changes.
Subgroup-7 was characterized by the copy number deletion on chromosome 9p21 (98.4 % CNA deletion; Fig. 4 and in Additional file 1: Figure S10). Genes located in this region include CDKN2A, CDKN2B, KLHL9, and MTAP as well as the IFNA gene family. More than half of GBM (60.6 %) were clustered in subgroup-7 with other significant enriched cancer types of HNSC, LUSC, and BLCA (Fig. 3a and Fig. 6b in Additional file 1). This subgroup demonstrates a typical cross-cancer similarity phenomenon that subsets of samples from different tumor types are characterized by the same genomic alterations on chromosome 9. The associations of the deletion of tumor suppressor genes CDKN2A, CDKN2B, and MTAP with the four significant enriched cancer types in this subgroup have been widely investigated and reported [30–34]. IFNA1, 2, 6, 8, 9, and 13 are members of the alpha-interferon genes cluster on chromosome 9. Interferons are encoded by IFNA genes in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. They activate immune cells, trigger the protective defenses of the immune system, and eradicate pathogens or tumors. As is known, viruses cause 10–15 % of all human cancers, and inflammation promotes oncogenesis in the evolution of cellular transformation [35, 36]. It was reported that human papilloma virus (HPV) types 16 and 18 were detected in HNSC and played an important role in carcinogenesis of this cancer . Similar discoveries show that HPV is the second most important cause of lung cancer after cigarette smoking . Shokeir et al.  showed that the carcinogenesis of bladder cancer is likely related to bacterial and viral infections. In addition, another study also suggested that HPV infection status could be considered as an independent prognostic factor for GBM and recognized as a causative agent in gliomagenesis . The lack of expression due to the deletion of IFNAs may be responsible for the HPV infection in carcinogenesis of these cancers; however, their relationships need to be further investigated. Subgroup-7 has shown distinct gene expression differences such as that of CDKN2A, CDKN2B, MATP, KLHL9, IFNA2, and IFNA6 with extremely low q-values, which could be explained by the ~100 % copy number deletion on chromosome 9 in subgroup-7 (Fig. 6b).
Subgroup-2 mainly consists of LUAD and BRCA tumors, which were characterized by the amplifications on chromosome 1 involving UBQLN4, SETDB1, MDM4, ENSA, and so forth (in Additional file 1: Figure S5). The largest patient group, subgroup-3 enriched with BRCA-basal, UCEC-serous, and OV tumors, was characterized by multiple recurrent chromosomal gains and losses (in Additional file 1: Figure S6A). The amplification of oncogene MYC occurs in 30.8 % of samples in subgroup-3. BRCA-basal, UCEC-serous, and OV patients in this cohort are associated with a high mutation rate of TP53 (88.4 %) (in Additional file 1: Figure S6B), which was consistent with previous observations [11, 14]. Amplification of 11q13 involving CCND1, ORAOV1, and ANO1 was dominated in subgroup-4, mainly consisting of luminal BRCA and HNSC (in Additional file 1: Figure S7). These estrogen-receptor positive luminal tumors are significantly enriched in this subgroup, while basal-like breast cancers are not. Amplification and overexpression of CCND1 would alter cell cycle progression and contribute to tumorigenesis. Previous studies have shown that luminal cancers harbor recurrent amplifications and overexpression of CCND1, whereas basal-like tumors harbor recurrent deletions and down-regulation of it [41, 42]. Subgroup-8, mainly consisting of LUSC, HNSC, and OV tumors, was characterized by 100 % copy number gain on chromosome 3q26 involving genes PIK3CA, KCNMB3, KCNMB2, MFN1, GNB4, MECOM, ZMAT3, SOX2, and KCNJ13 (in Additional file 1: Figure S11). Subgroup-9, mainly consisting of HNSC, OV, and COADREAD, was characterized by a distinct TP53 mutation rate (98.6 %, in Additional file 1: Figure S12).
In this paper, we adopted a network framework to integrate the alteration profile of 12 cancer types to reveal essential pan-cancer heterogeneity among diverse cancers. Without considering the primary tumor organ information, all tumors were clustered into pan-cancer subgroups, which allowed us to discover important cross-cancer commonalities. In a recent study, Ciriello et al.  revealed two major classes, the M class (dominated by mutation) and the C class (dominated by CNAs), and further derived a hierarchical classification of patients based on the binary event data by repeating the algorithm on each newly identified class. However, this process affects the identification of tumor heterogeneity and ignores the cross-cancer similarities embodied in pathways and networks. Our network-based stratification can conquer these limitations of the sparsity of the discrete binary data and the lack of information on neighboring genes.
Specifically, among the 11 classes identified by Hoadley et al., five show near one-to-one relationships with tissue of origin, while only one subgroup was found in our PC9 subgroups (KIRC specific subgroup-5; in Additional file 1: Figure S13). This repeated finding further confirms the highly exclusive molecular characteristics of KIRC compared to others. We also clustered BRCA luminal tumors and basal-like tumors into two separate classes (subgroup-3 and subgroup-4) as done by Hoadley et al., emphasising the intrinsic divergence of this tumor (in Additional file 1: Figure S13). The most important cross-cancer class in Hoadley et al.  is the squamous-like subtype, which consists of LUSC, BLCA, and some BLCA. Similar observations in our work can be found in subgroup-7 with additional enriched GBM samples (in Additional file 1: Figure S13). Both studies reported the loss of CDKN2A in this patient cohort; however, our subgroup-7 was characterized by the copy number deletion on chromosome 9p21 with nearly 100 % frequency. We also found that the loss of IFNA family genes in this group may be related to the virus infection in carcinogenesis of these tumors. Our results revealed the known cross-cancer similarities between basal-like and serous OV, however, which was failed to be clustered together in Hoadley et al. [12, 14] (in Additional file 1: Figure S13). In addition, our study reveals more cross-cancer similarities that were not reported in Hoadley et al. such as the hypermethylation of MGMT and other genetic characteristics shared by subsets of LAML and UCEC in subgroup-6 and the 100 % copy number gain on chromosome 3q26 in fractional OV, LUSC, and HNSC in subgroup-8 (in Additional file 1: Figure S13).
Finally, in order to evaluate the robustness of our classification to obtain the 9 pan-cancer subgroups, we performed random subsamplings of the samples and reclassified the reduced dataset into 9 classes with the same calculation procedure. The results demonstrate that our pan-cancer stratification is a robust grouping system that can uncover very consistent patient assignments (in Additional file 1: Figure S14 ).
In summary, our comprehensive network-based stratification of 12 cancer types reveals essential pan-cancer heterogeneity among diverse cancers without considering the primary tumor organ information. The uncovered similarities among cancers of different organs suggest important cross-cancer commonalities. These commonalities not only cover most of the recurrently reported cross-cancer similarities, but also identify several novel potential ones. The macro-scale pan-cancer subgroups demonstrate strong clinical relevance and reveal consistent clinical risk tendency among cancer types. The micro-scale stratification shows essential pan-cancer heterogeneity with subgroup-specific genomic network characteristics and molecular implementations of oncogenesis. We believe that the pan-cancer subgroups defined here are promising stratifications of tumors for deciphering the underlying mechanisms of cancer deeply. With the rapid accumulation of cancer genomics data, this pan-cancer subgrouping procedure can be adopted for a more comprehensive understanding of the pan-cancer heterogeneity. Moreover, it is known that mutations in the same gene can lead to different consequences depending on which domain interface is altered [43–45]. How to integrate such information into the pan-caner stratifications is of great interest and worth exploring in further study.
Functional genetic alterations data
We obtained the 479 selected functional events (SFEs) of three data types (copy number alterations, somatic mutations, and DNA hyper-methylations) that were filtered by statistical and functional significant analysis from thousands of genomic and epigenetic changes . The SFEs binary data were downloaded from http://cbio.mskcc.org/cancergenomics/pancan_tcga/. These data contain 479 functional genetic alterations, including 116 copy number gains, 151 copy number losses, 199 recurrently mutated genes, and 13 epigenetically silenced genes recorded across 3299 tumor samples from 12 cancer types (Additional file 1: Table S1). Three cancer types (breast, colorectal, and endometrioid tumors) were provided with molecular subtype information. The profile is represented by binary (1, 0) values, in which a “1” indicates that a certain genetic alteration has occurred in this tumor.
We first transformed the 479 functional genetic changes to genes. The genes located in the same region of recurrent copy number gain and loss were treated equally as altered events. Secondly, multiple alterations on the same gene (e.g., a gene was observed to harbor both copy number gain and mutation) were merged. This resulted in a binary matrix of 3299 samples with 1750 genes, where a “1” means the gene has been altered by some kind of genomic or epigenetic change. Finally, genes were projected onto a biological network STRING v.9  and gene symbols were mapped to Ensembl IDs for downstream analysis (in Additional file 2).
Identifying essential cancer subgroups using NBS
We adopted the NBS procedure  to integrate a genome-scale alteration profile with a gene interaction network (STRING v.9) to produce robust classifications of patients (in Additional file 2). Briefly, the NBS applies a network propagation method to spread the influence of each mutation over its network neighborhood and produce a network-smoothed profile to reflect the effect of each genetic alteration on network module or pathway levels with a continuous value. Next, the network-smoothed patient matrix is clustered into a predefined number of subgroups via a network-regularized non-negative matrix factorization approach. Finally, in order to ensure robust cluster assignments, consensus clustering was performed. We employed the MATLAB package “nbs_release_v0.2” (http://chianti.ucsd.edu/~mhofree/wordpress/?page_id=26) to implement NBS to stratify samples into k (k = 3 ~ 15) clusters (in Additional file 2: Table S2). All other parameters were set as defaults. We adopted the Pearson’s chi-squared test to determine the enrichment significance of a certain tumor type or subtype in a cluster. All P values were corrected for the FDR q value.
Clinical outcome association analysis
We test to see if the identified subgroups are associated with clinical features of a specific cancer type including patient survival, tumor grade, and stage. The clinical data of 12 cancer types were downloaded from the TCGA_Pancancer page on Synapse (https://www.synapse.org/#!Synapse:syn300013/). Patient survival time was extracted from the tab-separated .patient files and detailed AJCC TNM staging information was merged (e.g., Stage IIA/IIB/IIC was merged as Stage II). Patients with missing clinical variables were excluded from the correlation analysis for that feature. For each cancer type, the survival information of samples located in different cohorts (e.g., BRCAs in its enriched subgroup versus all other BRCAs) was compared using Kaplan-Meier survival curves with log-rank test. The association of tumor grade/stage annotation with identified tumor subgroups was evaluated by Fisher's exact test. We conducted these analyses for each cancer type individually. Survival analysis was conducted using the R package “survival” and “survcomp”.
Identifying differentially altered sub-networks for each pan-cancer subgroup
For patients in each subgroup, we identified significantly altered genes against the remaining samples based on the network-smoothed alteration data by SAM (SAM—significance analysis of microarrays—was originally designed for identifying differentially expressed genes) . The q-value was calculated using the SAM permutation scheme with 1,000 permutations. The top significantly altered genes (SAM score >15 and FDR q-value <0.05) in each subgroup were selected as “significant differentially influenced genes”, and were mapped to the STRING v.9 network for visualization using the Cytoscape software. The biological functional analysis of the “significant differentially influenced genes” in each subgroup was performed using DAVID (http://david.abcc.ncifcrf.gov/) and GeneMANIA (http://www.genemania.org/). Annotation categories were pre-selected as defaults in DAVID and only terms with q-values lower than 0.05 were selected.
Identifying genes with subgroup-specific mRNA expression changes
We adopted the normalized RNA Seq V2 RSEM data of the 3299 TCGA samples for identifying genes with significant subgroup-specific expression changes. The dataset was downloaded from the cBioPortal for Cancer Genomics (http://www.cbioportal.org/public-portal/index.do) using the R package “cgdsr.” For GBM and OV, we used Agilent microarray data instead since it covers more patients presented in the SFEs binary dataset. For each PC9 subgroup, gene expressions were compared using the Wilcoxon rank-sum test on patients in this subgroup and those in the remaining subgroups. We conducted this analysis for all differentially altered genes of each subgroup. P values were corrected to get the q-values using Benjamini and Hochberg correction .
The authors thank Profs Chris Sander and Giovanni Ciriello for providing us the selected functional events (SFEs) data and Prof Trey Ideker for providing us the NBS package.
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