Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

Specific Genomic Regions Are Differentially Affected by Copy Number Alterations across Distinct Cancer Types, in Aggregated Cytogenetic Data

Background

Regional genomic copy number alterations (CNA) are observed in the vast majority of cancers. Besides specifically targeting well-known, canonical oncogenes, CNAs may also play more subtle roles in terms of modulating genetic potential and broad gene expression patterns of developing tumors. Any significant differences in the overall CNA patterns between different cancer types may thus point towards specific biological mechanisms acting in those cancers. In addition, differences among CNA profiles may prove valuable for cancer classifications beyond existing annotation systems.

Principal Findings

We have analyzed molecular-cytogenetic data from 25579 tumors samples, which were classified into 160 cancer types according to the International Classification of Disease (ICD) coding system. When correcting for differences in the overall CNA frequencies between cancer types, related cancers were often found to cluster together according to similarities in their CNA profiles. Based on a randomization approach, distance measures from the cluster dendrograms were used to identify those specific genomic regions that contributed significantly to this signal. This approach identified 43 non-neutral genomic regions whose propensity for the occurrence of copy number alterations varied with the type of cancer at hand. Only a subset of these identified loci overlapped with previously implied, highly recurrent (hot-spot) cytogenetic imbalance regions.

Conclusions

Thus, for many genomic regions, a simple null-hypothesis of independence between cancer type and relative copy number alteration frequency can be rejected. Since a subset of these regions display relatively low overall CNA frequencies, they may point towards second-tier genomic targets that are adaptively relevant but not necessarily essential for cancer development.     

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

Background

Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.

Results

We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.

Conclusions

Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0454-7) contains supplementary material, which is available to authorized users.     

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution

Background

Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.

Results

We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma.

Conclusions

In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0433-z) contains supplementary material, which is available to authorized users.     

Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background

Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome.

Results

We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers.

Conclusions

Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-579) contains supplementary material, which is available to authorized users.     

Reporting Tumor Molecular Heterogeneity in Histopathological Diagnosis

Background

Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples.

Aim

To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity.

Methods

35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing.

Results

TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis.

Conclusions

TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.     

ENCAPP: elastic-net-based prognosis prediction and biomarker discovery for human cancers

Background

With the explosion of genomic data over the last decade, there has been a tremendous amount of effort to understand the molecular basis of cancer using informatics approaches. However, this has proven to be extremely difficult primarily because of the varied etiology and vast genetic heterogeneity of different cancers and even within the same cancer. One particularly challenging problem is to predict prognostic outcome of the disease for different patients.

Results

Here, we present ENCAPP, an elastic-net-based approach that combines the reference human protein interactome network with gene expression data to accurately predict prognosis for different human cancers. Our method identifies functional modules that are differentially expressed between patients with good and bad prognosis and uses these to fit a regression model that can be used to predict prognosis for breast, colon, rectal, and ovarian cancers. Using this model, ENCAPP can also identify prognostic biomarkers with a high degree of confidence, which can be used to generate downstream mechanistic and therapeutic insights.

Conclusion

ENCAPP is a robust method that can accurately predict prognostic outcome and identify biomarkers for different human cancers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1465-9) contains supplementary material, which is available to authorized users.     

Towards accurate characterization of clonal heterogeneity based on structural variation

Background

Recent advances in deep digital sequencing have unveiled an unprecedented degree of clonal heterogeneity within a single tumor DNA sample. Resolving such heterogeneity depends on accurate estimation of fractions of alleles that harbor somatic mutations. Unlike substitutions or small indels, structural variants such as deletions, duplications, inversions and translocations involve segments of DNAs and are potentially more accurate for allele fraction estimations. However, no systematic method exists that can support such analysis.

Results

In this paper, we present a novel maximum-likelihood method that estimates allele fractions of structural variants integratively from various forms of alignment signals. We develop a tool, BreakDown, to estimate the allele fractions of most structural variants including medium size (from 1 kilobase to 1 megabase) deletions and duplications, and balanced inversions and translocations.

Conclusions

Evaluation based on both simulated and real data indicates that our method systematically enables structural variants for clonal heterogeneity analysis and can greatly enhance the characterization of genomically instable tumors.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-299) contains supplementary material, which is available to authorized users.     

The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies

Background

Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.

Results

Based on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.

Conclusions

Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1450-3) contains supplementary material, which is available to authorized users.     

Automated Network Analysis Identifies Core Pathways in Glioblastoma

Background

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor in humans and the first cancer with comprehensive genomic profiles mapped by The Cancer Genome Atlas (TCGA) project. A central challenge in large-scale genome projects, such as the TCGA GBM project, is the ability to distinguish cancer-causing “driver” mutations from passively selected “passenger” mutations.

Principal Findings

In contrast to a purely frequency based approach to identifying driver mutations in cancer, we propose an automated network-based approach for identifying candidate oncogenic processes and driver genes. The approach is based on the hypothesis that cellular networks contain functional modules, and that tumors target specific modules critical to their growth. Key elements in the approach include combined analysis of sequence mutations and DNA copy number alterations; use of a unified molecular interaction network consisting of both protein-protein interactions and signaling pathways; and identification and statistical assessment of network modules, i.e. cohesive groups of genes of interest with a higher density of interactions within groups than between groups.

Conclusions

We confirm and extend the observation that GBM alterations tend to occur within specific functional modules, in spite of considerable patient-to-patient variation, and that two of the largest modules involve signaling via p53, Rb, PI3K and receptor protein kinases. We also identify new candidate drivers in GBM, including AGAP2/CENTG1, a putative oncogene and an activator of the PI3K pathway; and, three additional significantly altered modules, including one involved in microtubule organization. To facilitate the application of our network-based approach to additional cancer types, we make the method freely available as part of a software tool called NetBox.     

Deep sequencing of the X chromosome reveals the proliferation history of colorectal adenomas

Background

Mismatch repair deficient colorectal adenomas are composed of transformed cells that descend from a common founder and progressively accumulate genomic alterations. The proliferation history of these tumors is still largely unknown. Here we present a novel approach to rebuild the proliferation trees that recapitulate the history of individual colorectal adenomas by mapping the progressive acquisition of somatic point mutations during tumor growth.

Results

Using our approach, we called high and low frequency mutations acquired in the X chromosome of four mismatch repair deficient colorectal adenomas deriving from male individuals. We clustered these mutations according to their frequencies and rebuilt the proliferation trees directly from the mutation clusters using a recursive algorithm. The trees of all four lesions were formed of a dominant subclone that co-existed with other genetically heterogeneous subpopulations of cells. However, despite this similar hierarchical organization, the growth dynamics varied among and within tumors, likely depending on a combination of tumor-specific genetic and environmental factors.

Conclusions

Our study provides insights into the biological properties of individual mismatch repair deficient colorectal adenomas that may influence their growth and also the response to therapy. Extended to other solid tumors, our novel approach could inform on the mechanisms of cancer progression and on the best treatment choice.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0437-8) contains supplementary material, which is available to authorized users.     

Direct observation of genomic heterogeneity through local haplotyping analysis

Background

It has been an abiding belief among geneticists that multicellular organisms’ genomes can be analyzed under the assumption that a single individual has a uniform genome in all its cells. Despite some evidence to the contrary, this belief has been used as an axiomatic assumption in most genome analysis software packages. In this paper we present observations in human whole genome data, human whole exome data and in mouse whole genome data to challenge this assumption. We show that heterogeneity is in fact ubiquitous and readily observable in ordinary Next Generation Sequencing (NGS) data.

Results

Starting with the assumption that a single NGS read (or read pair) must come from one haplotype, we built a procedure for directly observing haplotypes at a local level by examining 2 or 3 adjacent single nucleotide polymorphisms (SNPs) which are close enough on the genome to be spanned by individual reads. We applied this procedure to NGS data from three different sources: whole genome of a Central European trio from the 1000 genomes project, whole genome data from laboratory-bred strains of mouse, and whole exome data from a set of patients of head and neck tumors. Thousands of loci were found in each genome where reads spanning 2 or 3 SNPs displayed more than two haplotypes, indicating that the locus is heterogeneous. We show that such loci are ubiquitous in the genome and cannot be explained by segmental duplications. We explain them on the basis of cellular heterogeneity at the genomic level. Such heterogeneous loci were found in all normal and tumor genomes examined.

Conclusions

Our results highlight the need for new methods to analyze genomic variation because existing ones do not systematically consider local haplotypes. Identification of cancer somatic mutations is complicated because of tumor heterogeneity. It is further complicated if, as we show, normal tissues are also heterogeneous. Methods for biomarker discovery must consider contextual haplotype information rather than just whether a variant “is present”.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-418) contains supplementary material, which is available to authorized users.     

A survey of tandem repeat instabilities and associated gene expression changes in 35 colorectal cancers

Background

Colorectal cancer is a major contributor to cancer morbidity and mortality. Tandem repeat instability and its effect on cancer phenotypes remain so far poorly studied on a genome-wide scale.

Results

Here we analyze the genomes of 35 colorectal tumors and their matched normal (healthy) tissues for two types of tandem repeat instability, de-novo repeat gain or loss and repeat copy number variation. Specifically, we study for the first time genome-wide repeat instability in the promoters and exons of 18,439 genes, and examine the association of repeat instability with genome-scale gene expression levels. We find that tumors with a microsatellite instable (MSI) phenotype are enriched in genes with repeat instability, and that tumor genomes have significantly more genes with repeat instability compared to healthy tissues. Genes in tumor genomes with repeat instability in their promoters are significantly less expressed and show slightly higher levels of methylation. Genes in well-studied cancer-associated signaling pathways also contain significantly more unstable repeats in tumor genomes. Genes with such unstable repeats in the tumor-suppressor p53 pathway have lower expression levels, whereas genes with repeat instability in the MAPK and Wnt signaling pathways are expressed at higher levels, consistent with the oncogenic role they play in cancer.

Conclusions

Our results suggest that repeat instability in gene promoters and associated differential gene expression may play an important role in colorectal tumors, which is a first step towards the development of more effective molecular diagnostic approaches centered on repeat instability.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1902-9) contains supplementary material, which is available to authorized users.     

Copy number alterations and allelic ratio in relation to recurrence of rectal cancer

Background

In rectal cancer, total mesorectal excision surgery combined with preoperative (chemo)radiotherapy reduces local recurrence rates but does not improve overall patient survival, a result that may be due to the harmful side effects and/or co-morbidity of preoperative treatment. New biomarkers are needed to facilitate identification of rectal cancer patients at high risk for local recurrent disease. This would allow for preoperative (chemo)radiotherapy to be restricted to high-risk patients, thereby reducing overtreatment and allowing personalized treatment protocols. We analyzed genome-wide DNA copy number (CN) and allelic alterations in 112 tumors from preoperatively untreated rectal cancer patients. Sixty-six patients with local and/or distant recurrent disease were compared to matched controls without recurrence. Results were validated in a second cohort of tumors from 95 matched rectal cancer patients. Additionally, we performed a meta-analysis that included 42 studies reporting on CN alterations in colorectal cancer and compared results to our own data.

Results

The genomic profiles in our study were comparable to other rectal cancer studies. Results of the meta-analysis supported the hypothesis that colon cancer and rectal cancer may be distinct disease entities. In our discovery patient study cohort, allelic retention of chromosome 7 was significantly associated with local recurrent disease. Data from the validation cohort were supportive, albeit not statistically significant, of this finding.

Conclusions

We showed that retention of heterozygosity on chromosome 7 may be associated with local recurrence in rectal cancer. Further research is warranted to elucidate the mechanisms and effect of retention of chromosome 7 on the development of local recurrent disease in rectal cancer.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1550-0) contains supplementary material, which is available to authorized users.