Comparative exome sequencing of metastatic lesions provides insights into the mutational progression of melanoma
© Gartner et al.; licensee BioMed Central Ltd. 2012
Received: 9 January 2012
Accepted: 30 August 2012
Published: 24 September 2012
Metastasis is characterized by spreading of neoplastic cells to an organ other than where they originated and is the predominant cause of death among cancer patients. This holds true for melanoma, whose incidence is increasing more rapidly than any other cancer and once disseminated has few therapeutic options. Here we performed whole exome sequencing of two sets of matched normal and metastatic tumor DNAs.
Using stringent criteria, we evaluated the similarities and differences between the lesions. We find that in both cases, 96% of the single nucleotide variants are shared between the two metastases indicating that clonal populations gave rise to the distant metastases. Analysis of copy number variation patterns of both metastatic sets revealed a trend similar to that seen with our single nucleotide variants. Analysis of pathway enrichment on tumor sets shows commonly mutated pathways enriched between individual sets of metastases and all metastases combined.
These data provide a proof-of-concept suggesting that individual metastases may have sufficient similarity for successful targeting of driver mutations.
Cancer is mainly a genetic disease with mutations arising that can either activate proto-oncogenes or inactivate tumor suppressor genes. The incidence of malignant melanoma is increasing worldwide. In fact, the most recent statistics predict approximately 69,000 new diagnoses and 8,700 deaths in the coming year in the United States alone . Once melanoma has metastasized it has an extremely poor prognosis, with 5 year relative survival of just 15% . In the past decade many genetic alterations have been discovered that influence tumor growth and spread. The knowledge gained during this time has led to the recent approval of the BRAF inhibitor PLX4032 (ZelborafTM) by the FDA for treatment of late–stage melanoma .
The recent advances in next-generation sequencing have allowed for the discovery of new causal variants and have also afforded us the opportunity to ask new questions which could help dictate future treatment strategies. In this study we use whole exome sequencing to investigate two sets of distinct metastases to determine similarity.
Exome sequencing and analysis
We performed whole exome sequencing of two distinct metastases from two individuals with melanoma (Additional file 1: Table S1). In both patients, metastatic deposits were present in multiple anatomic sites, as is typical for this form of cancer. Genomic DNA samples derived from these metastases underwent whole exome re-sequencing in parallel with their matched normal DNA. Exonic sequences were enriched with Agilent's SureSelect technology for targeted exon capture , targeting 50 Mb of sequence from exons and flanking regions in nearly 20,000 genes. Sequencing was performed with the Illumina GAII platform, and reads were aligned using ELAND (Illumina, Inc., San Diego, CA) followed by cross_match (http://www.phrap.org) to the reference human genome (Build 36.1). On average, 11.7 Gb of sequence were generated per sample to a mean depth of 103X to achieve exome builds with at least 87.5% of the targeted bases covered by high quality genotype calls. To eliminate common germ line mutations from consideration, we filtered variants observed in dbSNP130 or in a high quality set of common variants from the 1000 genomes project. To determine which of these alterations were somatic, we compared variants identified in the metastasis to their matched normal tissue and removed any variants found in the normal. From these putative alterations, 2356 potential somatic mutations in 1256 different genes were identified in the samples sequenced.
Summary of differences between metastases in 98 set
NS + S
Found in 14T only (NS)
Found in 14T only (S)
Found in 98T only (NS)
Found in 98T only (S)
Found in both Mets (NS)
Found in both Mets (S)
% found in both Mets (NS)
% found in both Mets (S)
% found in both Mets (NS + S)
Summary of differences between metastases in 130 set
NS + S
Found in 130T only (NS)
Found in 130T only (S)
Found in 133T only (NS)
Found in 133T only (S)
Found in both Mets (NS)
Found in both Mets (S)
% found in both Mets (NS)
% found in both Mets (S)
% found in both Mets (NS + S)
Copy number analysis
Percent copy number difference
% Copy number difference between the two metastases
Pathway enrichment analysis
Advances in next-generation Sequencing have allowed researchers to affordably generate vast amounts of data and address complex morbicentric genetics questions with incredible sensitivity and robustness. In the present study we used our data from whole exome sequencing of two distinct metastases from the same patient to elucidate the similarities and differences between the tumors. The answer to these questions could prove useful in selection of future treatment strategies.
Our data revealed similarities between the two paired metastases at the level of somatic mutation and copy number variations. These results would seem to indicate that in both sets the metastases were derived from the same parental clone that harbored the majority of the genetic alterations and chromosomal instability. The Clark model of melanoma progression holds that melanoma progression proceeds in a stepwise manner from melanocyte to melanoma, categorized by numerous molecular changes which facilitate the transition through each step . Our results suggest that once the transition from the vertical growth phase to malignant melanoma occurs, a limited number of molecular changes subsequently arise.
Despite these findings, further investigation is warranted. Future work should include a side by side genetic analysis of primary tumors pertaining to the metastatic lesions as well as the analysis of more metastases pertaining to the same patients derived from different regions to see whether these results hold true. Nonetheless, these findings provide a proof-of-concept that sequencing of a limited number individual metastases may be sufficient for targeting of melanoma driver mutations.
Tissue and melanoma cell lines used for this study were described previously . The clinical information associated with the melanoma tumors used in this study is provided in Additional file 1: Table S1.
Exome capture was performed using the Sure Select Human All Exon 50 Mb System (Agilent Technologies, Santa Clara, CA). The manufacturer’s protocol for Sure Select Human All Exon System (Illumina Paired-End Sequencing Library Prep), version 1.0.1 was used, with the following modifications. Bioanalyzer steps were either performed using agarose gel or omitted. The Illumina library preparation portion of the SureSelect protocol was performed using the SPRIworks Fragment Library System (Beckman Coulter Genomics, Danvers, MA, USA) according to manufacturer’s protocols.
Sequencing was performed on the Illumina GAIIx platform with version 5 chemistry and version 5 flowcells according to the manufacturer’s instructions. 76 and 101 base paired-end reads were generated summary of sequencing statistics can be seen in Additional file 6: Table S5.
Read mapping and variant analysis
Reads were initially aligned using ELAND (Illumina Inc., San Diego, CA). ELAND alignments were used to place reads in bins of approximately 100,000 base pairs. Unmapped reads were placed in the bin of the mate pair if the mate was mapped. Cross_match (Phil Green, http://www.phrap.org) was utilized to align the reads assigned to each bin to the corresponding ~100 kb of genomic sequence. Cross_match alignments were converted to the SamTools bam format, and then genotypes were called using bam2mpg (, http://research.nhgri.nih.gov). Bam2mpg was used to implement the Most Probable Genotype (MPG) algorithm, a Bayesian based method to determine the probability of each genotype given the data observed at that position. The quality score represents the difference of the log likelihoods of the most and second most probable genotype. The MPG was divided by the coverage at each position to calculate the MPG/coverage ratio.
To eliminate common germline mutations from consideration, alterations observed in dbSNP130 or in a high quality set of common variants from the 1000 genomes 11_2010 data release project were removed. To perform the 1000 genomes project filtering, low coverage genome data from 629 individuals was obtained from the November 2010 data release of the 1000 genomes project. From this list of variants we included those positions called by at least 3 of the 4 analysis methods used by the project. We further limited the list to those variants above 5% minor allele frequency. Polymorphisms were further removed by examination of the sequence of the gene in genomic DNA from matched normal tissue. Genotypes were annotated as described in .
Mutational analysis, confirmation, and determination of somatic status were carried out to validate all mutations found exclusively in one of the metastasis as previously described [8, 23]. Sequence traces of the Validation Screen were analyzed using the Mutation Surveyor software package and all genes had 93% coverage or above (SoftGenetics, State College, PA).
Copy number variation analysis
Where ri is the log2 ratio for the ith window and n is the total number of windows. The denominator is just a normalization parameter to make DLRS the same scale as the standard deviation (when calculated on a normally-distributed random variable).
Copy number validation
The results of CNV pseudo-CHG array were used to create a Nexus Copy Number file (BioDiscovery) and genes were randomly selected showing a CNV difference between either one or both of the samples and the normal. Primers for the gene indicated were designed using primer 3 with Line-1 primers used as controls for normalization (Additional file 8: Table S7). 1 ng of each sample gDNA was mixed with 2× Fast SYBR Green PCR mix at a final volume of 10 μl in triplicate (Applied Biosystems cat # 4355612). qPCR analysis was done using the ABI 7900HT Fast Real-Time PCR system (with a standard program of stage 1: 50°C for 2 min; stage 2: 95°C for 10 min; stage 3: 40 cycles of 95°C for 15 s and 60°C for 1 min). Results were analyzed using Microsoft Excel and SPSS.
CNV and SNV data were combined and IPA (Ingenuity ®Systems, http://www.ingenuity.com) was used to investigate pathway enrichment for each tumor and set. The resulting p-values were adjusted for multiple testing via the benjamini-hochberg procedure. Adjusted p-values less than 0.05 were considered significant. Common pathways between tumors were merged using SequelPro and MySQL. Pathways in common between two tumor types that were significant were plotted using the ggplot2 package in R.
We thank P. Cruz for the exome analyses of these data generated by NISC. This work supported by the Intramural Research Programs of the National Human Genome Research Institute, the National Cancer Institute, National Institutes of Health, USA and Company. This work is supported by a public-private partnership between the Intramural Research Programs of the National Human Genome Research Institute, the National Cancer Institute, and Eli Lilly and Company coordinated by the Foundation for the National Institutes of Health.
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