A recent systematic sequence analysis of well-annotated human protein coding genes or consensus coding sequences led to the identification of 189 genes displaying somatic mutations in breast and colorectal cancers. Based on their mutation prevalence, a subset of these genes was identified as cancer candidate (CAN) genes as they could be potentially involved in cancer. We evaluated the mutational profiles of 19 CAN genes in the highly aggressive tumors: glioblastoma, melanoma, and pancreatic carcinoma. Among other changes, we found novel somatic mutations in EPHA3, MLL3, TECTA, FBXW7, and OBSCN, affecting amino acids not previously found to be mutated in human cancers. Interestingly, we also found a germline nucleotide variant of OBSCN that was previously reported as a somatic mutation. Our results identify specific genetic lesions in glioblastoma, melanoma, and pancreatic cancers and indicate that CAN genes and their mutational profiles are tumor specific. Some of the mutated genes, such as the tyrosine kinase EPHA3, are clearly amenable to pharmacologic intervention and could represent novel therapeutic targets for these incurable cancers. We also speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition. [Cancer Res 2007;67(8):3545–50]

Cancer is a multistep, polygenic disease caused by accumulation of genetic alterations in oncogenes or tumor suppressor genes resulting in neoplastic transformation. Increasing evidence suggests that new and effective targets for diagnosis and therapy could be identified by mutation profiling of cancer genomes. A systematic analysis of 13,023 well-annotated human protein-coding genes, known as consensus coding sequences,7

was recently carried out by Sjöblom et al. (1). This strategy lead to the identification of 189 genes displaying somatic mutations. To distinguish genes likely to contribute to tumorigenesis from those in which passenger mutations occurred by chance, the authors used a statistical approach that allowed the definition of a subset of candidate cancer (CAN) genes. To determine the significance of these findings in cancers other then breast and colon, we analyzed a subset of CAN genes in glioblastoma multiforme, melanoma, and pancreatic ductal adenocarcinoma (PDAC). These tumor types are known to be highly malignant and resistant to treatment. For example, metastatic melanoma has a poor prognosis, with a median survival of 6 to 9 months (2). Glioblastoma multiforme is the most common brain tumor in adults and kills patients within a median of 14 months after diagnosis, even after surgical resection, radiotherapy, and concomitant chemotherapy (3). PDAC is highly aggressive and resistant to conventional and targeted therapeutic agents, resulting in a dismal 5-year survival rate of 3% to 5% (4).

In this study, we present the mutational profile of 19 CAN genes in glioblastoma multiforme, melanoma, and PDAC. The candidate genes were selected either because they displayed a mutation frequency above 10%, or because multiple mutations affecting a single amino acid residue were previously found in the same gene (1). Specifically, we examined the following genes and exons in which mutations have been recently described: ABCA1, ADAMTSL3, ATP8B1, CUBN, DIP2C, EGFL6, EPHA3, EPHB6, FBXW7, FLNB, GNAS, MACF1, MLL3, OBSCN, PKHD1, SPTAN1, SYNE1, TECTA, and ZNF668. Details on genes and exons analyzed can be found in Supplementary Table S1.

Twenty-three human glioblastoma multiforme samples and the matched normal DNA were obtained from the tumor bank maintained by the Departments of Neurosurgery and Neuropathology at the Academic Medical Center (Amsterdam, The Netherlands). The glioblastoma cell line U87MG, officially known as an astrocytoma grade 3 cell line, was provided by Dr. Chris van Bree (Department of Radiotherapy, Academic Medical Center, Amsterdam, The Netherlands). One of the changes we identified in EPHA3 (K500N) occurred in U87MG, for which no matched normal is available. Therefore, the somatic status of this mutation could not be ascertained. The melanoma and PDAC tumor samples and matched normals were obtained from the tumor banks maintained by the Department of Experimental Oncology, Instituto Nazionale Tumori, Milan, Italy and the Department of Pathology, Section of Anatomic Pathology, University of Verona, Verona, Italy, respectively (Table 1). Genomic DNA was isolated as previously described (5), except for the PDAC samples that were isolated using DNeasy Blood & Tissue kit (Qiagen, Milan, Italy). For samples in which mutations were found, matching between germline and tumor DNA was verified by direct sequencing of 26 single nucleotide polymorphism (SNP) at 24 loci (data not shown).

Table 1.

Detailed information on the samples included in this study

Tumor typeSampleAgeGenderGradeTumor sourceTumor DNA sourceMatched normal source
Glioblastoma multiforme T1 39 Primary tumor Primary tumor Blood 
 T03 52 Primary tumor Primary tumor Blood 
 T12 74 Primary tumor Primary tumor Blood 
 T37 69 Primary tumor Primary tumor Blood 
 T39 37 Primary tumor Primary tumor Blood 
 T52 72 Primary tumor Primary tumor Blood 
 T70 76 Primary tumor Primary tumor Blood 
 T71 66 Primary tumor Primary tumor Blood 
 T78 29 Primary tumor Primary tumor Blood 
 T79 54 Primary tumor Primary tumor Blood 
 T80 47 Primary tumor Primary tumor Blood 
 T83 52 Primary tumor Primary tumor Blood 
 T85 31 Primary tumor Primary tumor Blood 
 T86 73 Primary tumor Primary tumor Blood 
 T90 64 Primary tumor Primary tumor Blood 
 T91 64 Primary tumor Primary tumor Blood 
 T94 57 Primary tumor Primary tumor Blood 
 T99 62 Primary tumor Primary tumor Blood 
 T104 69 Primary tumor Primary tumor Blood 
 T105 41 Primary tumor Primary tumor Blood 
 T107 39 Primary tumor Primary tumor Blood 
 T111 46 Primary tumor Primary tumor Blood 
 T112 48 Primary tumor Primary tumor Blood 
 T113 40 Primary tumor Primary tumor Blood 
 U87MG 44 Primary tumor Cell line Not available 
Melanoma 2A 51 Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 4A 55 2B/3C Primary tumor Short-term cultures EBV immortalized B lymphocytes 
 5A 36 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 6A 43 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 7A 22 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 8A 55 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 9A 53 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 10A 52 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 11A 69 3A/3B Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 12A 70 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 13A 59 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 14A 56 Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 15A 30 4-M1c Colon metastasis Short-term cultures EBV immortalized B lymphocytes 
 16A 45 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 17A 51 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 18A 68 Cutaneous metastasis Short-term cultures Blood 
 19A 71 3C T4aN3M0 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 20A 24 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 21A 36 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 22A 39 3C Nodal metastasis Short-term cultures Blood 
 23A 56 4-M1c Lung metastasis Short-term cultures EBV immortalized B lymphocytes 
 24A 49 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 25A 49 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 26A 62 4-M1a Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
Pancreatic ductal Adenocarcinoma 360 76 T3N0/G2 Primary tumor Xenograft Duodenum 
 362 54 T3N1b/G3 Primary tumor Xenograft Pancreas 
 369 61 T3N0/G2 Primary tumor Xenograft Not available 
 370 52 T3N1b/G2 Primary tumor Xenograft EBV immortalized B lymphocytes 
 371 57 T3N0/G3 Primary tumor Xenograft Duodenum 
 374 62 T3N0/G3 Primary tumor Xenograft Pancreas 
 375 59 T3N1a/G3 Primary tumor Xenograft EBV immortalized B lymphocytes 
 377 52 T3N1a/G2 Primary tumor Xenograft Pancreas 
 379 62 T3N1/G2 Primary tumor Xenograft Spleen 
 380 57 T3N1a/G2 Primary tumor Xenograft Duodenum 
 382 44 T3N0/G2 Primary tumor Xenograft Duodenum 
 384 69 T3N0/G2 Primary tumor Xenograft Spleen 
Tumor typeSampleAgeGenderGradeTumor sourceTumor DNA sourceMatched normal source
Glioblastoma multiforme T1 39 Primary tumor Primary tumor Blood 
 T03 52 Primary tumor Primary tumor Blood 
 T12 74 Primary tumor Primary tumor Blood 
 T37 69 Primary tumor Primary tumor Blood 
 T39 37 Primary tumor Primary tumor Blood 
 T52 72 Primary tumor Primary tumor Blood 
 T70 76 Primary tumor Primary tumor Blood 
 T71 66 Primary tumor Primary tumor Blood 
 T78 29 Primary tumor Primary tumor Blood 
 T79 54 Primary tumor Primary tumor Blood 
 T80 47 Primary tumor Primary tumor Blood 
 T83 52 Primary tumor Primary tumor Blood 
 T85 31 Primary tumor Primary tumor Blood 
 T86 73 Primary tumor Primary tumor Blood 
 T90 64 Primary tumor Primary tumor Blood 
 T91 64 Primary tumor Primary tumor Blood 
 T94 57 Primary tumor Primary tumor Blood 
 T99 62 Primary tumor Primary tumor Blood 
 T104 69 Primary tumor Primary tumor Blood 
 T105 41 Primary tumor Primary tumor Blood 
 T107 39 Primary tumor Primary tumor Blood 
 T111 46 Primary tumor Primary tumor Blood 
 T112 48 Primary tumor Primary tumor Blood 
 T113 40 Primary tumor Primary tumor Blood 
 U87MG 44 Primary tumor Cell line Not available 
Melanoma 2A 51 Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 4A 55 2B/3C Primary tumor Short-term cultures EBV immortalized B lymphocytes 
 5A 36 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 6A 43 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 7A 22 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 8A 55 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 9A 53 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 10A 52 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 11A 69 3A/3B Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 12A 70 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 13A 59 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 14A 56 Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 15A 30 4-M1c Colon metastasis Short-term cultures EBV immortalized B lymphocytes 
 16A 45 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 17A 51 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 18A 68 Cutaneous metastasis Short-term cultures Blood 
 19A 71 3C T4aN3M0 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 20A 24 3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 21A 36 3B/3C Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
 22A 39 3C Nodal metastasis Short-term cultures Blood 
 23A 56 4-M1c Lung metastasis Short-term cultures EBV immortalized B lymphocytes 
 24A 49 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 25A 49 Cutaneous metastasis Short-term cultures EBV immortalized B lymphocytes 
 26A 62 4-M1a Nodal metastasis Short-term cultures EBV immortalized B lymphocytes 
Pancreatic ductal Adenocarcinoma 360 76 T3N0/G2 Primary tumor Xenograft Duodenum 
 362 54 T3N1b/G3 Primary tumor Xenograft Pancreas 
 369 61 T3N0/G2 Primary tumor Xenograft Not available 
 370 52 T3N1b/G2 Primary tumor Xenograft EBV immortalized B lymphocytes 
 371 57 T3N0/G3 Primary tumor Xenograft Duodenum 
 374 62 T3N0/G3 Primary tumor Xenograft Pancreas 
 375 59 T3N1a/G3 Primary tumor Xenograft EBV immortalized B lymphocytes 
 377 52 T3N1a/G2 Primary tumor Xenograft Pancreas 
 379 62 T3N1/G2 Primary tumor Xenograft Spleen 
 380 57 T3N1a/G2 Primary tumor Xenograft Duodenum 
 382 44 T3N0/G2 Primary tumor Xenograft Duodenum 
 384 69 T3N0/G2 Primary tumor Xenograft Spleen 

NOTE: Clinical information of the patient, tumor staging, and source of the genomic DNA used for the mutational analysis are indicated.

Abbreviations: F, female; M, male.

PCR and sequencing primers were designed using Primer 38

and synthesized by Invitrogen/Life Technologies, Inc. (Paisley, England; Supplementary Table S1). PCR primers were designed to amplify the selected exons and the flanking intronic sequences, including splicing donor and acceptor regions, of the 19 cancer genes. PCR products were ∼400 bp in length, with multiple overlapping amplimers for larger exons. PCRs were done in both 384- and 96-well formats in 5- or 10-μL reaction volumes, respectively, containing 0.25 mmol/L deoxynucleotide triphosphates, 1 mmol/L each of the forward and reverse primers, 6% DMSO, 1× PCR buffer, 1 ng/ μL DNA, and 0.01 unit/μL Platinum Taq (Invitrogen/Life Technologies). A touchdown PCR program was used for PCR amplification (Peltier Thermocycler, PTC-200, MJ Research, Bio-Rad Laboratories, Inc., Italy).

PCR conditions were as follows: 94°C for 2 min; three cycles of 94°C for 15 s, 64°C for 30 s, 70°C for 30 s; three cycles of 94°C for 15 s, 61°C for 30 s, 70°C for 30 s; three cycles of 94°C for 15 s, 58°C for 30 s, 70°C for 30 s; and 35 cycles of 94°C for 15 s, 57°C for 30 s, and 70°C for 30 s, followed by 70°C for 5 min and 12°C thereafter.

PCR products were purified using AMPure (Agencourt Bioscience Corp., Beckman Coulter S.p.A, Milan, Italy). Cycle sequencing was carried out using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) with an initial denaturation at 97°C for 3 min, for 28 cycles at 97°C for 10 s, 50°C for 20 s, and 60°C for 2 min. Sequencing products were purified using CleanSeq (Agencourt Bioscience, Beckman Coulter) and analyzed on a 3730 DNA Analyzer, ABI capillary electrophoresis system (Applied Biosystems). Sequence traces were analyzed using the Mutation Surveyor software package (SoftGenetics, State College, PA).

A recent large-scale sequencing effort led to the identification of somatic mutations in more than 150 CAN genes in breast and colon cancer (1). The detailed mutational status of these genes in other cancer types remains to be determined. In this study, we examined the mutational profile of 19 CAN genes (Supplementary Table S1) in a panel of 24 glioblastoma, 24 melanomas, and 12 pancreatic cancers samples. The clinical information associated with these tumors is described in Table 1. For each gene, all exons in which somatic mutations had previously been identified were analyzed. Exon specific primers were designed to amplify and sequence the coding region, and at least 15 intronic bases at both the 5′ and 3′ ends, including the splicing donor and acceptor sites (Supplementary Table S1).

A total of 7,798 PCR products, spanning 1.47 Mb of tumor genomic DNA, were generated and subjected to direct sequencing. A total of 2,743 nucleotide changes were identified during this initial screening. Changes previously described as SNPs were excluded from further analyses.9

To ensure that the observed mutations were not PCR or sequencing artifacts, amplicons were independently re-amplified and re-sequenced in the corresponding tumors. All verified changes were re-sequenced in parallel with the matched normal DNA to distinguish between somatic mutations and SNPs not previously described. This approach led to the identification of eight novel somatic mutations and one germline change in five CAN genes (Table 2). Three missense somatic mutations were found in EPHA3, two of which (K500N and A971P) were found in glioblastoma multiforme. The third EPHA3 mutation (G228R) was observed in a melanoma sample that also contained a missense mutation in OBSCN (E4574K). Two mutations were found in MLL3 in a glioblastoma multiforme (3614Ddel) and a PDAC sample (P1863A). In addition, one mutation each was found in FBXW7 (R473fs*23) and TECTA (P802S) in two melanoma samples. The observed mutation rate was higher than the expected passenger mutation rate (P < 0.01, binomial distribution; ref. 6). In addition, we found a germline variant of a previously reported somatic mutation (R4558H) in OBSCN in a glioblastoma multiforme patient.

Table 2.

Mutations identified in CAN genes

GeneNucleotide changeAmino acid changeZygositySample
EPHA3 c. 907 G>A p.G228R Heterozygous 23A 
EPHA3 c. 1725G>T p.K500N Heterozygous U87 
EPHA3 c. 3136 G>C p.A971P Heterozygous T78 
MLL3 c. 5767 C>G p.P1863A Homozygous 369 
MLL3 c.11020-11022delGAT p.3614Ddel Heterozygous T70 
TECTA c. 2404 C>T p.P802S Heterozygous 8A 
FBXW7c. 1566 del A p.R473fs*23 Heterozygous 19A 
OBSCN c.13791G>A p.E4574K Heterozygous 23A 
OBSCN c.13673G>A p.R4558H Heterozygous T1 
GeneNucleotide changeAmino acid changeZygositySample
EPHA3 c. 907 G>A p.G228R Heterozygous 23A 
EPHA3 c. 1725G>T p.K500N Heterozygous U87 
EPHA3 c. 3136 G>C p.A971P Heterozygous T78 
MLL3 c. 5767 C>G p.P1863A Homozygous 369 
MLL3 c.11020-11022delGAT p.3614Ddel Heterozygous T70 
TECTA c. 2404 C>T p.P802S Heterozygous 8A 
FBXW7c. 1566 del A p.R473fs*23 Heterozygous 19A 
OBSCN c.13791G>A p.E4574K Heterozygous 23A 
OBSCN c.13673G>A p.R4558H Heterozygous T1 

NOTE: The genes and the type of mutations found are listed alongside the samples in which they were found. The nucleotide position of each mutation corresponds to the position of that change in the coding sequence of each gene, where position 1 is the A of the ATG. Zygosity for the mutations is shown.

*

The deleted nucleotide is part of a codon formed by the last two bases of exon 9 and the first base of exon 10; therefore, a heterozygous deletion of the second last base of exon 9 causes a frameshift in exon 10 and leads to a premature stop codon.

This germline mutation in sample T1 in glioblastoma multiforme was previously reported as a somatic mutation (1).

One of the most interesting genes found mutated is the Ephrin receptor A3 (EPHA3). EPHA3 is a member of the Ephrin receptor family, which forms the largest subgroup of the receptor tyrosine kinases. Ephrin receptors and their ligands (Ephrins) are essential for a variety of biological processes and are implicated in tumor growth and survival (reviewed in ref. 7). EPHA3, or human eph/elk-like kinase, maps to chromosome 3p11.2, a region frequently affected in different cancers (8). Mutations in EPHA3 in have been described in lung and colon cancer (9, 10).10

Here, we report for the first time an EPHA3 mutation in melanoma. This mutation (G228R) occurs in a cysteine-rich linker region of the extracellular domain. Interestingly, this region is evolutionary conserved and may be important in determining the binding affinity to its particular ligand type. EphA2 receptor antagonists have remarkable antiangiogenic and antitumor effects, suggesting that the EphA signaling pathway represents an attractive novel target for cancer therapy (11). The tyrosine kinase activity of the Epha3 receptor may be therapeutically targeted. Therefore, for this gene only, we extended our analysis to all coding exons. Two additional EPHA3 mutations (K500N and A971P) were found in glioblastoma multiforme. The K500N affected the second fibronectin type-III domain, whereas A971P (Fig. 1A) occurred in the sterile α-motif region. Both domains are highly conserved throughout evolution, suggesting that the changes might affect critical functions of this gene.

Figure 1.

Examples of somatic mutations in EPHA3, TECTA, and OBSCN. Bottom, chromatogram of the sequence of a tumor sample; top, chromatogram of the matched normal. Arrows, location of missense somatic mutations. Nucleotide and amino acid alterations are below the traces. Numbers above the sequences are part of the software output. A, EPHA3 mutation in glioblastoma multiforme. B, TECTA mutation in melanoma. C, OBSCN mutation in melanoma.

Figure 1.

Examples of somatic mutations in EPHA3, TECTA, and OBSCN. Bottom, chromatogram of the sequence of a tumor sample; top, chromatogram of the matched normal. Arrows, location of missense somatic mutations. Nucleotide and amino acid alterations are below the traces. Numbers above the sequences are part of the software output. A, EPHA3 mutation in glioblastoma multiforme. B, TECTA mutation in melanoma. C, OBSCN mutation in melanoma.

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We report for the first time somatic mutations in the interdomain regions of mixed-lineage leukemia 3 (MLL3) gene in glioblastoma multiforme and PDAC cancer. MLL3, also designated as “homologous to ALR” (HALR), is a member of the TRX/MLL gene family and maps to 7q36, a chromosome region that is frequently deleted in myeloid leukemia (12). Members of the MLL family are often targets for translocations in leukemias, leading to oncogenic fusion proteins that are associated with an extremely poor prognosis (13). TRX/MLL members serve as tumor suppressors, act as chromatin regulators, and play an important role during development. Interestingly, amplification of MLL2 has been reported in glioblastoma and pancreatic carcinoma cell lines (14). Therefore, our results indicate that multiple members of the MLL family can be deregulated via different oncogenic mechanisms in these two cancer types.

The third gene in which we detected mutations was tectorin-α (TECTA) that was found altered in a melanoma sample. TECTA is the major non-collagenous component of the tectorial membrane of the inner ear. Mutations in TECTA have been shown to be responsible for autosomal dominant non-syndromic hearing impairments and a recessive form of sensorineural pre-lingual non-syndromic deafness (15). The identified mutation, P802S (Fig. 1B), lies in the second von Willebrand factor type D domain and has not been described in any type of deafness. No previous association between TECTA and cancer has been reported.

The mutational profiling of FBXW7 resulted in the identification of the R473fs*23 mutation in a melanoma sample. F-box and WD-40 domain protein 7 (FBXW7 or hCDC4) is part of an ubiquitin ligase complex that targets molecules, such as cyclin E, Notch, c-Jun, and c-Myc, for degradation (16). Inactivating mutations in FBXW7 have been previously described in a variety of human tumors and cancer cell lines. The nonsense mutation we found (R473fs*23) is located in the third of seven highly conserved WD40 repeats, known to serve as a protein binding platform. It gives rise to a premature stop codon, resulting in truncation of the protein, thereby potentially interrupting the binding to its substrate (17). This change may, therefore, have direct functional implications, especially considering the putative haploinsufficient nature of this tumor suppressor gene (16).

One of the most intriguing results of this analysis is the identification of novel somatic and germline mutations in OBSCN. OBSCN encodes a RhoGEF protein that interacts with cytoskeletal calmodulin and titin and is part of the giant sarcomeric signaling protein family of myosin light chain kinases. Different isoforms have been described, containing functionally interesting domains, including two serine-threonine kinase domains, with a potential role in signal transduction (18). The somatic mutation E4574K (Fig. 1C) affects the fibronectin type-III domain 3, which is highly conserved throughout evolution. Thus far, OBSCN has been mainly known for its role in cardiac and skeletal muscle, where it is required for the assembly and organization of sarcomeres and the sarcoplasmic reticulum (18). Mutations in other genes encoding the giant muscle proteins titin (TTN) and nebulin (NEB) have been associated with cardiac and skeletal myopathies in humans. Interestingly, OBSCN was reported to be interrupted by a t(1;7)(q42;p15) breakpoint, in a Wilms' tumor patient with thrombocytopenia-absent radius syndrome–like symptoms (19). The recent identification of OBSCN somatic mutations in multiple cancer types indicates that this gene may play a role in different diseases (1).10 It is also interesting to note that of the 189 CAN genes identified in colon and breast, only TP53 and OBSCN were common to both tumor types. The identification of mutations in OBSCN and TTN in multiple cancer types, which now include melanoma and glioblastoma, suggests that the cellular functions of these partner molecules could be related to a common tumor progression mechanism.

In addition to the E4574K somatic mutation, we also found another change (R4558H) in OBSCN in a glioblastoma multiforme sample. Exactly the same change had been previously found to be a somatic mutation by Sjöblom et al. (1). Surprisingly, however, we found that the same change was also present in the matched normal DNA (obtained from the blood of the same patient), indicating that it was a germline rather then a somatic mutation (Fig. 2). The R4558H mutation is not a common SNP as it is absent from the publicly available databases (20).11

To further exclude the possibility that R4558H was a rare SNP, we sequenced an additional 359 human DNA samples again without finding this allele. Our results, therefore, suggest that the R4558H could be a germline cancer mutation. Importantly, the glioblastoma multiforme patient carrying the R4558H allele does not have a history of cardiac or skeletal muscle anomalies. This suggests that the R4558H mutation is not associated with the previously reported role of OBSCN in muscle tissues. The identification of germline and somatic mutations in OBSCN resembles what has been previously observed in other important cancer genes such as PTEN, RET, and TP53. In those cases, the very same mutations involved in the development of sporadic tumors were found to be cancer-predisposing when present in the germline. It is conceivable that germline changes in OBSCN might also similarly predispose to cancer.

Figure 2.

Germline mutation in OBSCN. Sequence of the OBSCN gene in sample T1 where the germline mutation R4558H was found. Bottom, chromatogram of the sequence of the glioblastoma sample; top, chromatogram of the respective matched normal. Arrow, location of the mutation. Nucleotide and amino acid alterations are below the trace.

Figure 2.

Germline mutation in OBSCN. Sequence of the OBSCN gene in sample T1 where the germline mutation R4558H was found. Bottom, chromatogram of the sequence of the glioblastoma sample; top, chromatogram of the respective matched normal. Arrow, location of the mutation. Nucleotide and amino acid alterations are below the trace.

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In conclusion, our data identify novel genes and their specific molecular alterations involved in glioblastoma, melanoma, and pancreatic cancers. None of the somatic mutations described by Sjöblom et al. (1) were found in our analyses of glioblastoma multiforme, melanoma, and PDAC samples. These results, therefore, suggest that tumors have their own CAN genes, and only a few of the CAN genes are shared by different tumor types. In addition, the mutations themselves, rather than the genes, might be tumor specific. With the exception of P1863A in MLL3, all the mutations we found were in heterozygous state. Interestingly, this is similar to the mutation pattern observed by Sjöblom et al. The occurrence of heterozygous mutations is very common in oncogenes where they act in a dominant fashion. Alternatively, as previously shown for FBXW7, a haploinsufficient tumor suppressor, a heterozygous mutation may result in its inactivation. Because TECTA and OBSCN have not been previously associated with cancer, the effect of these mutations cannot presently be predicted. Some of the mutations we found affect genes (such as the tyrosine kinase EPHA3) that are clearly amenable to pharmacologic intervention and therefore could represent novel therapeutic targets for these untreatable cancers. Finally, we speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

A. Balakrishnan and F.E. Bleeker contributed equally to this study.

Grant support: Italian Association for Cancer Research (AIRC; A. Bardelli), Italian Ministry of Health (A. Bardelli), Italian Ministry of University and Research (A. Bardelli), Regione Piemonte (A. Bardelli), Compagnia di S. Paolo Foundation (A. Bardelli), Fondazione Cariverona (A. Scarpa), Fondazione Zanotto (A. Scarpa), Association for International Cancer Research UK (AIRC UK; A. Bardelli), European Union FP6, MCSCs contract 037297 (A. Bardelli) and PL018771 (A. Scarpa), Accelerate Brain Cancer Cure (A.A. van Tilborg), Netherlands Genomics Initiative Fellowship (F.E. Bleeker), and Stichting Jo Kolk Studiefonds (F.E. Bleeker).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. Dirk Troost (Department of Neuropathology, Academic Medical Center) for histologic verification of all glioblastoma multiforme samples, Catherine Tighe for article editing, Dr. Theo Hulsebos (Department of Neurogenetics, Academic Medical Center) and the members of the Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment for helpful discussions.

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Supplementary data