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Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed "fragment recruitment," addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed "extreme assembly," made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS.

Tryptophan degradation is an immune escape strategy shared by many tumors. However, cancer cells' compensatory mechanisms remain unclear. We demonstrate here that a shortage of tryptophan caused by expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) resulted in ATF4-dependent upregulation of several amino acid transporters, including SLC1A5 and its truncated isoforms, which in turn enhanced tryptophan and glutamine uptake. Importantly, SLC1A5 failed to be upregulated in resting human T cells kept under low tryptophan conditions but was enhanced upon cognate antigen T-cell receptor engagement. Our results highlight key differences in the ability of tumor and T cells to adapt to tryptophan starvation and provide important insights into the poor prognosis of tumors coexpressing IDO and SLC1A5. Cancer Res; 76(21); 6193-204. ©2016 AACR., Competing Interests: The authors disclose no potential conflicts of interest., (©2016 American Association for Cancer Research.)

Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify patients at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively parallel sequencing-based assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected postoperatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8 %) of 164 patients with negative ctDNA [hazard ratio (HR), 18; 95% confidence interval (CI), 7.9 to 40; P < 0.001]. In patients treated with chemotherapy, the presence of ctDNA after completion of chemotherapy was also associated with an inferior recurrence-free survival (HR, 11; 95% CI, 1.8 to 68; P = 0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence., (Copyright © 2016, American Association for the Advancement of Science.)

Melanoma is one of the major cancer types for which new immune-based cancer treatments have achieved promising results. However, anti-PD-1 and anti-CTLA-4 therapies are effective only in some patients. Hence, predictive molecular markers for the development of clinical strategies targeting immune checkpoints are needed. Using The Cancer Genome Atlas (TCGA) RNAseq data, we found that expression of ESRP1, encoding a master splicing regulator in the epithelial-mesenchymal transition (EMT), was inversely correlated with tumor-associated immune cytolytic activity. That association holds up across multiple TCGA tumor types, suggesting a link between tumor EMT status and infiltrating lymphocyte activity. In melanoma, ESRP1 mainly exists in a melanocyte-specific truncated form transcribed from exon 13. This was validated by analyzing CCLE cell line data, public CAGE data, and RT-PCR in primary cultured melanoma cell lines. Based on ESRP1 expression, we divided TCGA melanoma cases into ESRP1-low, -truncated, and -full-length groups. ESRP1-truncated tumors comprise approximately two thirds of melanoma samples and reside in an apparent transitional state between epithelial and mesenchymal phenotypes. ESRP1 full-length tumors express epithelial markers and constitute about 5% of melanoma samples. In contrast, ESRP1-low tumors express mesenchymal markers and are high in immune cytolytic activity as well as PD-L2 and CTLA-4 expression. Those tumors are associated with better patient survival. Results from our study suggest a path toward the use of ESRP1 and other EMT markers as informative biomarkers for immunotherapy. Cancer Immunol Res; 4(6); 552-61. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: APC mutations (APC-mt) occur in ∼70% of colorectal cancers (CRCs), but their relationship to prognosis is unclear., Methods: APC prognostic value was evaluated in 746 stage I-IV CRC patients, stratifying for tumour location and microsatellite instability (MSI). Microarrays were used to identify a gene signature that could classify APC mutation status, and classifier ability to predict prognosis was examined in an independent cohort., Results: Wild-type APC microsatellite stable (APC-wt/MSS) tumours from the proximal colon showed poorer overall and recurrence-free survival (OS, RFS) than APC-mt/MSS proximal, APC-wt/MSS distal and APC-mt/MSS distal tumours (OS HR⩾1.79, P⩽0.015; RFS HR⩾1.88, P⩽0.026). APC was a stronger prognostic indicator than BRAF, KRAS, PIK3CA, TP53, CpG island methylator phenotype or chromosomal instability status (P⩽0.036). Microarray analysis similarly revealed poorer survival in MSS proximal cancers with an APC-wt-like signature (P=0.019). APC status did not affect outcomes in MSI tumours. In a validation on 206 patients with proximal colon cancer, APC-wt-like signature MSS cases showed poorer survival than APC-mt-like signature MSS or MSI cases (OS HR⩾2.50, P⩽0.010; RFS HR⩾2.14, P⩽0.025). Poor prognosis APC-wt/MSS proximal tumours exhibited features of the sessile serrated neoplasia pathway (P⩽0.016)., Conclusions: APC-wt status is a marker of poor prognosis in MSS proximal colon cancer.

Background: Early indicators of treatment response in metastatic colorectal cancer (mCRC) could conceivably be used to optimize treatment. We explored early changes in circulating tumor DNA (ctDNA) levels as a marker of therapeutic efficacy., Patients and Methods: This prospective study involved 53 mCRC patients receiving standard first-line chemotherapy. Both ctDNA and CEA were assessed in plasma collected before treatment, 3 days after treatment and before cycle 2. Computed tomography (CT) scans were carried out at baseline and 8-10 weeks and were centrally assessed using RECIST v1.1 criteria. Tumors were sequenced using a panel of 15 genes frequently mutated in mCRC to identify candidate mutations for ctDNA analysis. For each patient, one tumor mutation was selected to assess the presence and the level of ctDNA in plasma samples using a digital genomic assay termed Safe-SeqS., Results: Candidate mutations for ctDNA analysis were identified in 52 (98.1%) of the tumors. These patient-specific candidate tissue mutations were detectable in the cell-free DNA from the plasma of 48 of these 52 patients (concordance 92.3%). Significant reductions in ctDNA (median 5.7-fold; P < 0.001) levels were observed before cycle 2, which correlated with CT responses at 8-10 weeks (odds ratio = 5.25 with a 10-fold ctDNA reduction; P = 0.016). Major reductions (≥10-fold) versus lesser reductions in ctDNA precycle 2 were associated with a trend for increased progression-free survival (median 14.7 versus 8.1 months; HR = 1.87; P = 0.266)., Conclusions: ctDNA is detectable in a high proportion of treatment naïve mCRC patients. Early changes in ctDNA during first-line chemotherapy predict the later radiologic response., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of "general" immune checkpoint drugs in this subset of patients.

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses., (©2014 American Association for Cancer Research.)

Cancer-testis (CT) antigens are potential targets for cancer immunotherapy because of their restricted expression in immune-privileged germ cells and various malignancies. Current application of CT-based immunotherapy has been focused on CT expression-rich tumors such as melanoma and lung cancers. In this study, we surveyed CT expression using The Cancer Genome Atlas (TCGA) datasets for ten common cancer types. We show that CT expression is specific and enriched within certain cancer molecular subtypes. For example, HORMAD1, CXorf61, ACTL8, and PRAME are highly enriched in the basal subtype of breast cancer; MAGE and CSAG are most frequently activated in the magnoid subtype of lung adenocarcinoma; and PRAME is highly upregulated in the ccB subtype of clear cell renal cell carcinoma. Analysis of CT gene expression and DNA methylation indicates that some CTs are regulated epigenetically, whereas others are controlled primarily by tissue- and subtype-specific transcription factors. Our results suggest that although for some CT expression is associated with patient outcome, not many are independent prognostic markers. Thus, CTs with shared expression pattern are heterogeneous molecules with distinct activation modes and functional properties in different cancers and cancer subtypes. These data suggest a cancer subtype-orientated application of CT antigen as biomarkers and immunotherapeutic targets.

Meningiomas are the most common primary intracranial tumors. Surgical resection remains the treatment of choice for these tumors. However, a significant number of tumors are not surgically accessible, recur, or become malignant, necessitating the repetition of surgery and sometimes radiation. Chemotherapy is rarely used and is generally not recognized as an effective treatment. Cancer/testis (CT) genes represent a unique class of genes, which are expressed by germ cells, normally silenced in somatic cells, but activated in various cancers. CT proteins can elicit spontaneous immune responses in patients with cancer and this feature makes them attractive targets for immunotherapy-based approaches. We analyzed mRNA expression of 37 testis-restricted CT genes in a discovery set of 18 meningiomas by reverse transcription PCR. The overall frequency of expression of CT genes ranged from 5.6% to 27.8%. The most frequently expressed was NY-ESO-1, in 5 patients (27.8%). We subsequently analyzed NY-ESO-1 protein expression in a larger set of meningiomas by immunohistochemistry and found expression in 108 of 110 cases. In some cases, NY-ESO-1 expression was diffused and homogenous, but in most instances it was heterogeneous. Importantly, NY-ESO-1 expression was positively correlated with higher grade and patients presenting with higher levels of NY-ESO-1 staining had significantly worse disease-free and overall survival. We have also shown that NY-ESO-1 expression may lead to humoral immune response in patients with meningioma. Considering the limited treatment options for patients with meningioma, the potential of NY-ESO-1-based immunotherapy should be explored., (©2013 AACR.)

Biallelic protein-truncating mutations in the adenomatous polyposis coli (APC) gene are prevalent in sporadic colorectal cancer (CRC). Mutations may not be fully inactivating, instead producing WNT/β-catenin signalling levels 'just-right' for tumourigenesis. However, the spectrum of optimal APC genotypes accounting for both hits, and the influence of clinicopathological features on genotype selection remain undefined. We analysed 630 sporadic CRCs for APC mutations and loss of heterozygosity (LOH) using sequencing and single-nucleotide polymorphism microarrays, respectively. Truncating APC mutations and/or LOH were detected in 75% of CRCs. Most truncating mutations occurred within a mutation cluster region (MCR; codons 1282-1581) leaving 1-3 intact 20 amino-acid repeats (20AARs) and abolishing all Ser-Ala-Met-Pro (SAMP) repeats. Cancers commonly had one MCR mutation plus either LOH or another mutation 5' to the MCR. LOH was associated with mutations leaving 1 intact 20AAR. MCR mutations leaving 1 vs 2-3 intact 20AARs were associated with 5' mutations disrupting or leaving intact the armadillo-repeat domain, respectively. Cancers with three hits had an over-representation of mutations upstream of codon 184, in the alternatively spliced region of exon 9, and 3' to the MCR. Microsatellite unstable cancers showed hyper-mutation at MCR mono- and di-nucleotide repeats, leaving 2-3 intact 20AARs. Proximal and distal cancers exhibited different preferred APC genotypes, leaving a total of 2 or 3 and 0 to 2 intact 20AARs, respectively. In conclusion, APC genotypes in sporadic CRCs demonstrate 'fine-tuned' interdependence of hits by type and location, consistent with selection for particular residual levels of WNT/β-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.

Purpose: PIK3CA and PTEN mutations are prevalent in colorectal cancer and potential markers of response to mitogen-activated protein/extracellular signal-regulated kinase inhibitors and anti-EGF receptor antibody therapy. Relationships between phosphoinositide 3-kinase (PI3K) pathway mutation, clinicopathologic characteristics, molecular features, and prognosis remain controversial., Experimental Design: A total of 1,093 stage I-IV colorectal cancers were screened for PIK3CA (exons 9 and 20), KRAS (codons 12-13), BRAF (codon 600) mutations, and microsatellite instability (MSI). PTEN (exons 3-8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data were integrated with 17 previous reports (n = 5,594)., Results: PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of colorectal cancers. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high (MSI-H), CIMP-high (CIMP-H), and BRAF mutation (P < 0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology, and KRAS mutation (P < 0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low (CIMP-L), and KRAS-mutated cancers (P ≤ 0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-H, CIMP-H, and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P ≤ 0.027). Disease-free survival for stage II/III colorectal cancers did not differ by PI3K pathway status., Conclusion: PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-H/CIMP-H/BRAF(mut)), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-L/KRAS(mut)) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct colorectal cancer subtypes.

Purpose: We aim to identify tumor-specific alternative splicing events having potential applications in the early detection, diagnosis, prognosis, and therapy for cancers., Experimental Design: We analyzed RNA-seq data on 470 clear cell renal cell carcinomas (ccRCC) and 68 kidney tissues to identify tumor-specific alternative splicing events. We further focused on the fibroblast growth factor receptor 2 (FGFR2) isoform switch and characterized ccRCCs expressing different FGFR2 isoforms by integrated analyses using genomic data from multiple platforms and tumor types., Results: We identified 113 top candidate alternatively spliced genes in ccRCC. Prominently, the FGFR2 gene transcript switched from the normal IIIb isoform ("epithelial") to IIIc isoform ("mesenchymal") in nearly 90% of ccRCCs. This switch is kidney specific as it was rarely observed in other cancers. The FGFR2-IIIb ccRCCs show a transcriptome and methylome resembling those from normal kidney, whereas FGFR2-IIIc ccRCCs possess elevated hypoxic and mesenchymal expression signatures. Clinically, FGFR2-IIIb ccRCCs are smaller in size, of lower tumor grade, and associated with longer patient survival. Gene set enrichment and DNA copy number analyses indicated that FGFR2-IIIb ccRCCs are closely associated with renal oncocytomas and chromophobe RCCs (chRCC). A reexamination of tumor histology by pathologists identified FGFR2-IIIb tumors as chRCCs and clear cell papillary RCCs (ccpRCC)., Conclusions: FGFR2 IIIb RCCs represent misdiagnosed ccRCC cases, suggesting FGFR2 isoform testing can be used in the diagnosis of RCC subtypes. The finding of a prevalent isoform switch of FGFR2 in a tissue-specific manner holds promise for the future development of FGFR2-IIIc as a distinct early detection biomarker and therapeutic target for ccRCC., (©2013 AACR.)

Activation of the canonical TGF-β signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP) microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-X-Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Starting from publicly-accessible datasets, we have utilized comparative and phylogenetic genome analyses to characterize the evolution of the human MAGE gene family. Our characterization of genomic structures in representative genomes of primates, rodents, carnivora, and macroscelidea indicates that both Type I and Type II MAGE genes have undergone lineage-specific evolution. The restricted expression pattern in germ cells of Type I MAGE orthologs is observed throughout evolutionary history. Unlike Type II MAGEs that have conserved promoter sequences, Type I MAGEs lack promoter conservation, suggesting that epigenetic regulation is a central mechanism for controlling their expression. Codon analysis shows that Type I but not Type II MAGE genes have been under positive selection. The combination of genomic and expression analysis suggests that Type 1 MAGE promoters and genes continue to evolve in the hominin lineage, perhaps towards functional diversification or acquiring additional specific functions, and that selection pressure at codon level is associated with expression spectrum.

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Purpose: Oncogene mutations contribute to colorectal cancer development. We searched for differences in oncogene mutation profiles between colorectal cancer metastases from different sites and evaluated these as markers for site of relapse., Experimental Design: One hundred colorectal cancer metastases were screened for mutations in 19 oncogenes, and further 61 metastases and 87 matched primary cancers were analyzed for genes with identified mutations. Mutation prevalence was compared between (a) metastases from liver (n = 65), lung (n = 50), and brain (n = 46), (b) metastases and matched primary cancers, and (c) metastases and an independent cohort of primary cancers (n = 604). Mutations differing between metastasis sites were evaluated as markers for site of relapse in 859 patients from the VICTOR trial., Results: In colorectal cancer metastases, mutations were detected in 4 of 19 oncogenes: BRAF (3.1%), KRAS (48.4%), NRAS (6.2%), and PIK3CA (16.1%). KRAS mutation prevalence was significantly higher in lung (62.0%) and brain (56.5%) than in liver metastases (32.3%; P = 0.003). Mutation status was highly concordant between primary cancer and metastasis from the same individual. Compared with independent primary cancers, KRAS mutations were more common in lung and brain metastases (P < 0.005), but similar in liver metastases. Correspondingly, KRAS mutation was associated with lung relapse (HR = 2.1; 95% CI, 1.2 to 3.5, P = 0.007) but not liver relapse in patients from the VICTOR trial., Conclusions: KRAS mutation seems to be associated with metastasis in specific sites, lung and brain, in colorectal cancer patients. Our data highlight the potential of somatic mutations for informing surveillance strategies., (©2011 AACR.)

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus: Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification.

Background: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype., Methodology/principal Findings: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1., Significance: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Voltage-gated Na(+) channels (VGSC) have been implicated in the metastatic potential of human breast, prostate, and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Na(v)1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiologic recordings. Na(+) channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by small interfering RNAs (siRNA) specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, with strong Na(v)1.5 protein staining found in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process, and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion.

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

The human microbiome refers to the community of microorganisms, including prokaryotes, viruses, and microbial eukaryotes, that populate the human body. The National Institutes of Health launched an initiative that focuses on describing the diversity of microbial species that are associated with health and disease. The first phase of this initiative includes the sequencing of hundreds of microbial reference genomes, coupled to metagenomic sequencing from multiple body sites. Here we present results from an initial reference genome sequencing of 178 microbial genomes. From 547,968 predicted polypeptides that correspond to the gene complement of these strains, previously unidentified ("novel") polypeptides that had both unmasked sequence length greater than 100 amino acids and no BLASTP match to any nonreference entry in the nonredundant subset were defined. This analysis resulted in a set of 30,867 polypeptides, of which 29,987 (approximately 97%) were unique. In addition, this set of microbial genomes allows for approximately 40% of random sequences from the microbiome of the gastrointestinal tract to be associated with organisms based on the match criteria used. Insights into pan-genome analysis suggest that we are still far from saturating microbial species genetic data sets. In addition, the associated metrics and standards used by our group for quality assurance are presented.

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.

Recent advances in DNA sequencing technology are providing unprecedented opportunities for comprehensive analysis of cancer genomes, exomes, transcriptomes, as well as epigenomic components. The integration of these data sets with well-annotated phenotypic and clinical data will expedite improved interventions based on the individual genomics of the patient and the specific disease.

Background: To identify potential tumor suppressor genes, genome-wide data from exome and transcriptome sequencing were combined to search for genes with loss of heterozygosity and allele-specific expression. The analysis was conducted on the breast cancer cell line HCC1954, and a lymphoblast cell line from the same individual, HCC1954BL., Results: By comparing exome sequences from the two cell lines, we identified loss of heterozygosity events at 403 genes in HCC1954 and at one gene in HCC1954BL. The combination of exome and transcriptome sequence data also revealed 86 and 50 genes with allele specific expression events in HCC1954 and HCC1954BL, which comprise 5.4% and 2.6% of genes surveyed, respectively. Many of these genes identified by loss of heterozygosity and allele-specific expression are known or putative tumor suppressor genes, such as BRCA1, MSH3 and SETX, which participate in DNA repair pathways., Conclusions: Our results demonstrate that the combined application of high throughput sequencing to exome and allele-specific transcriptome analysis can reveal genes with known tumor suppressor characteristics, and a shortlist of novel candidates for the study of tumor suppressor activities., (© 2010 Zhao et al.; licensee BioMed Central Ltd.)

Objectives: We tested the hypothesis that co-coordinated up-regulation or down-regulation of several ovarian cell surface kinases may provide clues for better understanding of the disease and help in rational design of therapeutic targets., Study Design: We compared the expression signature of 69 surface kinases in normal ovarian surface epithelial cells (OSE), with OSE from patients at high risk and with ovarian cancer., Results: Seven surface kinases, ALK, EPHA5, EPHB1, ERBB4, INSRR, PTK, and TGFbetaR1 displayed a distinctive linear trend in expression from normal, highrisk, and malignant epithelium. We confirmed these results using semiquantitative reverse transcription-polymerase chain reaction and tissue array of 202 ovarian cancer samples. A strong correlate was shown between disease-free survival and the expression of ERBB4. DNA sequencing revealed two novel mutations in ERBB4 in two cancer samples., Conclusions: A distinct subset of the ovarian surface kinome is altered in the transition from high risk to invasive cancer and genetic mutation is not a dominant mechanism for these modifications. These results have significant implications for early detection and targeted therapeutic approaches for women at high risk of developing ovarian cancer.

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements.

We have identified new genomic alterations in the breast cancer cell line HCC1954, using high-throughput transcriptome sequencing. With 120 Mb of cDNA sequences, we were able to identify genomic rearrangement events leading to fusions or truncations of genes including MRE11 and NSD1, genes already implicated in oncogenesis, and 7 rearrangements involving other additional genes. This approach demonstrates that high-throughput transcriptome sequencing is an effective strategy for the characterization of genomic rearrangements in cancers.

Background: Next generation sequencing (NGS) platforms are currently being utilized for targeted sequencing of candidate genes or genomic intervals to perform sequence-based association studies. To evaluate these platforms for this application, we analyzed human sequence generated by the Roche 454, Illumina GA, and the ABI SOLiD technologies for the same 260 kb in four individuals., Results: Local sequence characteristics contribute to systematic variability in sequence coverage (>100-fold difference in per-base coverage), resulting in patterns for each NGS technology that are highly correlated between samples. A comparison of the base calls to 88 kb of overlapping ABI 3730xL Sanger sequence generated for the same samples showed that the NGS platforms all have high sensitivity, identifying >95% of variant sites. At high coverage, depth base calling errors are systematic, resulting from local sequence contexts; as the coverage is lowered additional 'random sampling' errors in base calling occur., Conclusions: Our study provides important insights into systematic biases and data variability that need to be considered when utilizing NGS platforms for population targeted sequencing studies.

The HuRef Genome Browser is a web application for the navigation and analysis of the previously published genome of a human individual, termed HuRef. The browser provides a comparative view between the NCBI human reference sequence and the HuRef assembly, and it enables the navigation of the HuRef genome in the context of HuRef, NCBI and Ensembl annotations. Single nucleotide polymorphisms, indels, inversions, structural and copy-number variations are shown in the context of existing functional annotations on either genome in the comparative view. Demonstrated here are some potential uses of the browser to enable a better understanding of individual human genetic variation. The browser provides full access to the underlying reads with sequence and quality information, the genome assembly and the evidence supporting the identification of DNA polymorphisms. The HuRef Browser is a unique and versatile tool for browsing genome assemblies and studying individual human sequence variation in a diploid context. The browser is available online at http://huref.jcvi.org.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

There is much interest in characterizing the variation in a human individual, because this may elucidate what contributes significantly to a person's phenotype, thereby enabling personalized genomics. We focus here on the variants in a person's 'exome,' which is the set of exons in a genome, because the exome is believed to harbor much of the functional variation. We provide an analysis of the approximately 12,500 variants that affect the protein coding portion of an individual's genome. We identified approximately 10,400 nonsynonymous single nucleotide polymorphisms (nsSNPs) in this individual, of which approximately 15-20% are rare in the human population. We predict approximately 1,500 nsSNPs affect protein function and these tend be heterozygous, rare, or novel. Of the approximately 700 coding indels, approximately half tend to have lengths that are a multiple of three, which causes insertions/deletions of amino acids in the corresponding protein, rather than introducing frameshifts. Coding indels also occur frequently at the termini of genes, so even if an indel causes a frameshift, an alternative start or stop site in the gene can still be used to make a functional protein. In summary, we reduced the set of approximately 12,500 nonsilent coding variants by approximately 8-fold to a set of variants that are most likely to have major effects on their proteins' functions. This is our first glimpse of an individual's exome and a snapshot of the current state of personalized genomics. The majority of coding variants in this individual are common and appear to be functionally neutral. Our results also indicate that some variants can be used to improve the current NCBI human reference genome. As more genomes are sequenced, many rare variants and non-SNP variants will be discovered. We present an approach to analyze the coding variation in humans by proposing multiple bioinformatic methods to hone in on possible functional variation., Competing Interests: The authors have declared that no competing interests exist.

The completion of draft sequences of the human genome represented a remarkable achievement for automated DNA sequencing based on Sanger technology. However, the future requires substantial leaps in sequencing technology such that whole genome sequencing will become a standard component of biomedical research and patient care. In this review we describe current advances that are in early stages of development, but that point toward technology that will enable the onset of genomic medicine encompasses strategies for preventative medicine and intervention based on complete knowledge of an individual's genome.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Presented here is a genome sequence of an individual human. It was produced from approximately 32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2-206 bp), 292,102 heterozygous insertion/deletion events (indels)(1-571 bp), 559,473 homozygous indels (1-82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.

Although the number of protein-encoding human genes is more limited than many had estimated, the human transcript repertoire is much more diverse than anticipated. In part, transcript diversity is generated through the use of alternative promoters and alternate splicing. In addition, based on discoveries using technologies such as full-length cDNA libraries and whole genome tiling microarrays, it is now likely that non-protein-encoding transcripts comprise a substantial fraction of the human RNA population. Much attention is currently focused on understanding the role of alternative promoters in generating transcript diversity, both for non-protein-encoding (ncRNAs) and protein-encoding RNAs.

The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species.

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4x coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element-like and long interspersed element-like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.

Introduction: Genomic alterations have been observed in breast carcinomas that affect the capacity of cells to regulate proliferation, signaling, and metastasis. Re-sequence studies have investigated candidate genes based on prior genetic observations (changes in copy number or regions of genetic instability) or other laboratory observations and have defined critical somatic mutations in genes such as TP53 and PIK3CA., Methods: We have extended the paradigm and analyzed 21 genes primarily identified by expression profiling studies, which are useful for breast cancer subtyping and prognosis. This study conducted a bidirectional re-sequence analysis of all exons and 5', 3', and evolutionarily conserved regions (spanning more than 16 megabases) in 91 breast tumor samples., Results: Eighty-seven unique somatic alterations were identified in 16 genes. Seventy-eight were single base pair alterations, of which 23 were missense mutations; 55 were distributed across conserved intronic regions or the 5' and 3' regions. There were nine insertion/deletions. Because there is no a priori way to predict whether any one of the identified synonymous and noncoding somatic alterations disrupt function, analysis unique to each gene will be required to establish whether it is a tumor suppressor gene or whether there is no effect. In five genes, no somatic alterations were observed., Conclusion: The study confirms the value of re-sequence analysis in cancer gene discovery and underscores the importance of characterizing somatic alterations across genes that are related not only by function, or functional pathways, but also based upon expression patterns.

Cartilaginous fishes represent the living group of jawed vertebrates that diverged from the common ancestor of human and teleost fish lineages about 530 million years ago. We generated approximately 1.4x genome sequence coverage for a cartilaginous fish, the elephant shark (Callorhinchus milii), and compared this genome with the human genome to identify conserved noncoding elements (CNEs). The elephant shark sequence revealed twice as many CNEs as were identified by whole-genome comparisons between teleost fishes and human. The ancient vertebrate-specific CNEs in the elephant shark and human genomes are likely to play key regulatory roles in vertebrate gene expression.

We have described previously a cDNA library made from membrane-bound polysomal mRNA prepared from breast and prostate cancer cell lines. The library is highly enriched for cDNAs encoding membrane proteins, secreted proteins, and cytokeratins. To characterize this library, 25,277 cDNA clones were sequenced and aligned with various databases; 1,439 clones did not align with known genes. From this set of clones we identified a previously uncharacterized gene encoding a 334-aa protein. Although protein structural motif prediction programs indicate that the gene encodes a membrane protein comprising a signal sequence, a series of leucine-rich repeats, and a single transmembrane domain with a cytoplasmic tail, confocal microscopy of MCF7 breast cancer cells demonstrates that the protein is not directly associated with the plasma membrane or intracellular membranes but instead colocalizes with intermediate filaments and cytokeratins within the cell. Immunofluorescence studies also show that protein expression is increased greatly in mitotic MCF7 cells, and immunohistochemistry demonstrates its expression in human breast cancer cells. Analysis of mRNA levels in 25 different normal tissues by RT-PCR shows that this gene is expressed highly in normal prostate and salivary gland, very weakly in colon, pancreas, and intestine, and not at all in other tissues. RT-PCR studies on human cancer samples show that the RNA is expressed highly in many cancer cell lines and cancer specimens, including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3 colon cancers, and 3 of 3 pancreatic cancers. We name the protein CAPC, cytokeratin-associated protein in cancer.