Refinement of computational identification of somatic copy number alterations using DNA methylation microarrays illustrated in cancers of unknown primary.

High-throughput genomic technologies are increasingly used in personalized cancer medicine. However, computational tools to maximize the use of scarce tissues combining distinct molecular layers are needed. Here we present a refined strategy, based on the R-package 'conumee', to better predict somatic copy number alterations (SCNA) from deoxyribonucleic acid (DNA) methylation arrays. Our approach, termed hereafter as 'conumee-KCN', improves SCNA prediction by incorporating tumor purity and dynamic thresholding. We trained our algorithm using paired DNA methylation and SNP Array 6.0 data from The Cancer Genome Atlas samples and confirmed its performance in cancer cell lines. Most importantly, the application of our approach in cancers of unknown primary identified amplified potentially actionable targets that were experimentally validated by Fluorescence in situ hybridization and immunostaining, reaching 100% specificity and 93.3% sensitivity.

Predictors of Local Invasion in Infiltrative Basal Cell Carcinoma: Tumour Budding Outperforms the WHO Subtyping.

Tumour budding (TB) correlates with increased local invasion in various neoplasms. Certain basal cell carcinomas (BCCs) exhibit local aggressiveness. Detecting adverse prognostic factors in partial biopsies could aid in identifying cases with heightened local risk. The absolute number of TB (≤ 3 tumour cells) in excision specimens of 271 infiltrative BCCs (0: absent; 1: 1-2 foci; 2: ≥ 3 foci; 3: ≥ 10 foci), the histopathological subtype and depth of infiltration, perineural invasion, and other histological features were evaluated. A significant correlation was found between TB and both depth of infiltration (rho 0.445, p < 0.001) and perineural invasion (p = 0.009). In the multivariate analysis of depth and perineural invasion (multiple regression, stepwise), TB was identified as a significant covariate together with diameter, inflammation, and perineural invasion for the former, and depth for the latter. Conversely, no correlation existed between the WHO histological subtypes (infiltrating, sclerosing, and micronodular), and depth of infiltration or perineural invasion. This study demonstrates the value of TB as a biomarker for local invasiveness in BCC. In routine practice, a count of ≥ 3 TB foci in lesions incompletely excised or with narrow tumour-free surgical margins would be a straightforward and reproducible method to guide BCC treatment.

Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease.

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the "chronic inflammation-dysplasia-cancer" carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations.

The Challenge of Diagnosing Constitutional Mismatch Repair Deficiency Syndrome in Brain Malignancies from Young Individuals.

Biallelic germline mismatch repair (MMR) gene (MLH1, MSH2, MSH6, and PMS2) mutations are an extremely rare event that causes constitutional mismatch repair deficiency (CMMRD) syndrome. CMMRD is underdiagnosed and often debuts with pediatric malignant brain tumors. A high degree of clinical awareness of the CMMRD phenotype is needed to identify new cases. Immunohistochemical (IHC) assessment of MMR protein expression and analysis of microsatellite instability (MSI) are the first tools with which to initiate the study of this syndrome in solid malignancies. MMR IHC shows a hallmark pattern with absence of staining in both neoplastic and non-neoplastic cells for the biallelic mutated gene. However, MSI often fails in brain malignancies. The aim of this report is to draw attention to the peculiar IHC profile that characterizes CMMRD syndrome and to review the difficulties in reaching an accurate diagnosis by describing the case of two siblings with biallelic MSH6 germline mutations and brain tumors. Given the difficulties involved in early diagnosis of CMMRD we propose the use of the IHC of MMR proteins in all malignant brain tumors diagnosed in individuals younger than 25 years-old to facilitate the diagnosis of CMMRD and to select those neoplasms that will benefit from immunotherapy treatment.

Epigenetic Homogeneity Within Colorectal Tumors Predicts Shorter Relapse-Free and Overall Survival Times for Patients With Locoregional Cancer.

BACKGROUND & AIMS: There are few validated biomarkers that can be used to predict outcomes for patients with colorectal cancer. Part of the challenge is the genetic and molecular heterogeneity of colorectal tumors not only among patients, but also within tumors. We have explored intratumor heterogeneity at the epigenetic level, due to its dynamic nature. We analyzed DNA methylation profiles of the digestive tract surface and the central bulk and invasive front regions of colorectal tumors. METHODS: We determined the DNA methylation profiles of >450,000 CpG sites in 3 macrodissected regions of 79 colorectal tumors and 23 associated liver metastases, obtained from 2 hospitals in Spain. We also analyzed samples for KRAS and BRAF mutations, 499,170 single nucleotide polymorphisms, and performed immunohistochemical analyses. RESULTS: We observed differences in DNA methylation among the 3 tumor sections; regions of tumor-host interface differed the most from the other tumor sections. Interestingly, tumor samples collected from areas closer to the gastrointestinal transit most frequently shared methylation events with metastases. When we calculated individual coefficients to quantify heterogeneity, we found that epigenetic homogeneity was significantly associated with short time of relapse-free survival (log-rank P = .037) and short time of overall survival (log-rank P = .026) in patients with locoregional colorectal cancer. CONCLUSIONS: In an analysis of 79 colorectal tumors, we found significant heterogeneity in patterns of DNA methylation within each tumor; the level of heterogeneity correlates with times of relapse-free and overall survival.

Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis.

BACKGROUND: Cancer of unknown primary ranks in the top ten cancer presentations and has an extremely poor prognosis. Identification of the primary tumour and development of a tailored site-specific therapy could improve the survival of these patients. We examined the feasability of using DNA methylation profiles to determine the occult original cancer in cases of cancer of unknown primary. METHODS: We established a classifier of cancer type based on the microarray DNA methylation signatures (EPICUP) in a training set of 2790 tumour samples of known origin representing 38 tumour types and including 85 metastases. To validate the classifier, we used an independent set of 7691 known tumour samples from the same tumour types that included 534 metastases. We applied the developed diagnostic test to predict the tumour type of 216 well-characterised cases of cancer of unknown primary. We validated the accuracy of the predictions from the EPICUP assay using autopsy examination, follow-up for subsequent clinical detection of the primary sites months after the initial presentation, light microscopy, and comprehensive immunohistochemistry profiling. FINDINGS: The tumour type classifier based on the DNA methylation profiles showed a 99·6% specificity (95% CI 99·5-99·7), 97·7% sensitivity (96·1-99·2), 88·6% positive predictive value (85·8-91·3), and 99·9% negative predictive value (99·9-100·0) in the validation set of 7691 tumours. DNA methylation profiling predicted a primary cancer of origin in 188 (87%) of 216 patients with cancer with unknown primary. Patients with EPICUP diagnoses who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy (hazard ratio [HR] 3·24, p=0·0051 [95% CI 1·42-7·38]; log-rank p=0·0029). INTERPRETATION: We show that the development of a DNA methylation based assay can significantly improve diagnoses of cancer of unknown primary and guide more precise therapies associated with better outcomes. Epigenetic profiling could be a useful approach to unmask the original primary tumour site of cancer of unknown primary cases and a step towards the improvement of the clinical management of these patients. FUNDING: European Research Council (ERC), Cellex Foundation, the Institute of Health Carlos III (ISCIII), Cancer Australia, Victorian Cancer Agency, Samuel Waxman Cancer Research Foundation, the Health and Science Departments of the Generalitat de Catalunya, and Ferrer.

A cellular model reflecting the phenotypic heterogeneity of mutant HRAS driven squamous cell carcinoma.

Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV-unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor-promoting alterations such as the overexpression of the PADI2 oncogene in solid-type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case-to-case heterogeneity despite departing from the same set of oncogenes and the same genetic background.

Refining the role of PMS2 in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants.

BACKGROUND AND AIM: The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. METHODS: From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. RESULTS: Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. CONCLUSIONS: Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.

Gastric metaplasia as a precursor of nonconventional dysplasia in inflammatory bowel disease.

Gastric metaplasia in colonic mucosa with inflammatory bowel disease (IBD) develops as an adaptation mechanism. The association between gastric metaplasia and nonconventional and/or conventional dysplasia as precursors of colitis-associated colorectal cancer is unknown. To address this question, we retrospectively reviewed a series of 33 IBD colectomies to identify gastric metaplasia in 76 precursor lesions. We obtained 61 nonconventional and 15 conventional dysplasias. Among nonconventional dysplasia, 31 (50.8 %) were low-grade (LGD), 4 (6.5 %) were high-grade (HGD), 9 (14.8 %) had both LGD and HGD, and 17 (27.9 %) had no dysplasia (ND), while 14 (93 %) conventional dysplasias had LGD, and 1 (7 %) had LGD and HGD. Gastric metaplasia was assessed by concomitant immunoexpression of MUC5AC and loss of CDX2 staining. Expression of a p53-mut pattern was considered as a surrogate for gene mutation, and complete loss of MLH1 staining as presence of MLH1 hypermethylation. In nonconventional dysplasia, MUC5AC immunoexpression decreased as the degree of dysplasia increased, being 78 % in LGD and 39 % in HGD (p = 0.006). CDX2 was lost in epithelial glands with high expression of MUC5AC (p < 0.001). The p53-mut pattern was observed in 77 % HGD, 45 % LGD, and in 6 % with ND (p < 0.001). Neither nonconventional nor conventional dysplasia showed complete loss of MLH1 staining. Gastric metaplasia was also present in mucosa adjacent to nonconventional dysplasia with chronic changes or active inflammation. Our results show that gastric metaplasia appears in IBD-inflamed colon mucosa, it is the substrate of most nonconventional dysplasia and occurs prior to p53 alterations.

Changes In Serum CXCL13 Levels Are Associated With Outcomes of Colorectal Cancer Patients Undergoing First-Line Oxaliplatin-Based Treatment.

Metastatic colorectal cancer (mCRC) currently lacks reliable biomarkers for precision medicine, particularly for chemotherapy-based treatments. This study examines the behavior of 11 CXC chemokines in the blood of 104 mCRC patients undergoing first-line oxaliplatin-based treatment to pinpoint predictive and prognostic markers. Serum samples were collected before treatment, at response evaluation (EVAR), and at disease progression or last follow-up. Chemokines were assessed in all samples using a Luminex® custom panel. CXCL13 levels increased at EVAR in responders, while in non-responders it decreased. Increasing levels of CXCL13 at EVAR, independently correlated with improved progression-free survival (PFS) and overall survival (OS). Nanostring® analysis in primary tumor samples showed CXCL13 gene expression's positive correlation not only with gene profiles related to an immunogenic tumor microenvironment, increased B cells and T cells (mainly CD8+) but also with extended OS. In silico analysis using RNAseq data from liver metastases treated or not with neoadjuvant oxaliplatin-based combinations, and deconvolution analysis using the MCP-counter algorithm, confirmed CXCL13 gene expression's association with increased immune infiltration, improved OS, and Tertiary Lymphoid Structures (TLSs) gene signatures, especially in neoadjuvant-treated patients. CXCL13 analysis in serum from 36 oxaliplatin-treated patients from the METIMMOX study control arm, reported similar findings. In conclusion, the increase of CXCL13 levels in peripheral blood and its association with the formation of TLSs within the metastatic lesions, emerges as a potential biomarker indicative of the therapeutic efficacy in mCRC patients undergoing oxaliplatin-based treatment.

Human SMC2 protein, a core subunit of human condensin complex, is a novel transcriptional target of the WNT signaling pathway and a new therapeutic target.

Human SMC2 is part of the condensin complex, which is responsible for tightly packaging replicated genomic DNA prior to segregation into daughter cells. Engagement of the WNT signaling pathway is known to have a mitogenic effect on cells, but relatively little is known about WNT interaction with mitotic structural organizer proteins. In this work, we described the novel transcriptional regulation of SMC2 protein by direct binding of the ß-catenin·TCF4 transcription factor to the SMC2 promoter. Furthermore, we identified the precise region in the SMC2 promoter that is required for ß-catenin-mediated promoter activation. Finally, we explored the functional significance of down-regulating SMC2 protein in vivo. Treatment of WNT-activated intestinal tumor cells with SMC2 siRNA significantly reduced cell proliferation in nude mice, compared with untreated controls (p = 0.02). Therefore, we propose that WNT signaling can directly activate SMC2 transcription as a key player in the mitotic cell division machinery. Furthermore, SMC2 represents a new target for oncological therapeutic intervention.

Tumour Cell Seeding to Lymph Nodes from In Situ Colorectal Cancer.

Lymph node (LN) metastasis is an important prognostic factor in colorectal cancer (CRC). We aimed to demonstrate the presence of lymphatic vessels (LV) in the mucosa of in-situ (pTis) CRC, and of detectable tumour burden in regional LNs. This is an observational retrospective study of 39 surgically resected in situ CRCs. The number of LVs was evaluated in both pTis and normal mucosa using D2-40 immunostains. All LNs were assessed with both H&E and the One Step Nucleic Acid Amplification (OSNA) assay, and the results were correlated with clinicopathological features. D2-40 immunohistochemisty revealed LVs in the lamina propria of all pTis CRC (100%), being absent in normal mucosa. A median of 16 LNs were freshly dissected per patient, and all cases were pN0 with H&E. Molecular LN analysis with OSNA revealed the presence of low amounts of tumour burden in 11/39 (28%) cases (range 400 to 4270 CK19 mRNA copies/µL), which had no clinical consequences. This study demonstrates the presence of LVs in the lamina propria in 100% of pTis CRC, as well as the presence of low amounts of tumour burden in regional LNs, only detected by molecular methods. Given the prognostic value of LN tumour burden, its molecular quantification may help a patient's clinical management.

Validation of a DNA methylation microarray for 285,000 CpG sites in the mouse genome.

Mouse has been extensively used as a model organism in many studies to characterize biological pathways and drug effects and to mimic human diseases. Similar DNA sequences between both species facilitate these types of experiments. However, much less is known about the mouse epigenome, particularly for DNA methylation. Progress in delivering mouse DNA methylomes has been slow due to the currently available time-consuming and expensive methodologies. Following the great acceptance of the human DNA methylation microarrays, we have herein validated a newly developed DNA methylation microarray (Infinium Mouse Methylation BeadChip) that interrogates 280,754 unique CpG sites within the mouse genome. The CpGs included in the platform cover CpG Islands, shores, shelves and open sea sequences, and loci surrounding transcription start sites and gene bodies. From a functional standpoint, mouse ENCODE representative DNase hypersensitivity sites (rDHSs) and candidate cis-Regulatory Elements (cCREs) are also included. Herein, we show that the profiled mouse DNA methylation microarray provides reliable values among technical replicates; matched results from fresh frozen versus formalin-fixed samples; detects hemimethylated X-chromosome and imprinted CpG sites; and is able to determine CpG methylation changes in mouse cell lines treated with a DNA demethylating agent or upon genetic disruption of a DNA methyltransferase. Most important, using unsupervised hierarchical clustering and t-SNE approaches, the platform is able to classify all types of normal mouse tissues and organs. These data underscore the great features of the assessed microarray to obtain comprehensive DNA methylation profiles of the mouse genome.

[Update of the recommendations for the determination of biomarkers in colorectal carcinoma. National Consensus of the Spanish Society of Medical Oncology and the Spanish Society of Pathology]. / Actualización de la recomendación para la determinación de biomarcadores en el carcinoma colorrectal. Consenso Nacional de la Sociedad Española de Oncología Médica y de la Sociedad Española de Anatomía Patológica.

This update of the consensus of the Spanish Society of Medical Oncology (Sociedad Española de Oncología Médica - SEOM) and the Spanish Society of Pathology (Sociedad Española de Anatomía Patológica - SEAP), reviews the advances in the analysis of biomarkers in advanced colorectal cancer (CRC) as well as susceptibility markers of hereditary CRC and molecular biomarkers of localized CRC. Recently published information on the essential determination of KRAS, NRAS and BRAF mutations and the possible benefits of determining the amplification of human epidermal growth factor receptor 2 (HER2), the expression of proteins in the DNA repair pathway and the study of NTRK fusions are also evaluated. From a pathological point of view, the importance of analysing the tumour budding and poorly differentiated clusters and its prognostic value in CRC is reviewed, as well as the impact of molecular lymph node analysis on lymph node staging in CRC. The incorporation of pan-genomic technologies, such as next-generation sequencing (NGS) and liquid biopsy in the clinical management of patients with CRC is also outlined. All these aspects are developed in this guide which, like the previous one, will be revised when necessary in the future.

Antitumor Activity of the Novel BTK Inhibitor TG-1701 Is Associated with Disruption of Ikaros Signaling in Patients with B-cell Non-Hodgkin Lymphoma.

PURPOSE: Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored. EXPERIMENTAL DESIGN: A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTKC481S-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments. RESULTS: A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of "early-responders" from a group of "late-responders." This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo in late-responder patients and in BTKC481S, BTKKO, and noncanonical NF-κB models. CONCLUSIONS: These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.

A proteomic approach links decreased pyruvate kinase M2 expression to oxaliplatin resistance in patients with colorectal cancer and in human cell lines.

We aimed to gain further understanding of the molecular mechanisms involved in oxaliplatin resistance in colorectal cancer by using a proteomic approach. A 5-fold oxaliplatin-resistant cell line, HTOXAR3, was compared with its parental cell line, HT29, using two-dimensional PAGE. Mass spectrometry, Western blot, and real-time quantitative PCR confirmed the down-regulation of pyruvate kinase M2 (PK-M2) in HTOXAR3 cells. In a panel of eight colorectal cancer cell lines, we found a negative correlation between oxaliplatin resistance and PK-M2 mRNA levels (Spearman r=-0.846, P=0.008). Oxaliplatin exposure in both HT29 and HTOXAR3 led to PK-M2 mRNA up-regulation. PK-M2 mRNA levels were measured by real-time quantitative PCR in 41 tumors treated with oxaliplatin/5-fluorouracil. Tumors with the lowest PK-M2 levels attained the lowest response rates (20% versus 64.5%, P=0.026). High PK-M2 levels were associated with high p53 levels (P=0.032). In conclusion, the data provided clearly link PK-M2 expression and oxaliplatin resistance mechanisms and further implicate PK-M2 as a predictive marker of response in patients with oxaliplatin-treated colorectal cancer.

Complete Loss of EPCAM Immunoexpression Identifies EPCAM Deletion Carriers in MSH2-Negative Colorectal Neoplasia.

The use of epithelial cell adhesion molecule (EPCAM) immunohistochemistry (IHC) is not included in the colorectal cancer (CRC) screening algorithm to detect Lynch syndrome (LS) patients. The aim of the present study was to demonstrate that EPCAM IHC is a useful tool to guide the LS germ-line analysis when a loss of MSH2 expression was present. We retrospectively studied MSH2 and EPCAM IHC in a large series of 190 lesions composed of malignant neoplasms (102), precursor lesions of gastrointestinal (71) and extra-gastrointestinal origin (9), and benign neoplasms (8) from different organs of 71 patients suspicious of being LS due to MSH2 alterations. LS was confirmed in 68 patients, 53 with MSH2 mutations and 15 with EPCAM 3'-end deletions. Tissue microarrays were constructed with human normal tissues and their malignant counterparts to assist in the evaluation of EPCAM staining. Among 154 MSH2-negative lesions, 17 were EPCAM-negative, including 10 CRC and 7 colorectal polyps, and 5 of them showed only isolated negative glands. All lesions showing a lack of EPCAM expression belonged to patients with EPCAM 3'-end deletions. EPCAM IHC is a useful screening tool, with 100% specificity to identify LS patients due to EPCAM 3'-end deletions in MSH2-negative CRC and MSH2-negative colorectal polyps.

Increased levels of copper efflux transporter ATP7B are associated with poor outcome in colorectal cancer patients receiving oxaliplatin-based chemotherapy.

Recently, the copper efflux transporters ATP7B and ATP7A have been implicated in the transport of and resistance to platinum drugs in breast and ovarian cancers. Because of the extensive use of oxaliplatin in colorectal cancer (CRC), we examined the expression of both transporters in tumors from CRC patients treated with oxaliplatin/5FU and sought to determine whether their expression can predict clinical outcome in these patients. ATP7B and ATP7A levels were determined by quantitative real-time PCR in 50 primary tumors of previously untreated patients with advanced colorectal adenocarcinoma who were subsequently treated with oxaliplatin/5FU. Additionally, ATP7B protein expression was assessed by immunohistochemical staining using a tissue microarray. Patients with the lowest mRNA expression levels of ATP7B had a significantly longer time to progression (TTP) (p = 0.0009) than patients with the highest levels (12.14 months vs. 6.43 months) who also had an increased risk of progression (HR = 3.56; 95% CI, 1.6-7.9; p = 0.002). Furthermore, patients with low levels of both protein and mRNA of ATP7B derived the maximum benefit from oxaliplatin/5FU with the longest TTP as compared with patients with high levels of ATP7B protein and mRNA (14.64 months vs. 4.63 months, respectively, p = 0.01) and showed a nonsignificant trend toward a lower response rate (37.5% and 75%, respectively). In conclusion, ATP7B mRNA and protein expression in colorectal tumors is associated with clinical outcome to oxaliplatin/5FU. Prospective studies are required to evaluate the role of this marker in tailoring chemotherapy.