e15041 Background: Resistance to oxaliplatin is one of the main problems of colorectal cancer (CRC) treatment success. It is not clear if intrinsic and acquired resistance processes are developed by related mechanisms. In a previous work (Martinez-Cardús et al. Mol Cancer Ther, January 2009), we determined a profile of oxaliplatin-acquired resistance related genes by using an in vitro model. In the present work, we analyzed this genetic profile in paraffin-embedded primary adenocarcinomas from CRC patients treated with oxaliplatin-fluoropyrimidine. mRNA expression data was correlated with response rate and time to progression (TTP) in order to determine the role of these genes as markers of resistance to oxaliplatin-based treatment. Material and Method: mRNA levels were analyzed by using Real Time PCR. β-actin and 18s were used as housekeeping genes and, as a reference sample, we used commercial pool of mRNA from different human tumours. Chi- square and Fisher test were used in order to value differences in response rate to treatment. TTP was studied by using Kaplan Meyer curves and Log rank test. Median and percentile 33 and 66 were used as threshold values to determine both high and low expression level groups for each gene analyzed. We considered statistically significant a two-sided p-value lower than 0.05. Results: Forty-four advanced CRC patients treated with fluoropyrimidine plus oxaliplatin were analyzed. 54.5% of them were males; primary tumour was localized in colon in a 65.9% of cases. According to qRT-PCR analysis, the in vitro oxaliplatin acquired resistance related genes could be detected in the tumours but the expression of any of them correlated significantly with in vivo resistance to oxaliplatin-based treatment by using the three different threshold values to define groups. Conclusions: According to our results, these genes could not be used as markers of resistance to oxaliplatin-based treatment in non-treated tumours. Thereby, oxaliplatin resistance acquisition genes seems not to be involved in intrinsic drug resistance probably due to the fact that acquired and intrinsic oxaliplatin resistance are not related mechanisms. Further studies to typify oxaliplatin intrinsic resistance potential markers are guaranteed. No significant financial relationships to disclose.

Abstract Background/purpose: Despite remaining one of the most active drugs in the treatment of advanced colorectal cancer (aCRC), oxaliplatin (OXA) resistance still is a multifactorial and complex process to decipher. In a previous work we identified low levels of PKM2 (mRNA and protein) as a putative OXA-resistance marker in CRC cell lines and patients. The purpose of this work was to identify molecular mechanisms underlying PKM2 role in resistance to OXA. Material and methods: PKM2 transient gene silencing was achieved by transfecting specific siRNAs (Ambion) in HT29 and HCT116 CRC cell lines. Gene silencing was validated by qPCR and Western Blot with specific Taqman primers and probes (assay no. Hs00762869_s1) and antibody (Cell signaling), respectively. Cell proliferation was measured by the MTT assay (Roche Diagnostics). Inhibitory concentrations were determined in each cell line by the median-effect line method. Cell viability was studied with the use of trypan blue stain and apoptosis was detected with FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). PKM2 subcellular localization was studied by fluorescence microscopy in an Axiovision Z1 by using Apotome system at 40x immersion oil lens (Carl Zeiss, Heidelberg, Germany). Expression patterns of cell death genes were analyzed through Human Cell Death Pathway Finder PCR Array 384 HT (PAHS-212Z, SA Biosciences). Results: In order to assess how PKM2 expression influences OXA response in CRC cells, we silenced PKM2 by using specific siRNAs in HT29 and HCT116 cells. Cells were then treated with OXA at different concentrations and we observed that PKM2 silencing induced resistance in HT29 (p53 mutated) cells and sensitivity in HCT116 (p53 wild type) cells. Moreover, PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to OXA in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that OXA and PKM2 silencing altered cell death gene expression patterns including BMF, which was significantly increased in HT29 cells in response to OXA in a dose and time-dependent manner but not in siPKM2-HT29 and HTOXAR3 cells. Conclusion: Our data report new non-glycolytic roles of PKM2 in response to genotoxic damage, which depend on p53 mutational status and proposes BMF as a possible target gene of PKM2 to be involved in OXA response and resistance in CRC cells. Citation Format: Alba Ginés, Anna Martínez-Cardús, Vicenç Ruiz de Porras, Eva Musulén, José Luis Manzano, Laura Layos, Cristina Bugés, Albert Abad, Eva Martinez-Balibrea. PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in human colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3752. doi:10.1158/1538-7445.AM2014-3752

Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells.

We report a case of a 68-year-old man who developed an advanced Triple-Negative Breast Cancer (TNBC) presented as a Carcinoma of Unknown Primary (CUP) at diagnoses. After progressing to first line therapy, the molecular platform EPICUP® was performed and the results suggested breast cancer as origin, although non breast primary tumor was found by imaging techniques; and the patient was treated according to this origin. During the evolution of the disease a nodule appeared in the left breast, and a biopsy confirmed the diagnosis of an infiltrating ductal carcinoma. Male breast cancer (MBC) represents an infrequent and heterogeneous disease, but TNBC in men represents less than 0.02% of total breast cancer cases. Unfortunately, there is no standard of care for MBC, and due to the development of new treatment strategies are focused on women BC, there are less opportunities to participate in clinical trials for MBC. Through the review of a clinical case, we show how the molecular platform could help in finding the origin of the CUP. Moreover, further coordinated multicenter international collaboration is necessary to conduct larger and successful therapeutic clinical trials in MBC, and specifically, male TNBC trials to better understand this rare entity and guide future management strategies.

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.

In recent years, an increasing number of studies have shown that elevated expression of cyclin dependent kinase (Cdk5) contributes to the oncogenic initiation and progression of many types of cancers. In this study, we investigated the expression pattern of Cdk5 in colorectal cancer (CRC) cell lines and in a large number of tumor samples in order to evaluate its relevance in this pathogenesis and possible use as a prognostic marker. We found that Cdk5 is highly expressed and activated in CRC cell lines and that silencing of the kinase decreases their migration ability. In tumor tissues, Cdk5 is overexpressed compared to normal tissues due to a copy number gain. In patients with localized disease, we found that high Cdk5 levels correlate with poor prognosis, while in the metastatic setting, this was only the case for patients receiving an oxaliplatin-based treatment. When exploring the Cdk5 levels in the consensus molecular subtypes (CMS), we found the lowest levels in subtype 1, where high Cdk5 again was associated with a poorer prognosis. In conclusion, we confirm that Cdk5 is involved in CRC and disease progression and that it could serve as a prognostic and predictive biomarker in this disease.

One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.

A total of 5 full-scale Reinforced Concrete (RC) frames were tested to develop new models for predicting the structural behavior of corroded RC frames and to verify the accuracy of the models mostly developed for RC columns. Four corroded and one non-corroded RC frame were tested under cyclic loading. Corroded RC frames were tested under displacement-controlled cyclic loading at four corrosion levels and for a constant applied axial load ratio of 0.20, with the same concrete compression strength level and reinforcement bar layout. Actual corrosion ratios were obtained following the loading tests, breaking all RC columns and beams and extracting all reinforcement bars from concrete. The test results demonstrated that the corrosion level played an active role in the structural capacity of the RC frames. Initial corrosion crack widths had more significant effects on structural performance. Some models proposed for corroded RC columns can also be used for corroded RC frames in terms of yield and ultimate load capacities according to the corrosion levels of the columns and beam composing the RC frame. It was also concluded from the test results that the displacement-based ductility ratio for corroded RC frames could still be misleading if they were not computed according to energy-based or bilateral failure criteria. Structural properties such as initial/secant stiffness and energy absorption capacity calculated based on experimental measurements for RC frames had further disclosed corrosion effects

AIMS: Pathological evaluation of colorectal carcinoma (CRC) diagnosed at stage pT1 is challenging. Nevertheless, it is crucial for treatment guiding and to determine the patient's prognosis. This study aimed to assess the interobserver variability in the histopathological evaluation of pT1 CRC. METHODS AND RESULTS: A retrospective multicentre pT1 CRC cohort study was designed (EpiT1 consortium). A task force comprising 20 experienced pathologists conducted the histopathological evaluation using digitalized haematoxylin-eosin (H&E) slides. A pilot study was performed with 10 cases, and afterwards, a consensus meeting was held to assess interobserver variability. Then, a concordance study was performed by assessing 70 new pT1 CRC cases. We used percentage agreement and Gwet's Agreement Coefficient 1 for categorical variables, and intraclass correlation coefficient (ICC) for continuous variables. In the pilot study, histological grade and perineural invasion (PNI) demonstrated 100% agreement, with good concordance for lymphovascular invasion (LVI), tumour budding (TB), poorly differentiated clusters (PDC) and margin assessment. The concordance study showed high agreement (≥90%) on histological grade, PDC, PNI and LVI. Submucosal invasion depth showed excellent reliability in the concordance study (ICC = 0.97). Notably, in both studies, the agreement of PDC was higher than for TB. Lower concordance was observed on stromal lymphocytes and the status of muscularis mucosae. CONCLUSIONS: Our results emphasize the need for standardization in evaluating pT1 CRC to improve the concordance among pathologists, and the precision of digital measurements. Moreover, the addition of PDC assessment in pT1 CRC diagnostic guidelines could help to improve the accuracy of risk stratification and reliably predict prognosis.