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

PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in HT29 human colorectal cancer cell lines

Ajuts: Beca bianual de la Fundació Olga Torres 2008-2009Chemoresistance 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

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 search for lymphatic vessels (LVs) in the lamina propria of 39 surgically resected in situ CRC, as well as to detect the presence of tumour burden in the regional LNs. We identified the presence of LVs in the mucosa of all tumours (39/39; 100%) using D240 immunostains. LNs were analysed by both H&E and a RT-PCR-based molecular method. All cases were pN0 with H&E, and the molecular assay detected the presence of low amounts of tumour burden in the LNs of 11/39 (28%) cases, with no clinical consequences at 1 to 5 years of follow-up. The amount of tumour burden in LNs has proven to be a prognostic factor. Despite the fact that pTis is considered to have little or no risk of LN metastasis, our results enabled to quantify the amount of tumour burden within LNs, which may help clinical management. 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

Tumor expression of cyclin-dependent kinase 5 (Cdk5) is a prognostic biomarker and predicts outcome of oxaliplatin-treated metastatic colorectal cancer patients

Colorectal cancer; Cyclin-dependent kinase 5 (Cdk5); Prognostic and predictive biomarkerCáncer colorrectal; Quinasa dependiente de ciclina 5 (Cdk5); Biomarcador pronóstico y predictivoCàncer colorectal; Quinasa dependent de ciclina 5 (CDK5); Biomarcador pronòstic i predictiuIn 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.This work has been funded by the ISCIII grants from the Spanish Government, project numbers PI09/01334 and PI12/02228, and the Departament d’Innovació, Universitats i Empresa, Generalitat de Catalunya, project numbers 2014-SGR-1494, 2017-SGR-1705 and 2017-SGR-723. The group from Eva Martinez Balibrea is furthermore funded by the PIE16/00011 and the group from Diego Arango by the PI16/00540 and AC15/00066 grants

Tumor Expression of Cyclin-Dependent Kinase 5 (Cdk5) Is a Prognostic Biomarker and Predicts Outcome of Oxaliplatin-Treated Metastatic Colorectal Cancer Patients

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

Tumor expression of cyclin-dependent kinase 5 (Cdk5) is a prognostic biomarker and predicts outcome of oxaliplatin-treated metastatic colorectal cancer patients

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

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

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

In 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), 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 are reviewed. Recently published information on the essential determination of KRAS, NRAS and BRAF mutations and the convenience of determining the amplifcation 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 the pathological point of view, the importance of analysing the tumour budding and poorly diferentiated 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 remain open to any necessary revision in the future

Guidelines for biomarker testing in advanced colorectal carcinoma. A National Consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology

Este documento de consenso nace como una iniciativa conjunta de la Sociedad Española de Anatomía Patológica (SEAP) y de la Sociedad Española de Oncología Médica (SEOM) y propone recomendaciones diagnósticas y terapéuticas para el manejo del paciente con cáncer colorrectal (CCR) hereditario, localizado y avanzado basadas en la evidencia científica que existe en la actualidad sobre el uso de biomarcadores. Por tanto, este documento supone una oportunidad para mejorar la eficiencia de la actividad asistencial y la utilización de recursos, lo que redundará en un beneficio para estos pacientes. Con los datos disponibles en la actualidad, este grupo de expertos recomienda que en los pacientes con CCR localizado se determine la inestabilidad de microsatélites, ya que es un factor predictivo relevante para decidir el tratamiento adyuvante. Sin embargo, aunque las firmas de expresión genética ColoPrint® y Oncotype Dx® han demostrado tener valor pronóstico, no existe todavía consenso sobre su uso en la práctica clínica. En cuanto al CCR avanzado, la determinación del estado mutacional de KRAS es indispensable antes de administrar un tratamiento con anti-receptor del factor de crecimiento epidérmico (EGFR), como cetuximab y panitumumab. Sin embargo, la determinación de otros biomarcadores, como las mutaciones de BRAF, EGFR, PI3K y PTEN, no debe llevarse a cabo de forma rutinaria, ya que hoy por hoy no influye en la planificación del tratamiento. Otros aspectos importantes que incluye son los requisitos organizativos y los controles de calidad que deben existir para la adecuada determinación de estos biomarcadores, así como las implicaciones legales que se deben tener en cuenta

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