Search

Supplementary Figure 3 - PDF file 33K, GSK3B depletion does not abolish the resistance of HCT116p53KO cells to targeted drugs

Supplementary Figure 4 - PDF file 458K, Caspases are not activated after 5FU treatment in GSK3B-silenced HCT116p53KO

Supplementary Data - PDF file 93K, Supplementary Materials and Methods and Supplementary Legend to Figures

Supplementary Table 1 - PDF file 22K, Known genetic alterations of the different colon carcinoma cell lines used in the paper

Supplementary Figure 1 - PDF file 450K, Identification and validation of the genes supporting drug 4 resistance by shRNA-mediated phenotype screen

Supplementary Table 2 - PDF file 70K, Cohort of patients characterization

Supplementary Table 3 - PDF file 74K, Correlation between activated GSK3B and patients outcome

Supplementary Figure 5 - PDF file 219K, tAIF released from mitochondria upon 5FU treatment in GSK3B-silenced HCT116p53KO cells mediates drug-induced cell death

Supplementary Figure 6 - PDF file 2812K, GSK3B is activated in colon carcinoma samples

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression.Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients.Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR.

Supplementary Figure 2 - PDF file 42K, GSK3B silencing by siRNA abolishes drug resistance of p53-null colon carcinoma cell lines

Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of molecular analyses is strictly dependent on the quality of the biomolecules, which is highly influenced by pre-analytical processes. In this study, the effect of different fixatives was compared, including formalin, Bouin's solution, RCL2® and TAG-1™ fixatives, by stringent application of ISO standards in mouse liver tissue processing, including formalin-free transport of tissues and tissue grossing in a refrigerated environment. The effect of fixatives was studied in terms of nucleic acid quality at the time of tissue processing and after one year of tissue storage at room temperature in the dark. Furthermore, a microcomputed tomography (CT) scan analysis was applied to investigate the paraffin embedding. The results show that the application of ISO standards in tissue processing allows analysis of 400 bases amplicons from RNA and 1000 bases from DNA, even in extracts from formalin-fixed and paraffin-embedded tissues. However, after one year storage at room temperature in the dark, a degradation of the nucleic acids was observed. Nevertheless, extracts can still be analyzed, but for metachronous tests it is highly recommended to repeat the quantitation of housekeeping genes in order to standardize the extent of nucleic acid degradation.

When dealing with pre-analytics for tissues, it is often that case that tissue heterogeneity, and particularly tumor heterogeneity, is not taken into account as a preliminary condition for obtaining reproducible results in molecular analysis at the diagnostics and clinical research levels. It is well known that when sampling tumor tissues in different areas, for example the border or the central area of the tumor, different genes are expressed, and due to polyclonality in most tumors different areas can have different DNA and epigenetic alterations. For this reason, it is extremely important to establish and standardize specific tissue sampling protocols for molecular extraction as well as in situ molecular methods. A correct approach to heterogeneity is the basis for a more reproducible and exchangeable type of molecular analysis that can provide useful information at the prognostic and predictive levels. Heterogeneity should also be taken into consideration during cancer treatment, since therapy modifies the clonal composition of tumors. Here the different types of tumor heterogeneity and the improper pre-analytical conditions in tissue processing that can generate heterogeneous artefacts are described.

Lymph node metastatic involvement persists to be among the most important predictors of recurrence and survival in breast carcinoma (BC). This study is aimed at investigating possible gene expression differences in primary BC between patients with or without lymph node involvement at the time of diagnosis. In a retrospective study, we investigated the potential prognostic role of 9 candidate biomarkers at the mRNA level in a cohort of 305 breast cancer patients, 151 lymph node-negative (LN-) and 154 lymph node-positive (LN+) individuals. The analyzed genes belonged to the RAS pathway (RAF1, ERBB2, PIK3CB, AKT1, AKT2, and AKT3), RB pathway (RB1 and CDK2), and cellular differentiation (KRT8). Their expression profiles were investigated by RT-qPCR and were correlated to immunohistochemically based molecular subtypes and BC clinical and pathological features. The differential expression of several genes in the primary tumor tissue was related to the LN involvement. Some of those genes, including PIK3CB, RB1, and AKT3, were more expressed in LN- BC patients, while some others, notably ERBB2 and AKT1, in LN+ ones. Among the candidate biomarkers, the expression levels of AKT isoforms influenced also patients’ survival rates. In detail, higher expression levels of AKT1 and AKT2 negatively influenced overall patients’ survival, and in particular, AKT2 expression levels defined a group of luminal B BC patients with shorter cancer-specific survival. On the contrary, longer cancer-specific survival was recorded in luminal A BC patients with higher expression levels of AKT3. That finding was also confirmed by Cox multivariate analysis. The same AKT3 resulted to be a possible candidate predictive biomarker for Tamoxifen response. In conclusion, our study highlighted the complex regulation of the PI3K/AKT pathway in BC and its differences in BC patients with and without lymph node involvement.

Objective High grade serous ovarian carcinoma (HGSOC) is the most common type of malignant ovarian neoplasm and the main cause of ovarian cancer related deaths worldwide. Although novel biomarkers such as homologous recombination deficiency testing have been implemented into the clinical decision-making algorithm since diagnosis, morphological classification and immunohistochemistry analysis are essential for diagnostic purpose. This study aims at identifying histologic and clinical features that can be predictive of patients' prognosis. Methods Morphological and architectural characterization including SET (Solid-Endometroid-Transitional)/Classic features was carried out in a cohort of 234 patients analyzing 695 slides. From each slide tumor infiltrating lymphocyte (TILs), the presence of necrosis, the number of mitoses, the presence of psammoma bodies, giant cells and atypical mitoses were recorded. Morphological heterogeneity was quantified by the Shannon's diversity index (SDI) considering the percentage of each architectural pattern per patient's slide. Results The frequency of architectural patterns and morphological variables varied with respect of the surgical strategy (primary debulking surgery vs interval surgery after neoadjuvant chemotherapy). HGSOCs exhibiting SET features had a longer overall as well as progression free survival. Among SET features, pseudo-endometrioid and transitional like patterns had the best outcome, while it was heterogenous for solid pattern, that had better outcome for BRCA 1 negative and less heterogeneous tumors. In patients submitted to neoadjuvant chemotherapy a higher intratumor heterogeneity as defined by SDI was a negative independent prognostic factor. Conclusions A comprehensive histological examination considering architectural patterns and their heterogeneity can help in prognostication of HGSOCs.

Every human tissue collected from surgery or biopsy is formalin-fixed and paraffin-embedded (FFPE). A huge quantity of human tissues is preserved in hospitals, and it is possible to estimate that in Europe about three hundred million new specimens of tissues are stored in archives every year. These archive tissues (AT) represent any type of even rare pathological lesions with a number of cases sufficient for any possible study. Frequent evidence of acquired resistance to the new targeted therapies has posed clinicians new issues to be addressed, such as the need to go back to patients’ tissues. Increasingly more often we have to use ATs because of clinical research, which is now starting to be an integrated process with applied medicine. We cannot separate clinics from clinical research, it is a unique entity. This creates new necessities such as better quality ATs, more standardized methods, a careful choice of the tissues, and a better organization. The pathology archives of hospitals are clinical biorepositories that are different from a research biobank. The major difference is related to the clinical purpose of these archives; even though this may not seem to be an obstacle to their utilization for research purposes, it is perfectly fitted with the most recent necessities of a kind of clinical research that is strictly related to medicine and perfectly integrated into it. The use of AT biorepositories represents a peculiar type of tissue as a source that also needs a different bioethical approach.

Summary Next Generation Sequencing (NGS) is increasingly used in diagnostic centers for the assessment of genomic alterations to select patients for precision oncology. The Italian Society of Anatomic Pathology and Diagnostic Cytopathology (SIAPEC) through the Molecular Pathology and Predictive Medicine Study Group (PMMP) has been following the progressive development of centers that have adopted NGS technology in diagnostics over time. In July 2017, a study network on massive parallel sequencing was activated in Italy and recognized as the NGS SIAPeC National Network by the SIAPeC Scientific Society Board. Since then, activities have been implemented within the network that provide for alignment of laboratories through diagnostic concordance analysis and monitoring of centers adhering to the Network. Recently, considering the growing need for extended genomic analyses, the PMMP distributed a national survey to assess activities related to the use of genomic diagnostics in oncology within the NGS SIAPEC National Network. Thirty centers participated in the survey. Eighty percent of the centers are laboratories within Pathology Departments. The distribution of laboratories in the country, the diagnostic laboratory/population ratio, the staff dedicated, the type and number of sequencing and mechatronics platforms available, the genomic panels utilized, and the type and number of diagnostic tests carried out in the last year in each center, are reported. The centers were also asked whether they participated in a multidisciplinary Molecular Tumor Board (MTB) for management of patients. Thirty percent of the centers had a MTB that was ratified by regional decree. The professionals most frequently involved in the core team of the MTB are the pathologist, oncologist, molecular biologist, geneticist, pharmacologist, and bioinformatician. The data from this survey indicate that NGS diagnostics in Italy is still heterogeneous in terms of geographical distribution and the characteristics of laboratories and diagnostic test performed. The implementation of activities that favors harmonization, the logistics and the convergence of biological material in reference centers for molecular analyses is a priority for the development of a functional laboratory network.

Cutaneous melanoma (CM) incidence is rising worldwide and is the primary cause of death from skin disease in the Western world. Personal risk factors linked to environmental ultraviolet radiation (UVR) are well-known etiological factors contributing to its development. Nevertheless, UVR can contribute to the development of CM in different patterns and to varying degrees. The present study aimed at investigating whether altitude of residence can contribute to the development of specific types of CM and/or influence its progression. To this aim, 306 formalin-fixed and paraffin-embedded (FFPE) tissues from primary CM diagnosed in different geographical areas were submitted to B-RAF proto-oncogene serine/threonine kinase (BRAF) and N-RAS proto-oncogene GTPase (NRAS) mutational status detection and mRNA and miRNA profiling by qPCR. Genes were chosen for their functions in specific processes, such as immune response (CD2, PDL1, or CD274) and pigmentation (MITF, TYRP1, and TRPM1). Furthermore, four microRNAs, namely miR-150-5p, miR-155-5p, miR-204-5p, and miR-211-5p, were included in the profiling. Our results highlight differences in the gene expression profile of primary CM with respect to the geographical area and the altitude of residence. Melanoma-specific survival was influenced by the gene expression of mRNA and miRNAs and varied with the altitude of patients&rsquo, residence. In detail, TYRP1 and miR-204-5p were highly expressed in patients living at higher altitudes, unlike miR-150-5p, miR-155-5p, and miR-211-5p. Since miRNAs are highly regulated by reactive oxygen species, it is possible that different regulatory mechanisms characterize CMs at different altitudes due to the different environment and UVR intensity.

In clinical practice, patients&rsquo, tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin&rsquo, s solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, let-7e-5p, miR-423-3p, miR-92a-1-5p, miR-30d-5p, miR-155-5p, miR-200a-3p, and miR-429 were investigated in formalin and matched Bouin&rsquo, s solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin&rsquo, s-fixed specimens, but on average, higher Ct values and lower copies/&micro, L were found in Bouin&rsquo, s-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin&rsquo, s ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin&rsquo, s-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin&rsquo, s-fixed ones.

N/A

Metformin, the drug of choice in the treatment of type 2 diabetes mellitus (DM2), in addition to aspirin (ASA), the drug prescribed for cardioprotection of diabetic and non-diabetic patients, have an inhibitory effect on cancer cell survival. The present population-based study conducted in the province of Trieste (Italy), aimed to investigate the prevalence of DM2 in patients with colorectal adenocarcinoma (CRC) and survival for CRC in diabetic and nondiabetic patients. All permanent residents diagnosed with a CRC between 2004 and 2007 were ascertained through the regional health information system. CRC-specific and relative survival probabilities were computed for each group of patients defined by CRC stage, presence or absence of DM2 treated with metformin, and presence or absence of daily ASA therapy. A total of 515 CRC patients without DM2 and 156 with DM2 treated with metformin were enrolled in the study. At the time of CRC diagnosis, 71 (14%) nondiabetic and 39 (25%) diabetic patients were taking ASA daily. The five-year relative survival for stage III CRC was 101% [95% confidence interval (CI)=76–126] in the 18 patients with DM2 treated with metformin and ASA, 55% (95% CI=31–78) in the 23 without DM2 treated with ASA, 55% (95% CI=45–65) in the 150 without DM2 not taking ASA, and 29% (95% CI=13–45) in the 43 with DM2 treated with metformin, however not with ASA. The findings support the hypothesis of a possible inhibitory effect of metformin and ASA on CRC cells. Randomized controlled trials are required to verify this hypothesis.

This Pathobiology issue tries to better define the complex phenomenon of intratumor heterogeneity (ITH), mostly from a practical point of view. This topic has been chosen because ITH is a central issue in tumor development and has to be investigated directly in patient tissue and immediately applied in the treatment of the presenting patient. Different types of ITH should be considered: clonal genetic and epigenetic evolution, morphological heterogeneity, and tumor sampling, heterogeneity resulting from microenvironmental autocrine and paracrine interaction, and stochastic plasticity related to different functional cell efficiencies. For a higher level of reproducibility in clinical research and diagnostics, it is necessary to establish standardized analytical methods, including microdissection. In situ techniques can be pivotal to explore tumor microenvironment and can be improved with associated digital analysis. Liquid biopsies for plasma DNA analysis are at present the best method to study recurrent tumors with treatment adaptation, and widespread clinical use could be beneficial. The different types of tumor genomic instabilities could have pragmatic applications to rank ITH for clinical applications: treatment approaches differ in patients with a high nucleotide mutation rate and patients with high copy number alterations.

// Serena Bonin 1, * , Alessia Parascandolo 2, * , Cinzia Aversa 1 , Renzo Barbazza 1 , Nobuo Tsuchida 3 , Maria Domenica Castellone 4 , Giorgio Stanta 1 and Giancarlo Vecchio 5, 6, 7 1 Dipartimento di Scienze Mediche, Universita di Trieste-Cattinara, Trieste, Italy 2 IRCSS SDN, Naples, Italy 3 Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan 4 Istituto di Endocrinologia e Oncologia Sperimentale G.Salvatore, CNR, Naples, Italy 5 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli Federico II, Naples, Italy 6 Istituto Superiore di Oncologia, Naples, Italy 7 Istituto Superiore di Oncologia, Genoa, Italy * Co-first authors Correspondence to: Giancarlo Vecchio, email: vecchio@unina.it Keywords: breast cancer; luminal B; lymph node metastasis; α-L-Fucosidase-1; immunohistochemistry Received: October 06, 2017 Accepted: February 01, 2018 Epub: February 07, 2018 Published: March 16, 2018 ABSTRACT Background: The lysosomal enzyme α-L-Fucosidase-1 (FUCA-1) catalyzes the hydrolytic cleavage of terminal fucose residues. FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors as its inactivation perturbs the fucosylation of proteins involved in cell adhesion, migration and metastases. Results: Negativity to FUCA-1 was significantly related to the development of later recurrences in breast cancer patients with lymph node involvement at diagnosis. Cancer specific survival of luminal B LN+ patients was influenced by FUCA-1 expression as luminal B LN+ patients with positive expression had a longer cancer specific survival. FUCA-1 mRNA expression was inversely related to cancer stage and lymph node involvement. WB and qPCR analysis of FUCA-1 expression in breast cancer-derived cell lines confirmed an inverse relationship with tumor aggressiveness. Conclusions: This study shows that, within LN+ breast cancer patients, FUCA-1 is able to identify a sub-set of non recurrent patients characterized by the positive expression of FUCA-1 and that, within luminal B LN+ patients, the expression of FUCA-1 predicts longer cancer specific survival. Methods: We have analyzed FUCA-1 in 305 breast cancer patients by Immunohistochemistry (IHC), and by qPCR in breast cancer patients and in breast cancer cell lines.

Background Lack of early diagnosis methods and the development of drug resistance are among the main reasons for increased mortality rates within breast cancer patients. These two aspects are governed by specific pro-carcinogenic modifications, where TGBβ-induced EMT is one of the leading actors. Endowment of the epithelial cells with mesenchymal characteristics allows them to migrate and invade secondary tissues in order to form malignant sites and also confers chemoresistance. TGFβ which role switches from the tumor suppressor cytokine to the oncogenic one favoring the tumor microenvironment regulates this process. Scope of review This review aims to comprehensively present the updated TGFβ-induced EMT in breast cancer, including the regulatory role of the non-coding RNAs with focus on the miR-200 family and newly discovered lncRNAs such as HOTAIRM1. Additionally, a new phenotype, P-EMT, also modulated by miR-200 and miR-34 families that form complex feedback loops with TGFβ, SNAI1 and ZEB1/2 is presented under an updated form. Major conclusions The hallmarks of EMT are becoming increasingly associated with aggressive forms of breast cancer and low survival rates among patients. Considering that this phenotypical switch can trigger drug resistance, invasion and metastasis, inhibition of EMT could represent an important milestone in mammary cancer treatment. General significance The present review assembles the most recent data regarding TGFβ induced EMT, including the input of non-coding RNAs, contributing to the possible development of new targeted treatment strategies for cancer patients.

International audience

Introduction Breast carcinoma (BC) encompasses a heterogeneous group of tumours with a great variability at the molecular and morphological levels and clinical outcome. Material and methods In this retrospective study we investigated the potential prognostic role of 9 candidate biomarkers in a cohort of 305 breast cancer (BC) patients, both lymph node negative (151) and lymph node positive (154). The analysed genes belonged to the RAS pathway (RAF1, ERBB2, PIK3B, AKT1, AKT2, AKT3), RB pathway (RB1 and CDK2) and cellular differentiation (K8). The expression profiles were investigated by real-time qPCR in formalin-fixed and paraffin-embedded tissues, and correlated to immunohistochemical-based molecular classes, namely luminal A, luminal B, Her2 +and TN. The study was approved by the Ethical Committee of the University of Trieste. Results and discussions In our cohort lymph node involvement resulted to be related to the contribution of several genes at the primary tumour tissue level. Some of those genes resulted to be more expressed in LN negative BC, such as PIK3B, RB1 and AKT3, while some others were more expressed in LN positive BC, such as HER2 and AKT1. Our results show higher expression levels of PIK3B and AKT3 in less aggressive BC and higher expression levels of AKT1 in more aggressive BC highlighting the complex regulation of that pathway in BC. Shorter cancer specific survival was recorded in patients expressing higher levels of AKT1 and AKT2. Furthermore, better cancer specific survival was recorded in luminal A BC patients expressing higher levels of AKT3 (p=0.005 in LN- and p=0.01 in LN+). Conclusion By comparing gene expression in lymph node negative and lymph node positive breast cancers, we found that AKT3 is an independent favourable prognostic factor for luminal A BC patients. Our results showed that a high expression level of AKT3, but not AKT1 and AKT2 was associated to better outcome and longer cancer specific patients’ survival in those patients who display the luminal A molecular class irrespective of lymph node involvement.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.504.

Objective Stroke is one of the leading causes of disability and death in the world. The endocannabinoid (eCB) system is upregulated in several neurological diseases including stroke. A previous animal study demonstrated an increased expression of the endocannabinoid receptor 1 (CB1R) in the penumbra area surrounding the ischemic core, suggesting a crucial role in inflammation/reperfusion after stroke. Regarding the localization of CB1/CB2 receptors, animal studies showed that cortical neurons, activated microglia, and astroglia are involved. Our aim was to evaluate the cerebral expression of CB1R in the ischemic brain areas of 9 patients who died due to acute cerebral infarction in the middle cerebral artery territory. Methods The cerebral autoptic tissue was collected within 48 hours since death. Ischemic and contralateral normal-appearing areas were identified. After tissue preprocessing, 4-µm-thick cerebral sections were incubated with the primary CB1R antibodies (Cayman Chemical Company, Ann Arbor, MI). Thereafter, all cerebral sections were hematoxylin treated. In each section, the total cell number and CB1R-positive cells were counted and the CB1R-positive cell count ratio was calculated. For statistical analysis, Student's t-test was used. Results In normal tissue, CB1R-positive neurons were the majority; a few non-neuronal cells expressed CB1R. In the ischemic areas, a few neurons were detectable. A significant increase in total CB1R staining was found in the ischemic regions compared to contralateral areas. Conclusions We found an increase in CB1R expression in the ischemic region (neuronal and non-neuronal cell staining), suggesting the inflammatory reaction to the ischemic insult. Whether such response might mediate neuroprotective actions or excitotoxicity-related detrimental effects is still unclear.

Genetic factors are known to affect the efficiency of therapy with monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer (mCRC). At present, the only accepted molecular marker predictive of the response to anti-EGFR mAbs is the somatic mutation of KRAS and NRAS as a marker of resistance to anti-EGFR. However, only a fraction of KRAS wild-type patients benefit from that treatment. In this study, we show that the EGFR gene polymorphism rs1050171 defines, independently of RAS mutational status, a sub-population of 11 % of patients with a better clinical outcome after anti-EGFR treatment. Median PFS for patients with the GG genotype was 10.17 months compared to 5.37 of those with AG + AA genotypes. Taken together, our findings could be used to better define CRC populations responding to anti-EGFR therapy. Further studies in larger independent cohorts are necessary to validate the present observation that a synonymous polymorphism in EGFR gene impacts on clinical responsiveness.

Two major reasons compel us to study tumour heterogeneity: firstly, it represents the basis of acquired therapy resistance, and secondly it may be one of the major sources of the low level of reproducibility in clinical cancer research. The present review focuses on the heterogeneity of neoplastic disease, both within the primary tumour, and between primary tumour and metastases. We discuss different levels of heterogeneity and the current understanding of the phenomenon, as well as imminent developments relevant for clinical research and diagnostic pathology. It is necessary to develop new tools to study heterogeneity and new biomarkers for heterogeneity. Established and new in situ methods will be very useful. In future studies, not only clonal heterogeneity needs to be addressed, but also non-clonal phenotypic heterogeneity which might be important for therapy resistance. We also review heterogeneity established in major tumour types, in order to explore potential similarities that might help to define new strategies for targeted therapy.

Axillary lymph node status is one of the most important prognostic variables for breast cancer (BC). To investigate and understand the clinical, histopathological and biological factors that affect prognosis in node-positive young breast cancer patients, we compared the phenotype of 100 primary tumours with their corresponding loco-regional lymph node (LN) metastases using conventional immunohistochemistry (IHC) markers currently in use for molecular classification of breast cancer. By comparing the expression of ER, PR, HER-2, Ki67, K8, K5/6 and vimentin, we found that expression of HER-2, Ki67, K8 and vimentin is frequently lost in lymph node metastases. Between the primary tumour and corresponding lymph node metastases, expression of keratins K8 and K5/6 significantly changed. Expression of K8 in lymph node metastases, but not in primary tumours, segregates patients in two sub-groups with different outcomes. Survival of patients with K8-positive LN metastases at 5 years in comparison with patients with K8-negative LN metastases was 75 vs 48 %, at 10 years 62 vs 22 % and at 20 years 53 vs 14 % (p

A low level of reproducibility is the shortcoming of clinical studies where human tissues are used, especially in oncology (EGAAP, Evaluation of Genomic Applications and Prevention Working Group (2013) Recommendations from the EGAPP Working Group: can testing of tumor tissue for mutations in EGFR pathway downstream effector genes in patients with metastatic colorectal cancer improve health outcomes by guiding decisions regarding anti-EGFR therapy? Genet Med: Off J Am Coll Med Genet 15:517–527, Simon RM, Paik S, Hayes DF (2009) Use of archived specimens in evaluation of prognostic and predictive biomarkers. J Natl Cancer Inst 101:1446–1452). This could be due to the high variability of the pre-analytical conditions of tissue managing and preservation, but also to other two causes. One is the low level of standardization of the methods that is pertinent to the analytical phase. Heterogeneity of tissues is the other problem and it can be considered to be actually related to pre-analytical procedures. Indeed, it is a pre-condition that has to be taken into consideration before the specific analysis. Not all the consequences of heterogeneity can be more or less easily avoided by carefully choosing the tissues to be analyzed.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe., SCOPUS: ar.j, info:eu-repo/semantics/published

We carried out a cohort study in the Italian province of Trieste (2001 population, 242,000) to ascertain whether the risk of a subsequent primary cancer among 265 individuals diagnosed with a first malignancy at ages up to 25 years between 1971 and 1993 differed from that in the general population and to evaluate the effect of cancer family history, quantified by the family risk index (FRI), on the occurrence of second primaries. During the follow-up (median duration = 10 years; 25th–75th percentile = 2–16), 15 cohort members developed a second cancer vs. 1.60 expected for a standardized incidence ratio (SIR) of 9.4 (p < 0.0001). The overall SIR fell to 4.7 (p = 0.004) after excluding the 8 patients with well-known cancer-predisposing conditions (SIR = 300.0; p < 0.0001) and the 50 with a positive family history (FRI ≥ 1.0) of malignant tumors (SIR = 20.0; p < 0.0001). Among 114 patients treated with radiotherapy and chemotherapy for their first neoplasms and not affected by predisposing disorders, 23 with a positive family history of cancer showed a 6.4-fold excess risk of second primaries (p = 0.008) compared with 91 with a negative history (FRI < 1.0). It is imperative that clinicians carefully and regularly evaluate cancer family history of young patients with malignancies. This would enable them to identify possible individual and familial features in patients at higher risk of multiple primaries and to adopt more suitable preventive and therapeutic measures. © 2005 Wiley-Liss, Inc.

Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP–mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

Aside from ethical considerations, the primary requirement for usage of human tissues in basic or translational research is the thorough characterization of tissues. The second, but equally essential, requirement is that tissues be collected, processed, annotated, and preserved in optimal conditions. These requirements put the pathologist at the center of tissue banking activities and of research aimed at discovering new biomarkers. Pathologists not only provide information identifying the specimen but also make decisions on what materials should be biobanked, on the preservation conditions, and on the timeline of events that precede preservation and storage. This central position calls for increased recognition of the role of the pathologist by the biomolecular community and places new demands on the pathologist's workload and scope of scientific activities. These questions were addressed by an Expert Group Meeting of the European Biological and Biomolecular Research Infrastructure (BBMRI). While detailed recommendations are published elsewhere (Bevilacqua et al., Virchows Archivs, 2010, in press), this article outlines the strategic and technological issues identified by the Expert Group and identifies ways forward for better integration of pathology in the current thrust for development of biomarker-based "personalized medicine."

With the aging of the population, frailty has emerged as a new clinical entity. The frail person has exhausted any functional reserve. Current criteria for the recognition of frailty include age of over 85 years, dependence in one or more activities of daily living, three or more comorbid conditions, and the presence of one or more geriatric syndromes. It is calculated that there are approximately 6 million frail patients in the United States and approximately 400,000 of them have cancer. Management of cancer in the frail person is mainly comprised of palliation, which may include some forms of chemotherapy, such as navelbine, gemcitabine, or low-dose taxanes.

Background. There are little data on causes of death in extreme aged. We compared, using autopsy findings, main cause of death, overall disease status, and accuracy rate of clinical diagnoses in extreme aged and persons dying at younger ages. Methods. We reviewed the complete clinical and autopsy records of 114 consecutive inpatients (97 women, 17 men, age range 97-106, mean 99, median 98) who died in Trieste, Italy, and represented 99% of all extreme-aged person deaths in the hospital and 70% in the area. The control group included 151 patients (66 women, 85 men, age range 65-74, mean 70, median 70) who died during the same period in that hospital. Results. Vascular and respiratory diseases together caused 84% of deaths in extreme aged. The main causes of death were pneumonia (n =40,35%), pulmonary embolism (n =16, 14%), stroke (n = 12, I 1%), and myocardial infarction (n =8, 7%). Cancer was responsible for 6% (7/1 14) of deaths in extreme aged and 42% (64/15 I) in the control group. In 5% of extreme aged, autopsy findings did not explain death. The premortem diagnostic accuracy rate for clinical diagnoses was good in 44% of extreme aged, sufficient in 18%, poor in 28%, and not evaluable in 10%, and was significantly different from controls. Pneumonia, pulmonary embolism, and myocardial infarction were markedly underestimated by clinicians in both groups. Conclusions. Extreme aged die mainly of cardiovascular and respiratory diseases and, in most cases, of acute events. Senescence is a rare cause of death. Death from cancer is substantially lower than in persons dying at younger ages. In contrast to no autopsy studies, most extreme aged in our study were found to have specific diseases that explained their deaths.

Background In many tumors and also in melanomas a high expression of different growth factor receptors is sometimes present, and epidermal growth factor receptor (EGFr) and oncogene c-erbB2 are often involved. Recently a tumor suppressor gene, called MTS1, has been identified. MTS1 may have an important function in many tumors like those of the lung, breast, bone, ovary and skin melanomas. Objectives In our study we analyzed the expression at mRNA level of EGFr, c-erbB2 and MTS1 in biopsied paraffinembedded tissues of skin melanomas at different stages of Breslow and according to the presence or absence of metastatic spreading during follow-up. Methods The relative quantitation of the mRNA was performed from histological sections cut from formalin-fixed and paraffin-embedded tissues after RT-PCR. Eighteen patients with melanoma were studied at mRNA expression level of EGF-receptor, c-erbB2 oncogene and MTS1 tumor suppressor gene. The expression has been correlated with the Breslow stage of skin infiltration and the presence of recurrences during the follow-up period. Results The level of expression of EGFr was connected with the level of skin infiltration; in Breslow stages 1 and 2 the expression is low and clearly higher in Breslow 3. But a higher expression is not directly correlated with the presence of recurrences. On the other hand higher levels of c-erbB2 and MTS1 mRNA are related, particularly in stage 3 of skin infiltration melanomas, with absence of recurrences. In this case c-erbB2 seems to be a variable independent of Breslow stage. Conclusions Our study suggests in a preliminary way that a high level of expression of c-erbB2 or MTS1 could be connected with infiltrative melanomas, Breslow stage 3, that do not tend to cause recurrences. On the other hand the EGFr expression is related to the stage of the skin infiltration of the tumors.

Cancer in the oldest old is a novel problem, due to the recent burgeoning of the population aged 85 and older. This article addresses three critical questions related to cancer in the oldest old: Does the incidence of cancer increase after age 95? Is cancer a common cause of death for the oldest old? Is cancer accurately diagnosed in the oldest old? The authors analyzed a group of 507 autopsies of elderly, divided in three age groups, 75 90 years, 95 99, and over 99 (centenarians). The prevalence of cancer was 35% among the younger persons and 20% and 16% respectively for those aged 95 99 and for the centenarians. A fourth of the patients in the younger group died from cancer but only 9.5% of the people between 95 and 98 years and 7.1% of the centenarians died from cancer. The cancer was the direct cause of death for 67% of the younger persons and 41% of patients belonging to the two oldest groups. The prevalence of metastases was 63% for tumors occurring in persons aged 75 90, 32% in persons aged 95 98, and 29% in the centenarians. Cancer had been accurately diagnosed prior to death in 67.4% of persons aged 75 90, in 38.5% of those aged 95 99, and 29.4% of the centenarian. Cancer as cause of death had been underestimated in 16% of the cases in the younger persons and in almost 50% of cases of the oldest old. This study suggests that the incidence of cancer and the importance of cancer as a cause of death may decline after age 95 and that the clinical diagnoses underestimate significantly both the incidence of cancer and the prevalence of cancer deaths in the oldest old.