Tumour heterogeneity: principles and practical consequences

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

In stage II/III lymph node positive breast cancer patients less than 55 years of age, keratin 8 expression in lymph node metastases but not in the primary tumor is an indicator of better survival

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 outcome. 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<0.001). K8 immunostaining of tissue from the lymph node metastasis allows defining a sub-group of lymph node positive BC patients with a highly unfavourable outcome, for whom therapeutic options might have to be reconsidered

PI3K/AKT Signaling in Breast Cancer Molecular Subtyping and Lymph Node Involvement

Lymph node metastatic involvement persists to be among the most important predictors of recurrence and survival in breast carcinoma (BC). This study 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 mRNA level in a cohort of 305 breast cancer patients, 151 lymph node-negative (LN-) and 154 lymph node-positive (LN+). The analyzed genes belonged to the RAS pathway (RAF1, ERBB2, PIK3CB, AKT1, AKT2, AKT3), RB pathway (RB1 and CDK2) and cellular differentiation (KTR8). 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 AKTs isoforms influenced also patients\u2019 survival rates. In detail, higher expression levels of AKT1 and AKT2 negatively influenced overall patients\u2019 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

Reliability of miRNA Analysis from Fixed and Paraffin-Embedded Tissues

In clinical practice, patients\u2019 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\u2019s 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\u2019s 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\u2019s-fixed specimens, but on average, higher Ct values and lower copies/\u3bcL were found in Bouin\u2019s-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin\u2019s ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin\u2019s-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin\u2019s-fixed ones

Metformin and aspirin treatment could lead to an improved survival rate for Type 2 diabetic patients with stage II and III colorectal adenocarcinoma relative to non-diabetic patients

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\u2010diabetic patients, have an inhibitory effect on cancer cell survival. The present population\u2010based 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 informa\u2010 tion system. CRC\u2010speci c and relative survival probabilities were computed for each group of patients de ned 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\u2010year relative survival for stage III CRC was 101% [95% con dence interval (CI)=76\u2010126] in the 18 patients with DM2 treated with metformin and ASA, 55% (95% CI=31\u201078) in the 23 without DM2 treated with ASA, 55% (95% CI=45\u201065) in the 150 without DM2 not taking ASA, and 29% (95% CI=13\u201045) in the 43 with DM2 treated with metformin, however not with ASA. The ndings support the hypothesis of a possible inhibitory effect of metformin and ASA on CRC cells. Randomized controlled trials are required to verify this hypothesis

po 349 akt3 but not akt1 and akt2 confers a longer survival rate to less aggressive breast cancers

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

Liquid dynamic medicine and N-of-1 clinical trials: A change of perspective in oncology research

The increasing use of genomics to define the pattern of actionable mutations and to test and validate new therapies for individual cancer patients, and the growing application of liquid biopsy to dynamically track tumor evolution and to adapt molecularly targeted therapy according to the emergence of tumor clonal variants is shaping modern medical oncology., In order to better describe this new therapeutic paradigm we propose the term "Liquid dynamic medicine" in the place of "Personalized or Precision medicine". Clinical validation of the "Liquid dynamic medicine" approach is best captured by N-of-1 trials where each patient acts as tester and control of truly personalized therapies. \ua9 2017 The Author(s)

Reduced expression of \u3b1-L-FUCOSIDASE-1 (FUCA-1) predicts recurrence and shorter cancer specific survival in luminal B LN+ breast cancer patients

Background: The lysosomal enzyme \u3b1-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. 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. 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\u3000analysis 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