Investigating genomic, proteomic, and post-transcriptional regulation profiles in colorectal cancer: a comparative study between primary tumors and associated metastases.

INTRODUCTION: Approximately 50% of patients with primary colorectal carcinoma develop liver metastases. This study investigates the possible molecular discrepancies between primary colorectal cancer (pCRC) and their respective metastases. METHODS: A total of 22 pairs of pCRC and metastases were tested. Mutation profiling of 26 cancer-associated genes was undertaken in 22/22primary-metastasis tumour pairs using next-generation sequencing, whilst the expression of a panel of six microRNAs (miRNAs) was investigated using qPCRin 21/22 pairs and 22 protein biomarkers was tested using Reverse Phase Protein Array (RPPA)in 20/22 patients' tumour pairs. RESULTS: Among the primary and metastatic tumours the mutation rates for the individual genes are as follows:TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. The primary-metastasis tumour mutation status was identical in 54/60 (90%) loci. However, there was discordance in heterogeneity status in 40/58 genetic loci (z-score = 6.246, difference = 0.3793, P < 0.0001). Furthermore, there was loss of concordance in miRNA expression status between primary and metastatic tumours, and 57.14-80.95% of the primary-metastases tumour pairs showed altered primary-metastasis relative expression in all the miRNAs tested. Moreover, 16 of 20 (80%) tumour pairs showed alteration in at least 3 of 6 (50%) of the protein biomarker pathways analysed. CONCLUSION: The molecular alterations of primary colorectal tumours differ significantly from those of their matched metastases. These differences have profound implications for patients' prognoses and response to therapy.

IL-6 and IL-10 are associated with good prognosis in early stage invasive breast cancer patients.

Macrophage-associated cytokines play an important role in cancer metastasis; however, the functions of interleukins (IL) 6 and 10 in breast cancer (BC) progression and metastasis are not clear. In this study the roles of IL-6/IL-10 in regulating vascular invasion and their prognostic significance in BC are investigated. MDA-MB-231 and MCF-7 migration (± IL-6 or IL-10) was assessed by scratch wound assay. Cancer cell adhesion to IL-6/IL-10 stimulated blood and lymphatic endothelial cells (EC) was investigated. Expression of IL-6 /IL-10 was assessed using immunohistochemistry in an annotated cohort of early stage BC (n = 1380) and associations with clinicopathological variables and clinical outcome evaluated. IL-6 did not alter BC cell migration however a dose-dependent inhibition in MDA-MB-231 migration with IL-10 treatment was observed (P = 0.03). BC cells were more adhesive to blood vs lymphatic EC, however, IL-6/IL-10 had no effect on adhesion patterns. High expression of IL-6/IL-10 was associated with clinicopathological criteria (e.g. hormone receptor status, all P < 0.05), improved disease-free survival (DFS; P < 0.05) and improved BC-specific survival (BCSS; only IL-6, P = 0.017). However, neither IL-6 nor IL-10 expression were independent prognostic factors from multivariate analysis. In BC subgroups, IL-6 and IL-10 were good prognosticators in terms of DFS in non-basal, non-triple-negative (non-TN), ER-positive, PgR-positive (only IL-10), and Her-2-negative (only IL-6) BC (all P < 0.05). IL-6 was associated with improved BCSS in non-basal, ER-positive and non-TN BC (all P < 0.05).

Macrophage-derived interleukin-1beta promotes human breast cancer cell migration and lymphatic adhesion in vitro.

Lymphovascular invasion (LVI), encompassing blood and lymphatic vessel invasion, is an important event in tumourigenesis. Macrophages within the tumour microenvironment are linked to the presence of LVI and angiogenesis. This study investigates the role of macrophage-derived, caspase-1-dependent interleukin-1beta (IL-1ß) in an in vitro model of LVI. IL-1ß significantly augmented the adhesion and transmigration of breast cancer cell lines MCF7 and MDA-MB-231 across endothelial cell barriers. MDA-MB-231 and MCF7 showed a higher percentage of adhesion to lymphatic endothelial cells than blood endothelial cells following endothelial cell IL-1ß stimulation (P < 0.001 and P < 0.0001, respectively). Supernatants from activated macrophages increased the adhesion of tumour cells to lymphatic and blood endothelium. Secretion of IL-1ß was caspase-1 dependent, and treatment with caspase-1 inhibitor reduced IL-1ß production by 73% and concomitantly reduced tumour cell adhesion to levels obtained with resting macrophages. Transmigration of MDA-MB-231 cells across blood and lymphatic endothelial monolayers was significantly increased following IL-1ß stimulation. Furthermore, supernatants from activated macrophages increased transmigration of MDA-MB-231 cells across endothelial monolayers, which was abolished by caspase-1 inhibition. IL-1ß stimulation of tumour cells significantly increased their migratory ability and a significant increase in migration was observed when MDA-MB-231 cells were stimulated with macrophage conditioned media (two of three donors). Results demonstrate that macrophage production of IL-1ß plays an important role in the migration of breast cancer cells and their adhesion to, and transmigration across, blood and lymphatic endothelial cells. Results suggest that IL-1ß may play a role in the adhesion to lymphatic endothelial cells in particular.

Calpain-1 is associated with adverse relapse free survival in breast cancer: a confirmatory study.

AIMS: Calpain-1 is a ubiquitously expressed calcium-activated intracellular cysteine protease. Altered expression of calpain system proteins has been implicated in cancer progression and response to chemotherapy. METHODS AND RESULTS: The aim of the current study was to confirm previous data that suggested that calpain-1 expression is associated with relapse-free survival in trastuzumab-treated breast cancer patients (n = 93). An expanded patient cohort from Nottingham (n = 194; including 72 of the previous cohort) and an independent patient cohort from Newcastle (n = 87) were used. All patients received trastuzumab following adjuvant therapy according to local guidelines with expression of calpain-1 investigated using standard immunohistochemistry. Results show that calpain-1 expression is associated with relapse-free survival in both the Nottingham (P = 0.01) and Newcastle (P = 0.019) cohorts, with high expression associated with adverse relapse-free survival. Expression was also associated with poor relapse-free survival when patient cohorts were combined (n = 281, P = 0.01). Calpain-1 remained, from multivariate analysis, an independent marker for relapse-free survival in the Newcastle cohort [hazard ratio (HR) = 5.169; 95% confidence interval (CI) 1.468-18.200; P = 0.011]. CONCLUSIONS: Calpain-1 expression is associated with poor relapse-free survival in breast cancer patients treated with trastuzumab. Further work is warranted to standardize and develop methodology with a view to potentially introducing assessment of this important biomarker into clinical practice.

Targeted Next-Generation Sequencing Validates the Use of Diagnostic Biopsies as a Suitable Alternative to Resection Material for Mutation Screening in Colorectal Cancer.

BACKGROUND: Mutation testing in the context of neoadjuvant therapy must be performed on biopsy samples. Given the issue of tumour heterogeneity, this raises the question of whether the biopsies are representative of the whole tumour. Here we have compared the mutation profiles of colorectal biopsies with their matched resection specimens. METHODS: We performed next-generation sequencing (NGS) analysis on 25 paired formalin-fixed, paraffin-embedded colorectal cancer biopsy and primary resection samples. DNA was extracted and analysed using the TruSight tumour kit, allowing the interrogation of 26 cancer driver genes. Samples were run on an Illumina MiSeq. Mutations were validated using quick-multiplex-consensus (QMC)-polymerase chain reaction (PCR) in conjunction with high resolution melting (HRM). The paired biopsy and resection tumour samples were assessed for presence or absence of mutations, mutant allele frequency ratios, and allelic imbalance status. RESULTS: A total of 81 mutations were detected, in ten of the 26 genes in the TruSight kit. Two of the 25 paired cases were wild-type across all genes. The mutational profiles, allelic imbalance status, and mutant allele frequency ratios of the paired biopsy and resection samples were highly concordant (88.75-98.85%), with all but three (3.7%) of the mutations identified in the resection specimens also being present in the biopsy specimens. All 81 mutations were confirmed by QMC-PCR and HRM analysis, although four low-level mutations required a co-amplification at lower denaturation temperature (COLD)-PCR protocol to enrich for the mutant alleles. CONCLUSIONS: Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy.

High nuclear MSK1 is associated with longer survival in breast cancer patients.

PURPOSE: Mitogen- and stress-activated kinases (MSKs) are important substrates of the mitogen-activated protein kinase (MAPK)-activated protein kinase family. MSK1 and MSK2 are both nuclear serine/threonine protein kinases, with MSK1 being suggested to potentially play a role in breast cancer cell proliferation, cell cycle progression, cell migration, invasion and tumour growth. The aim of the current study was to assess MSK1 protein expression in breast cancer tumour specimens, evaluating its prognostic significance. METHODS: A large cohort of 1902 early stage invasive breast cancer patients was used to explore the expression of MSK1. Protein expression was examined using standard immunohistochemistry on tissue microarrays. RESULTS: Low MSK1 protein expression was associated with younger age (P = 0.004), higher tumour grade (P < 0.001), higher Nottingham Prognostic Index scores (P = 0.007), negative ER (P < 0.001) and PR (P < 0.001) status, and with triple-negative (P < 0.001) and basal-like (P < 0.001) phenotypes. Low MSK1 protein expression was significantly associated with shorter time to distant metastasis (P < 0.001), and recurrence (P = 0.013) and early death due to breast cancer (P = 0.01). This association between high MSK1 expression and improved breast cancer-specific survival was observed in the whole cohort (P = 0.009) and in the HER2-negative and non-basal like tumours (P = 0.006 and P = 0.024, respectively). Multivariate analysis including other prognostic variables indicated that MSK1 is not an independent marker of outcome. CONCLUSIONS: High MSK1 is associated with improved breast cancer-specific survival in early stage invasive breast cancer patients, and has additional prognostic value in HER2-negative and non-basal like disease. Although not an independent marker of outcome, we believe such findings and significant associations with well-established negative prognostic factors (age, grade, Nottingham Prognostic Index, hormone receptor status, time to distant metastasis, recurrence and triple-negative/basal-like status) warrant further examination and validation in independent patient cohorts.

Immunohistochemical Assessment of Leukocyte Involvement in Angiogenesis.

Angiogenesis is a hallmark of cancer and is important for tumor growth, development, and metastasis. Leukocytes, including neutrophils, eosinophils, basophils, lymphocytes, and monocytes, are found invading many solid tumors, and this inflammation is often associated with tumorigenesis. Tumor-associated macrophages have been shown to be involved in tumor migration and metastasis and are modulators of tumor vascularization. Tumor-associated macrophages are a source of angiogenic factors, and pro-inflammatory cytokines involved in angiogenesis, lymphangiogenesis, and metastasis. Here we describe a method of quantifying the number of macrophages and their class within tumor tissue which can be compared with tumor blood and lymphatic microvessel density as a measure of angiogenesis and lymphangiogenesis. Although not described in depth, application of the methodology is described for other leukocyte populations, such as tumor-infiltrating lymphocytes.