The presence of clustered circulating tumor cells (CTCs) and circulating cytokines define an aggressive phenotype in metastatic colorectal cancer.

PURPOSE: Colon carcinoma is a malignant tumor showing a marked preference to metastasize to distant organs. The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. However, whether circulating cytokines are linked to the circulation of tumor cells, as individual cells or clusters, remain unclear. In this study, we investigated the circulating levels of TGF-beta, CXCL1, VEGF and PAI-1 as potential bioindicators of the presence of CTCs in patients with metastatic colon cancer. METHODS: Circulating tumor cells (CTCs) were isolated from peripheral blood by immunomagnetic separation and phenotypically characterized in a cohort of 103 patients with metastatic colon cancer. TGF-beta, CXCL1, VEGF and PAI-1 concentrations were determined by immunoassay in plasma samples from the same patients. RESULTS: We detected two different populations of CTCs, single cells or clusters in patients with metastatic colon cancer. Importantly, we found that the presence of clustered CTCs is significantly associated with elevated circulating levels of TGF-beta and CXCL1 and with reduced overall survival. Finally, we observed that circulating levels of cytokines are differently associated with the two populations of CTCs. CONCLUSIONS: Taken together, these findings show that detection of clustered CTCs represents a negative prognostic factor in patients with metastatic colon cancer. The presence of clustered CTCs is associated with elevated circulating levels of cytokines such as TGF-beta and CXCL1. This suggests an additional role for circulating cytokines as predictive tool for cancer prognosis and diagnosis of minimal residual disease as well as assessment of tumor sensitivity to anticancer therapy.

Serum tryptase, mast cells positive to tryptase and microvascular density evaluation in early breast cancer patients: possible translational significance.

BACKGROUND: Tryptase is a serine protease released from mast cells that plays a role in tumor angiogenesis. In this study we aimed to evaluate serum tryptase levels in 105 female early breast cancer patients before (STLBS) and after (STLAS) radical surgical resection, mast cell density positive to tryptase (MCDPT) and microvascular density (MVD). METHODS: STLBS and STLAS were assessed using the UniCAP Tryptase Fluoroenzyme immunoassay. Tumor sections were immunostained with a primary anti-tryptase antibody and an anti-CD-34 antibody by means of immunohistochemistry. RESULTS: The mean ± 1 standard deviation STLBS and STLAS was 7.18 ± 2.63 µg/L, and 5.13 ± 2.21 respectively and a significant difference between mean levels was found (p = 0.0001) by student t-test. A strong correlation between STLBS and MVD (r = 0.81, p = 0.0001); STLBS and MCDPT (r = 0.69, p = 0.003); and MCDPT and MVD (r = 0.77; p = 0.0001) was found. CONCLUSIONS: Results demonstrated higher STLBS in breast cancer patients, indicating an involvement of MC tryptase in breast cancer angiogenesis. Therefore, serum tryptase levels may play a role as a novel surrogate angiogenic marker predictive of response to radical surgery in breast cancer patients. In this patients setting, it's intriguing to hypothesize that tryptase inhibitors might be evaluated in clinical trials.

Anticancer Effects of Nutraceuticals in the Mediterranean Diet: An Epigenetic Diet Model.

Epidemiological and clinical studies support the association between nutrition and development or progression of different malignancies such as colon, breast, and prostate cancer, defining these tumors as diet-associated cancer. The Mediterranean diet shows inverse associations with metabolic diseases, cardiovascular pathologies and various types of cancer. Many bioactive nutrients of the Mediterranean diet have been identified as factors protective against these types of pathologies. The epigenome has been identified as the primary goal of modulations in gene expression related to these molecular nutrients. In fact, they can modify the epigenome and can be incorporated into the 'epigenetic diet', which translates into a diet regimen that can be used therapeutically for health or preventative purposes. Most epigenetic changes are influenced by lifestyle and nutrition. Epigenetic therapy is a new area for the development of nutraceuticals whose absence of toxicity can represent a valid asset in cancer prevention strategies. Recent advances in understanding the mechanisms of nutrigenomics, nutrigenetics and nutraceuticals have led to the identification of superfoods capable of favorably conditioning gene expression. In this review, we highlight the importance of nutraceuticals present in the Mediterranean diet as epigenetic modifiers both in the mechanisms of tumor onset and as protective agents.

The Role of Circulating Adiponectin and SNP276G>T at ADIPOQ Gene in BRCA-mutant Women.

BACKGROUND: Environmental factors may influence the lifetime risk of cancer (penetrance) in women with a BRCA mutation. MATERIALS AND METHODS: In 89 BRCA-mutant women, affected or unaffected by breast/ovarian cancer, we explored serum levels of adipokines and their relation with the polymorphism SNP276G>T as modulators of BRCA penetrance. RESULTS: Affected women had significantly lower adiponectin than healthy women. Affected women with rs1501299 TT had significantly lower adiponectin and higher leptin than GT and GG genotypes. GT genotype was significantly associated with the disease status [odds ratio (OR)=3.24, 95% confidence interval (95% CI)=1.03-10.17]. Women in the lower tertile of serum adiponectin had a RR of BRCA-associated cancer of 2.80, 95% CI=1.1-7.1 (p for trend=0.03) compared with women in the higher tertile. CONCLUSION: In the SNP rs1501299 the T allele was significantly associated with lower serum levels of adiponectin in affected women, suggesting that the T allele might be related to cancer.

Synergism of Adipocytokine Profile and ADIPOQ/TNF-α Polymorphisms in NAFLD-associated MetS Predict Colorectal Liver Metastases Outgrowth.

BACKGROUND/AIM: The aim of this study was to evaluate whether the altered profile of adipocytokine and genetic fingerprint in NAFLD-associated metabolic syndrome "cluster" represents synergistic risk factors predicting onset of liver colorectal cancer metastases. MATERIALS AND METHODS: A total of 165 colorectal cancer patients were enrolled, 56,3% were with metabolic syndrome/NAFLD. Serum samples were assayed for ADIPOQ, leptin and TNF-a levels by ELISA. ADIPOQ rs266729 C/G and TNF-308 A/G genotypes were analyzed in DNA isolated from whole blood. RESULTS: Reduction in adiponectin levels and increase in leptin and TNF-α was shown in patients with liver metastases. This trend was influenced by BMI, MetS/NAFLD, and insulin resistance. ADIPOQ G rs266729 and TNF- 308 A allele are associated with obesity, MetS/NAFLD and insulin resistance. ADIPOQ CG/GG and GA/AA TNF-alpha genotypes confer susceptibility to liver metastases. CONCLUSION: Obesity and hepatic steatosis significantly favor the development of colorectal cancer liver metastases and the individual adipocytokines genetic profile may play an important predictive role.

Circulating Levels of VEGF and CXCL1 Are Predictive of Metastatic Organotropismin in Patients with Colorectal Cancer.

Colorectal cancer is the most common cancer of the gastrointestinal system and has a marked preference to metastasize to distant organs. In this study, we investigated whether levels of circulating serum pro-angiogenic cytokine such as chemokine (C-X-C motif) ligand 1 (melanoma growth-stimulating activity, alpha; CXCL1) and vascular endothelial growth factor (VEGF) have a role in favoring the colonization of metastatic cells at preferential sites and determined their prognostic significance in a cohort of 103 patients with metastatic colorectal cancer. Importantly, we found that the presence of elevated circulating levels of VEGF and CXCL1 are predictive of liver and lung metastasis, respectively. Moreover, the presence of a high serum VEGF level represents a negative prognostic factor for patients with liver metastases, with a worse prognosis than patients with lung metastasis. This suggests an additional role for circulating cytokines as a predictive tool for cancer prognosis and diagnosis, as well as for assessment of tumor sensitivity to anticancer therapy.

Assessment of Nutritional and Inflammatory Status to Determine the Prevalence of Malnutrition in Patients Undergoing Surgery for Colorectal Carcinoma.

BACKGROUND/AIM: Colorectal Cancer is the fourth most frequent cause of cancer death worldwide and its incidence increases from 50 years of age. It is often associated with protein-caloric malnutrition and 20% of cancer deaths occur due to this event. The aim of this study was to assess the prevalence of malnutrition and inflammatory status in 78 patients undergoing surgery for colorectal carcinoma. PATIENTS AND METHODS: Nutritional Status was assessed by Mini Nutritional Assessment (MNA). Serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA, while albumin, C-reactive protein (CRP) and transferrin (TRF) were tested using an immunometric assay. RESULTS: The mean MNA score in colorectal patients was 20.4±8.4, while 23/78 patients (29.4%) were well nourished, 36/78 (46.1%) were at risk of malnutrition and 19/78 (24.3%) were malnourished, reporting in the previous six months from the date of diagnosis a significant weight loss (>10 kg), muscle mass loss and severe reduction of food intake due to loss of appetite and altered taste perception. The serum means of IL-6, TNF-α and CRP, were significantly higher in colorectal patients compared to the control group (p<0.001, p<0.0001, p<0.0001, respectively) while lower TRF, albumin and HCT serum levels in cancer patients vs. healthy subjects (p<0.0001; p<0.0001 and p<0.0001) were found. CONCLUSION: more than 50% of colorectal cancer patients were malnourished or at risk of malnutrition and reported an imbalance between nutritional and inflammatory status. They, therefore, require a nutritional intervention before treatment in order to have a more effective response and improve quality of life.

Circulating Levels of PAI-1 and SERPINE1 4G/4G Polymorphism Are Predictive of Poor Prognosis in HCC Patients Undergoing TACE.

Although several molecular markers have been proposed as prognostic of disease progression in Hepatocellular carcinoma (HCC), predictive markers of response to treatment are still unsatisfactory. Here, we propose a genetic polymorphism as a potential predictive factor of poor prognosis in HCC patients treated with transcatheter arterial chemoembolization (TACE). In particular, we show that the guanosine insertion/deletion polymorphism in the promoter region of SERPINE1 gene at the -675 bp position, named 4G/4G, predicts poor prognosis in a cohort of 75 patients with HCC undergoing TACE. By a combination of ELISA and SERPINE1 promoter study, we found that the presence of elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) in patients with 4G/4G genotype is significantly associated with reduced overall survival compared to patients with 5G/5G or 4G/5G genotype in HCC patients after TACE. Our analysis provided evidence that variation in SERPINE1 gene plays a role in defining the outcome in patients treated with TACE. In addition to a poor disease outcome, the 4G/4G variant represents an unfavorable predictive factor for response to chemotherapy as well.

Clinical and prognostic role of circulating MMP-2 and its inhibitor TIMP-2 in HCC patients prior to and after trans-hepatic arterial chemo-embolization.

BACKGROUND AND AIMS: Trans-hepatic arterial chemo-embolization is the most commonly used treatment for unresectable hepatocellular carcinoma. The prognostic impact of tumor biomarkers has not therefore been evaluated in this treatment. Imbalance between matrix metalloproteinase-2 and tissue inhibitor metalloproteinase-2 is considered to play an important role in extracellular matrix remodeling and degradation. Higher serum levels of MMP-2 have been shown to predict a poor prognosis and shorter overall survival in HCC after TACE. The objective of this study was to evaluate the serum levels of MMP-2 and TIMP-2 in HCC patients before and after TACE to evaluate their clinical significance and usefulness as prognostic biomarkers. METHODS: MMP-2 and TIMP-2 levels were measured by ELISA in 75 HCC patients and 30 healthy controls. Sera MMP-2 and TIMP-2 were correlated with clinico-pathological features. RESULTS: The mean serum MMP-2 and TIMP-2 levels of HCC patients before TACE were 1700±71ng/mL and 89±45ng/mL respectively, significantly higher than that of the control group: 771±60ng/mL (p<0.0001, t-test) and 25.7±20ng/mL respectively (p<0.0001, t-test). A significant decrease of MMP-2 levels after 1 and 3months compared to baseline time was observed (p<0.0001), while with TIMP-2 a gradual increase in serum before and after TACE (p<0.01) was detected. No significant correlation between serum MMP-2 levels and other clinico-pathological features was observed. Patients with serum MMP-2 >1500ng/mL (median value) had worse overall and recurrence-free survival compared with those with serum MMP-2 levels <1500ng/mL before treatment. CONCLUSION: Higher serum MMP-2 levels and MMP-2/TIMP-2 ratio could predict poor prognosis after TACE, suggesting prognostic role of these biomarkers in HCC.

Molecular profiling of thin-prep FNA samples in assisting clinical management of non-small-cell lung cancer.

The discovery of new target treatments for NSCLC has led to a search for new genetic and epigenetic markers able to selectively predict response to these new drugs. Somatic mutations in EGFR and KRAS genes are routinely analyzed to predict response to tyrosine kinase inhibitors (TKIs), used in the treatment of NSCLC patients, whose efficacy depend on the presence or the absence of specific mutations. MicroRNA (miRNA) expression evaluation has been recently analyzed because of the involvement of these molecules in lung cancer pathogenesis and in drug resistance. Only 30 % of NSCLC patients present a resectable stage at time of diagnosis so tissue samples cannot be the only starting material for genetic and epigenetic analysis. Therefore, the possibility to use cytological sampling already used for diagnosis also for molecular testing is emerging. The aim of this study was to evaluate for the first time in lung cancer the use of liquid-based cytology both for EGFR and KRAS mutational testing and for the expression trend of some miRNAs involved in lung cancer pathogenesis: miR-21, miR-155, miR-7, and let7a. We enrolled 20 fine-needle aspirate (FNA) samples diagnosed as NSCLC, 10 FNAs without neoplastic cells, and tissue samples coming from 5 of the 20 patients who underwent surgery after FNA NSCLC diagnosis. All Thin-Prep processed FNA samples were evaluable for DNA and RNA analysis and results were compared with those of the small group of patients whose matched tumor histology was available. The mutational status of the EGFR and KRAS genes and the expression profile of the selected miRNA showed comparable results between FNA samples and histological tissues. Our results underline that cytological samples could give the same genetic information as that obtained from histological specimens and so could be collected to create a nucleic acids bank.

Circulating levels of transforming growth factor-ßeta (TGF-ß) and chemokine (C-X-C motif) ligand-1 (CXCL1) as predictors of distant seeding of circulating tumor cells in patients with metastatic breast cancer.

BACKGROUND: The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. Transforming growth factor-ßeta (TGF-ß) and Chemokine (C-X-C Motif) Ligand-1 (CXCL1) are cytokines involved in the colonization of distant sites by CTCs in several pre-clinical animal models. However, their role is poorly-investigated in patients with metastatic cancer. Here, we investigated whether circulating levels of TGF-ß and CXCL1 are predictors of CTC seeding in preferential distant sites in patients with metastatic breast cancer. MATERIALS AND METHODS: CTCs were isolated from the peripheral blood of 61 patients with metastatic breast cancer by immunomagnetic separation. Plasma samples were collected from the same patients and assayed for TGF-ß and CXCL1 by enzyme-linked immunoassay. RESULTS: Patients were grouped in CK1+/- (N<10), CK2+ (N ≥ 10<50) and CK3+ (N ≥ 50), according to the number (N) of cytokeratin 7/8-positive CTCs: the highest number of CK7/8-positive CTCs was detected in patients with negative Human epidermal growth factor receptor-2 (HER-2/NEU) status (p<0.0001) antigen, identified by the monoclonal antibody Ki-67 (Ki-67) ≥ 15% (p=0.003), Carcinoma antigen 15-3 (CA-15.3) ≥ 40 U/ml (p=0.004) and those with lung metastases (p=0.01). We found that elevated plasma concentrations of TGF-ß and CXCL1 are predictive for the detection of CTCs. In particular, patients with CK3+ CTCs and plasma concentrations of TGF-ß and CXCL1 higher than the median value had a poor prognosis in comparison to patients with CK1+/- CTCs and TGF-ß and CXCL1 concentrations below the median value. CONCLUSION: Our study shows that elevated circulating levels of TGF-ß and CXCL1 are associated with a poor prognosis, and higher detection of CTCs and propensity of these cells to seed lung metastases in patients with breast cancer.

Circulating hTERT DNA in early breast cancer.

BACKGROUND: The objective of the study was to quantify the human telomerase reverse transcriptase (hTERT) gene in the circulating DNA of patients with primary breast cancer (BC) and to test its correlation with clinical parameters of the disease. PATIENTS AND METHODS: One hundred and twenty-one BC patients, 30 patients with fibroadenoma (NBC) and 50 healthy women were enrolled. RESULTS: The level of hTERT in the plasma was significantly different in BC, NBC and controls (p<0.01), showing a sensitivity of 50% and specificity of 90% in the ability to detect malignancy. The circulating hTERT DNA was significantly different in the estrogen receptor (ER)(+)/progesterone receptor (PgR)(+) compared to the ER(-)/PgR(-) patients (p=0.03). Higher hTERT levels were associated with higher human epidermal growth factor receptor (HER)-2/Neu expression: score 0-1 vs. score 2+ (p=0.01) and vs. score 3+ (p=0.02). Finally, hTERT was significantly inversely correlated with the carbohydrate antigen (CA) 15.3 serum level (p=0.001). CONCLUSION: Circulating hTERT DNA has a better diagnostic value than CA 15.3 in early breast cancer disease and could be a possible candidate as a tumor marker in patients with infiltrating ductal carcinoma positive to steroid hormonal receptor and with amplification of HER-2/Neu.