Marco Bisoffi

Telomeres: prognostic markers for solid tumors.

Solid tumors continue to affect millions of people worldwide. Increasingly sophisticated diagnostic tools contribute to the high incidence rates for some tumor types, and treatment options continue to expand. However, the progression of solid tumors represents a challenge for the appropriate treatment of individual patients because of the relative inaccuracy of current prognostic markers, including the widely used Tumor‐Nodes‐Metastasis (TNM) staging system, to predict the course of disease. As a result, both over‐ and undertreatment are clinical realities in the management of patients diagnosed with solid tumors. Therefore, population‐based screening programs that increase the overall cancer incidence rates are controversial, as they may do little to improve the patients quality of life. Consequently, there is a strong need to develop novel and independent markers of prognosis. In this context, we review the use of telomeres as prognostic markers for solid tumors, including cancers from lung, breast, prostate, colon, brain and head and neck. Telomeric sequences, the repetitive DNA at the end of human chromosomes, are mediators of genomic stability and can undergo length alterations during tumor initiation and progression. In a number of studies reviewed here, these alterations, measured as telomere attrition and elongation, have been shown either to be associated with clinical markers of disease progression or to be independent markers of cancer prognosis. We conclude from these studies that careful assessment of telomere length or its proxies, such as telomere DNA content, will be part of novel risk assessment and prognostic modalities for patients with solid tumors.

Mammary field cancerization: molecular evidence and clinical importance

The term “field cancerization” originally denoted the presence of histologically abnormal tissue/cells surrounding primary tumors of the head and neck. Similar concepts with different and continuously changing definitions have been used for other types of tumors including breast adenocarcinoma, where field cancerization presently denotes the occurrence of molecular alterations in histologically normal tissues surrounding areas of overt cancer. Human mammary tissue morphology lends itself to the proposed concepts of field cancerization, which may include the gradual accumulation of genetic and other aberrations in stationary epithelial cells with intact morphology, or the spread of histologically normal yet genetically aberrant epithelial cells within mammary tissue. In this report, we review published molecular genetic, epigenetic, and gene expressional data in support of field cancerization in human mammary tissues. We then discuss the clinical implications of mammary field cancerization, including its source for potential biomarkers with diagnostic/prognostic potential, and its relationship to surgical margins and disease recurrence. We conclude with a future outlook on further research on mammary field cancerization addressing experimental methods, as well as the development of possible models and integrated approaches to gain a better understanding of the underlying mechanisms with the ultimate goal of developing clinical applications.

Detection of bioagents using a shear horizontal surface acoustic wave biosensor

Viruses are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325MHz with the specificity provided by antibodies for the detection of viral agents. A lithium tantalate-based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against either Coxsackie virus B4 or the category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 5 x 10(5)-fold more sensitive for the detection of SNV. For both pathogens, the sensors selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable to selectively detect SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing. This is the first study that reports on the detection of viral agents using an antibody-based SAW biosensor that has the potential to be used as a hand-held and self-contained device for rapid viral detection in the field.

Telomere DNA content and allelic imbalance demonstrate field cancerization in histologically normal tissue adjacent to breast tumors

Cancer arises from an accumulation of mutations that promote the selection of cells with progressively malignant phenotypes. Previous studies have shown that genomic instability, a hallmark of cancer cells, is a driving force in this process. In the present study, two markers of genomic instability, telomere DNA content and allelic imbalance, were examined in two independent cohorts of mammary carcinomas. Altered telomeres and unbalanced allelic loci were present in both tumors and surrounding histologically normal tissues at distances at least 1 cm from the visible tumor margins. Although the extent of these genetic changes decreases as a function of the distance from the visible tumor margin, unbalanced loci are conserved between the surrounding tissues and the tumors, implying cellular clonal evolution. Our results are in agreement with the concepts of “field cancerization” and “cancer field effect,” concepts that were previously introduced to describe areas within tissues consisting of histologically normal, yet genetically aberrant, cells that represent fertile grounds for tumorigenesis. The finding that genomic instability occurs in fields of histologically normal tissues surrounding the tumor is of clinical importance, as it has implications for the definition of appropriate tumor margins and the assessment of recurrence risk factors in the context of breast‐sparing surgery.

Targeting and cellular trafficking of magnetic nanoparticles for prostate cancer imaging.

Antibody-conjugated iron oxide nanoparticles offer a specific and sensitive tool to enhance magnetic resonance (MR) images of both local and metastatic cancer. Prostate-specific membrane antigen (PSMA) is predominantly expressed on the neovasculature of solid tumors and on the surface of prostate cells, with enhanced expression following androgen deprivation therapy. Biotinylated anti-PSMA antibody was conjugated to streptavidin-labeled iron oxide nanoparticles and used in MR imaging and confocal laser scanning microscopic imaging studies using LNCaP prostate cancer cells. Labeled iron oxide nanoparticles are internalized by receptor-mediated endocytosis, which involves the formation of clathrin-coated vesicles. Endocytosed particles are not targeted to the Golgi apparatus for recycling but instead accumulate within lysosomes. In T1-weighted MR images, the signal enhancement owing to the magnetic particles was greater for cells with magnetic particles bound to the cell surface than for cells that internalized the particles. However, the location of the particles (surface vs internal) did not significantly alter their effect on T2-weighted images. Our findings indicate that targeting prostate cancer cells using PSMA offers a specific and sensitive technique for enhancing MR images.

Conjugated linoleic acid or omega 3 fatty acids increase mitochondrial biosynthesis and metabolism in skeletal muscle cells

BackgroundPolyunsaturated fatty acids are popular dietary supplements advertised to contribute to weight loss by increasing fat metabolism in liver, but the effects on overall muscle metabolism are less established. We evaluated the effects of conjugated linoleic acid (CLA) or combination omega 3 on metabolic characteristics in muscle cells.MethodsHuman rhabdomyosarcoma cells were treated with either DMSO control, or CLA or combination omega 3 for 24 or 48 hours. RNA was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was quantified by measuring extracellular acidification and oxygen consumption rates.ResultsOmega 3 significantly induced metabolic genes as well as oxidative metabolism (oxygen consumption), glycolytic capacity (extracellular acidification), and metabolic rate compared with control. Both treatments significantly increased mitochondrial content.ConclusionOmega 3 fatty acids appear to enhance glycolytic, oxidative, and total metabolism. Moreover, both omega 3 and CLA treatment significantly increase mitochondrial content compared with control.

Telomere content correlates with stage and prognosis in breast cancer

SummaryPurposeTo evaluate the hypothesis that telomere DNA content (TC) in breast tumor tissue correlates with TNM staging and prognosis.Experimental designSlot blot assay was used to quantitate TC in 70 disease-free normal tissues from multiple organ sites, and two independent sets of breast tumors containing a total of 140 samples. Non-parametric Rank–Sums tests, logistic regression and Cox proportional hazards models were used to evaluate the relationships between TC and tumor size, nodal involvement, TNM stage, 5-year survival and disease-free interval.ResultsTC in 95% of normal tissues was 75–143% of that in the placental DNA standard, whereas only 50% of tumors had TC values in this range. TC was associated with tumor size (p=0.02), nodal involvement (p<0.0001), TNM stage (p=0.004), 5-year overall survival (p=0.0001) and 5-year disease-free survival (p=0.0004). A multivariable Cox model was developed using age at diagnosis, TNM stage and TC as independent predictors of breast cancer-free survival. Relative to the high TC group (>123% of standard), low TC (<101% of standard) conferred an adjusted relative hazard of 4.43 (95% CI 1.4–13.6, p=0.009). Receiver operating characteristic curves using thresholds defined by the TC distribution in normal tissues predicted 5-year breast cancer-free survival with 50% sensitivity and 95% specificity, and predicted death due to breast cancer with 75% sensitivity and 70% specificity.ConclusionsTC in breast cancer tissue is an independent predictor of clinical outcome and survival interval, and may discriminate by stage.

Characterization of the metabolic effects of irisin on skeletal muscle in vitro

This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes.

Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors.

Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT‐PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient‐matched, tumor‐adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN‐1 and TAHN‐5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra‐lobular fibroblasts expressing MMP2, SPARC, TGF‐β3, which are inducers of EMT, were both prevalent in TAHN‐1 tissues, sparse in TAHN‐5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α‐smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN‐1 tissues. These results identify cellular processes that are differentially activated between TAHN‐1 and TAHN‐5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors.

Telomere DNA Content Predicts Breast Cancer–Free Survival Interval

Background: Telomeres are nucleoprotein complexes that protect chromosome ends from degradation and recombination. Critically shortened telomeres generate genomic instability. It has been postulated that the extent of telomere DNA loss is related to the degree of genomic instability within a tumor and therefore may presage clinical outcome. The objective of this investigation was to evaluate the hypothesis that telomere DNA content (TC) in breast tumor tissues predicts breast cancer–free survival interval. Materials and Methods: Slot blot titration assay was used to quantitate TC in 530 archival breast tumor tissues in a population-based cohort. The relationships between TC, 12 risk factors for breast cancer adverse events (i.e., death due to breast cancer, breast cancer recurrence, or development of a new primary breast tumor), and breast cancer–free survival interval were evaluated by Fishers exact test, log-rank analysis, and univariate and multivariate Cox proportional hazards models. Results: TC was independent of each of the 12 risk factors. Ethnicity, tumor-node-metastasis stage, estrogen receptor, progesterone receptor, and p53 status, chemotherapy sequence, adjuvant therapy, and TC each conferred significant relative hazards. The best overall multivariate Cox proportional hazards model included TC, p53 status, tumor-node-metastasis stage, and estrogen receptor status as independent predictors of breast cancer–free survival interval (P < 0.00005). Low TC (≤200% of standard), relative to the high-TC group (>200% of standard), conferred an adjusted relative hazard of 2.88 (95% confidence interval, 1.16-7.15; P = 0.022) for breast cancer–related adverse events. Conclusions: TC in breast cancer tissue is an independent predictor in this group of breast cancer–free survival interval.