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Breast cancer remains one of the leading causes of death among women across the world. The last few decades have seen significant reduction in mortality owing to earlier detection and better adjuvant treatments that were developed based on clinical staging and morphological features. As these treatments have evolved, the heterogeneity of breast cancer poses a new challenge, since there is no standard gold-therapy suitable for all tumors of the mammary gland. Therefore, contemporary management and research efforts are directed toward specific prognostic and predictive molecular signatures that can guide targeted individualized therapy. The goal of ongoing research in this field is to identify specific molecular targets for developing novel therapeutic approaches. These targets can also serve to improve screening of breast cancer. This review focuses on the role of cancer testis antigens (CTAs) in breast carcinogenesis and explores the potential for development of targeted screening and therapeutic approaches. Normally found in the testes, these antigens are highly correlative with cancers of the breast, skin, and ovaries. These implications have been further corroborated through uncovering the interaction of CTAs with genes and proteins involved in tumor suppression and homeostasis like p53. There is some evidence that these genes can be targeted for early detection in addition to being candidates for cancer immunotherapy.

Hepatitis C virus (HCV) is still one of the major causes of chronic viral infection worldwide, and hepatic steatosis is a frequent pathological finding in patients with chronic HCV-related diseases. It is unclear whether the steatosis is associated with host factors or the virus itself, although a consistent relationship has been found between steatosis and a necro-inflammatory reaction with the increased secretion of immuno-regulators. A primary sources of inflammatory mediators are mast cells (MCs) bone marrow-derived cells that are detected in both normal and diseased livers. We determined MC density and correlated it with the fibrosis, inflammatory reaction and steatosis observed in the liver biopsies of patients affected by HCV with or without steatosis. All the histological features were assessed using a computer-aided image analysis system. There was a statistically significant difference in MC density between the HCV-infected patients with and without steatosis, with the lower mean value being detected in those without (P < 0.02). Furthermore, a nonstatistically significant difference in fibrosis and inflammation between the two patient groups was found. In conclusion, this is the first study showing a significant increase in MC density in the tissues of patients with chronic HCV infection and histologically documented steatosis.

Mast cells (MCs) have been indicated as a source of various inflammatory cytokines, chemokines and growth factors. This study evaluates liver tissue MC density as a quantitative marker of acute liver inflammation in 2- and 19-month old rats treated with carbon tetrachloride (CCl4) to assess the relationships between MC density, hepatocellular damage, mRNA encoding TGF- β1, hepatic stellate cell (HSC) activation and collagen levels. Consecutive histological sections from each age group were stained with toluidine blue to identify granulated MCs, Direct Red 80 to recognize collagen matrix, and by immunohistochemistry to identify activated hepatic stellate cells (HSCs), which were subsequently counted by means of a computer-aided image analysis. Histology showed hepatocellular necrosis with inflammatory cell infiltration and collagen matrix deposition. Two and 24 hours after intoxication, MC density had considerably increased in the younger rats, but less in those aged 19 months. Although the untreated older rats had a larger area occupied by activated HSCs than the untreated younger rats, the increase in the number of HSCs was greater in the younger rats both two and 24 hours after intoxication. The greater MC density in younger rats suggests that older rats have a reduced immune response or recruit fewer MCs. The activated HSCs and TGF- β1 transcripts did not increase significantly during the study period, thus indicating that these are later events in chemically induced hepatic toxicity. In conclusion, MC density may be an index of acute liver inflammation after CCl4 intoxication.

In geometrical terms, tumor vascularity is an exemplary anatomical system that irregularly fills a three-dimensional Euclidean space. This physical characteristic, together with the highly variable vessel shapes and surfaces, leads to considerable spatial and temporal heterogeneity in the delivery of oxygen, nutrients and drugs, and the removal of metabolites. Although these biological features have now been well established, quantitative analyses of neovascularity in two-dimensional histological sections still fail to view tumor architecture in non-Euclidean terms, and this leads to errors in visually interpreting the same tumor, and discordant results from different laboratories. A review of the literature concerning the application of microvessel density (MVD) estimates, an Euclidean-based approach used to quantify vascularity in normal and neoplastic pituitary tissues, revealed some disagreements in the results and led us to discuss the limitations of the Euclidean quantification of vascularity. Consequently, we introduced fractal geometry as a better means of quantifying the microvasculature of normal pituitary glands and pituitary adenomas, and found that the use of the surface fractal dimension is more appropriate than MVD for analysing the vascular network of both. We propose extending the application of this model to the analysis of the angiogenesis and angioarchitecture of brain tumors.

Background: We aimed at characterizing the aging gingiva analyzing: i) collagen content and turnover in human gingival tissues and fibroblasts obtained from healthy young and aging subjects. ii) the effect of cyclosporin A administration in human cultured gingival fibroblasts obtained from aging compared to young subjects. Methods: Morphological analysis was performed on haematoxylin-eosin and Sirius red stained paraffin-embedded gingival biopsies from young and aging healthy subjects. The expression of the main genes and proteins involved in collagen turnover were determined by real time PCR, dot blot and SDS-zymography on cultured young and aging gingival fibroblasts, and after cyclosporin A administration. Results: Our results suggest that in healthy aged people, gingival connective tissue is characterized by a similar collagen content and turnover. Collagen turnover pathways are similarly affected by cyclosporin A treatment in young and aging gingival fibroblasts. Conclusions: Cyclosporin A administration affects gingival collagen turnover pathways in young and aging fibroblasts at the same extent, suggesting that during aging cyclosporin A administration is not related to relevant collagen turnover modifications.

Background Cancer remains one of the most complex diseases affecting humans and, despite the impressive advances that have been made in molecular and cell biology, how cancer cells progress through carcinogenesis and acquire their metastatic ability is still widely debated. Conclusion There is no doubt that human carcinogenesis is a dynamic process that depends on a large number of variables and is regulated at multiple spatial and temporal scales. Viewing cancer as a system that is dynamically complex in time and space will, however, probably reveal more about its underlying behavioural characteristics. It is encouraging that mathematicians, biologists and clinicians continue to contribute together towards a common quantitative understanding of cancer complexity. This way of thinking may further help to clarify concepts, interpret new and old experimental data, indicate alternative experiments and categorize the acquired knowledge on the basis of the similarities and/or shared behaviours of very different tumours.

No experimental animal model employing a primary human ovarian carcinoma (OC) cell line is presently available that tracks the progression of this cell line with an identifiable marker. This hinders investigations related to developing new approaches for treating OC. Here, we describe the development of a tumor model in NOD/SCID mice for human OC that makes use of the endogenously expressed tumor specific sperm protein 17 (Sp17) cancer testis antigen. In this model, human SKOV-3 OC cell lines were intra-peritoneally seeded. Subsequently viable SKOV-3 cells were recovered from primary organ cell cultures from the liver ovaries, abdomen, and ascitic fluid, and their presence was confirmed by the detection of Sp17 mRNA by RT-PCR and Sp17 protein by immunocytochemistry and FACS analysis. When SKOV-3 tumor cells were administered intravenously the mice developed primarily lung tumor foci. This model makes it possible to evaluate new immunotherapeutic strategies for the treatment of human OC based on the biomarker Sp17.

Complexity is the main feature of every biological system, including human organs, tissues, cells and sub-cellular components. This property of the organized biological matter cannot be quantified by means of the classical Euclidean geometry, which is able to measure regular object, practically unknow in Nature. The aims of this paper werea)to underline the importance of theshapeof a biological structure andb)to evaluate theoutline roughnessand theinternal irregularityof the extracellular matrix collagen seen on liver biopsy specimens during the chronic diseases. The quantification of these two parameters gives a measure of two qualitative properties of the liver extracellular matrix.

Irregularity and complexity are the main features of every biological system, including human tissues, cells and sub-cellular components. These two properties of the organized biological matter cannot be quantified by means of the classical Euclidean geometry, which is able to measure regular object, practically unknown in Nature. The aims of our paper were a) to underline the importance of the shape of a biological structure, b) to investigate the fractal geometry for quantifying the liver histo-pathological structures, and c) to explain the significance of several terms used in the fractal analysis of complex biological systems.

The aim of this study was to describe a computer-assisted method based on fractal dimension and the coefficient of roundness, two mathematical descriptors of irregularly shaped objects, in order to discriminate benign and malignant mammographic lesions.Twenty-three digitized mammograms were classified as containing benign lesions (n=12) or as containing malignant lesions (n=11) on the basis of readings by two independent radiologists. Morphometrical and fractal analysis of the breast lesions were automatically performed by a computer-assisted image analysis system.The method shows, for the first time, two parameters that allow the classification of irregularly shaped objects, such as benign and malignant breast lesions. A significant increase was obtained when comparing the fractal dimension and the coefficient of roundness of benign versus malignant mammographic lesions.Benign and malignant breast lesions are characterized by complex morphologies. Complexity is the main property of all biological systems, including natural and pathological structures. In this study, we showed that fractal geometry allows quantitative measurement of the complex morphology of benign and malignant mammographic lesions. Furthermore, this mathematical approach may be helpful for the study of the non-linear dynamic processes involved in breast carcinogenesis.

The aim of this chapter is to propose a computerized simulation of the parenchymal liver tissue formation process. The simulation program considers two anatomical structures: the hepatic vascular system and the parenchymal tissue made up by ephitelial hepatic cells, called hepatocytes. On the basis of previous theoretical rules, we have considered two dynamical mechanisms: the aggregation of the hepatic cells around the centrolobular vein of the vascular system and the self-replication of the hepatic cells. The simulation is able to furnish images very similar to those commonly seen, at microscopy level, by means of histological specimens. This simulated structure may be of help to reveal more information about the liver morphology, and it will be the basis for future simulations of the hepatic functionality and of viral pathologies.

To evaluate the usefulness of a reliable and reproducible mathematical scoring system based on fractal geometry for quantifying the irregular pattern in fibrosis commonly seen in liver biopsy specimens from chronic liver diseases.The study used 26 standard liver biopsy specimens obtained from patients with chronic hepatitis C virus-related liver disease. The degree of fibrosis in each specimen was estimated using a quantitative scoring system based on the computer-assisted evaluation of both the fractal and spectral dimensions of deposited collagen. The fractal dimension was then compared with the percent area of collagen measured using an image analysis system.The fractional dimension of its irregular shape defines fibrosis as a natural fractal structure. The complex distribution of its collagenous components (unmeasurable by means of the usual morphometric parameters) can be optimally quantified using a single numerical score that seems to be a better alternative to the semiquantitative methods adopted so far. The proposed method is reproducible, rapid and inexpensive; furthermore, supported by specific software, its mathematical approach excludes subjectivity and eliminates the external factors capable of influencing staging and classification.This study demonstrated that it is possible to quantify the irregularity of the structures of the liver in an objective manner and that the box-counting fractal dimension does not depend on the amount of collagen deposited on the slide. Furthermore, as has been found in other fields of investigation, study of the fractal properties of the liver is likely to reveal more about its structure and the pathogenesis of liver diseases.

Purpose An experimental pulmonary metastatic model for ovarian cancer has still not been developed. Here we introduce tumor models in NOD/SCID mice for ovarian cancer incorporating a tumor-specific Sp17/CT antigen. Methods Ovarian adenocarcinoma cells (SKOV-3) were intraperitoneally injected in an attempt to demonstrate a seeding of the organs located in the peritoneal cavity. Examination of the ovaries for the progression of the disease was marked by an increase of the size of the growing tumor. Following intravenous injection, viable SKOV-3 cells were recovered by primary organ cell culture in the liver, lungs, ovaries, abdomen, and ascites. Summary The presence of SKOV-3 cells in these tissues was confirmed by RT-PCR and immunocytochemistry using the sperm protein 17 (Sp17). The number and size of tumoral foci were quantified using a computer-aided model. Tumoral foci were observed in the lungs of tumor-bearing mice and they are dependent on the dose of injected SKOV-3 cells and time after injection. Conclusion The quantification of the number of lung tumoral foci and the detection of distant metastasis might provide specific reference points for examining the mechanism(s) of the immune response to Sp17 and for evaluating immunologically based therapies within this novel ovarian murine tumor model.

Purpose Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen-loading of dendritic cells (DC) generates significant and rapid cytotoxic T lymphocyte (CTL) responses in vitro. As a more extensive analysis of the rAAV system, we used a self-antigen, Her-2, expressed in many cancers including breast and ovarian cancer, in particular, lymphoma. Methods The AAV vectors were found to be able to transduce up to 85% of DC and the transduced DC displayed higher levels of CD80, CD83, CD86, and CD1a over controls. Autologous PBMC/LCL targets and Her-2/neu positive lymphoma primary cancer cell. Generation of CTL and test their function by Cr(51) release assay against LCL/Her-2/neu target and relative controls. Summary We used the Her-2/neu gene, divided into 3 overlapping segments for insertion into AAV. The three Her-2 subgenes are aa 153-653, 403-906, and 76-1255. Using only one stimulation, significant MHC class I-restricted, anti-Her-2-specific CTL killing was demonstrated against a Her-2/neu-positive lymphoma primary cancer cell line. Using synthetic antigen-positive target cells with the three Her-2 subgenes and the primary lymphoma positive for Her-2/neu for a target, we stimulated highest CTL killing. The killing was done using specific Her-2(403-903)CTL against a target present in the particular peptide and inserted by rAAV AAV/Her-2(403-906)/Neo in the LCL. Conclusion These data suggest that AAV-based antigen loading of DC is highly effective for generating a CTL response against lymphoma tumor.

Purpose Human sperm protein 17 (Sp17) is a highly conserved protein originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. Recently, Sp17 has been included in the cancer/testis (CT) antigens family and shown to be expressed in multiple myeloma and ovarian cancer. Here we investigate the immunolocalization of Sp17 in specimens of nervous system (NS) malignancies in order to establish the usefulness of this antigen as a target for tumor-vaccine strategies for NS tumors. Methods The authors assessed the expression pattern of Sp17 in formalin-fixed and paraffin-embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumors (6 astrocytomas, 16 glioblastomas multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumors, and 5 peripheral nerve sheath tumors (4 schwannoma, and 1 neurofibroma) using a standardized immunohistochemical procedure. Summary A number of neuroectodermal (21%) and meningeal tumors (4%) were found heterogeneously immunopositive, and it was prevalently expressed in female patients (71%). No case among peripheral nerve sheath tumors was found to be immunopositive for Sp17. The expression pattern was found heterogeneous in all of the positive samples and it does not correlate with the degree of malignancy. Conclusions The obtained frequency of expression and the heterogeneous cell distribution of Sp17 suggest the inability of this antigen to be used as a unique target for immunotherapeutic strategies in NS cancer. However, the present study firstly shows the immunolocalization of Sp17 in a proportion of cells of NS tumors but not in their normal counterpart. The emergent complex function of Sp17 renders necessary further studies to understand the link between immunopositive cells and this protein.

Purpose Many cases of chronic hepatitis C virus (HCV) infection are resistant to conventional therapies. Such cases might be treated by cell-mediated immunotherapy as cytotoxic T lymphocytes (CTL) are the main mechanism by which viral infections are cleared. The HCV core gene, being well conserved among HCV types, may be an appropriate antigen for targeting HCV-infected cells. Methods Here a series of five recombinant adeno-associated virus (rAAV) vectors carrying the full length (aa1-190) or autoimmune domain-depleted (AIDD) versions of core were used to load dendritic cells (DC), which, in turn, stimulated anti-core CTL. The AAV vectors were found to be able to transduce 88 to 95% of DC and the transduced DC displayed higher levels of CD80, CD83, CD86, and CD1a over controls. One vector, AAV/core(49-180)/Neo, with both autoimmune domains deleted from core, stimulated comparable core-positive target killing to the other versions yet stimulated significantly lower levels of "self " killing of core negative-targets, either of autologous PBMC targets (p = .002) or HLA-matched HepG2 liver cancer cells (p = .001). Results The resulting CTL populations displayed higher IFN-g expression, higher CD8:CD4 ratios, and lower CD56:CD8 ratios than controls. The rAAV loading-derived CD8+ T cells had more CD69+ cells and the CD4+ T populations had fewer CD25+ cells than controls. Summary and Conclusion AAV/core(49-180)/Neo, containing a dual AIDD core gene, may be particularly useful for clinical treatments as it stimulates lower "self " recognition but stimulates robust anticore CTL activity.

Purpose Multiple myeloma (MM) is difficult to cure by conventional chemotherapeutic modalities but may be amenable to immunotherapeutic approaches. In past studies we have investigated the use of recombinant adeno-associated virus (AAV)-based vectors for their ability to effectively deliver viral antigen and cytokine genes into dendritic cells (DCs). Surprisingly rAAV/viral antigen-pulsing of DC stimulated significant cytotoxic T lymphocytes (CTL) with only one stimulation. Methods Generation of the AAV-Spanxb virus stock, generation and infection of the DC to prime the na•ve T cell and by Facs analysis and Cr (51) to characterized the anti-Span-xb CTL. Summary The generation of CTL against Span-xb antigens represents a more difficult challenge. In this study it is shown that rAAV/Span-xb antigen gene delivery into DC, with 7 days of T cell stimulation, resulted in (1) high MHC class I-restricted, antigen-specific CTL killing of target cells in 51 chromium release assays, (2) high levels of intra-T cell interferon g (IFN-g) expression, and (3) high levels of CD80, CD86, and CD40 expression on DC. Both synthetic, autologous Span-xb-positive targets and primary MM cell lines were good targets for killing by the anti-Span-xb CTL, while Span-xb-negative targets were not significant targets. Conclusion These data suggest that rAAV/antigen gene pulsing of DC is a surprisingly effective technique for generating anti-self-antigen CTL as it is for viral antigens. Furthermore, these data suggest that Span-xb may be an effective antigen for targeting anti-MM CTL in gene therapy.

Purpose SPANX/(CTp11) is a recently identified cancer-testis (CT) antigen in multiple myeloma, melanoma, and other tumors of unrelated histological origin. We here show that it is expressed at mRNA in neoplastic cells from up to 35% of patients affected by liver cancer. Since the expression of this tumor antigen has been demonstrated to be restricted in normal tissues, it seems to be an excellent target for tumor vaccine of liver cancer. Methods PBMCs were separated from heparinized venous blood by density gradient centrifugation. DCs were differentiated from PBMC. Results SPANX (CTp11) has been found to be expressed at mRNA level and at protein level by immunohistochemistry in neoplastic cells from up to 35% of patients affected by liver cancer. It contains functional cytotoxic T cell (CTL), supporting its use as a tumor vaccine. In this study, we determined the ability to generate CTp11-specific HLA-class I restricted CTLs from PBL of 5 patients, 3 consecutive SPANX (CTp11)+ patients and two SPANX (CTp11)-patients. Summary Despite previous chemotherapy and the immunosuppression so often associated with liver cancer, CTL generation was successful in all 3 patients, suggesting the presence in the immune repertoire of SPANX (CTp11)-specific T cells in these patients, irrespective of the SPANX (CTp11) status of their tumors. Conclusion These observations provide the basis for a clinical study aimed at inducing a cellular immune response directed at SPANX (CTp11)-positive liver cancer patients.

Purpose The correlation between expression of tapasin and HLA class I was studied in patients refractory to conventional treatment in multiple myeloma (MM). Regulation of tapasin and HLA class I expression could be suitable markers for prognostic factors of MM patients. Methods Expression of mRNA for tapasin and HLA class I was evaluated via reverse transcription-polymerase chain reaction (RT-PCR) for 20 primary MM tumors and 6 MM cell lines. Protein analysis was performed using Western blots for protein expression of tapasin and HLA class I. Summary HLA class I and tapasin were analyzed in a panel of 20 fresh primary MM tumors and 6 MM cell lines. In primary tumors, 50% do not express tapasin at the protein level but are positive for mRNA. Following standard treatment of MM, 50% do not express tapasin at the protein level and 30% do not express mRNA or protein. Restoration by IFN-g further suggests tapasin expression in MM is alternately due to dysregulation rather than structural alterations by mutation. Conclusion These data indicate that absence of tapasin plays a central role in down-regulation of HLA class I in relapse MM patients. Tapasin and HLA class I down-regulation was prominent in relapsing patients compared to control. Deficiency of tapasin in tumor cells appears to be a frequent event in human tumors. Furthermore, tapasin and HLA class I down-regulation in MM was extensively associated with reduced survival of patients, therefore playing a role in poor prognosis of disease.

Purpose g-Irradiation of normal cells causes an increased synthesis of specific proteins, including MHC class I/II antigens and ICAM-I. However, few studies have described the effects of high doses of irradiation on the expression of cell surface antigens in tumor cells. The aim of this study was to analyze the effects of high doses of g-irradiation on the surface antigen expression of MHC class I, class II, and ICAM-I in human multiple myeloma (MM) cell lines, including ARP-1, ARK-RS and 10 multiple myeloma primary tumors. Methods The expression of surface antigens on this MM by FACS analysis at different time points following the exposure to high doses of g-irradiation was documented. The ARP-1, ARK-RS, cells lines, and 10 MM primary tumor cells expressed variable baseline levels of MHC class I/II antigens, ICAM-I. Summary While doses of 10,000 cGy were not sufficient to totally block cell replication in both cell lines and primary tumors, and exposure to 15,000 was also unable to stop cell replication, only at 18,000 cGy was cell replication completely inhibited. Low doses (10,000 cGy) and lethal doses of irradiation (ie, 15,000 and 18,000 cGy) drastically and consistently increased the expression of all surface antigens present on the cells prior to irradiation. Conclusion This up-regulation was shown to be dose dependent, with higher radiation doses resulting in higher antigen expression. Furthermore, when the kinetics of this up-regulation were studied 3 and 6 days after irradiation, there was a constant increase in antigen expression in multiple myeloma cells.

AIM: To introduce a computer-aided morphometric method for quantifying the necro-inflammatory phase in liver biopsy specimens using fractal geometry and Delaunay’s triangulation. METHODS: Two-micrometer thick biopsy sections taken from 78 chronic hepatitis C virus-infected patients were immunohistochemically treated to identify the inflammatory cells. An automatic computer-aided image analysis system was used to define the inflammatory cell network defined on the basis of Delaunay’s triangulation, and the inflammatory cells were geometrically classified as forming a cluster (an aggregation of a minimum of three cells) or as being irregularly distributed within the tissue. The phase of inflammatory activity was estimated using Hurst’s exponent. RESULTS: The proposed automatic method was rapid and objective. It could not only provide rigorous results expressed by scalar numbers, but also allow the state of the whole organ to be represented by Hurst’s exponent with an error of no more than 12%. CONCLUSION: The availability of rigorous metrical measures and the reasonable representativeness of the status of the organ as a whole raise the question as to whether the indication for hepatic biopsy should be revised by establishing clear rules concerning the contraindications suggested by its invasiveness and subjective interpretation.

One of the major liver fibrogenic activators is the cellular iron overload that can severely damage parenchymal and non-parenchymal cells. The aim of this study was to investigate a histochemical staining technique that allows the simultaneous detection of the irregular deposition of matrix collagen and both the amount and distribution of iron in liver cells on the same histological section. The method was evaluated using 3-microm histological sections obtained from ten standard liver biopsy specimens taken from patients with hepatitis C virus-related diseases and simultaneous liver cell iron overload. The results indicate that the double-staining technique is simple, sensitive and rapid, and can be routinely applied to liver biopsy specimens for diagnostic purposes. Furthermore, it may also facilitate the study of the complex relationship between hepatic fibrosis and iron overload during common genetic or secondary liver metabolic disorders.