BM micrometastases and circulating tumor cells in breast cancer patients: where have we been, where are we now and where does the future lie?

Over the past 15 years early tumor cell dissemination has been detected in patients with breast cancer using sensitive immunocytochemical and molecular assays based on the use of MAb and PCR, respectively. Clinical studies involving more than 4,000 breast cancer patients have now demonstrated that the presence of disseminated tumor cells in BM identified with immuncytochemical assays at primary diagnosis is a strong and independent prognostic factor. The published studies for the detection of disseminated tumor cells in BM fulfill the highest level of evidence as prognostic markers in primary breast cancer. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have been developed recently and some studies suggest a potential clinical relevance of this parameter as a prognostic and predictive factor. Comparative analyzes indicate that the prognostic information derived from BM and blood screening seems to be complementary and not redundant. Advanced methods for molecular characterization of single tumor cells and the surrounding environment have been developed lately, and this approach allows new insights into the metastatic cascade and characterization of targets for therapeutic approaches. Taken together, these findings provide the basis for the implementation of disseminated tumor cells in BM or blood as markers for stratification and assessment of therapies in prospective clinical trials. The valuable information derived from these trials should help to improve future treatment of breast cancer patients.     

Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity.     

Biomarkers of Residual Disease,Disseminated Tumor Cells,and Metastases in the MMTV-PyMT Breast Cancer Model

Cancer metastases arise in part from disseminated tumor cells originating from the primary tumor and from residual disease persisting after therapy. The identification of biomarkers on micro-metastases, disseminated tumors, and residual disease may yield novel tools for early detection and treatment of these disease states prior to their development into metastases and recurrent tumors. Here we describe the molecular profiling of disseminated tumor cells in lungs, lung metastases, and residual tumor cells in the MMTV-PyMT breast cancer model. MMTV-PyMT mice were bred with actin-GFP mice, and focal hyperplastic lesions from pubertal MMTV-PyMT;actin-GFP mice were orthotopically transplanted into FVB/n mice to track single tumor foci. Tumor-bearing mice were treated with TAC chemotherapy (docetaxel, doxorubicin, cyclophosphamide), and residual and relapsed tumor cells were sorted and profiled by mRNA microarray analysis. Data analysis revealed enrichment of the Jak/Stat pathway, Notch pathway, and epigenetic regulators in residual tumors. Stat1 was significantly up-regulated in a DNA-damage-resistant population of residual tumor cells, and a pre-existing Stat1 sub-population was identified in untreated tumors. Tumor cells from adenomas, carcinomas, lung disseminated tumor cells, and lung metastases were also sorted from MMTV-PyMT transplant mice and profiled by mRNA microarray. Whereas disseminated tumors cells appeared similar to carcinoma cells at the mRNA level, lung metastases were genotypically very different from disseminated cells and primary tumors. Lung metastases were enriched for a number of chromatin-modifying genes and stem cell-associated genes. Histone analysis of H3K4 and H3K9 suggested that lung metastases had been reprogrammed during malignant progression. These data identify novel biomarkers of residual tumor cells and disseminated tumor cells and implicate pathways that may mediate metastasis formation and tumor relapse after therapy.     

Focus: A Multifaceted Battle Against Cancer: The Benefit and Burden of Cancer Screening in Li-Fraumeni Syndrome: A Case Report

Li-Fraumeni syndrome is a rare cancer predisposition syndrome classically associated with remarkably early onset of cancer in families with a typical spectrum of malignancies, including sarcoma, breast cancer, brain tumors, and adrenocortical carcinoma. Because the risks of cancer development are strikingly high for Li-Fraumeni syndrome, aggressive cancer surveillance is often pursued in these individuals. However, optimal screening methods and intervals for Li-Fraumeni syndrome have yet to be determined. In addition, there may be a significant psychosocial burden to intensive cancer surveillance and some prevention modalities. Here, we describe a case of a young woman with a de novo mutation in TP53 and multiple malignancies, with her most recent cancers found at early, curable stages due to aggressive cancer screening. The potential benefits and risks of intensive cancer surveillance in hereditary cancer syndromes is discussed.     

The other side of the coin: dissecting molecular mechanisms behind hereditary breast cancer in search of therapeutic opportunities

Over the last quarter century several genetic alterations have been implicated in hereditary breast cancer (HBC). Two papers recently published in the New England Journal of Medicine explored the mutation prevalence in breast cancer predisposition genes across a large population of affected and unaffected subjects. These analyses designated ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C and RAD51D as the core set of genes associated with a significantly increased risk of developing breast cancer. A deeper understanding of the biological role of these genes unearths an intricate mechanism involving DNA repair and cell cycle regulation. Exploiting these inherited alterations for targeted treatments, as is currently the case with PARP inhibitors, may provide additional therapeutic opportunities for HBC patients.     

A new approach to phenotyping disseminated tumor cells: methodological advances and clinical implications

At the time of primary therapy (surgery, systemic chemotherapy and/or radiation), disseminated tumor cells in the bone marrow can be found in almost one-third of patients with cancer of the breast, ovary, esophagus, stomach, colon, and other solid tumors. Whereas the prognostic impact of the mere presence of these cells is still a matter of debate, it has been shown that expression of tumor-associated antigens in disseminated tumor cells is linked to more aggressive disease. Therefore, further characterization of disseminated tumor cells at the protein and gene level has become increasingly important. To date, the most common detection method for disseminated tumor cells in the bone marrow is an immunocytochemical approach using cytokeratin-directed antibodies for detection of epithelial cells and the APAAP system for their visualization. We have established a new double immunofluorescence technique enabling simultaneous detection, phenotyping, and antigen quantification of disseminated tumor cells. Mononuclear cells from bone marrow are enriched by Ficoll gradient centrifugation and cytospins are prepared. Double immunofluorescence is performed using antibodies against cytokeratins 8/18/19 (mAb A45B/B3) and the uPA receptor CD87 (pAb HU277). CD87 expression is recorded by confocal laser scanning microscopy (CLSM) using fluorescence labeled latex beads as the reference; staining intensities of all the scans are then summed and quantified (extended focus). This protocol, originally designed for disseminated tumor cells in bone marrow, can also be applied to disseminated tumor cells in blood, to leukapheresis cells or to cells present in malignant ascites or other malignant effusions. The tumor cells detected may be used for gene and mRNA analyses. Furthermore, disseminated tumor cells also represent interesting targets for clinical studies on patient prognosis or prediction of therapy response as well as for specific tumor-biological therapies.     

Targeting the Wnt pathway in cancer: The emerging role of Dickkopf-3

Aberrant activation of the Wnt signaling pathway is a major trait of many human cancers. Due to its vast implications in tumorigenesis and progression, the Wnt pathway has attracted considerable attention at several molecular levels, also with respect to developing novel cancer therapeutics. Indeed, research in Wnt biology has recently provided numerous clues, and evidence is accumulating that the secreted Wnt antagonist Dickkopf-related protein 3 (Dkk-3) and its regulators may constitute interesting therapeutic targets in the most important human cancers. Based on the currently available literature, we here review the knowledge on the biological role of Dkk-3 as an antagonist of the Wnt signaling pathway, the involvement of Dkk-3 in several stages of tumor development, the genetic and epigenetic mechanisms disrupting DKK3 gene function in cancerous cells, and the potential clinical value of Dkk-3 expression/DKK3 promoter methylation as a biomarker and molecular target in cancer diseases.In conclusion, Dkk-3 rapidly emerges as a key player in human cancer with auspicious tumor suppressive capacities, most of all affecting apoptosis and proliferation. Its gene expression is frequently downregulated by promoter methylation in almost any solid and hematological tumor entity. Clinically, evidence is accumulating of Dkk-3 being both a potential tumor biomarker and effective anti-cancer agent. Although further research is needed, re-establishing Dkk-3 expression in cancer cells holds promise as novel targeted molecular tumor therapy.     

Animal models of ovarian cancer

Ovarian cancer is the most lethal of all of the gynecological cancers and can arise from any cell type of the ovary, including germ cells, granulosa or stromal cells. However, the majority of ovarian cancers arise from the surface epithelium, a single layer of cells that covers the surface of the ovary. The lack of a reliable and specific method for the early detection of epithelial ovarian cancer results in diagnosis occurring most commonly at late clinical stages, when treatment is less effective. In part, the deficiency in diagnostic tools is due to the lack of markers for the detection of preneoplastic or early neoplastic changes in the epithelial cells, which reflects our rather poor understanding of this process. Animal models which accurately represent the cellular and molecular changes associated with the initiation and progression of human ovarian cancer have significant potential to facilitate the development of better methods for the early detection and treatment of ovarian cancer. This review describes some of the experimental animal models of ovarian tumorigenesis that have been reported, including those involving specific reproductive factors and environmental toxins. Consideration has also been given to the recent progress in modeling ovarian cancer using genetically engineered mice.     

Targeting LKB1 signaling in cancer

The serine/threonine kinase LKB1 is a master kinase involved in cellular responses such as energy metabolism, cell polarity and cell growth. LKB1 regulates these crucial cellular responses mainly via AMPK/mTOR signaling. Germ-line mutations in LKB1 are associated with the predisposition of the Peutz–Jeghers syndrome in which patients develop gastrointestinal hamartomas and have an enormously increased risk for developing gastrointestinal, breast and gynecological cancers. In addition, somatic inactivation of LKB1 has been associated with sporadic cancers such as lung cancer. The exact mechanisms of LKB1-mediated tumor suppression remain so far unidentified; however, the inability to activate AMPK and the resulting mTOR hyperactivation has been detected in PJS-associated lesions. Therefore, targeting LKB1 in cancer is now mainly focusing on the activation of AMPK and inactivation of mTOR. Preclinical in vitro and in vivo studies show encouraging results regarding these approaches, which have even progressed to the initiation of a few clinical trials. In this review, we describe the functions, regulation and downstream signaling of LKB1, and its role in hereditary and sporadic cancers. In addition, we provide an overview of several AMPK activators, mTOR inhibitors and additional mechanisms to target LKB1 signaling, and describe the effect of these compounds on cancer cells. Overall, we will explain the current strategies attempting to find a way of treating LKB1-associated cancer.     

Development of near-infrared imaging agents for detection of junction adhesion molecule-A protein

IntroductionProstate and breast cancer are the most prevalent primary malignant human tumors globally. Prostatectomy and breast conservative surgery remain the most common definitive treatment option for the >500,000 men and women newly diagnosed with localized prostate and breast cancer each year only in the US. Morphological examination is the mainstay of diagnosis but margin under-sampling of the excised cancer tissue may lead to local recurrence. In despite of the progress of non-invasive optical imaging, there is still a clinical need for targeted optical imaging probes that could rapidly and globally visualize cancerous tissues.MethodsElevated expression of junctional adhesion molecule-A (JAM-A) on tumor cells and its multiple pro-tumorigenic activity make the JAM-A a candidate for molecular imaging. Near-infrared imaging probe, which employed anti-JAM-A monoclonal antibody (mAb) phthalocyanine dye IR700 conjugates (JAM-A mAb/IR700), was synthesized and used to identify and visualize heterotopic human prostate and breast tumor mouse xenografts in vivo.ResultsThe intravenously injected JAM-A mAb/IR700 conjugates enabled the non-invasive detection of prostate and breast cancerous tissue by fluorescence imaging. A single dose of JAM-A mAb/IR700 reduced number of mitotic cancer cells in vivo, indicating theranostic ability of this imaging agent. The JAM-A mAb/IR700 conjugates allowed us to image a specific receptor expression in prostate and breast tumors without post-image processing.ConclusionThis agent demonstrates promise as a method to image the extent of prostate and breast cancer in vivo and could assist with real-time visualization of extracapsular extension of cancerous tissue.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

New perspectives on hereditary influences in metastatic progression

Metastasis, the process by which cancer cells spread to distant sites and form secondary tumors, depends upon the ability of cells to escape the primary tumor, and colonize and proliferate in a novel microenvironment. Many mechanisms have been proposed to explain this phenomenon although no theory has comprehensively explained all biological observations. There is growing evidence that host hereditary factors modulate the ability of tumor cells to form metastatic lesions, and host genetic polymorphism could be a significant variable in this process. This review is intended to illustrate the role of hereditary variation in metastatic progression, how this integrates with currently proposed metastatic mechanisms, and the potential clinical impact on this frequently fatal consequence of cancer.     

Resveratrol inhibits tumor progression by down-regulation of NLRP3 in renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most common urologic malignant tumors. Current chemotherapy is not effective in RCC and results in some side effects. Resveratrol (RSV) has been reported to exert antitumor effects in some cancer cells; however the mechanism is not fully understood. Herein, we aimed to determine the anticancer effect of RSV on RCC and further explore the underlying molecular mechanism in this process. We found that RSV inhibited tumor cells proliferation, migration and invasion and increased apoptosis of RCC either in vivo or in vitro. RSV significantly down-regulated expressions of NLRP3 and its downstream genes. Inhibition of NLRP3 by NLRP3 small interfering RNA mimicked the effects of RSV on RCC cells. These results suggested that RSV could exert antitumor effect by depressing activity of NLRP3, and NLRP3 would be a promising clinical therapeutic strategy for RCC.     

DNA mismatch repair and Lynch syndrome

The evolutionary conserved mismatch repair proteins correct a wide range of DNA replication errors. Their importance as guardians of genetic integrity is reflected by the tremendous decrease of replication fidelity (two to three orders of magnitude) conferred by their loss. Germline mutations in mismatch repair genes, predominantly MSH2 and MLH1, have been found to underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary predisposition for cancer. Lynch syndrome affects predominantly the colon and accounts for 2–5% of all colon cancer cases. During more than 30 years of biochemical, crystallographic and clinical research, deep insight has been achieved in the function of mismatch repair and the diseases that are associated with its loss. We review the biochemistry of mismatch repair and also introduce the clinical, diagnostic and genetic aspects of Lynch syndrome.     

Conquering cancer through discovery research

Cancer is a leading cause of death worldwide. Cancer research improves our understanding of cancer biology, which leads to the discoveries of novel detection approaches and effective therapies for cancer. Translational cancer medicine changes essential science breakthroughs to the practice of medicine and uses clinical result to supply back into basic research. This article covers some studies in the field of translational cancer medicine including the identification of 319 driver genes and the 12 core cancer pathways, the use of MammaPrint in breast cancer, the development of OncoMap, the progress in genome-wide association studies, as well as the generation of microRNA networks in cancer and leukemia. Apart from cancer genome, cancer stem cells, immune and tumor microenvironment are also discussed. In addition, some innovations in translational cancer medicine are introduced. A number of targeted agents have been developed, such as the histone deacetylases inhibitors, poly(ADP-ribose) polymerase inhibitors, anti-mammalian target of rapamycin agents, and PI3K pathway inhibitors. There are also reports of the results from some important clinical trials, including the STAR P-2 trial, NeoBIG program, and BATTLE trial. This review focuses on discussions that emphasize the marriage between curiosity-driven basic research and patient care-focused clinical investigations. With highlights on the most up-to-date molecular, cellular, clinical, and therapeutic cancer research findings, this article tends to provide a wealth of insights into better understanding of the complexity of cancer.     

Manganese superoxide dismutase in breast cancer: From molecular mechanisms of gene regulation to biological and clinical significance

Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently reliable predictive biological markers, and this is a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation, which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O₂^•−) and hydrogen peroxide (H₂O₂). Particularly, they report how these reactive oxygen species may activate some signaling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests a way to define precisely the clinical interest of MnSOD as a prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well-known biomarkers, in addition to the typical clinical parameters.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

A mathematical model of combined bacillus Calmette-Guerin (BCG) and interleukin (IL)-2 immunotherapy of superficial bladder cancer

We report a mathematical model that describes the growth of superficial bladder cancer and the effect thereupon of immunotherapy based on the administration of Bacillus Calmette-Guerin (BCG) combined or not with interleukin-2 (IL-2). Intravesical instillations of BCG performed after surgical removal of tumors represents an established treatment with approximately 50% success rate. So far, attempts to improve this efficiency have not led to essential changes. However, convincing clinical results have been reported on the combination of IL-2 to BCG, even though this is still not applied in current practice. The present model provides insights into the dynamical outcomes arising in the bladder from the interactions of immune cells with tumor cells in the course of BCG therapy associated or not with IL-2. Specifically, from the simulations performed using seven ordinary and non-linear differential equations we obtained indications on the conditions that would result in successful bladder cancer treatment. We show that immune cells -effector lymphocytes and antigen-presenting cells-expand and reach a sustainable plateau under BCG treatment, which may account for its beneficial effect, resulting from inflammatory “side-effects” which eliminate residual or eventual newly arising tumor cells, providing thus protection from further cancer development. We find, however, that IL-2 does not actually potentiate the effect of BCG as regards tumor cell eradication. Hence, associating both under the conditions simulated should not result in more efficient treatment of bladder cancer patients.     

Colorectal cancer prevention: Immune modulation taking the stage

Prevention or early detection is one of the most promising strategies against colorectal cancer (CRC), the second leading cause of cancer death in the US. Recent studies indicate that antitumor immunity plays a key role in CRC prevention. Accumulating evidence suggests that immunosurveillance represents a critical barrier that emerging tumor cells have to overcome in order to sustain the course of tumor development. Virtually all of the agents with cancer preventive activity have been shown to have an immune modulating effect. A number of immunoprevention studies aimed at triggering antitumor immune response against early lesions have been performed, some of which have shown promising results. Furthermore, the recent success of immune checkpoint blockade therapy reinforces the notion that cancers including CRC can be effectively intervened via immune modulation including immune normalization, and has stimulated various immune-based combination prevention studies. This review summarizes recent advances to help better harness the immune system in CRC prevention.