Possible Mechanisms of Resistance Development to Photodynamic Therapy (PDT) In Vulvar Cancer Cells.

Photodynamic therapy (PDT) is a low-invasive treatment method that can be used to treat VIN patients. A photosensitizer (PS) applied to a patient is activated with use of the appropriate wavelength of light, which in an oxygen environment leads to the formation of a reactive oxygen species (ROS) that destroys the tumor. However, cells can protect themselves against these cytotoxic products by increasing their antioxidant mechanisms and repair capacity. Changes in the cytoskeleton may also influence resistance to PDT. Our results revealed that PDT-resistant cells changed the amount of ROS. Cells resistant to PDT A-431 exhibited a decreased ROS level and showed higher viability after oxidizing agent treatment. Resistant Cal-39 cells exhibited a decreased O2- level but increased other ROS. This provides protection from PDT but not from other oxidizing agents. Moreover, PDT leads to alterations in the cytoskeleton that may result in an epithelial-mesenchymal transition (EMT) or increased adhesion. Both EMT and cell adhesion may activate signaling pathways involved in survival. This means that resistance to PDT in vulvar cancer may be at least in part a result of changes in ROS level and alterations in the cytoskeleton.

Mechanisms of Resistance to Photodynamic Therapy (PDT) in Vulvar Cancer.

Photodynamic therapy (PDT) is a valuable treatment method for vulvar intraepithelial neoplasia (VIN). It allows for the treatment of a multifocal disease with minimal tissue destruction. 5-Aminolevulinic acid (5-ALA) is the most commonly used prodrug, which is converted in the heme pathway to protoporphyrin IX (PpIX), an actual photosensitizer (PS). Unfortunately, not all patients treated with PDT undergo complete remission. The main cause of their failure is resistance to anticancer therapy. In many cancers, resistance to various anticancer treatments is correlated with increased activity of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1). Enhanced activity of drug pumps may also affect the effectiveness of therapy. To investigate whether multidrug resistance mechanisms underlie PDT resistance in VIN, porphyrins were isolated from sensitive and resistant vulvar cancer cells and their culture media. APE1 activity was measured, and survival assay after PDT combined with APE1 inhibitor was performed. Our results revealed that resistant cells accumulated and effluxed less porphyrins than sensitive cells, and in response to PDT, resistant cells increased APE1 activity. Moreover, PDT combined with inhibition of APE1 significantly decreased the survival of PDT-resistant cells. This means that resistance to PDT in vulvar cancer may be the result of alterations in the heme synthesis pathway. Moreover, increased APE1 activity may be essential for the repair of PDT-mediated DNA damage, and inhibition of APE1 activity may increase the efficacy of PDT.

Photodynamic therapy ­ significance in oncology / Terapia fotodynamiczna ­ znaczenie w onkologii.

Photodynamic therapy (PDT) is one of the least toxic methods causing the death of cancer cells. Photosensitizer (PS) applied to a patient accumulates in the tumor, where under the appropriate wavelength and insensitivity of light is activated. Activated PS in the presence of oxygen produces reactive oxygen species (ROS), which make significant damage leading to the destruction of cancer cells by apoptosis, necrosis or autophagic process. Moreover, PDT causes an acute local inflammatory response that is involved in removing dead cells, restoring normal tissue homeostasis, and sometimes leads to the development of systemic immunity. However, some cells may survive treatment and develop resistance. Mechanisms, which lead to decrease of the level of PS in cells may be involved in the cytoprotection of cancer cells from PDT. Furthermore, increased activity of antioxidant mechanisms, overexpression of molecular chaperones and activation of survival pathways can protect cells from PDT.

How to Predict Metastasis in Luminal Breast Cancer? Current Solutions and Future Prospects.

Breast cancer metastasis is the main cause of breast cancer mortality. Luminal breast cancer represents the majority of breast cancer cases and, despite relatively good prognosis, its heterogeneity creates problems with a proper stratification of patients and correct identification of the group with a high risk of metastatic relapse. Current prognostic tools are based on the analysis of the primary tumor and, despite their undisputed power of prediction, they might be insufficient to foresee the relapse in an accurate and precise manner, especially if the relapse occurs after a long period of dormancy, which is very common in luminal breast cancer. New approaches tend to rely on body fluid analyses, which have the advantage of being non-invasive and versatile and may be repeated and used for monitoring the disease in the long run. In this review we describe the current, newly-developed, and only-just-discovered methods which are or may become useful in the assessment of the probability of the relapse.

Circulating Tumor Cells in Early and Advanced Breast Cancer; Biology and Prognostic Value.

Breast cancer metastasis is the leading cause of cancer deaths in women and is difficult to combat due to the long periods in which disseminated cells retain a potential to be re-activated and start the relapse. Assessing the number and molecular profile of circulating tumor cells (CTCs) in breast cancer patients, especially in early breast cancer, should help in identifying the possibility of relapse in time for therapeutic intervention to prevent or delay recurrence. While metastatic breast cancer is considered incurable, molecular analysis of CTCs still have a potential to define particular susceptibilities of the cells representing the current tumor burden, which may differ considerably from the cells of the primary tumor, and offer more tailored therapy to the patients. In this review we inspect the routes to metastasis and how they can be linked to specific features of CTCs, how CTC analysis may be used in therapy, and what is the current status of the research and efforts to include CTC analysis in clinical practice.

Resistance to endocrine therapy in breast cancer: molecular mechanisms and future goals.

INTRODUCTION: The majority of breast cancers (BCs) are characterized by the expression of estrogen receptor alpha (ERα+). ERα acts as ligand-dependent transcription factor for genes associated with cell survival, proliferation, and tumor growth. Thus, blocking the estrogen agonist effect on ERα is the main strategy in the treatment of ERα+ BCs. However, despite the development of targeted anti-estrogen therapies for ER+ BC, around 30-50% of early breast cancer patients will relapse. Acquired resistance to endocrine therapy is a great challenge in ER+ BC patient treatment. DISCUSSION: Anti-estrogen resistance is a consequence of molecular changes, which allow for tumor growth irrespective of estrogen presence. Those changes may be associated with ERα modifications either at the genetic, regulatory or protein level. Additionally, the activation of alternate growth pathways and/or cell survival mechanisms can lead to estrogen-independence and endocrine resistance. CONCLUSION: This comprehensive review summarizes molecular mechanisms associated with resistance to anti-estrogen therapy, focusing on genetic alterations, stress responses, cell survival mechanisms, and cell reprogramming.

Longitudinal analysis of circulating tumor cell numbers improves tracking metastatic breast cancer progression.

Hormone-responsive breast cancer represents the most common type and has the best prognosis, but still approximately 40% of patients with this type can develop distant metastases, dramatically worsening the patient's survival. Monitoring metastatic breast cancer (mBC) for signs of progression is an important part of disease management. Circulating tumor cell (CTC) detection and molecular characteristics gain importance as a diagnostic tool, but do not represent a clinical standard and its value as a predictor of progression is not yet established. The main objective of this study was to estimate the prognostic value of not only the CTC numbers, but also the dynamics of the CTC numbers in the same patient during the continuous evaluation of CTCs in patients with advanced breast cancer. The other objective was to assess the molecular changes in CTCs compared to primary tumor samples by genetic analysis of the seven genes associated with estrogen signaling pathway, mutations in which are often responsible for the resistance to endocrine therapy, and subsequent progression. This approach was taken to evaluate if genetic analysis of CTCs can be used in tracking the resistance, signaling that hormonal therapy should be replaced. Consequently, this report presents the results of a longitudinal CTC study based on three subsequent blood collections from 135 patients with metastatic breast cancer, followed by molecular analysis of the isolated single CTCs. CTCs were detected and isolated using an image-based, EpCAM-independent system CytoTrack; this approach allowed evaluation of EpCAM expression in detected CTCs. Isolated CTCs were subjected to NGS analysis to assess mutational changes. The results confirm the importance of the status of the CTC for progression-free survival and overall survival and provide new data on the dynamics of the CTC during a long monitoring period and in relation to clinical progression, highlighting the advantage of constant monitoring over the single count of CTC. Furthermore, high genetic and phenotypic inter- and intrapatient heterogeneity observed in CTCs suggest that metastatic lesions are divergent. High genetic heterogeneity in the matching CTC/primary tumor samples may indicate early dissemination. The tendency towards the accumulation of activating/oncogenic mutation in CTCs, leading to anti-estrogen resistant disease, was not confirmed in this study.

SOX11 expression as a MRD molecular marker for MCL in comparison with t(11;14) and IGH rearrangement.

The main cause of death in mantle cell lymphoma (MCL) patients is relapse due to undetermined minimal residual disease (MRD) and therefore monitoring MRD is crucial for making the best treatment decisions. The gold standard method for MRD analysis is the quantitative polymerase chain reaction. The most commonly used molecular markers for measuring MRD in MCL are: t(11;14)(q13;p32) translocation or CCND1 expression and IGH rearrangement. Such markers can, however, be found in other B cell non-Hodgkin lymphomas. Recent studies demonstrate that SOX11 expression is highly specific for MCL and could be used as a marker for measuring MRD. Moreover, evidence shows that SOX11 level could be predictive for overall survival (OS) and progression-free survival (PFS). We have measured MRD level in follow-up samples from 27 patients diagnosed with MCL using the molecular markers: t(11;14), IGH rearrangement and SOX11 expression. We compared all markers by their sensitivity, utility and quantitative range. We also examined the predictive value of SOX11 expression for OS and PFS. SOX11 expression was found to have better specificity, quantitative range and utility than the t(11;14). The predictive value of SOX11 expression was confirmed. At diagnosis, patients with high SOX11 expression had shorter PFS than patients with low SOX11 expression (p = 0.04*); differences between OS being statistically insignificant. To our best knowledge this is a first study comparing SOX11 with t(11;14) and IGH rearrangement as markers of MRD level. Moreover, in this study we confirmed that SOX11 is useful in cases when other molecular markers cannot be used.

Usefulness of real-time PCR in long-term follow-up of follicular lymphoma patients.

The aim of this study was to evaluate the usefulness of quantitative real-time PCR (RQ-PCR) for the monitoring of molecular remission in follicular lymphoma (FL) patients during long-term follow-up. RQ-PCR by the use of TaqMan detection system is a sensitive tool to monitor minimal residual disease (MRD) in FL through amplification of the t(14;18) fusion gene during and post-therapy. In most cases the breakpoint region occurs within the major breakpoint region (MBR). Among 75 patients diagnosed with FL, cells harboring the fusion gene BCL2/JH were found in peripheral blood of 31 patients (41%). We further monitored 30 of these patients in a period varying from 6 months to 5 years by RQ-PCR. In our study the level indicating the possibility of the presence of MRD was established at more than five t(14;18)-positive cells in the background of 83,000 normal cells. The results of this work also confirmed that the presence of MRD detected by RQ-PCR is an indication for careful observation of patients because of a higher risk of disease recurrence.

CTC clusters in cancer progression and metastasis.

Circulating tumor cells (CTC) in the blood of cancer patients are regarded as potential metastatic seeds, and their detailed characterization holds great promises for more exact prognosis, better diagnosis and therapy of the metastatic cancer. Circulating tumor cell clusters represent different class of CTCs, with specific properties, including high metastatic potential. In this review, we present current opinions on differences between single CTCs and CTC clusters, their mode of dissemination, methods of detection and clinical importance in various types of cancer.

Lipid peroxidation in face of DNA damage, DNA repair and other cellular processes.

Exocyclic adducts to DNA bases are formed as a consequence of exposure to certain environmental carcinogens as well as inflammation and lipid peroxidation (LPO). Complex family of LPO products gives rise to a variety of DNA adducts, which can be grouped in two classes: (i) small etheno-type adducts of strong mutagenic potential, and (ii) bulky, propano-type adducts, which block replication and transcription, and are lethal lesions. Etheno-DNA adducts are removed from the DNA by base excision repair (BER), AlkB and nucleotide incision repair enzymes (NIR), while substituted propano-type lesions by nucleotide excision repair (NER) and homologous recombination (HR). Changes of the level and activity of several enzymes removing exocyclic adducts from the DNA was reported during carcinogenesis. Also several beyond repair functions of these enzymes, which participate in regulation of cell proliferation and growth, as well as RNA processing was recently described. In addition, adducts of LPO products to proteins was reported during aging and age-related diseases. The paper summarizes pathways for exocyclic adducts removal and describes how proteins involved in repair of these adducts can modify pathological states of the organism.

Retracing Circulating Tumour Cells for Biomarker Characterization after Enumeration.

BACKGROUND: Retracing and biomarker characterization of individual circulating tumour cells (CTCs) may potentially contribute to personalized metastatic cancer therapy. This is relevant when a biopsy of the metastasis is complicated or impossible to acquire. METHODS: A novel disc format was used to map and retrace individual CTCs from breast-cancer patients and nucleated cells from healthy blood donors using the CytoTrack platform. For proof of the retracing concept, CTC HER2 characterization by immunofluorescence was tested. RESULTS: CTCs were detected and enumerated in three of four blood samples from breast-cancer patients and the locations of each individual CTCs were mapped on the discs. Nucleated cells were retraced on seven discs with 96.6%±8.5% recovery on five fields of view on each disc. Shifting of field of view for retracing was measured to 4-29 µm. In a blood sample from a HER2-positive breast-cancer patient, CTC enumeration and mapping was followed by HER2 characterization and retracing to demonstrate downstream immunofluorescence analysis of the CTC. CONCLUSION: Mapping and retracing of CTCs enables downstream analysis of individual CTCs for existing and future cancer genotypic and phenotypic biomarkers. Future studies will uncover this potential of the novel retracing technology.

Detection of circulating breast cancer cells in peripheral blood by a two-marker reverse transcriptase-polymerase chain reaction assay.

The aim of this study was to use a two-marker assay for the detection of breast cancer cells circulating in patients' blood. We have applied a PCR-based methodology to follow up the possibility of the development of metastatic disease in stage I and II patients who had undergone curative surgery. Since the number of circulating cancer cells in peripheral blood is very low, the technique for their detection needs to be not only highly sensitive, but also very specific. The reverse transcriptase-polymerase chain reaction (RT-PCR) technique may improve the sensitivity of breast cancer cell detection up to only a few cells per one million. The principle of the RT-PCR assay is to amplify a messenger RNA characteristic for breast epithelial cells in a blood sample. Since we do not expect such cells to be circulating in peripheral blood of healthy subjects, detection of the characteristic mRNA should indicate the presence of circulating breast cancer cells. We analyzed the usefulness of three mRNA markers: cytokeratin 19 (CK19), mammaglobin (hMAM) and beta subunit of human chorionic gonadotropin (beta-hCG) for this test. Blood samples (112) were obtained from 55 patients, in stages I and II, with or without metastasis to regional lymph nodes (N0 or N1). We found that a two-marker assay increases the sensitivity of detection of breast cancer cells in comparison with a single-marker one. Combination of two tumor-specific mRNA markers, hMAM/CK19 or beta-hCG/CK19, allowed the detection of circulating breast cancer cells in 65% of N1 patients and 38% of N0 patients. By comparison, the combination hMAM/beta-hCG allowed the detection of circulating breast cancer cells in the blood of 68% of N1 patients and 46% of N0 patients. Addition of the third marker did not significantly increase the detection sensitivity.

Evaluation of prognostic significance of circulating tumor cells detection in rectal cancer patients treated with preoperative radiotherapy: prospectively collected material data.

The aim of this study was to evaluate the prognostic value of circulating tumor cells (CTC) in nonmetastatic rectal cancer patients treated with short-term preoperative radiotherapy. In this single-center trial, 162 patients with rectal cancer after preoperative short-term radiotherapy (5 × 5 Gy) were recruited from January, 2008 to September, 2011. Clearance of CTC was determined in 91 patients enrolled in the molecular analysis. CTC presence was evaluated with real-time reverse transcription polymerase chain reaction assay (qPCR) based on the expression of three tumor genetic markers: carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and/or cancer stem cells marker CD133 (CEA/CK20/CD133). We found that CTC detection 7 days after surgery was of prognostic significance for the local recurrence (P value = 0.006). CTC detected preoperatively and 24 hours after resection had no prognostic value in cancer recurrence; however, there was a significant relationship between CTC prevalence 24 hours after surgery and lymph node metastasis (pN1-2). We also confirmed a significant clearance of CTC in peripheral blood (PB) 24 hours after surgery. Preoperative sampling is not significant for prognosis in rectal cancer patients treated with short-term radiotherapy. Detection of CTC in PB 7 days after surgery is an independent factor predicting local recurrence in this group of patients.

Polymorphisms of DNA repair and oxidative stress genes in B-cell lymphoma patients.

The purpose of the study was to investigate the possible association between ERCC2 rs28365048, ERCC5 rs17655, XRCC3 rs861539 and NOS2A rs2297518 polymorphisms with B-cell lymphoma. The study was conducted on 189 patients with CD20+ B-cell lymphoma and 193 controls. The genotype frequencies were compared in the patient and control groups using quantitative polymerase chain reaction, based on allelic discrimination analysis. Our results indicated that variation in NOS2A may be significant in B-cell lymphoma in a population ≥50 years old (OR=2.15; 95% CI, 1.17-3.92; P=0.013). No association was observed between variations in ERCC2, ERCC5, XRCC3 and B-cell lymphoma in the studied population. Our finding of an association between age and NOS2A polymorphisms in lymphoma is unique and requires additional studies. The results concerning ERCC2, ERCC5 and XRCC3 variations add additional data to studies on genetic polymorphisms in the DNA repair pathway.