Special Issue "Advances in Molecular Pathology"

Guest Editor
Prof. Dr. Paul Fisch
Institute of Pathology, University Hospital Freiburg, Breisacher Strasse 115a, 79106 Freiburg, Germany
E-Mail: paul.fisch@uniklinik-freiburg.de

Dear Colleagues,

Molecular pathology is a medical subspecialty, increasingly localized between pathology and clinical medicine. Why may this somewhat provocative view be justified? Molecular pathology got its start in the last 10 to 20 years of the last century as a field with the primary goal of aiding pathologists in making the right diagnosis in difficult cases or to find out whether a lesion is neoplastic or reactive. Initially, PCR-based methods were developed that worked on paraffin material allowing the determination of clonality of immunoglobulin or T-cell receptor genes in lymphoid neoplasia, or even in nonlymphoid tissues such as myeloid proliferations or endometriosis using HUMARA. In parallel, RT-PCR and later Q-PCR from cDNA or FISH- based techniques became extremely useful methods to look for gene-fusions such as BCR-ABL and EWS-FLI1 in leukemias and sarcomas, or Q-PCR from DNA to find JAK2 mutations in myeloproliferative and myelodysplastic syndromes. Besides hematology and oncology, a considerable overlap has developed with microbiology, when looking for mycobacteria and other infectious agents in human tissues by PCR, and even with medical genetics, when looking for microsatellite instability in HNPCC or for the diagnosis of hemochromatosis. “Molecular pathology” in recent years is also becoming a tool for basic scientists and, lately for clinicians to look for minimal residual disease, determine prognosis and chose the best therapy. Examples for the latter are the analysis of mutations of KRAS in colon cancer, EGFR in lung cancer or CKIT in GIST as predictive markers. Novel technologies such as oligonucleotide arrays, whole genome sequencing and cDNA microarrays allow genomic analysis and gene expression profiling of tumor entities. They have the potential to revolutionize disease nomenclature, to better understand disease pathogenesis, help in treatment decisions and more accurately determine prognosis. On the other hand, rate limiting steps are the costs of these novel technologies in research and clinical applications and the need for effective cooperation between the departments of pathology, basic sciences, microbiology, virology, genetics, biometry/statistics, as well as clinical medicine.

Prof. Dr. Paul Fisch
Guest Editor

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• microarrays
• whole genome sequencing
• predictive markers
• minimal residual disease
• PCR
• FISH
• disease pathogenesis
• predictive markers

Type of Paper: Review
Title: Epigenetic Disregulation in Oral Cancer
Authors: Stefania Staibano, Maria Siano, Gennaro Ilardi and Massimo Mascolo
Affiliation: Department of Biomorphological and Functional Sciences, Pathology Section, School of Medicine, University Federico II of Naples, Naples, Italy; E-Mail: staibano@unina.it (S.S.)
Abstract: Squamous cell cancer of the oral region (OSCC) shows a continuous positive trend of incidence worldwide, with high rates of death for disease. There is the lack of reliable molecular prognostic molecular markers for this tumor. Although significant efforts have been made to identify molecular signatures of the clinical outcome of oral cancers, still neither single nor combination of markers is accepted in clinical practice. Current scientific evidence indicates that transition from normal epithelium to pre-malignancy condition and finally to oral carcinoma is the result of accumulation of genetic and epigenetic alterations in a multistep process. Epigenetic changes have been linked to OSCC initiation and progression, but they have been currently only partially characterized . It is urgent in particular to study the significant alterations in multiple genes expression simultaneously in biopsy sample from large cohort of subjects. The aim of this study was to review the up-graded knowledge concerning the contribute of  epigenetic modifications in early and later phases of oral malignant transformation, exploring also their role in epigenetic reprogramming in response to microenvironmental stresses involved in the establishment of the malignant phenotype. Particular attention will be devoted to the current evidence for a role of epigenetic marks as novel markers for early diagnosis, prognosis as well as potential therapeutic targets in oral cancer.

Type of Paper: Article
Title: Evaluation of K-RAS Gene Mutations in Serum of Patients with Non-Small Cell Lung Cancer
Authors: Anetta Sulewska ^1,2, Aleksandra Serwicka ², Jolanta Kowalewska ³, Radoslaw Charkiewicz ², Wojciech Naumnik ⁴, Jacek Niklinski ² and Lech Chyczewski ³
Affiliations: ¹ Departments of Thoracic Surgery, Medical University of Bialystok, Poland; E-Mail: anettasul@poczta.onet.pl
² Departments of Clinical Molecular Biology, Medical University of Bialystok, Poland
³ Departments of Pathomorphology, Medical University of Bialystok, Poland
⁴ Departments of Lung Diseases and Tuberculosis, Medical University of Bialystok, Poland
Abstract: Various mutations of K-RAS gene are frequently observed in adenocarcinoma (AC) and large cell carcinoma (LCC) of the lung. Analysis of free circulating tumor DNA has led to hypothesis that K-RAS mutation found in serum DNA could be a marker for tumor detection. This correlation has not been clearly confirmed. The aim of our study was to detect mutations in codon 12 of K-RAS gene and to determine the correlation between the presence of this mutation in tumor cells and serum free DNA. The study material consisted of specimens obtained from 89 patients with non-small cell lung cancer (54 AC and 35 LCC). Mutations were determined utilizing enriched polymerase chain reaction restriction fragment length polymorphism (enriched PCR-RFLP). DNA from the cases in which the mutation was found was sequenced. In AC group mutation occurred in 15 (28% of all AC) tumors and 10 (19% of all AC) sera, however, only in 2 cases there was simultaneous occurrence of mutation in tumor cells and serum. Sequencing did not confirm the mutation in one case. In the second case, different substitution was found in the tumor cells and in the serum. In LCC group the mutation was detected in 7 (20% of all LCC) tumors and 8 (23 % of all LCC) sera. The coexistence of mutations in tumor and paired serum was noted in one case, but sequencing showed a different type of substitution. Our study showed that mutations in the K-RAS gene while occurring  in both tumor cells and serum of patients with AC and LCC do not coexist in majority cases, and if they do, they show different pattern of mutation. It appears that the analysis of serum free DNA is not a useful test for tumor detection.
Keywords: lung cancer; serum; K-RAS mutation; enriched PCR-RFLP; sequencing

Type of Paper: Review
Title: Advanced Technology in Laser Capture Microdissection and Its Downstream Applications: A Review
Author: Tomoaki Tanaka
Affiliation: Department of Urology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan;
E-Mail: tomoaki826@msic.med.osaka-cu.ac.jp
Abstract: Fundamentally, normal or diseased tissues are complicated structures composed of many different types of cell populations. The molecular analysis of individual or enriched cell populations in their original tissues is crucial to elucidate biological actions of target molecules in their native microenvironment. For instance, in vitro culturing cell populations collected from fresh organs is an option in order to reduce cell contamination. However, the cultured cells are divided from the diverse tissue factors (e.g., hormonal factors, cytokines, extracellular matrix molecules). Thus, it is possible that cultured cells have different nature as compared with the same kind of cells existing in specific tissues. The developmental technologies of laser tissue microdissection have largely resolved the aforementioned problem. The use of sophisticated laser capture dissection (LCM) contributes to the preservation of molecules in homogenous cell populations gathered from heterogeneous tissues if investigators require accuracy for quantitative analysis, such as differential display, microchip microarray and proteomics, to discover candidates associated with normal tissue developing or diseased tissue series. For example, it is possible to find out the key molecules associated with interactions between tumor and stromal tissues in tumor microenvironment in which prostate cancer progresses. Technically, two different methods have been developed for laser microdissection systems, that is, laser capture microdissection and laser cutting microdissection. Both these technologies have until recently been refined to be appropriately applied for further examinations. It is the aim of this review to present the characteristics and the clinical availability of the commonly utilized laser microdissection systems and the downstream applications followed by them.

Type of Paper: Review
Title: Tumor Heterogeneity: Mechanisms and Bases for a Reliable Application of Molecular Marker Design
Author: Salvador J. Diaz-Cano
Affiliation: Dept Histopathology, King’s College Hospital and King’s Health Partners, London, UK; E-Mail: sjdiazcano@me.com
Abstract: Tumor heterogeneity is a confusing finding in the assessment of neoplasms, potentially resulting in inaccurate diagnostic, prognostic and predictive tests. This tumor heterogeneity is not always a random and unpredictable phenomenon, whose knowledge helps designing better tests. The biologic reasons for this intratumoral heterogeneity would then be important to understand both the natural history of neoplasms and the selection of test samples for reliable analysis. The main factors contributing to intratumoral heterogeneity induce gene abnormalities or modify its expression, including: the gradient ischemic level within neoplasms, the action of tumor microenvironment (bidirectional interaction between tumor cells and stroma), mechanisms of intercellular transference of genetic information (episomes), and differential mechanisms of sequence-independent modifications of genetic material and proteins. The intratumoral heterogeneity is at the origin of tumor progression and it is also the byproduct of the selection process during progression. Any analysis of heterogeneity mechanisms must be integrated within the process of segregation of genetic changes in tumor cells during the clonal expansion and progression of neoplasms. The evaluation of these mechanisms must also consider the redundancy and pleiotropism of molecular pathways, for which appropriate surrogate markers would support the presence or not of heterogeneous genetics and the main mechanisms responsible. This knowledge would constitute a solid scientific background for future therapeutic planning.
Keywords: neoplasm; tumor heterogeneity; episome; tumor microenvironment; intratumoral ischemia; tumor progression; clonal expansion; cell segregation

Title: Gef Gene Expression in MCF-7 Breast Cancer Cells is Associated with a Better Prognosis and Induction of Apoptosis by p53-Mediated Signalling Pathway
Authors: Houria Boulaiz ^1,3, Jose Prados ^1,3, Juan Marchal ^1,3, Consolación Melguizo ^1,3, Pablo Álvarez ^1,3, Macarena Peran ^2,3, Esmeralda Carrillo ^1,3, Fernando Rodríguez ^1,3, Alberto Ramírez ^1,3, Raúl Ortíz ^1,3 and Antonia Aránega ^1,3
Affiliations: ¹ Basic Cardiovascular Research Section, Department of Anatomy and Human Embriology, School of Medicine, University of Granada, E-18012 Granada, Spain
² Department of Health Sciences, University of Jaén, E-23071 Jaén, Spain
³ Biopathology and Medicine Regenerative Institute (IBIMER), Granada, Spain
Abstract: Breast cancer research has developed rapidly in the past few decades, leading to longer survival times for patients and opening up the possibility of developing curative treatments for advanced breast cancer. Our increasing knowledge of the biological pathways associated with the progression and development of breast cancer, alongside the failure of conventional treatments, has prompted us to explore gene therapy as an alternative therapeutic strategy. We previously reported that gef gene from E. coli has shown considerable cytotoxic effects in breast cancer cells. However, its action mechanism has not been elucidated. Indirect immunofluorescence technique using flow cytometry and immunocytochemical analysis were used to detect breast cancer markers: estrogen (ER) and progesterone (PR) hormonal receptors, human epidermal growth factor receptor-2 proto-oncogene (c-erbB-2), ki-67 antigen and p53 protein. gef gene induces an increase in ER and PR expressions and a decrease in ki-67 and c-erbB-2 gene expressions, indicating a better prognosis and response to treatment and a longer disease‑free interval and survival. It also increased p53 expression, suggesting that gef‑induced apoptosis is regulated by a p53-mediated signalling pathway. These findings support the hypothesis that the gef gene offers a new approach to gene therapy in breast cancer.
Keywords: Breast cancer; gef gene; tumour markers; loss of tumour malignancy