Research Of Pre-Operative Diagnosis With Percutaneous Biopsy With Needle As A Necessary Process In Breast Cancer Management

Breast cancer is a primary cancer with one of the highest incidences in women worldwide with 1,700,000 new cases, a rate that steadily increases every year (Siegel, Ma et al. , 2014). In Europe, breast cancer is responsible for about 20% of cancer deaths with a number exceeding 120,000 (McSherry et al. , 2007). In Greece, approximately 4,000 new cases of breast cancer are treated annually, with the incidence of this cancer rising dramatically in the last 20 years, mainly in women aged 45-55 years (Markopoulos, 2004). It is generally accepted that all malignant transformations represent a complicated and complex process where genetic factors (family history, genetic mutations, other diseases), biological factors (sex, menstruation and menopause age, childbearing age, body weight) and environmental factors (drugs, nutrition, radiation), may alter cellular pathways of molecular signaling leading to excessive cell growth which is the key-point in carcinogenesis (Band, 1995; "Familial breast cancer: collaborative reanalysis of individual data from 52 epidemiological studies including 58,209 women with breast cancer and 101,986 women without the disease," 2001). In any case of suspicious breast lesion, biopsy confirmation of the malignancy is necessary before any decision for treatment.

In the last decades, many important developments in terms of understanding the biological behavior of cancer and its treatment have occurred. The systematic preventive screening detects very small tumors or even pre-cancerous lesions (in situ carcinoma). Selective surgical techniques have been developed (tumor excision, lymph node biopsy etc). The value of the pre-operative or introductory systematic treatment has been established (chemotherapy, immunotherapy, hormonotherapy) in many patient groups (locally extensive inoperative tumors, inflammatory cancer, triple negative tumors, HER-2 positive tumors etc). From the above, the pre-operative diagnosis with percutaneous biopsy with needle is now a necessary diagnostic process.

Today, Fine Needle Aspiration is a widely accepted diagnostic process, that has replaced open biopsy in many cases and has made possible the establishment of the final treatment pre- operatively. It is a simple invasive technique where a fine needle is inserted near the lesion and the material is aspirated for cytological diagnosis or culture when inflammation is suspected. In detail, lesions can be aspirated with fine needle (23G or smaller), while it is possible to aspirate even smaller lesions with ultrasound assistance. The “triple” ultrasound approach, which involves palpation, ultrasound findings and FNA, is applied in benign, pre-neoplasmatic, limited malignancy or malignant breast lesions. Breast FNA is extremely successful, with sensitivity between 80%-100% and specificity >99%. Also, in routine, apart from the accurate diagnosis, it can be used for the analysis of the biological behavior of the tumor by assessing the level of estrogen/progesterone receptors (ER/PR), cellular growth markers, and the determination of prognostic markers and molecular analyses as well. Various mutation, either hereditary or new ones may result finally in carcinogenesis. Mutations may occur in various genes causing their loss of function which may lead directly or indirectly to carcinogenesis. Such genes are called tumor suppressive genes (TSGs) and code for proteins vital for the function and phenotype of cells whose loss may result in destabilization of the cell growth, inhibition of cell cycle and apoptosis (cellular immortalization) and destabilization of the DNA replication and repair mechanisms (Oren 1992, Weinberg 1992, Hinds and Weinberg 1994, Fearnhead, Britton et al. 2001, Haase 2005).

For many decades there was the question whether the initiation and evolution of cancer was only caused due to mutations or and due to “epigenetic” changes, changes that in contrast to mutations do not affect the genetic sequence. The term "epigenetics" involves all those hereditary changes in genetic expression in somatic cells that are a result of changes of the DNA sequence (Galm, Herman et al. 2006). The two main epigenetic mechanisms are: a) DNA methylation and b) the post-translational histone changes (Verma and Srivastava 2002). Destabilization of the balance between the epigenetic mechanisms may have a severe impact on chromatin structure and transcriptional activity. The epigenetic changes may bridge with the best possible way the exposure to environmental factors and the genetic substrate in the process of carcinogenesis (Esteller 2003). Cellular differentiation is a physical phenomenon observed in all alive organisms. In eukaryotic human cells, only the cells that are specific for a cell type are expressed while the vast majority of the genes remains inactive. These genes are deactivated through epigenetic mechanisms with DNA methylation as the most important of all. DNA methylation causes a series of changes in chromatin structure and the control of genetic transcription leading finally in silencing of specific genes (Li, Bestor et al. 1992, Herman and Baylin 2003, Ng and Gurdon 2008). The process of gene silencing is carried out through specific enzymes called DNA Methyltransferases (DNMTs) which add a methyl-group in cytokines (C) that are connected through phosphodiester bond with the 5 ́ positions of the deoxyribose with a guanine (G), consisting part of a dinucleotide CpG (Bestor 2000, Hermann, Gowher et al. 2004, Feil and Fraga 2012).

From the existing CpG dinucleotides almost 60-90% are methylated and most of them are dispersed in the human genome, however some of them are placed in high concentrations in areas called CpG islands. CpG islands include genomic areas with length 500- 2000bp and correspond to the areas of genetic promoters that control the gene expression (Holliday and Grigg 1993, Laird and Jaenisch 1994, Ramsahoye, Davies et al. 1996). Methylation causes a series of changes in the double helix of DNA in the promoter region that attracts proteins that connect to the methylated DNA and finally inhibit the expression of the gene that is connected with this promoter (Lewis and Bird 1991, Nan, Ng et al. 1998). Methylation is a normal cellular mechanism through which cells keep a large part of their genome inactive, since its expression could be destructive for them (Bird and Wolffe 1999). Methlyation either alone or as a combination with some mutation has been described as a pathologic damage for multiple TSGs and oncogenes and has proved value as a risk marker for cancer development and evolution and as a prognostic marker (Stirling and Chandler 1976, Lagios, Rose et al. 1980). Methylation changes may favor the development of malignant neoplasias with various ways such as the total loss of methyl-cytokines (DNA hypomethylation) which may cause genomic instability or through hypermethylation.

Also, through point muations, the methylated cytokines may be subjected to automatic deamination to thymine (Τ) and be converted into endogenous mutagenic factors (Gronbaek, Hother et al. 2007). Studies have shown that cancer cells show changes in the methylation profile of the promoters of many genes that are associated with cell cycle, DNA repair mechanism, cellular adhesion and the cellular apoptosis mechanism (Battagli, Uzzo et al. 2003, Ahmed 2010, Hoffman and Cairns 2011, Jia, Yu et al. 2016, Ma, Cao et al. 2016, Tekcham and Tiwari 2016, Yang, Niu et al. 2016, Zheng, Li et al. 2016, Yokoi, Yamashita et al. 2017, Zhan, Zhang et al. 2017) At first, it should be noted that genetic methylation is one of the most important epigenetic mechanisms of cells. It has been proved that the various epigenetic mechanisms and mainly the methylation profile are affected in a significant degree from various environmental factors and the exposure to them in combination with the genetic background of the study population. Such environmental factors are the nutrition, stress, working habits, smoking habits, alcohol consumption and lifestyle (Bollati and Baccarelli 2010, Alegria-Torres, Baccarelli et al. 2011, Christensen and Marsit 2011, Cortessis, Thomas et al. 2012, Feil and Fraga 2012, Lim and Song 2012, Giuliani, Sazzini et al. 2016).

Also, studies have shown certain genetic methylation profiles specific for some genetic populations, while other different profiles characterize different populations (population-specific methylation analysis) (Fraser, Lam et al. 2012, Hernando-Herraez, Garcia-Perez et al. 2015, Rotti, Mallya et al. 2015). From the above, the proposed study, that involves the molecular analysis of clinical samples from Greek population, a population unique both geographically and in term of environmental and lifestyle parameters that characterize it and distinguish it from the study population in other studies. Ιt should be noted that the proposed study is based on the molecular analysis of clinical cytological FNA samples, while the international literature mainly focuses on histological samples.

Finally, Next Generation Sequencing (ΝGS) will be the main analytical method in this study, a technology not used in the international literature so far for this purpose since almost all the studies are based on more conventional techniques such as the Real-time PCR and simple PCR with the all their disadvantages. In conclusion, the investigation of correlation between the methylation profile of the genetic promoter and the occurrence/evolution of the breast cancer in clinical FNA samples from Greek population is considered as highly important and innovative in comparison with the international literature since the later one focuses on different study populations, outside Greece, on the one hand, with specific genetic background and environmental parameters and on the on the other hand is based on the molecular analysis of other clinical samples different from histological material while it involves the use of ΝGS, the state-of-the-art of modern Molecular Biology.