EDITORIAL EDITORIAL Marius Raica Department of Microscopic Morphology/Histology, Angiogenesis Research Center, „Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania Piata Eftimie Murgu 2, 300041 Timisoara, Romania. E-mail: raica@umft.ro Dear reader, It is a privilege for me and the Editorial Board to introduce a new medical journal, Research and Clinical Medicine. This journal was born after long discussions on its necessity and utility. The story of medical journals is longer than 200 years. In last decades, there were published many new journals, as a consequence of the signiicant increase of basic and clinical research. Nowadays, it is often dificult to have a precise landscape on a given narrow topic, because articles on the same topic are published in different journals. The type of the journal is also very important, but its rating gives only partial and often unclear information about the quality and value of published articles. Given the large number of journals, the peer-review process performed on voluntary basis in most of the cases, is more “permissive” than some decades ago. Research has been dramatically changed in last years, particularly because the introduction of new methods of investigation, like molecular biology, gene analysis, high accuracy imagistic methods, or laparoscopic and robotic surgery. We live in the era of the genome, but on the other hand, there is a strong need to translate the basic research into the practice as soon as possible. This leads to the better knowledge of normal biological processes, development of new diagnostic procedures and introduction of new therapeutic strategies. Translational medicine is now a well-known concept, but on the other hand, it is not easy to be achieved. In this journal you will have the opportunity to meet researchers, experienced clinicians, PhD students, and they all could beneit from each others’ experience and possibilities to apply their results into the clinical practice that is in fact, the ultimate goal of the research. To translate research into practice is an old dream that became reality. Actually, it is the main goal of research to produce knowledge, to improve diagnostic and therapeutic procedures, to introduce new experimental models that could help to understand the many hidden faces of pathology. We strongly encourage submission of manuscripts relecting not only general and cohort studies, but particularly those focused on personalized medicine and therapy. Nowadays, when “the case” became “the patient”, it is essential to perform a profound investigation with particular importance, like the individual characterization of the lesions, behavior and parameters that can predict the response to therapy. I know it is very dificult to introduce and maintain a new journal on the real competition of the scientiic market. On the other hand, based on the high level of expertise in the ield of the members of the Editorial Board, I am sure that Research and Clinical Medicine will ind the targeted niche in the medical literature. The open-access of the journal offers the possibility of a rapid and worldwide spread of information, and therefore, authors from all the world are welcome to join us. Marius Raica Editor in Chief _____________________________ M. Raica 1 EDITORIAL LIMITS OF ANTI-ANGIOGENIC THERAPY Domenico Ribatti*1,2, Beatrice Nico1 1 Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy, 2 National Cancer Institute “Giovanni Paolo II”, Bari, Italy * Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Policlinico - Piazza G. Cesare, 11, 70124 Bari, Italy. E-mail: domenico.ribatti@uniba.it Angiogenesis is controlled by the balance between molecules that have positive and negative regulatory activities and this concept has led to the notion of the angiogenic switch, which depends on an increased production of one or more positive regulators of angiogenesis [1]. Most human tumors arise and remain in situ without angiogenesis for a long time before switching to an angiogenic phenotype, through a pre-neoplastic stage as occurs in breast and cervical carcinomas, which becomes neovascularized before the malignant tumor appears. Activation of the angiogenic switch has been attributed to the synthesis or release of angiogenic factors, and accordingly to the balance hypothesis, the level of angiogenesis inducers and inhibitors regulates angiogenesis in physiological conditions. This balance is altered in pathological conditions, including tumors, as a consequence of an increase bioavailability or activity of the inducer proteins, or reducing the concentrations of endogenous angiogenesis inhibitors. Restore of this balance may induce a normalization of structure of blood vessels. The concept of “normalization” of tumor blood vessels by anti-angiogenic drugs was introduced by Rakesh Jain in 2001 [2]. Accordingly, anti-vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) therapies induce morpho-functional normalization of tumor blood vessels, favoring an increase in blood low and release of cytotoxic drugs. However, in non small cell lung cancer (NSCLC) anti-angiogenic therapy decreases cytotoxic drug delivery to tumors [3]. The state of normalization is probably transient and dependent on the dose and duration of the treatment. Beginning in the 1980’s, the industry exploited the ield of anti-angiogenesis for creating new therapeutic molecules in angiogenesis-dependent diseases. However, experimental drugs that in preclinical early stage prevention or intervention trials have been proven to eficiently impair the onset of tumor angiogenesis may not exert any anti-angiogenic activity when tested in late stage intervention or regression trials, as usually performed in the clinics. _____________________________ 2 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 At present anti-angiogenic therapy is essentially anti-VEGF)/VEGFR therapy and has yet fulilled its promise in the clinic. Bevacizumab (Avastin) was the irst angiogenesis inhibitor approved by the Food and Drug Adminstration (FDA) for the treatment of colorectal cancer in February 2004, administered in combination with irinotecan, 5-luorouracil and leucovirin [4]. It was subsequently approved for use, in combination with cytotoxic chemotherapy, in other cancers. Actually, inhibition of VEGF/VEGFR axis is obtained by targeting VEGF ligand with antibodies or receptor traps, or its receptors with small-molecule tyrosine kinase inhibitors (TKI). Clinical beneits are obtained when ligand-blocking drugs are combined with chemotherapeutic agents or radiotherapy, while clinical studies testing various TKIs combined with chemotherapy have failed because of increased cytoxicity. Depending on cancer type, these anti-angiogenic treatments can lead to a 3-6 months increase in progression-free survival, but fail to provide enduring clinical responses, with transitory improvements being followed by a relapse phase in tumor angiogenesis and subsequent tumor growth. Removal of VEGF inhibition causes tumor regrowth due to the fact that pericytes provide a scaffold for the rapidly re-growing of tumor vessels [5]. The occurrence of pericytes expressing alpha smooth muscle actin (α-SMA) has been considered as a biomarker for tumors refractory to therapy [6]. Pericytes have been indicated as putative targets in the pharmacological therapy of tumors by using the synergistic effect of anti-endothelial and anti-pericytic molecules. Removal of pericyte coverage leads to exposed tumor vessels, which may explain the enhanced effect of combining inhibitors that target both tumor vessels and pericytes. Bergers et al. (2003) showed that combined treatment or pre-treatment with anti-platelet derived growth factor (PDGF-B)/platelet derived growth factor receptor beta (PDGFBR-β) reducing pericyte coverage increases the success of anti-VEGF treatment in the mouse RIP1TAG2 model [7]. PDGFR inhibition has been developed in the context of combined inhibition of VEGFR and PDGFR with dual-speciicity small-molecule inhibitors, including sunitinib, sorafenib, and pazopanib. The results from clinical trials have not shown the dramatic antitumor effects that were expected following preclinical studies, which revealed a much higher eficacy of these type of agent in animal models. Patients with different types of tumors respond differently to antiangiogenic therapy. While colorectal, lung and breast cancer patients have responded, pancreatic cancer patients have not shown survival advantages when treated with anti-angiogenic monotherapy or combinations of anti-angiogenic agents with chemotherapy. Moreover, responses to anti-angiogenic drugs vary between primary tumors and their metastases [8]. Additionally, preclinical and clinical data have shown the possibility that tumors may acquire resistance to antiangiogenic drugs or may escape anti-angiogenic therapy via compensatory mechanisms. Most of the FDAapproved drugs, as well as those in phase III clinical trials, target a single pro-angiogenic protein. However, multiple angiogenic molecules may be produced by tumors, and tumors at different stages of development may depend on different angiogenic factors for their blood supply. Therefore, blocking a single angiogenic molecule might have little or no impact on tumor growth. In 2011, it has been introduced the use of dual ibroblast growth factor receptor (FGFR)/VEGF TKI brivanib, that inhibits VEGFR1-3 and disrupt FGFR1-3, overcoming resistance to VEGF-selective therapy, and blocking FGFdependent tumor proliferation [9]. Finally, it has been demonstrated that angiogenesis inhibitors make some tumors more aggressive in different animals, increasing invasion and lymphatic or hematogeneous metastasis [10,11]. REFERENCES 1. Ribatti D, Nico B, Crivellato E, et al. The history of the angiogenic switch concept. Leukemia. 2007;21:44-52. 2. Jain RK. Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med. 2001;7:987-89. 3. Van der Veldt AA, Lubberink M, Bahce I, et al. Rapid decrease in delivery of chemotherapy to tumors after anti-VEGF therapy: implications for scheduling of anti-angiogenic drugs. Cancer Cell. 2012;21:82-91. 4. Hurwitz F, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, and leucovurin for metastatic colorectal cancer. N Engl J Med. 2004;350:2335-42. 5. Mancuso MR, Davis R, Norberg SM, et al. Rapid vascular regrowth in tumors after reversal of VEGF inhibition. J Clin Invest. 2006;116:2610-21. 6. Franco M, Paez-Ribes M, Cortez E, et al. Use of a mouse model of pancreatic neuroendocrine tumors to find perciyte biomarkers of resistance to anti-angiogenic therapy. Horm Metab Res. 2011;43:884-89. 7. Bergers G, Song S, Mayer-Morse N, et al. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest. 2003;111:1287-95. 8. Jin K, Lan H, Cao F, et al. Differential response to EGFR- and VEGFR- targeted therapies in patient-derived tumor tissue xenograft models of colon carcinoma and related metastases. Int J Oncol. 2012;41:583-8. 9. Allen E, Walters IB, Hanahan D. Brivanib. A dual FGF/VEGF inhibitor, is active both first and second line against mouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance to VEGF inhibition. Clin Cancer Res. 2011;17:5299-310. 10. Paez-Ribes M, Allen H, Hudock J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15:220-31. 11. Ebos JM, Lee CR, Cruz-Munoz W, et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell. 2009;15:232-9. _____________________________ D. Ribatti, B. Nico 3 HISTORICAL REVIEW WHO APPEARED FIRST IN OTOLARYNGOLOGY: CLINICIANS, ANATOMISTS OR HISTOLOGISTS? A QUESTIONABLE ISSUE! Anca Maria Cîmpean1*, Caius Doroş2, Petru Matusz 3, Marius Raica1 1 Department of Microscopic Morphology/Histology, Angiogenesis Research Center, 2 Department of Surgery I-ENT, Department of Anatomy and Embryology, ”Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania * Department of Microscopic Morphology/Histology, Angiogenesis Research Center, Piata Eftimie Murgu 2, 300041 Timisoara, Romania. E-mail: ancacimpean1972@yahoo.com 3 ABSTRACT Otolaryngology is mainly associated with clinical practice. Despite of this actual evidence, otolaryngology can be considered, from historical point of view as a complex speciality made up of a mixture of several preclinical specialities as anatomy, histology, pathology and physiology. Several scientists who studied these specialities first, became then otolaryngologists and others were known in the medical literature because of their studies in other specialities than otolaryngology. Most of the historical papers were focused on the ear, other regions being neglected. This review presents the forgotten part of otolaryngology, especially its preclinical facts with importance in etiology and pathogenesis of various disease of the ear, nose and throat structures and thus, present work can be considered as a particular overview of „forgotten” otolaryngology. Key words: otolaryngology, anatomy, histology, pathology AMAZING MEDICAL DISCOVERISES ANCIENT CIVILISATIONS OF In 1926, Charles Cumston said that “we can never be in full possession of a science until we know the history of its development” [1]. This „universal phrase” also characterises the otolaryngology development from its begining to nowadays. The most ancient of the medical specialities seems to be rhinology. Around 3500 B.C., many years before the Edwin Smith Papyrus started to be written, Sekhet’enanch treated King Sahura by healing his nostrils [2]. Evidence of this is a drawing of the doctor and his wife, found in the tomb of the king who ordered to perfom an inscription to a limstone as a testimony and also as a gratitude for cure of his nose [3]. Inscriptions in the tomb describe how he “healed the king’s nostrils.” What Sekhet’ enanch did to King Sahura’s nostrils, or what was wrong with them, historians don’t know but Sekhet’ enanch became the irst known physician in the world. Edwin Smith Papyrus, the earliest surgical writing, dating back to about 1600 B.C. remained one of the most comprehensive treatise of medicine from ancient times. Ancient surgical methods, therapies and natural drugs were described in different section of the papyrus. The head and neck represented the favourite body part for „research” ield of Ancient Egypt. This evidence is supported by a high number of cases (33 _____________________________ 4 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 out of 48) described in Edwin Smith Papyrus, localized on head and neck region [4]. According to the popular belief that the brain represents an unimportant organ, the ancient egyptians discarded it during the embalming process. The mummiiers knew how to insert a special instrument to a nostril , penetrating the brain case and remove the brain [5]. But, more important, the case presentations concerning nose, ear, lips, cheeks and throat were wery well documented and their description contained data about clinical and therapeutic approach of their lesions. For nose, varying lesions from trauma to foreign bodies were mentioned in the papyrus, each of them having a particular description of examination, diagnosis, treatment and peculiarities. Lips and cheek disease were also accurately described [6]. Moreover, a rigorous and mandatory step of treating ENT lesions was represented by a carefully cleaning of the wound, especially by removal of blood clots and damaged tissues before applying „drugs”. Despite of their ine clinical sense, Egyptians used almost the same treatment for all diseases of the ENT region: „ bind with fresh meat in the irst day, followed by grease, honey and lint everyday until they recovers” [7]. Despite of the generally accepted belief that Hippocrates mentioned for the irst time the tympanic membrane [8], a short sentence in the Edwin Smith Papyrus contradicts this. According with Ebbell translation (1937) [9], of the Ancient Egyptian Ebers Papyrus, „the ear that contain inside the tympanic membrane would be deaf from the eye vessels”. In the same writing, it was discovered for the irst time the empirical description of the future Eustacian Tube as it follows: „there are four vessels to his two ears together with the ear canal, namely two on his right side and two on his left side. The breath of life enters into the right ear, and the breath of death enters into the left year” [10]. Ozaena, nasal cattarh, secretory and suppurative otitis media were also known, described and treated by Egyptians [10]. In the past, in India, amputation of the nose was a frequent form of punishment for such crimes as adultery. Later, around 600 B.C., surgical reconstruction of the nose was irst developed in ancient India by Sushruta together with earlobes reconstruction [11]. He has enlisted 29 ear disorders, 18 diseases related to nose and 75 diseases related to mouth cavity. The real name of this indian surgeon was not Sushruta. The name Sushruta derived from the Sanskrit, means „good listener” and anticipated his future work in the ancient otolaryngology ield (http://www.sanskrit.nic.in). But, the most amazing inding involving head and neck structures was reported by Wells in 1963 [12]. He accurately described the irst multiple myeloma changes in the skull dating back from 3rd to 5th dynasty. Erosions of the left maxilla bone, destruction of the same side hard palate and posterior wall of maxillary sinus together with the vault of the skull with multiple translucent areas characterized the irst plasmacytoma from medical history. Two evidences about tracheostomy in Ancient Egypt was found in Abydos and Sakara. Drawings with two seated slabs with arms placed behind them to produce hyperextension of the neck and with a lancet directed to their trachea were the irst proves of tracheostomy usage for breath problems [10]. Malignant pathology was also recognized by the studies done on egyptian mummies. Strouhal (1978) [13] described a case of nasopharyngeal carcinoma in a skull from the 5th Dynasty to the XIIth Dynasty in Upper Egypt. Derry’s case, with destructive lesions involving the cribriform plate, ethomids and sphenoids was, probably, the irst decription of a nasal carcinoma [14]. Ancient Arabian World, by their physicians as Rhases, Avicenna, Ali In Abbas, Abdol Latif al Baghdady or Ibn al Nais developed early otolaryngology diagnosis and ENT surgical methods. Their observations were grouped in chapters from several important muslim medical treatises [15-18]. ANATOMY, HISTOLOGY, PATHOLOGY AND OTOLARYNGOLOGY Together with its ine clinical sense, Avicenna had good skills in ENT anatomy. He accurately described the ear anatomy, by giving data about external auditory canal, eardrum and also the larynx and pharynx anatomical components (as cartilages, ligaments, joints and the small muscles of the larynx, and their role in performing the different laryngeal functions) [15]. Ali Ibn Abbas al-Baghdady [19] and Ibn al-Nais proved that there are two separate cranial nerves for ear and face. Aristotle (384-322 B.C.) provided basic information about anatomy and embryology for several organs by dissecting various animals. He also dissected the ear and speciied that „...of animals possessed of ears, man is the only one that cannot move this organ”. From Leonardo da Vinci, who made new descriptions of frontal and maxillary sinuses to modern anatomy, many anatomists were interested by the ear, nose and throat structures. Nathaniel Highmore (1613-1685) was a british surgeon not so famous for his surgical performance but remembered for his anatomical studies including an accurate description of maxillary sinus which used to be more popularly referred to as the antrum of Highmore [20, 21] Niels Stensen (1638-1686), a danish student and the favourite pupil of Thomas Bartholin, accidentally discovered parotid duct by dissecting goat, lambs and rabbits heads. First, he considered this duct as to be a vessel but later, he described the saliva inside it „as a secretion derived from the blood but not similar with it”. Now, this excretory duct is known as Stenon’s duct [22]. Caspar Bartholin The Elder (the father of Thomas Bartholin) was a polymathic person, being 11 years professor of medicine at University of Copenhagen and then professor of divinity at the same university. In his book Anatomicae Institutiones Corporis Humani (1611), he observed and described for the irst time the olfactory nerve [23]. Claude Perrault (1613-1688), the well known architect of the east wing of the Louvre Palace in Paris, was also a physician and now, unfortunately, one of the „forgotten” anatomist. His work entitled „Du Bruit” (On Noise) mentioned for the irst time the „spiral membrane” and gave us an accurate description of it as „a soft and lexible membrane attached to the modiolus but not to the opposite wall”. He also had several hypothesis about nerve ibers of the ear but he was not able to prove them [24]. Recognized as a scientist with deep involvement in the study of ear anatomy, Antonio Scarpa (17521832) also gave details about the nose-palatine nerve [25]. His work was continued with an extensive and accurate description of nasopalatine nerve plexus done by Domenico Cotugno (1736-1822), who also found the labyrinthic luid and formulated a theory of resonance and hearing [26]. Despite of his descriptive studies in otolaryngology, Cotugno remains famous because of seminal work in the neurology ield, being credited with the discovery of “liquor cotunnii” (known today as cerebrospinal luid) [27]. _____________________________ A.M. Cimpean et al 5 Marie François Xavier Bichat (1771 –1802), french anatomist and physiologist is considered also the father of histophysiology and descriptive anatomy [28, 29]. Based on „tissue theory” derived from British Medical school (previous theories launched by John Hunter) but working without microscope, Bichat was the irst to make correlation between tissue types and their different reaction to external stimuli primarly observed on nasal and pharyngeal mucosa. He started to prove his theory about connection of a structure with a speciic function by performing numerous dissections and macroscopic observation of texture and colour for different parts of the body. He divided mucous membranes (mucosa from modern histology) into two types: irst type included those which lies in the interior of nose, mouth, pharynx, larynx and reacted by inlammation to cold exposure and, the second type, those mucosa lining urethra, ureter, kidneys and prostate or vagina which do not react by inlammation to cold exposure [30]. Bichat considered tissues as anatomical entities “carrying” speciic properties. German histologist, pathologist and anatomist, Friedrich Gustav Jakob Henle is well known in the medical literature because he is credited with the discovery of the loop of Henle in the kidney. But, few people know that, in 1861, Henle described the supra-meatal spine that serves as a landmark in the mastoid area, named today Henle’s spine [31]. Joseph Toynbee (1815-1866) was an English otologist but, his carreer was dedicated mostly to pathological and anatomical studies of the ear.It is believed that he was the irst scientist who found a link between stapes ixation and hearing loss [32]. His passion for otology experimental ield was fatal for him. He was found dead in the consulting room at the age of 50, after accidentally inhalation of a mixture of prussic acid and chloroform during an experimetal test of a new remedy for tinnitus [33]. Pupil of Bartolomeo Panizza and Joseph Hyrtl, and close friend with Kolliker and Virchow, Alfonso Giacomo Gaspare Corti (1822-1876) can be considered as a proeminent igure of „preclinical” otolaryngology. He dedicated his scientiic life to the study of the cohlea and learned to use methods to preserve several preparations of the cochlea in the laboratory of Professors Jacobus Schroeder van der Kolk and Pieter Harting in Utrecht [34, 35]. He was the irst who described the microscopic structure of the organ of Corti, including the sensory epithelium spiral ganglia and stria vascularis, also. He also identiied the Hensen cells but he did not give them a special attention.These cells were recognized and completely characterized later by the founder of biological oceanography, the german zoologist, embryologist and anatomist, Victor Hensen [36]. Wilhelm Kiesselbach (1838-1902), came from a family of doctors and businessmen. He was research assistant for ear clinical examinations at the surgical _____________________________ 6 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 clinic in Erlangen and, then, Associate Professor of Otolaryngology (http://www.luise-kiesselbach.de/wilhelmkiesselbach ). He was interesed in the studies of nosebleeds and characterized a special portion of the nasal mucosaKiesselbach plexus- riched in branched and anastomosed capillaries, which is responsible for more than 90% of nasal bleedings. His interest in this nasal vascular plexus, and his fame as otolaryngologist specialised in the treatment of nose bleedings and ear diseases, favoured him to know his future wife, Louise, who was 24 years younger than him. Right at the irst consultation Louise (who came initially for an ear ailment) had also a nose bleeding and Kiesselbach exclaimed: “Oh, you’re bleeding nose, no, what is the kind of you, I’m working on it and I appreciate any case quite formidable”. Kiesselbach made also extensive studies on the the mucus content of nasal polyps (Kiesselbach, 1888), laryngo-tracheal stenosis [37], papillary epithelioma of the middle nasal mussels [38] and treatment of diseases of the nose and pharynx [39]. Wilhelm His (1831-1904, a swiss anatomist, inventor of the microtome and well known researcher of cell and tissues under the microscope) together with Adam Politzer (hungarian and austrian physician, one of the otology founders), examined the skull of Johann Sebastian Bach in order to explain the relationship between Bach talent and structural peculiarities of his ear and temporal bones structures. They found a particularly pronounced development of the temporal bones and in addition to this „the abnormally large size of the fenestra rotunda (diameter of 2.5 mm. as opposed to a normal of 1.5 mm); the extraordinary thickness and irmness of the mastoid process, particularly in its cortical part; the remarkable width of the incisura mastoidea; the prominence of the petrous ridge; the unusual hiatus subarcuatus” as it is stated in the work of Baer (1956) [40]. The same author reported the large size of the irst coil of the cochlea as a sign for an unusual development of the cochlear ganglion and, accordingly, of the higher sensory centers. Despite of its irst description in humans long time ago, by Friedrich Ruysch (1703) in a 2-year-old child’s nasal septum [41], the vomeronasal organ (VNO, aslo known as Jacobson organ) remains one of the most mysterious structure in humans. The controversies start even from the person who described it in humans. Who was the irst? Ruysch, Jacobson or Kollinger? A huge paradox „governs” this organ with emphasis to the irst scientist who observed it in humans. It is widely accepted today that Ludwig Levin Jacobson never described human vomeronasal organ; he described it only in animals, especially in birds! But the most known name of this nasal structure even rudimentary in humans is ...Jacobson organ! Ruysch anatomical and accurate macroscopic description was later completed by von Kollinger excellent histologic assessement of the VNO. Rudolf Albert Kölliker, swiss anatomist, histologist and physiologist, is likely known for his histologic studies and discoveries of muscle and nervous system but his accurate descriptions of prenatal and postnatal VNO histology is less known [42, 43]. Today, molecular methods are applied to study vomeronasal organ. The genes which code for VNO receptor proteins are nonfunctional in humans. In addition, no accessory olfactory bulbs, which receive information from the vomeronasal receptor cells, are found. Thus, some controversies still persist concerning the structural and functional changes of human VNO [44]. In close relationship with VNO, Grüneberg ganglion is an olfactory subsystem responsible for the detection of alarm pheromons and cold temperature. He was reported for the irst time in 1973 , by a british genetician, Hans Grüneberg who described it in rodents [45]. According to him, this structure is also present in human beings nose, but this theory failed to be proved till now. Grüneberg was also interested in the study of Chievitz organ, another medical controversial issue [46]. Juxtaoral organ of Chievitz, was irst described by Johan Heinrik Chievitz in a 10 weeks old human embryo (Pantanowitz and Tschen, 2004) [47]. Since its irst description, juxtaoral organ of Chievitz was considered exclusively an embryonic neuroepithelial structure for a long time (almost 100 years), till Zenker, in 1953 observed and reported it in adult humans [48]. Unfortunately, Chievitz had also an unpleasant relationship with otolaryngologic pathology. In the last years of his life, he developed a laryngeal tuberculosis which restricted his academic and scientiic duties. 7. Johnson A. A short history of wound dressings: from animal grease and lint to hydrocolloids and alginates. Ostomy Wound Manage. 1992; 38:36-40. 8. Stevenson RS, Guthrie D. A History of Otolaryngology. Edinburgh: Livingstone, 1949. 9. Ebbell B. The Papyrus Ebers. Levin & Munksgaard (Editors). Copenhagen: Ejnar munksgaard, 1937. 10. David AR. Proceedings of the ‘science in Egyptology’. In: Symposia, 1979 and 1984. Manchester University Press, 1986. p. 243-250. 11. Saraf SS. Rhinoplasty in 600 B.C. 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Bulletin of the Medical Library Association. 1956;39:206. 48. Zenker W, Halzl L. Development of Chievitz’s organ in man. Z Anat 41. Bhatnagar KP, Reid KH. The human vomeronasal organ. I. Historical Entwicklungsgesch. 1953;117:215-236. _____________________________ 8 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 REVIEW ARTICLE THERAPEUTIC POTENTIAL OF ANGIOGENESIS INHIBITOR MIXTURES IN CANCER TREATMENT Patrycja Nowak-Sliwinska, Arjan W. Griffioen* Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands * VU University Medical Center, Dept. of Medical Oncology, Angiogenesis Laboratory, De Boelelaan 1118, 1081 HV Amsterdam, The Netherlands. E-mail: aw.griffioen@vumc.nl ABSTRACT It is generally understood that improvement of cancer therapies is achieved by the combination of drugs. This approach will improve efficacy, while toxicity and the risk for drug resistance may be decreased. In this review, the efforts on combining drugs for the treatment of two tumor types is highlighted, followed by a description of the challenges that are faced in designing optimal combination therapies. INTRODUCTION Over the recent years signiicant advances have been made in the treatment of advanced stage tumors. Cytotoxic chemotherapy regimens have improved and overall survival in patients has increased. But most therapies are associated with severe systemic toxicities [1,2]. Angiogenesis inhibitors such as bevacizumab/ Avastin®, panitumumab/Vectibix®, sunitinib/Sutent®, sorafenib/Nexavar® and erlotinib/Tarceva® are used in clinical practice in many tumor types as single drugs or in combination with chemotherapeutics [1,3-6]. These agents target and perturb critical cellular signaling pathways that regulate tumor angiogenesis. The eficacy of such combinations strongly depends on many factors, among which are the treatment schedule, the genetic background of the patient and the choice for the combination of drugs [5,7]. To date, the clinical beneit of anti-angiogenic agents has been variable, depending on the tumor type, and in most cases only a limited survival increase was noted. Major causes for this limited success are the occurrence of side effects and the induction of resistance. It is generally believed that a major progress in cancer treatment can be achieved by inding optimal combination therapy strategies. This made the search for optimal drug combinations among the fastest growing topic in the ield of cancer research. However, concerns regarding toxicity and drug resistance still constitute barriers to be overcome [8]. We have recently published an overview on current efforts of combining anti-angiogenesis drugs with other treatment modalities such as such as chemo-, radio-, immuno- or photodynamic therapy (PDT) and several advances in these efforts were reported [9]. This review explores the emerging issue on the therapeutic potential of optimized angiostatic drug combinations by analyzing clinical data for two tumor types, hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). ANTI-ANGIOGENIC DRUG COMBINATIONS BASED ON CLINICAL PRACTICE. THE LESSONS WE LEARNED The selection of drugs for use in combination therapies in the clinic is often based on the previous success of individual drugs as monotherapies [10]. This problem was well illustrated by the following quote of Robert Weinberg [11]: “Traditionally, new drugs have been evaluated as single agents during pre-clinical development and Phase I clinical trials. This practice contrasts with the growing belief of cancer researchers that most monotherapies are unlikely to yield curative treatments and that, with rare exceptions, truly successful clinical outcomes will depend on the use of combinations of anti-cancer drugs”. Moreover, in most cases new molecules tested in the irst-line setting need to be combined with the standard of care compound to demonstrate superiority. It is clear that such an approach seems to rely on trial and error and does not take into consideration the drug-drug interactions and their inluence to the tumor microenvironment. In the following section, we analyze the clinical results of targeted treatments in two examples of advanced malignancies. _____________________________ P. Nowak-Sliwinska, A.W. Griffioen 9 Hepatocellular carcinoma (HCC) is a highly vascularized tumor type that is treated with antiangiogenic agents. The only systemic drug approved for treatment of unresectable HCC is sorafenib [12]. This treatment modality causes, nevertheless, major toxicities, such as hand-foot syndrome, skin reactions, diarrhea and fatigue [13], while it induces both primary and acquired resistance [14]. The eficacy of sorafenib is probably due to a good balance between targeting cancer cells and their microenvironment, as a result of blocking multiple kinases (including VEGF, PDGF, C-KIT, and B-RAF). However, with this treatment the life expectancy of these patients is still only around 12 months, indicating a clear need for better treatment strategies. The reason for failure could be the large heterogeneity of both patients and tumors, as well as the lack of understanding of critical drivers of tumor progression or dissemination. Numerous clinical trials have been conducted to evaluate a large number of molecularly targeted drugs for treating HCC in the irst-line (brivanib, sunitinib, erlotinib, linifanib or tivantinib) or second-line (brivanib, everolimus), but most drugs exhibited less eficacy and/ or higher toxicity as compared to sorafenib (see Sun et al. for a comprehensive review [15]. Bevacizumab showed promise as an effective and tolerable treatment for advanced HCC. The reported eficacy of bevacizumab appeared to compare favorably with that of sorafenib [16]. Despite the fact that anti-endoglin therapy with TRC105 in phase II studies in patients with advanced HCC - post-sorafenib - brought evidence of clinical activity, it was not promoted to the second stage of investigation [17]. Tivantinib, a selective oral inhibitor of MET, has shown promising antitumor activity in HCC as monotherapy. In a randomized phase II study, time to progression was longer in patients treated with tivantinib as compared to placebo group. This result was unfortunately accompanied with grade 3, or worse, neutropenia and anaemia [18]. Everolimus, the mTOR inhibitor, was used in a randomized phase III trial (EVOLVE-1) in the second-line treatment in advanced HCC patients who experienced failure of sorafenib treatment. No signiicant overall survival beneit was observed in comparison with the placebo group [19]. These examples of treatment failure in the management of HCC add further insight to the need to change the current mindset of trial design. Different treatment roads should be sought and urgently translated into the design of early clinical trials in order to maximize the chances of positive clinical outcomes. There are only a few clinical trials with an arm of evaluating the simultaneous administration of two anti-angiogenic agents in HCC. The combination of bevacizumab and erlotinib was reported to have high clinical activity for HCC by a phase II study conducted in the USA, however, it showed modest activity in an Asian cohort [20]. This might very well be that genetic/racial _____________________________ 10 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 backgrounds play a signiicant role in treatment design and outcome. Refametinib, an allosteric MEK inhibitor, has been reported to possess antitumor activity in combination with sorafenib in preclinical models [21]. In a phase II study evaluating eficacy and safety of this combination HCC it appeared to be clinically active, although the high incidence of dose modiications may have compromised the eficacy results. Interestingly, the subgroup of patients with RAS mutations seemed to respond the most from this treatment [22]. Another pivotal example of the clinical use of anti-angiogenic therapy is the treatment of advanced renal cell carcinoma (RCC). The kidney is a heavily vascularized organ and it is probably for this reason that RCC is among the most vascularized malignancies [23]. Anti-angiogenic agents demonstrated signiicantly greater antitumor effects in RCC compared with the previous standard irst-line therapy with interferon-a (IFNa). Also for RCC, as was the case for HCC, the limited activity of single drug therapies is mainly due to disease and patient heterogeneity [24], toxicity [25-28] and possible enhancement of metastasis [29] and drug resistance [30-32]. Many preclinical and clinical efforts have focused on combining existing agents or sequencing of them to maximize their impact on clinical outcomes. A comprehensive systematic review of sequencing and combinations of systemic therapy in metastatic renal cancer has recently been presented by Albiges et al. [33]. Often, the choice of therapy is based on a patient and physician decision, which is based on comorbidities, toxicity proiles and costs. The treatment selection from the multiple choices that exist now for the irst- and second-line treatment of patients with mRCC became substantially complex by the possibility of combining drugs and by their sequencing options [6]. When applied in combination, next to – or instead of - enhanced eficacy, substantial toxicity has been a recurrent observation in these studies [33,34], even when designed to target complementary pathways [35]. Large studies such as the RECORD, INTORACT, CALGB and TORAVA studies [36-39] investigated irst-line combination regimens and did not prove, in most cases, superior outcome over single agents. The activity of sorafenib, temsirolimus, and bevacizumab administered in doublet combinations (e.g. the BEST trial) did not signiicantly improve median progression-free survival in comparison with bevacizumab monotherapy [40]. The list of available clinical trials is very long [41] and the current (2015) European Association of Urology (EAU) guidelines for treatment of mRCC recommend nivolumab (a programmed death 1 (PD-1) checkpoint inhibitor) [42] and cabozantinib (targets VEGFR, as well as MET and AXL) [43] used in sequence over the previous standard of care (everolimus) in patients who have failed one or more lines of VEGF targeted therapy [40]. BACK TO THE DRAWING BOARD: HOW TO DESIGN MORE EFFECTIVE MULTIDRUG TREATMENTS? The information given in the two above mentioned tumor types, shows that the trial and error approach in combining previously tested promising agents is by far not a systematic way of inding the best combination treatment. Although it is logical to assume that drugs with a positive anti-tumor effect will synergize with other drugs that have shown promise. However, drugs may also synergize or have synergies without proof of eficacy as a monotherapy. Not to talk yet about which drug doses should be used. There is simply not enough time, neither there are enough cancer patients, to search systematically for optimal combination treatments. Nevertheless, the above clearly demonstrates that one of the keys for improvement of cancer therapy in general, is the combination of existing drugs. Three main challenges exist in the effort of optimizing multi-drug combinations: (i) the complex nature of a biological system, which makes it virtually impossible to predict optimal drug combinations based on empirical information, (ii) the large number of possible drug combinations that exist when multiple drugs are considered at multiple concentrations [44], and (iii) optimization of drug administration in a pre-designed sequence in order to minimize the impact of drug resistance or escape mechanisms. In clinical settings, a maximum tolerated dose (MTD) approach was used to deine tolerable drug concentrations, but several of the trials reported that the MTD was not well tolerated and lower doses may have been more appropriate. Moreover, little consistency with the establishment of MTD based on dose limiting toxicities (DLTs) was observed as several of the trials measured DLTs. The sequential use of presently available molecularly targeted agents has become a standard in everyday clinical practice, especially in RCC. The available data for novel therapies in mRCC suggest they can be used sequentially across multiple lines of treatment to extend survival (see the current EAU guidelines for mRCC in a chapter above). Up to date, the results from both combination and sequencing clinical trials suggest that these approaches can be feasible, but prospective data on expected beneit are still missing, and new treatment tailoring methods are needed. Many groups have investigated methods to optimize drug combinations, including techniques to model cancer progression and predict cell responses [45,46], systematic searches [47], as well as technologies using various deterministic and stochastic search algorithms [48-50] or sequential decoding algorithms [51]. In previous research, we have used the feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate in the experimental parametric space of nine angiostatic drugs applied at four concentrations. For many biological applications, the FSC scheme is ideal, because it requires no knowledge of the mechanisms involved in determining the cellular response to a given drug stimulus or input. It only requires an output value that describes the overall cellular activity in response to a drug combination. This output is fed into the closedloop feedback system, in order for the search algorithm to determine the next iteration of drug combinations to be tested on the cell system 44. FSC has already been successfully used in various complex biological systems, including the inhibition of viral infection [52], maintenance of human embryonic stem cells [53], the differentiation of mesenchymal stem cells [54] or nanodiamond-modiied drugs [55]. By an iterative approach of in vitro testing for endothelial cell (EC) viability and applying a secondorder linear regression analysis, we used this procedure of elimination of less effective drugs and identify the optimal synergistic, low-dose drug combination. Drug doses were reduced by 5- to 11-fold, as compared to optimal single drug concentrations. The three remaining drugs were found to target distinct and complementary signaling pathways. One might expect that the differences in pharmacokinetics and pharmacodynamics between the components of the drug mixture may interfere with the in vivo treatment outcome. However, the lat response surfaces, which are the graphic illustration of drug combination-drug eficiency landscapes, allow for a much simpliied translation from the optimal in vitro combination to in vivo application (i.e. moderate changes in drug ratios do not signiicantly change the output). Thus, successful “ratiometric” translation of the synergistic anti-cancer activity of this drug combination was proven in two preclinical in vivo tumor models, i.e. human ovarian carcinoma and colorectal carcinoma. Indeed, in two tumor models we have observed an effective tumor growth inhibition clearly driven via antiangiogenic mechanism [56]. Summarizing, the three major advantages of the proposed personalized technology (i.e. rapid development of a patient-speciic optimal drug mixture, that is adjusted during the course of treatment while the cancer progresses) are: (i) the possibility to reduce side effects, (ii) the decreased probability of developing drug resistance and (iii) the relative speed of the FSC-based optimization process which would facilitate its clinical application. We believe that FSC can also be the gateway to a reliable method for the design of personalized optimal combination treatments. CONCLUSIONS AND FUTURE DIRECTIONS Clinical trials are currently evaluating whether therapeutic beneit can be improved by combining agents that block multiple levels of the same or different pathways, thus providing additive or even synergistic effects. It remains to be determined if systematic _____________________________ P. Nowak-Sliwinska, A.W. Griffioen 11 computer guided identiication of optimized reduceddose drug combinations would provide maximum eficacy and tolerability beneits in patients. The attractiveness of inding optimal drug combinations, using targeted compounds, for the inhibition of angiogenesis, is the broad application opportunities. Treatment of many cancer types may be improved, but also application in other angiogenic diseases, such as atherosclerosis [57,58], rheumatoid arthritis [59] and endometriosis [60, 61] is feasible. ABBREVIATIONS EC: endothelial cell; EAU: European Association of Urology; FSC: feedback system control; HCC: hepatocellular carcinoma; IFNa: interferon-a; MTD: maximum tolerated dose; PDT: photodynamic therapy; PD-1: programmed death 1; RCC: renal cell carcinoma. ACKNOWLEDGEMENTS This work was inancially supported by the Dutch Cancer Foundation to PNS and AWG (KWF: VU2014-7234) and the European Union to PNS (PIEFGA-2013-626797). REFERENCES 1. 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Angiogenesis inhibitors endostatin or TNP-470 reduce intimal neovascularization and plaque growth in apolipoprotein E-deficient mice. Circulation. 1999;99:1726-1732. 59. Thairu N, Kiriakidis S, Dawson P, Paleolog E. Angiogenesis as a therapeutic target in arthritis in 2011: learning the lessons of the colorectal cancer experience. Angiogenesis. 2011;14:223-234. 60. Nap AW et al. Angiostatic agents prevent the development of endometriosis-like lesions in the chicken chorioallantoic membrane. Fertil Steril. 2005;83:793-795. 61. Rocha AL, Reis FM, Taylor RN. Angiogenesis and endometriosis. Obstet Gynecol Int. 2013; 859619. _____________________________ P. Nowak-Sliwinska, A.W. Griffioen 13 ORIGINAL ARTICLE THE GENERAL PROTOCOL FOR THE S10 TECHNIQUE Mircea Constantin Șora Zentrum für Anatomie und Molekulare Medizin, Sigmund Freud Private University, Vienna, Austria Freudplatz 1, 1020 Vienna, Austria. E-mail: constantin.sora@med.sfu.ac.at ABSTRACT Plastination is a process of preservation of anatomical specimens by a delicate method of forced impregnation with curable polymers like silicone, epoxy or polyester resins, with vast applications in medical fields of study. In this process, water and lipids in biological tissues are replaced by curable polymers (silicone, epoxy, polyester) which are hardened, resulting in dry, odorless and durable specimens. Today, after more than 30 years of its development, plastination is being applied in more than 400 departments of anatomy, pathology, forensic sciences and biology all over the world. The standard S10 silicone technique produces flexible, resilient and opaque specimens. After fixation and dehydration, the specimens are impregnated with silicone S10 and in the end the specimens are cured. The key element in plastination is the impregnation step and therefore depending on the polymer used the optical quality of the specimens differ. The S10 silicone technique is the most common technique used in plastination. It is used worldwide for beginners, but also for experimented plastinators. The S10 plastinated specimens can be easily stored at room temperature, are non-toxic and odorless. The S10 specimens can be successfully used, especially in teaching, as they are easy to be handled and display a realistic topography. Plastinated specimens are also used for displaying whole bodies, or body parts in exhibition. Key Words: plastination, S10, anatomy, silicon INTRODUCTION Anatomy remains one of the core courses of medical school. Anatomy is where students learn the basic language of medicine, learn to develop understanding through experimentation, and develop skills in solving problems in a three-dimensional space [1]. The nature of anatomy education has changed substantially over the past decade due to both a new generation of students who learn differently from those of past years and the enormity of advances in anatomical imaging and viewing [2]. Signiicant advancements in the teaching and study of anatomy have been made, thanks to a new method of preserving human organs through plastination. Water and fats in tissues are replaced with silicone in a process which takes about one month. After plastination, the resulting tissue is safe to handle (i.e., toxic ixatives are eliminated), the tissue has no odor and it is extremely durable, and do not disintegrate. The end product is an anatomical specimen practically indistinguishable from the original. Plastination is the most important recently developed technique for the preservation of biological specimens. Plastination is carried out in many institutions worldwide and has obtained great acceptance particularly because of the durability of the plastinated specimens, the possibility for direct comparison to CTand MR-images, and the high teaching value plastinated specimens have. _____________________________ 14 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 Plastination has been developed for teaching as well as for research. Back in 1977, at the department of Anatomy of Heidelberg University, Dr. von Hagens invented plastination as a ground-breaking technology for preserving anatomical specimens with reactive polymers [3]. The process was patented between 1978 and 1982. This method has proved to be the superior method for preservation of gross specimen. Nowadays, the method is applied in more than 300 institutes for Human Anatomy, Clinical Pathology, Biology and Zoology worldwide [4-7]. Optical properties - opaque or transparent - and mechanical properties - smooth and lexible or hard - can be chosen by appropriate composition of plastination resins. The Sl0 technique is the standard technique in Plastination. Specimen impregnation with BIODUR ® Sl0 results in opaque, more or less lexible, and natural looking specimens. The procedure consists of the four main steps of plastination, besides the specimen preparation and dissection before. FIXATION Biological material must be ixed before plastination to prevent putrefaction and stop the action of other enzymes. Proper ixation of an organ or tissue sample is very important to the inal quality of a plastinated specimen. The usual ixatives, such as 5-20% formalin, may be used; however, exposure must be kept to an absolute minimum if natural color is to be preserved. All embalming luids containing long chain alcohols (e.g., glycerol) have to be removed before dehydration, as they easily spoil the inal specimens. Hollow organs must be dilated during ixation as well as during dehydration and gas curing. Fixation can be achieved by all usual ixatives as formaldehyde solution, Kayserling solution etc. Hollow organs must be dilated during ixation as well as during dehydration and gas curing. Types of ixation: immersion, iniltration, injection, perfusion, dilatation. The best for plastination is the use of formaldehyde solution (at decreased concentrations). If one desire, he could use other ixation agents as Kaiserling’s solution that yields a red-brown color to the specimens, while McCormick’s, Klotz, or Jore’s solutions may help preserve color. Immersion: This is the easiest of them all; you just put the specimens into the ixative. A 5% solution of formalin is used most often. Occasionally 1% or 2% solution can be used, but a concentration higher than 5% is not advisable. Immersion is especially suitable for thin specimens such as aortas. A lower temperature (+1 to +5 °C) will retard putrefaction and hardly affect the rate of penetration of the ixative. Furthermore, it favors color preservation. Injection: This term is used to denote the introduction of ixative luid via vessels. In most cases the arteries are used, seldom the veins. A plastic cannula or piece of lexible tubing is inserted into the vessel and serves as its extension. Injection is especially suitable for whole bodies or extremities. Perfusion: This is deined as the continuous lushing of the vascular system of whole bodies, organs or extremities with luid. Perfusion liquid is injected into a main artery and lows through the capillaries and veins. Perfusion with tap water is used to rinse blood out of the vessels. Tap water perfusion must always precede ixative perfusion or else the blood will clot in the vessels and block further perfusion. In plastination, tap water perfusion is very commonly used to remove blood from organs such as kidney, heart and liver. Iniltration: In plastination, this is deined as “direct injection of ixative into tissue.” The vessels are not used. Iniltration is employed for specimens that are too thick for immersion and cannot be injected. It is also handy for specimens whose vascular system has been damaged or injected with colored material. Optional: Bleach if necessary to brighten stained or darkened tissue, by using a 1-2% peroxide solution for 3 days. DEHYDRATION Dehydration removes the specimen luid, as well as some fat. In this step, tissue luid is replaced with an organic solvent. Either alcohol [8] or acetone may be used as dehydrating agent for plastination. Acetone is used in most cases because acetone also serves as the intermediary solvent during the next step - forced impregnation. To minimize specimen shrinkage, dehydration is done in cold (- l5 °C to 25 °C) acetone. The standard dehydration procedure for plastination is freeze substitution at -25 °C. This method saves time, requires less labor compared with ethanol dehydration, and causes less shrinkage. During freeze substitution, the specimens freeze immediately on immersion into the cold acetone, thereby stabilizing the specimens shape instantly. Within the next 3-5 weeks, the specimens become dehydrated. Fig. 1. Freezer with dehydration container If the removal of fat is also desired the dehydrated specimen must be kept in acetone (or methylene chloride) at room temperature for some time (this however, must not be done with nervous tissue, particularly with brains). An acetone amount of l0 times the specimen weight is best for good results. Dehydration is inished when the water content is less than l %. Equipment: deep freezer (explosion proof or motor and compressor removed and placed in a different room), acetonometer (to measure the content of water). Dehydrate until you reach 99% - 100% acetone (3-6 weeks): • Cold acetone; • Ethanol. Protocol: a. - Pre-cool the specimen (5 ºC, 12h in water); b. - Acetone baths (-25 ºC): • Acetone 97% (2-3 weeks); • Acetone 100% (1 week). After two or three days in each acetone bath, the purity is checked with an acetonometer after stirring the acetone bath. Make the acetone changes at -25 ºC, it will produce less evaporation and you are working under inlammation point (-19 ºC). FORCED IMPREGNATION Forced impregnation is the central step of plastination and it’s done under vacuum in a vacuum chamber [9]. In this step, the intermediary solvent (acetone) is replaced with a curable polymer (Biodur® Sl0). The silicone polymer S l0 is mixed with a curing agent Biodur® S3 (l part S3 and l00 parts Sl0), which _____________________________ M.C. Șora 15 commences the process of end-to-end linkage of the molecules. This linking is enhanced at room temperature; however, it is very slow when kept in the freezer at -l5 °C to -25 °C. Fig. 4. Normal impregnation speed, slow bubble formation Fig. 2. S10 silicon and S3 hardener Fig. 5. Vacuum manometer The dehydrated specimen is submerged in the cold (-l5° C to -25 °C) polymer mixture. After some days of immersion, vacuum is applied to it. Vacuum is applied slowly, as determined by the rate of gas bubble, building at the surface of the polymer. Vacuum is increased gradually to boil the intermediary solvent (acetone), which has a lower boiling point, out of the specimen. Fig 3. Vacuum chamber Fig. 6. Final vacuum is reached (4 mmHg) Impregnation is monitored by watching the bubble formation on the surface of the mixture and by a vacuum gauge. Vacuum is complete when the pressure is around 4 mm Hg [10]. _____________________________ 16 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 Equipment: deep freezer (explosion proof or motor and compressor removed and placed in a different room), vacuum chamber (e.g. Heidelberg plastination kettle), vacuum pump. Fig. 7. Standard Heidelberger vacuum kettle Protocol: a. Impregnation: Silicone polymer (Biodur S10) plus catalyst and chain extender (Biodur S3) (100:1 ratio) In a deep freezer at -15 °C is preferred. • Gradually decrease absolute pressure (increase vacuum) from one atmosphere (3-5 week period) until 5 mmHg is reached; • Room temperature impregnation is possible and is completed much faster (2-3 weeks). b. When impregnation is inished: • Drain polymer excess from impregnated specimens, while in deep freezer, back into plastination chamber. be cured (hardened). This is achieved by exposing the impregnated specimen to a gaseous hardener (Biodur® S6). S6 is a liquid that vaporizes at room temperature [9]. The impregnated specimen and a lask illed with S6 is placed in a tightly closed chamber for several weeks. To keep the environment for curing dehumidiied, a lask with a desiccant (e.g. anhydrous calcium chloride) is also placed in the curing chamber. To enhance the curing procedure, air may be bubbled through the luid S6. For complete curing inside the specimen, this should be kept in a plastic bag for several weeks [11]. Equipment: (plastic or stainless steel) box, stretch foil, membrane (aquarium) pump. Protocol: a. Slow Curing: • Room temperature (1 - 6 months) then use gas cure (Biodur S6). b. Fast Curing: • Gas cure (3-7 days) vaporize S6. Enhance volatilization by using an aquarium pump to bubble air through the S6 or a fan to circulate the S6; • Wipe excess polymer daily; • Bag specimen for 1-3 months. Each of the curing methods has advantages and disadvantages. The slow curing gives you a more lexible specimen, but it take more time, the fast curing produces stiffer specimens, but is more quick. Fig. 9. Gas curing unit. Fig.8. Impregnation timetable Impregnation time for S10: after the irst week the impregnation pressure should reach 1/3 of the starting pressure (different for acetone and methylene chloride), after 2 weeks it should reach 2/3 of the initial pressure and after 3 weeks it should reach at least 4 mmHg. GAS - CURING (HARDENING) Finally, the polymer inside the specimen has to After curing, the specimens are inished and can be stored at room temperature for a long time. Impregnated Biodur S10 silicone plastinated specimens have the same ultimate quality. Surface clarity is perfect and some lexibility is associated with thinner specimens. All specimens are durable, free of offensive odor and dry. They make excellent teaching aids both in the classrooms and the clinical setting [12]. As they are accumulated, they can become a fantastic library of specimens for normal, exotic and pathological anatomy. They may be used as teaching aids both in the class room and the clinical setting [12-15]. _____________________________ M.C. Șora 17 Fig. 10. Dissected brain plastinated with S10 Fig. 13. Kidney Fig. 11. Head & neck REFERENCES Fig. 12. Abdominal cavity 1. Bouffard C, Bouffard M. Spectacular anatomy: plastination and salutary dread. Lancet. 2012;379:704-5. 2. Jones DG, Whitaker MI. Engaging with plastination and the Body Worlds phenomenon: a cultural and intellectual challenge for anatomists. Clin Anat. 2009;22:770-6. 3. von Hagens G. Impregnation of soft biological specimens with thermosetting resines and elastomers. Anat Rec. 1979;194:43-50. 4. Durand M, Pourchez J, Louis B, Pouget JF, Isabey D, Coste A, Prades JM, Rusch P, Cottier M. Plastinated nasal model: a new concept of anatomically realistic cast. Rhinology. 2011;49:30-6. 5. König HE, Hagen J, Macher R, Donoso S, Probst A. Synoviale Einrichtungen an der Rinderzehe [Synovial facilities at the cattle toe]. Wiener Tierarztliche Monatsschrift. 2013;100:34-8. 6. Latorre RM, García-Sanz MP, Moreno M, Hernández F, Gil F, López O, Ayala MD, Ramírez G, Vázquez JM, Arencibia A, Henry RW. How useful is plastination in learning anatomy? J Vet Med Educ. 2007;34:172-6. 7. Sora MC, Jilavu R, Matusz P. Computer aided three-dimensional reconstruction and modeling of the pelvis, by using plastinated cross sections, as a powerful tool for morphological investigations. Surg Radiol Anat. 2012;34:731-6. 8. Pereira-Sampaio MA, von Horst C, Marques-Sampaio BPS, Smodlaka H, Favorito LA, Sampaio FJB, Henry RW. Theoretical considerations and preliminary studies on alcohol as an intermediary solvent. J Int Soc Plastination. 2006;21:27-8. 9. DeJong K, RW Henry. Silicone plastination of biological tissue: cold temperature technique Biodur™ S10/S15 technique and products. J Int Soc Plastination. 2007;20:36-7. 10. Henry RW. Vacuum and vacuum monitoring during silicone plastination. J Int Soc Plastination. 2005;20:37. 11. Henry RW. Silicone impregnation and curing. J Int Soc Plastination. 2005;20:36-7. 12. Bickley HC, von Hagens G, Townsend FM. An improved method for the preservation of teaching specimens. Arch Pathol Lab Med. 1981;105:674-6. 13. Sakamoto Y, Miyake Y, Kanahara K, Kajita H, Ueki H. Chemically reactivated plastination with Shin- Etsu Silicone KE-108. J Int Soc Plastination. 2006;21:11-6. 14. Latorre R, Rodríguez MJ. In search of clinical truths: equine and comparative studies of anatomy. Equine Vet J. 2007;39:263-8. 15. Gómez A, Del Palacio JF, Latorre R, Henry RW, Sarriá R, Albors OL. Plastinated heart slices aid echografic interpretation in the dog. Vet Radiol Ultrasound. 2012;53:197-203. _____________________________ 18 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 REVIEW ARTICLE IS THERE STILL A PLACE FOR LIVER BIOPSY FOR THE EVALUATION OF CHRONIC LIVER DISEASES IN THE ERA OF NON-INVASIVE METHODS? Ioan Sporea Department of Gastroenterology and Hepatology, “Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania 156 L. Rebreanu Blvd., 300736 Timisoara, Romania, E-mail: isporea@umft.ro Chronic liver diseases are quite frequent in daily medical activity. Chronic viral hepatitis (B or C), alcoholic or non-alcoholic liver diseases (ASH or NASH) are not rarely encountered and the evaluation of such patients is an important part of a hepatologist’ activity. Confronted with such a patient, correct disease assessment is important for the decision of treatment (especially in chronic viral hepatitis) and for prognosis. But what does assessment mean? Usually we want to ind out the severity of necro-inlammation (grading) and the stage of ibrosis (staging) in the liver. In some liver diseases, the presence of typical histological signs of disease should be assessed (such as in ASH, in primary biliary cirrhosis - PBC, in autoimmune hepatitis). For many years liver biopsy (most frequently performed percutaneously) was the “gold standard” for evaluation in patients with chronic liver disease. By using echoguided liver biopsy (LB) or blind LB, a hepatic sample is obtained for staging and grading the disease. But this classic assessment modality has some limitation: not always the liver fragment is large enough for a correct evaluation of ibrosis [1], not all the patients accept this invasive procedure and, last but not least, sometimes LB is followed by minor or major complications [1]. More than 10 years ago, the hepatologists started to use non-invasive means for the evaluation of liver diseases. Using biological tests such as FibroTestActiTest, FibroMax, APRI, Lock, ELF or others, we are able to make a quite correct evaluation of activity (necro-inlammation) and ibrosis in the liver (FibroTestActiTest) or of steatosis, NASH and ASH (FibroMax). These types of tests were developed irstly in France and subsequently spread around the world. The cost of such tests exceeds in some cases 100 Euros, but the advantage is that they are totally non-invasive. Quite at the same time with the biological tests, the irst ultrasound based elastographic method for ibrosis assessment - Transient Elastography (TE FibroScan®, Echosens, Paris, France) was developed. The principle of this method is that mechanical excitation of the hepatic tissue induces shear-waves into the liver, whose speed, quantiied by ultrasound, is indicative of ibrosis severity. Hundreds of thousands of patients were already evaluated by TE, the accuracy ranging from 80 to 95%. At least 3 meta-analyses [2-4] proved the value of this method for liver ibrosis assessment and showed a strong direct correlation between TE values expressed in kPa and the severity of ibrosis. However this method has some limitations: valid measurement can be obtained only in approximately 85% of subjects [5] or less [6], liver assessment by TE is impossible when ascites is present and TE measurements are irrelevant in patients with high values of aminotranspherases, with obstructive jaundice, or with congestive heart failure. TE has been proven to be accurate irstly in patients with C chronic hepatitis, and subsequently in other pathological conditions, such as chronic B viral infection, NASH, ASH or PBC. Thus, TE was included in the Guidelines of the European Association for the Study of the Liver (EASL), as a standard method for liver ibrosis assessment in HCV patients. Approximately 5 years ago, another ultrasound based elastographic method appeared on the market - “point” shear wave elastography (ARFI). By this method, using ultrasound imaging, a precise region is selected in real time into the liver. By pushing a button, an acoustic push pulse is generated by the transducer that induces shear-waves into the liver, whose speed, also quantiied by the system, expressed in m/s, is indicative of ibrosis severity. Several studies showed a good correlation between the ARFI values and the severity of ibrosis [7], both in C and B chronic hepatitis [8]. Two meta-analyses [9, 10] showed that the accuracy of this method ranges from 80 to 95% for ibrosis assessment and that the accuracy increases with ibrosis severity. The study of Bota et al [10] also showed that ARFI is noninferior as compared with TE. Another “point” shear wave elastographic method is ElastPQ. Preliminary results showed the same good correlation between the values obtained by ElastPQ and liver ibrosis [11]. _____________________________ I. Sporea et al 19 In the last three years, a new ultrasound based elastographic method was developed – 2D shear wave elastography (SWE). It is a color coded method that also displays numeric values. Published papers showed promising results of this method for liver ibrosis assessment [12-14]. Also in the last years, several studies were performed using strain elastography (a color coded method), with promising results regarding the evaluation of liver ibrosis severity. Thus, since so many non-invasive modalities are available, the question that arises is if there still is a place for LB in chronic liver disease patients. In clinical practice, at least in Europe and Romania [15] a dramatic reduction in the number of LB was observed. This reduction is not so important in USA, where TE or ARFI were only recently accepted by FDA. In Romania there are university centers [15], in which LB was totally abandoned in favor of non-invasive means of evaluation. But in which cases can we renounce to perform LB? In patients with C chronic hepatitis, in which we must treat the infection, regardless of ibrosis severity. If we want to stratify, to prioritize these patients, the elastographic methods (TE, ARFI or SWE) or FibroTest are good enough for this discrimination. In ASH or NASH patients, using biological tests such as FibroMax, or elastographic methods, we can estimate the severity of ibrosis or steatosis (FibroMax). In other diseases, such as B hepatic chronic infection or autoimmune hepatitis, liver biopsy can still be needed. Why? In HBV infection, not only ibrosis severity matters, inlammation also seems to be important, which can be evaluated by ActiTest or by LB. On the other hand, in autoimmune hepatitis, morphological signs such as interface hepatitis are important for diagnosis and prognosis. On the other hand, some studies have shown that elastographic methods can underestimate the severity of liver disease and can deprive the patient of an adequate therapy. Thus, when signiicant ibrosis is demonstrated by elastographic methods, therapy can be initiated. In cases with elastographic values smaller than the cut-off proposed for signiicant ibrosis, maybe a LB should be performed. Some authors [16] propose a combination of biological and elastographic tests to evaluate chronic liver disease patients. In concordant cases, LB can be avoided. For the others, LB is still an option. Finally, to answer the question from the title, I believe that in the next future the demand for liver biopsy will decrease everywhere, but a number of patients will still need to undergo this procedure for a correct staging of the liver disease. Future developments of ultrasound based elastographic methods (maybe strain elastography or 2D-SWE) or MRI elastography will make liver biopsy obsolete. _____________________________ 20 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 REFERENCES 1. Cholongitas E, Senzolo M, Standish R, Marelli L, Quaglia A, Patch D, Dhillon AP, Burroughs AK. A systematic review of the quality of liver biopsy specimens. Am J Clin Pathol. 2006;125:710-21. 2. Talwalkar JA, Kurtz DM, Schoenleber SJ, West CP, Montori VM. Ultrasound-Based Transient Elastography for the Detection of Hepatic Fibrosis: Systematic Review and Meta-analysis. Clin Gastroenterol Hepatol. 2007;5:1214-1220. 3. Friedrich-Rust M, Ong MF, Martens S, Sarrazin C, Bojunga J, Zeuzem S, Herrmann E. Performance of transient elastography for the staging of liver fibrosis: a meta-analysis. Gastroenterology. 2008;134:960-974. 4. Tsochatzis EA, Gurusamy KS, Ntaoula S, Cholongitas E, Davidson BR, Burroughs AK. Elastography for the diagnosis of severity of fibrosis in chronic liver disease: a meta-analysis of diagnostic accuracy. J Hepatol. 2011;54:650-659. 5. Castera L, Foucher J, Bernard PH, Carvalho F, Allaix D, Merrouche W, Couzigou P, de Ledinghen V. Pitfalls of Liver Stiffness Measurement: A 5-Year Prospective Study of 13,369 Examinations. Hepatology. 2010;51:828-835. 6. Șirli R, Sporea I, Bota S et al. Factors influencing reliability of liver stiffness measurements using transient elastography (M-probe)monocentric experience. Eur J Radiol. 2013; 82:313-316. 7. Sporea I, Șirli R, Deleanu A et al. Acoustic radiation force impulse elastography as compared to transient elastography and liver biopsy in patients with chronic hepatopathies. Ultraschall Med. 2011;32:46-52. 8. Sporea I, Șirli R, Bota S et al. Comparative study concerning the value of Acoustic Radiation Force Impulse elastography (ARFI) in comparison with Transient Elastography (TE) for the assessment of liver fibrosis in patients with chronic hepatitis B and C. Ultrasound in Med & Biol. 2012;38:13101316. 9. Friedrich-Rust M, Nierhoff J, Lupsor M, Sporea I, FierbinteanuBraticevici C, Strobel D, Takahashi H, Yoneda M, Suda T, Zeuzem S, Herrmann E. Performance of Acoustic Radiation Force Impulse imaging for the staging of liver fibrosis: a pooled meta-analysis. J Viral Hepat. 2012;19:212-219 10. Bota S, Herkner H, Sporea I, Salzl P, Sirli R, Neghina AM, PeckRadosavljevic M. Meta-analysis: ARFI elastography versus transient elastography for the evaluation of liver fibrosis. Liver Int. 2013;33:11381147. 11. Sporea I, Bota S, Grădinaru-Taşcău O et al. Comparative study between two point Shear Wave Elastographic techniques: Acoustic Radiation Force Impulse (ARFI) elastography and ElastPQ. Ultrasound in Med & Biol. 2014 In press 12. Bavu E, Gennisson J-L, Couarde M, Bercoff J, Mallet V, Fink M, Badel A, Vallet-Pichard A, Nalpas B, Tanter M, Pol S. Noninvasive in vivo liver evaluation using supersonic shear imaging: a clinical study on 113 hepatitis C virus patients. Ultrasound Med Biol. 2011;37:1361-1373. 13. Ferraioli G, Tinelli C, Dal Bello B, Zicchetti M, Filice G. Accuracy of realtime shear wave elastography for assessing liver fibrosis in chronic hepatitis C: A pilot study. Hepatology. 2012;56:2125-33. 14. Sporea I, Bota S, Jurchiș A et al. Acoustic Radiation Force Impulse and SuperSonic Shear Imaging versus Transient Elastography for liver fibrosis assessment. Ultrasound in Med & Biol. 2013;39:1933-1941. 15. Sporea I, Popescu A, Gheorghe L, Cijevschi Prelipcean C, Sparchez Z, Voiosu R. “Quo vadis” liver biopsy? A multi-centre Romanian study regarding the number of liver biopsies performed for chronic viral hepatitis. J Gastrointestin Liver Dis. 2012;21:326. 16. Castéra L, Sebastiani G, Le Bail B, de Ledinghen V, Couzigou P, Alberti A. Prospective comparison of two algorithms combining non-invasive methods for staging liver fibrosis in chronic hepatitis C. J Hepatol. 2010;52:191-198 ORIGINAL ARTICLE EXPRESSION OF CK5 BASAL CYTOKERATIN DURING METASTATIC DEVELOPMENT OF BREAST CARCINOMA Veaceslav Fulga1*, Amalia Raluca Balica2, Anca Maria Cîmpean2, Lilian Șaptefrați1, Marius Raica2 2 1 Department of Histology, Cytology and Embryology, State Medical University “Nicolae Testemitanu”, Chisinau, Republic of Moldova Department of Microscopic Morphology/Histology, Angiogenesis Research Center, “Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania * Department of Histology, Cytology and Embryology, State University of Medicine and Pharmacy “Nicolae Testemițanu”, Ștefan cel Mare Str. 165, MD-2004, Chișinău, Republic of Moldova. E-mail: vmfulga@usmf.md ABSTRACT Objective. Breast cancer is a one of the most common cancers in females worldwide. Basal cytokeratin CK5 represent the marker of progenitors for glandular and myoepithelial lineages of mammary epithelium. During epithelial differentiation there is a gradual decrease of CK5 expression. The purpose of this study was to compare the expression of basal cytokeratin CK5 vs hormone receptors, HER2, Ki67 and molecular subtypes immunohistochemically defined in the primary breast carcinoma of NST type and axillar lymph node metastasis. Material and Methods. We processed immunohistochemically 91 invasive breast carcinomas of NST type and their ipsilateral axillar lymph node metastasis (LNM). Results. The majority of primary tumors were evaluated as CK5 negative (78 cases/85.7%). The majority of cases were evaluated as Luminal B (50 cases/54.9%) and Luminal A (28 cases/30.8%) tumors. The HER2 subtype was confirmed in 8 cases/8.8%, 5NP in 3 cases/3.3% and Basal-like in 2 cases/2.2%. The parallel comparison of CK5 expression at both sites, primary and metastatic, revealed that this marker is not stable during metastatic progression. The molecular subtypes were not stable during metastatic process in 21 cases/23.1%. Conclusions. The majority of NST invasive ductal breast carcinomas are CK5 negative. The molecular subtypes and CK5 are not stable during metastatic process. Cancerous cells prefer to lose this marker in the lymph node environment. The presence of cases with simultaneous expression of CK5 and hormone receptors is an open field to debate the existence of other, transient molecular subtypes. We expect a further confirmation in larger study groups. Key Words: molecular subtypes, invasive carcinoma NST type, basal cytokeratin. INTRODUCTION Breast cancer is a one of the most common cancers in women worldwide. The new concept purpose to identify at least ive molecular subtypes: two hormone positive types (Luminal A and Luminal B) and three hormone negative types (HER-2 overexpressed, Basallike, and Normal breast-like). Every molecular subtype has distinct clinical features, and different prognosis [1]. Nielsen et al. (2004) purposed to differentiate immunohistochemically these subtypes by a panel of four antibodies (ER, HER1, HER2, and cytokeratin 5), point of view sustained later also by Goldhirsch et al. [2, 3]. Actually, the secretory portion of the breast is considered to be made of ive distinct cell populations: committed stem (progenitor) cells CK5 positive, glandular precursor cells which express all spectrum of cytokeratins (CK5+/CK8/18/19+), glandular end cells, positive for luminal cytokeratins (CK8/18/19+), myoepithelial precursor cells positive for CK 5/6+ and SMA+ (smooth muscle actin), and myoepithelial mature cells, SMA positive. By Böcker et al. (2005) the CK5 positive cells represent progenitors for both glandular and myoepithelial lineages of mammary epithelium [4]. During epithelial differentiation there is a gradual decrease of CK5 expression. After spreading from its primary situs, metastases arise as solid tumors in distant organs. But the microenvironment of the tissue in which they have landed it’s quite different from which they have originated. Weigelt et al. (2003) afirmed that primary breast tumors are similar to the distant metastases in lung, ovary, skin, lymph nodes [5]. Authors consider that metastatic capacity of breast cancer is an inherent feature and the metastatic proiciency of a tumor is preprogrammed from its beginning [6]. By the other side a series of publications showed intriguing results about different immunohistochemically deined subtypes at primary site and LNM [7-10]. Authors concluded that molecular proiles of breast cancer are not stable throughout tumor progression. The aim of this study was to compare the expression of basal cytokeratin CK5 vs hormone receptors, HER2, Ki67 and molecular subtypes immunohistochemically deined in the primary breast carcinoma of NST type and axillar LNM. _____________________________ V. Fulga et al 21 In the present work we found, that basal cytokeratin CK5 and molecular subtypes of invasive breast carcinoma of no special type are not stable during metastatic process. MATERIAL AND METHODS Patients Ninety one cases of breast carcinomas and their LNM collected during 2013-2014 from the Oncological Institute, Republic of Moldova. No drug therapy preceded and all of patients (33-86 years old) underwent radical mastectomy and lymph nodes dissection. Tissue processing and immunohistochemistry The specimens were ixed in 10% phosphate buffered formalin for 24-48 h and parafin (Paraplast High Melt, Leica Biosystems) embedded as traditionally. To avoid any misunderstandings about tissue processing, the specimens (primary tumor and lymph node) of the same patient have been embedded in a single parafin block and sections were processed on the same slide (Fig.1). For histopathological assessment 3-5 μm sections were cut (Shandon HM355S Automatic Microtome, Thermo Scientiic, USA) and stained with hematoxylin Harris (HHS32, SigmaAldrich) and eosin CS701 (Dako, Denmark). Histological grade was scored by the ScarffBloom-Richardson grading system. All cases were morphologically conirmed as invasive ductal breast carcinomas of NST type with developed axillar LNM. The immunohistochemical assessment included 5 markers: for estrogen (ER), progesterone (PR), human epidermal growth factor receptor 2 (HER2), marker of proliferation Ki67 and basal cytokeratin CK5 (Table 1). Specimens were processed automatically on Leica Bond-Max autostainer (Leica Microsystems GmbH, Wetzlar, Germany). The hematoxylin Mayer, Lille modiied (HMM500, ScyTek Laboratories, Inc.) was used for counterstaining. Microscopic evaluation. Hormone receptors, as well Ki67 marker were counted by using a semi-quantitative method Suciu et al. (2014) purposed [11]. In accordance with Goldhirsch et al. (2011) recommendations for Ki67 marker we used a 14% threshold as a limit to distinguish positive/negative cases [12]. For estrogen and progesterone evaluation we followed the guidelines purposed by Allred et al. [13]. The cases scored as +1 – +3 were considered positive. The threshold of positivity was 10% (Fig. 2). The HER2 status was interpreted in accordance with ASCO (American Society of Clinical Oncology) recommendations [14]: HER2+0 – if no staining observed or weak, barely perceptible membrane staining until 10% of cells; HER2+1 – in case of a weak membrane staining of >10%; HER2+2 – in case of incomplete, weak/moderate circumferential membrane staining of more than 10% of tumor cells or complete circumferential intense staining less than 10% of cells; HER2+3 – in case of intense, circumferential staining of more than 10% of tumor cells. Cases with HER2 scored as +2 and +3 were considered positive. The positive cells of normal ducts served as internal control. The CK5 expression was interpreted in accordance with Azoulay et al. recommendations: 0 – no tumor cells stained; +1 – less of 10% of tumor cells stained; +2 – 10-50% of positive tumor cells; +3 – more than 50% of tumor cells stained [15]. Expression was scored as Fig.1. Invasive breast carcinoma of NST type, primary tumor and LNM processed on the same slide, hematoxylin-eosin, x6. _____________________________ 22 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 Table 1. Immunohistochemical supplies: source and dilution of antibodies, systems of detection and retrieval, time of incubation. Fig. 2. Invasive ductal breast carcinoma NST type, G3 stained for Ki67. The tumor was appreciated as Ki67 positive (61%). Counterstaining with Lille modiied hematoxylin. On the right side is represented the inverted image by Nis-elements 2.30 imaging software, x400. possibility which facilitates the ER, PR and Ki67 numeric evaluation. A MS Access 2007 database was used to store and group the data. Statistical analysis The WINSTAT 2012.1 (R. Fitch Software, Bad Krozingen, Germany) software was used for a descriptive statistics: the mean value, standard error of mean and median were determined for Ki67. For all the tests a p≤0.05 was considered signiicant. A Pearson’s correlation (r) was used to determine the relationship between different variables. The CK5 expression from primary tumor and LNM was compared by t-Student and linear regression tests. positive (>0) if any cytoplasmic and/or membranous staining tumor cells were observed. The results were grouped in 7 subtypes: ER-, PR-, HER2-, CK5- as 5NP (ive negative phenotype); ER-, PR-, HER2-, CK5+ as Basal-like; ER-, PR-, HER2+, CK5- as HER2+ (HER2 overexpressed); ER+, PR+, Her2+, CK5-, Ki67<14 as Luminal B/Her2; ER+, PR+, Her2+, CK5-, Ki67>14 as Luminal B/Her2/Ki67; ER+, PR+, Her2-, CK5-, Ki67>14 as Luminal B/Ki67; ER+, PR+, Her2-, CK5-, Ki67<14 as Luminal A. The Luminal B subtype can be deined by ER, PR expressions simultaneously with HER2 and/or Ki67 positivity threshold over than 14%. As the Luminal A and B subtypes are different by prognosis, as well as the tendency of modern medicine is to personalize the treatment for every patient apart we considered opportune to highlight in the end of the name of Luminal B subtype the marker(s) which switched Luminal A case to Luminal B. Image acquisition and data processing Slides were examined on Nikon Eclipse 80i microscope with Nikon DS-Fi1 installed camera by using Nis-elements 2.30 imaging software (Nikon Instruments Europe BV). By this software we inverted images, Ethics This study has been approved by the Ethics Committee of the “Nicolae Testemitanu” University of Medicine and Pharmacy from Chisinău, Republic of Moldova (approval number 21/13/31.03.2014). RESULTS The G1 grade of differentiation was determined in 4 cases/4.4%, G2 in 51 cases/56% and G3 in 36 cases/39.6%. The majority of primary tumors were evaluated as CK5 negative (78 cases/85.7%), of which 3 cases/3.3% had G1 grade of differentiation, 45 cases/49.5% were G2 and 30 cases/33% were evaluated as G3. The CK5 positive tumors had predominantly G2 (6 cases/6.6%) and G3 (6 cases/6.6%) grades. Only one case evaluated with +2 for CK5 had a G1 grade. The majority of cases were evaluated as Luminal B (50 cases/54.9%) and Luminal A (28 cases/30.8%) tumors. The Luminal B structure was as follow: Luminal B/Ki67 in 38 cases/41.8%, Luminal B/HER2/Ki67 in 9 cases/9.9% and Luminal B/HER2 in 3 cases/3.3%. The HER2 subtype was conirmed in 8 cases/8.8%, 5NP in 3 cases/3.3% and Basal-like in 2 cases/2.2%. By comparing CK5 expression with molecular subtypes, we determined that 70 cases/76.9% evaluated as CK5 negative were diagnosed as Luminal one (A or B), 5 cases/5.5% as HER2 and 3 cases/3.3% as 5NP. _____________________________ V. Fulga et al 23 Table 2. CK5 expression in primary tumor and LNM vs molecular subtype. The CK5 positive cases (13 cases/14.3%) could enface features of any molecular subtype, except 5NP (Table 2). The statistical assay demonstrated a positive correlation between CK5 expression from primary tumor and CK5 from metastatic site, HER2 and Ki67 from both sites. A negative correlation was determined when CK5 was associated with ER and PR expression in primary tumor and LNM. More, CK5 value from metastatic side depends of CK5 expression from primary tumor, statement proved by linear regression and t-Student (t=1.30, p<0.20) tests (Fig.3). The parallel comparison of CK5 expression at both sites, primary and metastatic, revealed that this marker is not stable during metastatic progression: 2 cases/2.2% (both G3) evolved with CK5 acquiring in metastatic _____________________________ 24 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 place and 6 cases/6.6% (2 cases were G2 and 4 cases as G3) with loosing CK5 in LNM. Fig. 3. The relationship between CK5 expression from primary tumor and lymph node metastasis: the simple linear regression test. Table 3. CK5 expression vs ER, PR, HER2, Ki67, patients age and tumor grade of differentiation: Pearson correlation. The molecular subtypes were not stable during metastatic process in 21 cases/23.1%: Basal-like change to 5 NP in 1 case/1.1%, HER2 to Luminal B/HER2/ Ki67 in 1 case/1.1%, Luminal A to 5 NP in 2 cases/2.2%, Luminal A to Luminal B/HER2 in 1 case/1.1%, Luminal A to Luminal B/Ki67 in 2 cases/2.2%, Luminal B/ HER2 to Luminal A in 1 case/1.1%, Luminal B/ HERses/3.3% to Basal-like, HER2 and Luminal A, Luminal B/Ki67 resulted in 1 case/1.1% with Basallike and in 9 cases/9.9% evolved in Luminal A. The 15 cases of subtypes transfers were CK5 negative tumors and 6 cases were CK5 positive at primary site. Have to mention, that 2 cases, where CK5 changed from negative status in primary tumor to positive in LNM had a stable molecular subtype (Luminal A and Luminal B/Ki67). All other shifted cases had a stable CK5 negative status at both sites. From 6 CK5 positive cases, 3 loosed this marker at LNM site and changed molecular subtype as follow: Basal-like to 5NP, Luminal B/Ki67 to Luminal A and Luminal A to 5 NP. DISCUSSION Cancer is a leading cause of death worldwide and the breast cancer is the most common reason of cancer death in women. Classical pathology has segregated this carcinoma into multiple groups, based on their overall morphology. The most common reported is invasive ductal carcinoma NST type. Beside of well known classical histological features, as tumor size, histological type and grade, vascular invasion, identifying of new molecular factors has become the objective of many research studies [1]. A common feature of malignant tumors is the ability to invade the nearby tissues and spread to more distant parts of the body through the bloodstream or lymphatic system. After spreading from its primary site, metastases arise as solid tumors in distant organs [16]. But the microenvironment of the tissue in which they have landed it’s quite different from which they have originated and possibly creates a pressure on these cells requiring them to develop an adaptive responses which allow them to grow as a tumor in these nodes. Plus, was demonstrated the role of extracellular matrix components in breast cancer progression and metastasis [17]. In the present study we analyzed the evolution of another basic marker, CK5. By contemporary opinion this marker is expressed by basal/myoepithelial cells and together with CK14, CK8/18, p63 is used to distinct the basal group of breast carcinomas. CK5 positive cells in fact represent progenitors for both glandular and myoepithelial lineages of mammary epithelium. During epithelial differentiation there is a gradual decrease of CK5 expression, associated with an increase in expression of CK8/18/19 in the glandular cells, and smooth muscle actin in the myoepithelial cells along the pathways of differentiation. By the Bocker W et al. (2005) data, in the lactating breast, there is a segregation of epithelial structures into CK8/18 expressing secretory zone and the proliferative zone which harbors cells of both glandular (CK8/18+) and basal /myoepithelial (CK 5/6+) type [4]. In case of benign lesions the luminal tumors present a high number of CK5/6 positive cells due to a high proliferation of both glandular and basal cells. In Heatley M et al. (1995) opinion, the majority of malignancies which are derived from differentiated glandular cells line do not reveal immunohistochemical staining with CK5/6 leading, by this explaining and CK5 negativity in most lesions of atypical hyperplasia and ductal carcinoma in situ [18]. In Laakso et al. (2005) opinion CK5/14 positive tumors encounter about 9% and majority of them are of histological grade 3 [19]. In our study this subtype was slightly higher, 14,3% and CK5 positive cases were equally G2 and G3. By this article we conirmed the Laakso et al. (2005) results that primary tumor and LNM are statistically concordant for CK5 expression. But in our study, the parallel comparison of CK5 expression revealed that this marker is not stable during metastatic progression: 2 cases/2.2% (both G3) evolved with CK5 acquiring in metastatic place and 6 cases/6.6% (2 cases were G2 and 4 cases as G3) with loosing of CK5 in LNM. The parallel comparison of CK5 with expression of surrogate markers included in study, conirmed its basal localization: inverse correlation with ER, PR and positive _____________________________ V. Fulga et al 25 association with Ki67, HER2. The last correlation value, with HER2 is contradictory to Laakso et al. (2005) which reported a negative value of CK5 expression with HER2 ampliication. Plus, CK5 basal origin was disturbed by the fact, that CK5 positive cases (13 cases/14.3%) could enface features of any molecular subtype, inclusive Luminal and except 5NP. By the present moment, are few data which compared the molecular proiles of primary tumor and its metastasis. Falck et al. (2010) initially have not determined a statistically signiicant skewness by comparing the fraction of ER, PR, HER2 and Ki67 positive cells from both sites, but a fraction of 7% (ER), 16% (PR), 3% (HER2) discordant pairs were reported [7]. The same authors, showed also discordance between primary tumors and lymph node metastasis in breast cancer patients according to a multiple molecular phenotype [8]. The discordant 16% of cases were shifted from Luminal A to a subtype where survival analysis showed an impaired prognosis compared to this subgroup. Moreover authors determined that when a shift in subtype between primary tumor and metastatic lymph node was happened, the prognosis (for 10 years) seemed to follow the subtype of the lymph node [9]. It means that the prognostic information for individual patients appears to be available only after synchronous analysis of biomarker expression in metastatic lymph nodes. In the present study the molecular subtypes were not stable during metastatic process in 21 cases/23.1%. Have to mention that majority of shifted cases had a stable CK5 negative status at both sites. Montel et al. (2005, 2006) demonstrated several differences in the expression signatures of tumors derived from cloned weakly/non-metastatic human cell lines and from their isogenic metastatic counterparts of the same patient [20, 21]. Urquidi et al. (2002) have provided direct proof that individual cancer cells, co-exist within a given tumor, but differ greatly in metastatic capability, moreover some of them are non-metastatic [22]. It seems that primary cancers contain many types of tumor cells which can differ by expression proiles and their metastatic activity. Plus, as the metastatic ability of the cell population increases, the receptors proiles changes concomitantly. These are demonstrating conclusively that the malignant phenotype and its molecular signature are not pre-determined and static, but continue to evolve in a tumor throughout its life history [23]. Raica et al. (2014) reported about 20% of cases with switch of molecular subtypes during metastatic development, especially because of ER, which seems to be the most unstable parameter during invasive process [10]. This evidence supported the most frequent switching from the luminal A subtype in the primary tumor to the luminal B subtype in the matched LNM. Authors concluded that primary cancer probably contain many types of tumor cells, which can differ by their expression proiles and their metastatic potential. _____________________________ 26 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 The Basal-like breast carcinoma was reported to have the worst prognosis [24]. These tumors are described as negative for hormone receptors and HER2, and positive for CK5 and/or HER1 [25]. Basal-like tumors are also the most common type of tumors in patients with germline BRCA1 mutations. Such tumors are considered as high proliferative one, with a low cellular differentiation [26, 27]. Such results are conirmed by the present study, where majority of tumors were conirmed as G2 and G3. In accordance to Sood et al. (2014) the CK5 did not show statistically signiicant correlation with age, tumor size and stage, histological type, the state of tumor margins, presence of lymphoid iniltrate and necrosis, lymph node status, and Ki67 positivity [27]. Authors reported a single signiicant correlation, CK5 vs tumors’ grade, result debated by Rao et al. (2013) and conirmed by us: CK5 correlated signiicantly with all studied markers, except grade of differentiation [26]. Some authors debate in the literature whether triple-negative tumors (negative for hormone receptors and HER2, CK5 positive) are synonym with Basal-like carcinoma [28]. Cheang et al. (2008) by using additional markers identiied patients with a signiicantly worse outcome in the group of triple-negative tumors, which it means that breast carcinoma is not homogenous even inside of the molecular subtypes [29]. With present study we entwine a series of our previous publications concerning breast cancer stability throughout during metastatic development. We tested the surrogate markers which are commonly accepted by the oncologist to stratify the molecular subtypes of this tumor. We entitled the studies from stability position and didn’t answer from tumors cells homogeneity. By this we raise the question to research communities: another proile of metastatic cells is due to inhomogeneous cellular structure of primary tumor or these cells are changing receptors kit in metastatic ambiance? CONCLUSION The majority of NST invasive ductal breast carcinomas are CK5 negative. The molecular subtypes and CK5 are not stable during metastatic process. Cancerous cells prefer to lose this marker in the lymph node environment. The presence of cases with simultaneous expression of CK5 and hormone receptors is an open ield to debate the existence of other, transient molecular subtypes. We expect a further conirmation in larger study groups. ACKNOWLEDGEMENTS This work was sustained by UEFISCDI_ Bilateral Cooperation Romania-Moldova grant 684/2013 of Romanian Ministry of Education and Research. AUTHOR CONTRIBUTION MR and AMC participated in developing the study design, data interpretation and manuscript editing. VF collected and processed the data, wrote the primary version of manuscript. LS participated in manuscript editing. ARB processed imunohistochemically all cases and interpreted results. The inal version of manuscript was read and approved by all authors. 12. Goldhirsch A, Wood WC, Coates AS, et al. Strategies for subtypes— dealing with the diversity of breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast. Cancer Ann Oncol. 2011; 22:1736-47. 13. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998;11:155-168. 14. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists CONFLICT OF INTERESTS The authors declare that there is no conlict of interests regarding the publication of this paper. REFERENCES 1. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature. 2000; 406:747–752. 2. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004;10:5367–5374. 3. Goldhirsch A, Winer EP, Coates AS, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol. 2013; 24:2206-23. 4. Böcker W, Bürger H, Buchwalow IB, Decker T. Ck5-positive cells are precursor cells of glandular and myoepithelial cell lineages in the human breast epithelium. A new cell concept as a basis for a better understanding of proliferative breast disease? Verh Dtsch Ges Pathol. 2005; 89:45-7. 5. Weigelt B, Glas AM, Wessels LF, et al. Gene expression profiles of 6. van der Vijver MJ, He YD, van’t Veer LJ, et al. A gene-expression 7. Falck AK, Fernö M, Bendahl PO, Rydén L. Does Analysis of Biomarkers 8. Falck AK, Bendahl PO, Ingvar C, et al. Analysis of and prognostic primary breast tumors maintained in distant metastases. Proc Natl Acad Sci USA. 2003;100:15901–15905. signature as a predictor of survival in breast cancer. N Engl J Med. 2002;347:1999–2009. in Tumor Cells in Lymph Node Metastases Give Additional Prognostic Information in Primary Breast Cancer? World J Surg. 2010;34:1434-41. information from disseminated tumour cells in bone marrow in primary breast cancer: a prospective observational study. BMC Cancer. 2012;12:403. 9. Falck AK, Bendahl PO, Chebil G, Olsson H, Fernö M, et al. Biomarker expression and St Gallen molecular subtype classification in primary tumours, synchronous lymph node metastases and asynchronous relapses in primary breast cancer patients with 10 years’ follow-up. Breast cancer Res Treat. 2013;140:93-104. 10. Raica M, Cîmpean AM, Ceaușu RA, et al. Hormone receptors and HER2 expression in primary breast carcinoma and corresponding lymph node metastasis: do we need both? Anticancer Res. 2014; 34:1435-40. 11. Suciu C, Mureșan AM, Cornea R, Suciu O, Dema A, Raica M. Semi automated evaluation of Ki 67 index in invasive ductal carcinoma of the breast. Oncol Lett. 2014; 7:107-114. Clinical Practice Guideline Update. J Clin Oncol. 2013; 31:3997-4013. 15. Azoulay S, Laé M, Fréneaux P, et al. KIT is highly expressed in adenoid cystic carcinoma of the breast, a basal-like carcinoma associated with a favorable outcome. Mod Pathol. 2005;18:1623-31. 16. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002;2:563–572. 17. Oskarsson T. Extracellular matrix components in breast cancer progression and metastasis. Breast. 2013;22:S66-72. 18. Heatley M, Maxwell P, Whiteside C, Toner P. Cytokeratin intermediate filament expression in benign and malignant breast disease. J Clin Pathol. 1995;48:26-32. 19. Laakso M, Loman N, Borg A, Isola J. Cytokeratin 5/14-positive breast cancer: true basal phenotype confined to BRCA1 tumors. Mod Pathol. 2005;18:1321-8. 20. Montel V, Huang TY, Mose E, Pestonjamasp K, Tarin D. Expression profiling of primary tumors and matched lymphatic and lung metastases in a xenogeneic breast cancer model. Am Pathol. 2005;166:1565–1579. 21. Montel V, Mose ES, Tarin D. Tumor–stromal interactions reciprocally modulate gene expression patterns during carcinogenesis and metastasis. Int J Cancer. 2006;119:251-63. 22. Urquidi V, Sloan D, Kawai K, et al. Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis. Clin Cancer Res. 2002;8:61–74. 23. Suzuki M, Tarin D. Gene expression profiling of human lymph node metastases and matched primary breast carcinomas: Clinical implications. Mol Oncol. 2007;1:172-180. 24. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA. 2003;100:8418-8423. 25. Collins LC, Martyniak A, Kandel MJ, et al. Basal cytokeratin and epidermal growth factor receptor expression are not predictive of BRCA-1 mutation status in women with triple-negative breast cancers. Am J Surg Pathol. 2009;33:1093-1097. 26. Rao C, Shetty J, Kishan Prasad HL. Morphological profile and receptor status in breast carcinoma: an institutional study. J Cancer Res Ther. 2013;9:44-9. 27. Sood N, Nigam JS. Correlation of CK5 and EGFR with Clinicopathological Profile of Triple-Negative Breast Cancer. Patholog Res Int. 2014;14:1864. 28. Rakha E, Ellis I, Reis-Filho J. Are triple-negative and basal-like breast cancer synonymous? Clin Cancer Res. 2008;14:618-9. 29. Cheang MC, Voduc D, Bajdik C, et al. Basal-like breast cancer defined by five biomarkers has superior prognostic value than triple-negative phenotype. Clin Cancer Res. 2008;14:1368-1376. _____________________________ V. Fulga et al 27 ORIGINAL ARTICLE OVEREXPRESSION OF VEGF AND VEGFR2 IN CHRONIC HEPATITIS AND LIVER CIRRHOSIS Raluca Amalia Ceauşu, Anca Maria Cîmpean*, Pușa Gaje, Marius Raica Department of Microscopic Morphology/Histology, Angiogenesis Research Center, „Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania * Piata Eftimie Murgu 2, 300041 Timisoara, Romania. E-mail: ancacimpean1972@yahoo.com ABSTRACT VEGF (vascular endothelial growth factor) and the receptor for VEGF- Flk-1 (fetal liver kinase 1) are important players of normal and pathologic angiogenesis. Also, it was proved that they are involved in tumor progression and metastasis in many tumors types by overexpression in cancer cells. Liver malignances and premalignant lesions represent controversial issues concerning VEGF and VEGFR2 (vascular endothelial growth factor receptor 2) expression and their potential involvement in the progression of inflammatory and cirrhotic lesions and also in malignant transformation is virtually unknown. The aim of this work was to describe the differentiate expression and distribution of VEGF and VEGFR2 in chronic hepatitis and liver cirrhosis, and according to these findings to better characterize the molecular profiling of liver disease with malignant transformation potential. We investigated 20 cases with chronic hepatitis and cirrhosis on specimens taken during surgery. Immunohistochemistry was performed in all cases for VEGF, VEGFR2, and FVIII related antigen (Von Willebrand factor). We found significant correlation between HAI (histological activity index) value, VEGF and VEGFR2 expression and factor FVIII related antigen in central part of specimens with chronic hepatitis. Liver cirrhosis lacks this correlation. Our findings suggested that VEGF dependent angiogenesis is more active in chronic hepatitis in the center of the lesion compared with cirrhosis where MVD (microvessel density) is higher at the periphery of the nodules. We hypothesize that the involvement of VEGF and VEGFR2 complex in development of chronic hepatitis and liver cirrhosis could be considered for the use of anti VEGF antibodies as adjuvant therapy in early stages of these diseases. Key Words: VEGF, VEGFR 2, liver cirrhosis, chronic hepatitis INTRODUCTION Vascular endothelial growth factor, also known as vascular permeability factor, is the most potent angiogenic factor. Cirrhotic livers had signiicantly higher MVD and VEGF expression compared with noncirrhotic livers. It has been shown that expression of the potent angiogenic factor, VEGF and its receptors, VEGFR-1(vascular endothelial growth factor receptor 1) and VEGFR-2, increase during the development of liver ibrosis in murine model [1]. Although current treatments for ibrotic diseases such as idiopathic pulmonary ibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular ibrosis typically target the inlammatory response, there is accumulating evidences that the mechanisms driving ibrogenesis are distinct from those regulating inlammation [2]. These changes appear to be due in part to hypoxia produced by collapsed hepatic sinusoids following the liver regeneration processes. An up-regulation of VEGF was found in the cirrhotic liver of patients with or without HCC (hepatocellular carcinoma), suggesting that this factor might be responsible for cirrhosis-associated angiogenesis [3] VEGF-positive expression was signiicantly higher _____________________________ 28 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 in surrounding cirrhotic liver tissues than in HCC, so VEGF may play an important role in the angiogenesis and prognosis of HCC, and in the angiogenesis of liver cirrhosis as well. Capsular iniltration, vascular invasion and hepatic metastasis were observed more frequently in patients with VEGF-positive expression than in those with VEGF negative expression [4]. Few data are available about expression of VEGF and VEGFR2 in chronic hepatitis and liver cirrhosis without malignant transformation. Most of the recent studies used animal models for VEGF/VEGFR2 expression in induced liver cirrhosis. The characterization of human liver tissue from liver cirrhosis and chronic hepatitis without associated hepatocellular carcinoma is a dificult approach due to the lack of biopsy material. A better understanding of VEGF/VEGFR2 expression in liver lesions without neoplastic transformation could explain some of the controversial issues concerning the involvement of these markers in tumor progression and malignant transformation and also their possible use in monitoring the effects of therapy. We hypotesized that distribution and expression pattern of VEGF and VEGFR2 in hepatocytes are different between liver cirrhosis and chronic hepatitits. Quantiication of this particular features might be useful to explain the controversial results from the literature concerning the utility of VEGF and VEGFR2 as prognostic and follow up markers, and also might be a startpoint to introduce the anti VEGF therapies in liver lesions with malignant transformation potential. MATERIAL AND METHODS We included in our study 20 patients diagnosed with liver chronic diseases diagnosed by imagistic investigations and modiied laboratory tests. All patiens had history of alchoolic intake or C hepatitits. No specimens had associated hepatocelular carcinoma or any type of metastasis. Resected liver specimens obtained by open surgery during gallbladder ablation were ixed in 10 % bufferred formalin for 48 hours and parrain embedded. Sections from each case were stained with routine haematoxylin and eosin method for histopathologic evaluation. Immunohistochemical study included antibodies against VEGF, Flk 1 and FVIII antigens. Imunohistochemical technical details are summarized in Table 1. We used normal renal parenchyma as positive control for VEGF and also the staining of blood vessels endothelium as positive control for VEGFR2. Evaluation of the specimens included the intensity and distribution of positive reaction of VEGF and correlation with F VIII positive vessels density. Microscopic observation was performed by two independent observers using Nikon Eclipse E600. cases showed macrovesicular steatosis either for chronic hepatitis and cirrhosis. The Histologic Activity Index (HAI) was evaluated for all cases with chronic hepatitis and liver cirrhosis. Nine from ten cases of liver cirrhosis had a HAI value over 14 and only one case was scored as 18. Immunohistochemistry for VEGF revealed differences of expression between chronic hepatitis and cirrhosis. In chronic hepatitis we found a weak to moderate expression for VEGF with homogeneous distribution of positive reaction in the entire hepatic parenchyma. A particular aspect was found in hepatocytes disposed close to inlammatory iniltrate. Few cells in these area were intensely stained for VEGF with cytoplasmic granular pattern (Fig. 1). We noticed that VEGF expression corelated with VEGFR 2 expression without signiicant diferences beetwen chronic hepatitis and liver cirrhosis. No coorelastion was found between VEGF and VEGFR2 expression and HAI in chronic hepatitis. The lack of such correlation was also observed in liver cirrhosis (p=0,134). Figure 1. VEGF expression in chronic hepatitis. Note the intense reaction for VEGF of hepatocytes close to the inlammatory zone. IHC, VEGF, x400. RESULTS Microscopic examination of 20 specimens revealed 10 cases with chronic hepatitits and 10 cases with cirrhosis. Histopathology of liver cirrhosis included classic nodular pattern showing nodules of liver tissue surrounded by ibrous septae. Chronic hepatitis cases were microscopically characterised by classic hepatocyte changes and severe portal inlamation with lymphoid aggregates in some cases. Association of hepatic steatosis were observed in 4 patients with cirrhosis. Most of the Liver cirrhosis showed particular aspects of VEGF expression. The intensity of reaction increased with HAI. All hepatocytes from the nodular structures expressed VEGF but with heterogeneous pattern linked Table 1. Technical details of antibodies and working systems used for immunohistochemistry _____________________________ R.A. Ceaușu et al 29 to the intensity and distribution of positive reaction. The strongest expression was noticed in at the periphery of liver nodules. In the nodules the reaction for VEGF was moderate in intensity with homogeneous distribution (Fig. 2). Figure 2. Detailed view of intense cytoplasmic granular reaction for VEGF of the peripheral hepatocytes from cirrhotic nodules. IHC, VEGF, x400. cases. Some particular aspects were found concerning the distribution of positive reaction. In chronic hepatitis VEGFR 2 was present as weak to moderate staining with diffuse pattern and increased peripheral intensity in isolated cases. A particular feature of the positive reaction for VEGFR 2 in chronic hepatitis was represented by the presence of intense staining in the hepatocytes located around portal spaces. This aspect was found predominantly in chronic hepatitis with low HAI value. Isolated hepatocytes with intense staining was observed scattered in hepatic parenchyma. VEGFR 2 expression had moderate intensity in cirrhotic lessions with differences of reaction intensity in hepatocyte of perypheric and central areas from cirrhotic nodules. In the case with highest value of HAI (18) all hepatocytes from nodular lesions intensely expressed VEGFR2 (Fig. 5). Figure 4. Correlation between HAI and FVIII-MVD in center of nodular areas of chronic hepatitis. FVIII related antigen expression was restricted to the endothelium of blood vessels.We found a double FVIII-MVD value in the central area compared with periphery in 8 cases of chronic hepatitis (80 % of total cases). Cases with HAI value ranged between 6 and 8 had the highest microvessel density in central area (Fig. 3) which was corelated with FVII-MVD values (Fig. 4). We found a signiicant correlation between HAI and FVIIIMVD values in central area of chronic hepatitis lesions (p=0.031). In liver cirrhosis we report a higher MVD for FVIII in peripheric areas of the nodules. Globally, no signiicant correlation was found between FVIII-MVD and HAI values in liver cirrhosis (p= 0,787). VEGF expression in entire cirrhotic nodule found in only one case with highest HAI (18) was correlated with high FVIII positive microvessel density. Figure 5. Particular aspect of VEGFR2 positive staining in cirrhotic nodules from high value HAI case. Note the lack of differential expression between periphery and center of the nodule. IHC, VEGFR2, x200. Figure 3. Distribution of cases according with HAI value. DISCUSSION Receptor 2 for VEGF (Flk 1) was also found in chronic hepatitits and cirrhotic nodules. Both lesions shared positive reaction for VEGFR 2 in most of the _____________________________ 30 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 Angiogenic factors represent important players involved in normal [5] and pathologic angiogenesis [68]. Vascular endothelial growth factor (VEGF) is a potent factor involved in vascular permeability and has strong mitogenic properties for endothelial cells [9]. It acts through its speciic receptors Flt1, Flk1, and Flt 4. Also, it was demonstrated that the complex VEGFVEGFR2 is involved in tumorigenesis by the expression of ligand [10] and receptors [11-13] in many tumor types. VEGF and its receptors are intensely studied in hepatocelular carcinoma [14,15]. Few data are available concerning the expression of VEGF and VEGFR2 in hepatic lesions with premalignant potential, especially on animal models induced cirrhosis [16]. Most of these studies relected the involvement of VEGF-VEGFR2 complex in vascular changes and angiogenesis process from cirrhosis and hepatocellular carcinoma [17] and did not explain the potential role of such complex in malignant transformation. Our data support the angiogenic effect of VEGF in liver lesions by correlation between the strong expression of VEGF and high micro vessel density for F VIII in cirrhosis. It was reported that serum level of VEGF tend to increases in acute and chronic hepatitis and decreases in liver cirrhosis [18] and that VEGF serum levels was not correlated with the progression of the disease. These divergent results could be explained in part by our observation concerning the distribution of VEGF and VEGFR2 in chronic hepatitis and liver cirrhosis. The disagreements concerning the positive rates of VEGF protein and mRNA level could be explained by microscopic distribution of VEGF positive hepatocytes in chronic hepatitits (close to inlammatory zones) and liver cirrhosis (limited to the peripheral hepatic cells of the nodules). Controversies could derive from the use of aleatory hepatic tissues specimens. The weak to moderate expression of VEGFR2 in chronic hepatitis with particular strong expression around portal spaces correlated with VEGF expression predominantly in hepatocytes close to inlammatory iniltrate suggested an autocrine and paracrine mechanism of VEGF action in this type of liver disease. In chronic hepatitis we can hypothesize that ibrosis could be generated by a crosstalk between VEGF secreting hepatocytes and stromal cells of the portal space connective tissue. This is in accord with many data which demonstrated the VEGF involvement in the progression of liver ibrosis and regeneration [19,20]. This autocrine and paracrine mechanisms seems to be kept in liver cirrhosis but with different patterns. The intense expression of VEGF in the periphery of the cirrhotic nodules suggested the existence of a particular subpopulation of VEGF secreting hepatocytes which are also positive for VEGFR2. These hepatocytes might have a neoplastic transformation potential. These indings are supported by a recent study of Coradinni et al [15] which compared the VEGF-A protein level in hepatocellular carcinoma (HCC), surrounding cirrhotic liver and cirrhosis at distance from the HCC. They reported a higher expression of VEGF-A protein in HCC and surrounding cirrhotic liver compared with cirrhosis lesions distant from carcinoma. In a murine model of hepatocellular carcinoma, Kornek et al [21] demonstrated that the elevated status of VEGF-A and VEGFR2 accelerated tumor growth probably by a pro-angiogenic mechanism. Bockhorn et al [22] demonstrated that anti-VEGF almost completely suppressed and VEGF markedly enhanced hepatic proliferation in the irst 24 h after partial liver resection. We hypotesize that in liver cirrhosis, overexpression of VEGF and VEGFR2 could induce a similar liver regeneration but possibly associated with a neoplastic transformation of hepatocytes. This hypotesis could be supported by the overexpression of VEGF and VEGFR 2 in other types of cancer cells. Corelation between FVIII and HAI ( p=0,031) sugests that angiogenic process is more active in chronic hepatitis compared with liver cirrhosis. CONCLUSIONS VEGF and VEGFR2 overexpression represent an early event in the development of the liver lesions with inlammatory or premalignant potential. Both markers are expressed in hepatocytes from chronic hepatitis and liver cirrhosis but the pattern and distribution of positive cells are different between these two lesions. We conclude that VEGF-VEGFR2 complex play an important role not only as angiogenic factors and promoters of liver ibrosis but also as a keyplayer of neoplastic transformation. These indings could be a evidences for the use of anti VEGF therapies as an adjuvant treatment for chronic hepatitis and cirrhosis in order to diminish the risk for malignant transformation. ACKNOWLEDGEMENTS This study was supported by TD CNCSIS 527/ 2007 and PN II 41- 054/ 2007 of Romanian Education and Research Ministry. REFERENCES 1. Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut. 2003;52:1347–1354. 2. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol. 2008;214:199-210. 3. El-Assal ON, Yamanoi A, Soda Y, et al. Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: Possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology. 1998; 27:1554-1562. 4. Bangoura G, Yang LY, Huang GW, Wang W. Expression of HIF- 2alpha/EPAS1 in hepatocellular carcinoma. World J Gastroenterol. 2004;10:525-530. 5. Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater. 2008;15:100-114. 6. Rmali KA, Puntis MC, Jiang WG. Tumour-associated angiogenesis in human colorectal cancer. Colorectal Dis. 2007;9:3-14. 7. Breen EC. VEGF in biological control. J Cell Biochem. 2007;102:1358-67. _____________________________ R.A. Ceaușu et al 31 8. Ferrara N. Role of vascular endothelial growth factor in physiologic 15. Corradini SG, Morini S, Liguori F, et al. Differential VEGF-A protein Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell 2009;29:103-112. and pathologic angiogenesis: therapeutic implications. Semin Oncol. 2002;29:10-14. 9. survival functions of vascular endothelial growth factor (VEGF). J Cell Mol Med. 2005;9:777-794. 10. Raica M, Mogoantă L, Cîmpean AM, et al. Immunohistochemical expression of vascular endothelial growth factor (VEGF) in intestinal type gastric carcinoma. Rom J Morphol Embryol. 2008;49:37-42. 11. Cimpean AM, Raica M, Encica S, Cornea R, Bocan V. Immunohistochemical expression of vascular endothelial growth factor A (VEGF), and its receptors (VEGFR1, 2) in normal and pathologic conditions of the human thymus. Ann Anat. 2008;190:238-245. expression between small HCC and cirrhosis at distance from the tumor correlates with serum VEGF-A and alpha-fetoprotein. Liver Int. 16. Xu H, Shi BM, Lu XF, Liang F, Jin X, Wu TH, Xu J. Vascular endothelial growth factor attenuates hepatic sinusoidal capillarization in thioacetamide-induced cirrhotic rats. World J Gastroenterol. 2008;14:2349-2357. 17. Amarapurkar AD, Amarapurkar DN,Vibhav S, Patel ND. Angiogenesis in chronic liver disease. Ann Hepatol. 2007;6:170-173. 18. Shi B, Wang X, Yang Z. Vascular endothelial growth factors and liver diseases. Hepatogastroenterology. 2001;48:1145-1148. 19. Novo E, Cannito S, Zamara E, et al. Proangiogenic cytokines as 12. Brychtova S, Bezdekova M, Brychta T, Tichy M. The role of vascular hypoxia-dependent factors stimulating migration of human hepatic endothelial growth factors and their receptors in malignant melanomas. stellate cells. Am J Pathol. 2007;170:1942-1953. Neoplasma. 2008;55:273-279. 13. La Rosa S, Uccella S, Finzi G, Albarello L, Sessa F, Capella C. Localization of vascular endothelial growth factor and its receptors in digestive endocrine tumors: correlation with microvessel density and clinicopathologic features. Hum Pathol. 2003;34:18-27. 14. Iavarone M, Lampertico P, Iannuzzi F, et al. Increased expression of vascular endothelial growth factor in small hepatocellular carcinoma. Journal of Viral Hepat. 2007;14:133-139. _____________________________ 32 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 20. Ueno T, Nakamura T, Torimura T, Sata M. Angiogenic cell therapy for hepatic fibrosis. Med Mol Morphol. 2006;39:16-21. 21. Kornek M, Raskopf E, Tolba R, et al. Accelerated orthotopic hepatocellular carcinomas growth is linked to increased expression of pro-angiogenic and prometastatic factors in murine liver fibrosis. Liver Int. 2008;28:509-518. 22. Bockhorn M, Goralski M, Prokofiev D, et al. VEGF is important for early liver regeneration after partial hepatectomy. J Surg Res. 2007;138:291-9. ORIGINAL ARTICLE HETEROGENEITY OF C ERB B FAMILY MEMBERS EXPRESSION IS RELATED TO CELL MORPHOLOGY AND IMMUNOPROFILE IN PITUITARY ADENOMAS Anca Maria Cîmpean1*, Eugen Melnic2, Ana Corlan3, Amalia Raluca Ceaușu1, Marius Raica1 Department of Microscopic Morphology/Histology, Angiogenesis Research Center, „Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania, 2 Department of Pathology, Nicolae Testemițanu University of Medicine and Pharmacy Chișinău, Moldova, 3 Department of Endocrinology, Vasile Goldiș University, Arad, Romania * Department of Microscopic Morphology/Histology, Angiogenesis Research Center, Piata Eftimie Murgu 2, 300041 Timisoara, Romania. E-mail: ancacimpean1972@yahoo.com 1 ABSTRACT Purpose. Epidermal growth factor receptor (EGFR, HER1) and human epidermal receptor 2 (HER2) assessement in pituitary adenomas related to hormone profile. Design and methods. For 60 retrospective cases of pituitary adenomas, we established the histopathologic diagnosis by using morphological stains, followed by case selection for immunoprofile and EGFR and HER 2 assessement. Results. More than one third of the studied pituitary adenomas (33,33%) were positive for HER2, with membranar pattern in basophilic cells and with predominantly cytoplasmic, granular pattern for acidophils cells. HER2 immuno-expression characterized PRL secreting adenomas (p=0.005) and associations between FSH-LH (p< 0.001) TSH-FSH (p=0,024) and TSH-LH (p=0.028). In situ hybridization confirmed HER2 gene amplification in 33,34% out of all positive cases for HER2 by immunohistochemistry. EGFR positivity was found significantly for GH-prolactin (p=0.000) and prolactin-ACTH (p=0.045) co-expressing pituitary adenomas, peritumoral macrophages and folliculostellate cells. Conclusions. Differential HER2 and EGFR expression related to hormone profile heterogeneity can define different subclasses of pituitary adenomas and could explain clinical, prognostic and therapeutic heterogeneity which are observed in clinical practice. Our results support re-classification of pituitary adenomas based on molecular approach which should include markers with well certified prognostic and therapeutic impact. Key Words: HER2, EGFR,pituitary adenomas, targeted therapy INTRODUCTION Targeted therapies represent the “gold standard” of various malignancies treatment intensely tested in experimental models and clinical trials [1-3], especially for those cancers with a high incidence and agressiveness as breast, renal or lung cancers [4-6]. Actual modern trends in clinical practice are focus on the discovery and testing of various thyrosine-kinase receptors family. Between them, epidermal growth factor receptor (EGFR, ErbB-1, HER1 in humans) and human epidermal growth factor receptor 2 (HER2, ErbB-1) are already therapeutic targets approved in clinical practice.Their ampliication or overexpression is rarely encountered in normal tissues but is present in the development and progression of different types of malignant tumors (mamary, colonic, gastric), being considered a poor prognostic factor for metastasis and due to this fact an agressiveness factor. HER2 is already accepted by FDA and used as a therapeutic target in case of metastatic breast cancer [7]. In gastric cancer, HER2 overexpression is widely accepted but the interpretation and the evaluation of its expression are still controversial and due to this aspect, the transtuzumab based therapy is not approved for clinical application [8]. Another receptor that is recognized as a therapeutic target and as a prognostic factor in malignant tumors is EGFR or HER1. Its identiication as an oncogene has led to the development of a targeted therapy based on geitinib and erlotinib that are applied in lung cancer [9, 10] and also cetuximab that is used in colon cancer [11]. In pituitary adenomas, EGFR and HER2 expressions are sporadically studied and, for most of the cases literature data reporting results that derive from in vitro or in vivo experimental models. Except prolactinomas, there is a totally lack of correlations between the expression of HER2, EGFR, pituitary adenomas morphologic type and hormone proile. Moreover, the data regarding the potential role of HER2 and EGFR in the development and progression of pituitary adenomas was sporadically studied and the indings were not integrated into clinical or therapeutic context, neither in a potential molecular classiication of pituitary adenomas which is more and more frequently discuss nowadays. MATERIAL AND METHODS A retrospective study has been designed by randomly choosing and including a total number of 80 _____________________________ A.M. Cîmpean et al 33 cases of pituitary adenomas which were collected by open surgery or by transsphenoidal approach and processed in a similar manner following routine standard protocols for parafin embedding. A histopathologic re-evaluation was performed by three independent experienced pathologist and a inal number of 60 cases were considered proper for the future immunohistochemical and molecular methods. We focused irst on the identiication of hormone immunoproile for growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), folliculostimulating hormone (FSH) and luteinizing hormone (LH). There were performed immunohistochemical procedures to highlight all six pituitary hormones such as growth hormone (GH, polyclonal rabbit anti-human, dilution 1 : 300), prolactin (PRL, polyclonal rabbit antihuman, dilution 1 : 250), adrenocorticotropin (ACTH, monoclonal mouse anti-human, clone 02A3, dilution 1 : 50), thyroid stimulating hormone (TSH, monoclonal mouse anti-human, clone 0042, dilution 1 : 50), luteinizing hormone (LH, monoclonal mouse anti-human, clone C93, dilution 1 : 50), and follicle stimulating hormone (FSH, monoclonal mouse anti-human, clone C10, dilution 1 : 50). All primary antibodies were supplied by Dako Cytomation, Carpinteria, USA. HER2 protein was irst quantify by immunohistochemistry using fully automated assay Bond Oracle HER2 IHC System, interpretation being performed according with the protocol provided in the speciication sheet of the assay. Because of the lack of data regarding HER2 expression in pituitary adenomas, we consider that HER2 immunohistochemistry is needed to be validated by in situ hybridisation performed with SPoT-Light® CISH Polymer Detection Kit (Invitrogen, USA). For epidermal growth factor receptor (EGFR), a mouse anti human monoclonal antibody clone EGFR.25 has been used together with Bond Reine Detection System DAB, also from Leica Microsystems. Microscopic evaluation was made by two independent observers using Nikon Eclipse E600 microscope (Nikon Corporation Japan). The images were captured and processed using Lucia G system. Statistical analysis was made using the SPSS system, version 17. The statistic methods included correlation tests such as: Pearson, Kendall and Spearman. The correlatios were considered statistically signiicant if the p value was less than 0.05. RESULTS Our evaluation included both immunohistochemical expression and gene ampliication of HER2 in pituitary adenomas. The reaction presented two patterns, membraneous (ig. 1a) and cytoplasmic (ig.1b), but the membranous one predominated. The restricted membraneous expression of HER2 was observed in the basophilic cells from pituitary adenomas. In _____________________________ 34 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 case of the acidophilic cells from pituitary adenomas, the immunohistochemical expression of HER2 was predominantly cytoplasmatic and presented a granular pattern . For most of the cases, the membraneous reaction presented a discontinuous pattern, with dotted-like contour (ig.1c), located along the circumference of the cell membrane. The cytoplasmic expression was homogenous, with a granular pattern. Regarding the distribution, positive cells presented a focal layout being organized as small groups or islands of positive tumor cells. After the quantiication of HER2 immunohistochemical expression, we assessed HER2 presence with histopathologic types and growth patterns of pituitary adenomas. From the total number of HER2 positive cases, a percentage of 40 % were mixed pituitary adenomas. The chromophobe areas were present in all mixed pituitary adenomas being most frequently associated with acidophilic type areas (in 62,5 % of cases) and basophilic type areas (37,5 % of cases). The chromophobe areas had a greater intensity of protein HER2 expression compared to the acidophilic or basophilic areas. Regarding the growth pattern, papillary type was frequently encountered, being observed in 45 % of HER2 positive cases. HER2 positive papillary adenomas were heterogeneously stained. About half of them (55,5 %) were papillary type adenomas with basophilic cells, 33,3 % were papillary adenomas with acidophilic cells, the rest (11, 2 %) being chromophobe. The compact (solid) growth pattern was also present in 45 % HER2 positive cases. The distribution of cell types was as follows : 33, 3 % were constituted of acidophilic cells, 44, 4 % were of mixed type with cromophobe cells that were associated with acidophilic or basophilic cells and 22,3 % were composed of basophilic cells. The rest of 10 % of HER2 positive pituitary adenomas presented a trabecular pattern with basophilic (50 %) and chromophobe cells (50 %). There was no statistically signiicant correlation registered between HER2 expression and the GH secreting pituitary adenomas (p=0,162). On the other hand, the PRL type hormone proile presented a statistically signiicant correlation with HER2 immunohistochemical expression. For the other types of hormones no statistically signiicant correlations were registered with HER2, the values for each of these associations being represented as follows : TSH-HER2 (p=0,232), ACTHHER2 (p=0,727), FSH-HER2 (p=0,949) and LH-HER2 (p=0,364). The study of HER2 expression in pituitary adenomas continued with the evaluation of the correlation between the pituitary hormones in order to determine whether in case of HER2 positive pituitary adenomas, hormone associations differ compared to those obtained for pituitary adenomas secreting one hormone only. Thus, in HER2 positive pituitary adenomas, GH-PRL co-expression was registered, and it had a statistically signiicant value (p=0,005). Also, a partial Kendall type correlation (p=0,036) and Spearman correlation (p=0, 032) was registered for PRL-LH co-expression. For TSH-FSH and TSH-LH, in the cases that were HER2 positive, the co-expression correlation was statistically signiicant for both cases, having a p=0,028 and a p=0,024 respectively. Another statistically signiicant correlation was registered between FSH-LH (p=0,000) for HER2 positive adenomas. Half of the HER2 positive pituitary adenomas (50%) got a +2 score when microscopically interpreting the immunohistochemical reaction, 45 % were clasiied as +1 score and only 5 % presented an intense HER2 expression which was noted +3. Due to these reasons, in order to respect the evaluation protocol for HER2 applied in mamary tumors, all +2 positive cases were reevaluated in order to detect the genetic ampliication by means chromogenic in situ hybridization (CISH). The genic ampliication conirmed the immunohistochemical expression of HER2 protein in 33,34 % of cases noted as + 2 by immunohistochemsitry. Cases with genic ampliication were extremely heterogenous, on one hand in that which considers the number of tumor cells and, on the other hand regarding the ampliication score evidenced by the nuclear presence of three or more dot-like distinct signals or clusters of positive signals(ig.2 a-d) Analysis of EGFR expression in pituitary adenomas has revealed a percentage of 48,33 % of positive cases for this marker. Three patterns of expression have been observed : the classical one, restricted to the cell membrane (M, ig.1d) with a continuous aspect and a homogenous intensity, a membraneous (continuous or discontinuous) plus cytoplasmatic one (with a granular aspect) (MC, ig. 1e) and cytoplasmic only (C, ig. 1f) with a granular or diffuse aspect. About 51,72 % of cases presented a +2 or +3 score, the rest being noted as +1. The solid growth pattern was observed in 45 % of cases, , while the papillary pattern was observed in 35 % of EGFR positive cases. The rest were, in variable proportions, trabecular type pituitary adenomas, alveolar type, fusiform type and with a mixed growth pattern. As we have done before for HER2, the study continued with the identiication of the correlations between the hormone proile and EGFR expression. Unlike HER2 quantiication in which a statistically signiicant correlation has been demostrated between HER2 expression and PRL secretion, in case of EGFR there was no co-expression demostrated for none of the evaluated pituitary hormones that were quantiied separately in comparison with EGFR expression. In order to identify the most frequent hormone associations in the EGFR positive groups the co-expressions of the pituitary hormones were quantiied. In case of GH it has been demonstrated that the most frequent co-expression was established with PRL, supported by the statistically signiicant correlation for GH-PRL secreting pituitary adenomas and EGFR (p=0,000). Partial correlation has been registered for PRLTSH secretion (Kendall=0,053, Spearman=0,047) and a complete correlation for PRL-ACTH (Pearson=0,045, Kendall=0,008 and Spearman=0,009). EGFR expression was not restricted to tumor epithelial cells only in pituitary adenomas. Other two cell types were frequently observed as being EGFR positive in the present study. Some pituitary adenomas were characterized by a massive acumulation of EGFR positive macrophages situated at the tumor periphery. EGFR expression in pituitary adenomas did not correlate with the expression of other growth factors such as VEGF but neither did it correlate with the overexpression of protein HER2. EGFR negative pituitary adenomas contain cells with a stellate morphology, having cytoplasmic processes inserted amongst tumor cells (ig.1f, inset). Due to the morphology and the pattern of distribution of these EGFR positive cells, we interpreted them as being EGFR positive folicular stellate cells. DISCUSSIONS HER1 and HER2 are recognized as being important therapeutic targets in several malignancies as breast [12, 13], lung [14, 15] or gastric cancer [16, 17]. For such tyrosin-kinase receptors tageted therapies were developed, the most known being trastuzumab, approved and used on a large scale in metastatic breast cancer [18], but also targeted anti EGFR therapies [19]. Studies regarding two receptors in pituitary adenomas are scattered. In the past 10 years less than 10 articles focused on the implication of oncoprotein HER2/ neu in pituitary adenomas were published, the majority being of an experimental type (http://www.ncbi.nlm.nih. gov/pubmed/?term=c+erbB2%2C+pituitary+adenomas). Except a single article about EGFR expression and the expression of oncoprotein HER2/neu is described in detail as an expression found in lactotrophic and corticotrophic cells [20], nowadays the prognostic role of these factors in the evolution and progression of pituitary adenomas has not been certiied. Unlike what has been described in the previously mentioned article, we did nor observe the expression of protein HER2/ neu in the normal pituitary tissue. On the other hand, the percentage of pituitary adenomas that were positive for HER2/neu oncoprotein was similar to the one encountered in case of the metastatic breast cancer, about one third of the analysed cases respectively [21]. Pituitary adenomas had a membraneous expression pattern and a differentiated cytoplasmic one depending on the cell types from pituitary adenomas structure. In basophilic cells the expression of oncoprotein HER2/neu was restricted to the membrane, in a similar manner to that observed and quantiied in tumor cells from mamary neoplasms _____________________________ A.M. Cîmpean et al 35 [22], while in acidophilic cells the expression of HER2/ neu was predominantly granular cytoplasmatic, with or without membraneous intensiication, in a similar manner to that described in the majority of gastric cancer that are positive for this marker [23]. The cytoplasmic expression of HER2/neu in gastric cancer was signiicantly correlated from a statistical point of view with the age and the expression of HER3, a well known receptor that presents the afinity to highly heterodimerize with HER2 and, through this action, being a poor prognostic factor in mamary cancer by determining Transtuzumab therapy resistance [24]. In pituitary adenomas with acidophilic cells the cytoplasmic expression was predominant, an aspect that may suggest a strong hetherodymerization of HER2 with HER3 that may constitute a poor prognostic factor in pituitary adenomas with acidophilic cells also. Unlike other studied regarding the expression of protein HER2 in pituitary adenomas, the present study observed its overexpression, also with a cytoplasmic pattern in chromophobe cells pituitary adenomas, an aspect that was not previously mentioned. Without depending on the solid or the papillary type, HER2 was positive in about 66 % of cases with acidophilic cells. In pure pituitary adenomas, HER2 overexpression was correlated with PRL secretion but not with GH secretion. This correlation sustains even more the implication of HER2 with HER3 hetherodymerization in pituitary adenomas that secrete PRL, an aspect based on the fact that there are previously published data that demonstrated the poor prognostic role of HER2/HER3 association as a factor of high agressiveness in pituitary adenomas that secrete PRL and suggested the fact that the targeted inhibition of this overexpression may be a useful alternative therapy in case of prolactinomas with an unfavourable evolution and resistant to conventional therapy [25]. In order to support this hypothesis the data published by Zhao and Ren may be taken into consideration; they have demonstrated that Neuregulin secretion (a ligand for HER3) acts as a regulator of PRL synthesis possibly by means of a paracrine/juxtacrine mechanism [26]. On the other hand, in case of pituitary adenomas with a double hormone proile, the simultaneous expression of GH-PRL was signiicantly correlated from a statistical point of view with HER2 expression. These data are accordingly to the previously published ones [27], which demonstrated the fact that, more than half of the GH-PRL pituitary adenomas cases were positive for HER2 oncoprotein and from these, half presented the overexpression of protein p53, partially ovelapped with a high proliferation index. A particular aspect of our study is represented by types TSH-LH and TSH-FSH hormone associations which were signiicantly correlated with protein HER2 overexpression. This association was not reported before in literature and may be studied as a prognostic factor. Another pair of hormones that expressed themselves in tandem in the HER2 positive pituitary adenomas was the one constituted by FSH and LH, their co-expression having a complete correlation with the overexpression of protein HER2 in pituitary adenomas. This correlation conirmed the high expression of HER2 in chromophobe cells from the studied pituitary adenomas. The high serum levels of FSH and LH were incriminated as poor prognostic factors associated with (or possible inductors) HER2 Figure 1. HER2 (a, b, c) and EGFR (d, e, f) immunohistochemical expression in pituitary adenomas. Both members have different and heterogeneous expression patterns membranar restricted (a, d) combined membranar and cytoplasmic (b, e) and cytoplasmic restricted with granular pattern (c, f). Note that EGFR expression was also present in folliculostellate cells in those case where tumor cells were EGFR negative (f, inset). _____________________________ 36 Research and Clinical Medicine, 2016, Vol. 1, Nr. 1 overexpression in menopause patients who developed breast cancer with a high proliferation rate compared to the HER2 negative ones [28]. These data may be considered indirect evidences of the fact that, FSH-LH association with HER2 overexpression is a poor prognostic factor that characterizes a subtype of pituitary adenomas. We did not notice any literature data regarding the correlation between protein HER2 and FSH/LH expression for pituitary adenomas. Also, our study revealed the fact that the associations of TSH-FSH and TSH-LH hormone expression signiicantly correlated HER2 overexpression. There is no data available in literature until now regarding these particularities of expression of the hormone proile linked with HER2 expression. If for HER2, there are few literature data, direct and indirect evidences sustain its possible implication in a well deined group of pituitary adenomas with acidophilic cells. The epidermal growth factor has, besides the well known HER2, other three receptors, HER1, HER3 and HER4 respectively. HER1, also known as EGFR, represents an intensely studied therapeutic target in malignant tumors, being the support of targeted therapy development such as Cetuximab, already applied in colorectal and lung cancers [29, 30]. The irst description of EGFR expression in pituitary adenomas was published by Cahiadarun and collaborators in 1994 [31]. The authors have noticed the fact that EGFR was expressed in 5-10 % of the cells belonging to the normal pituitary gland, especially in case of gonadotrophic and tirotrophic cells. In our study, EGFR expression in the normal pituitary gland was low and inconstant, restricted to the chromophobe cells, an aspect that may be accordingly with the already published data. All these authors observed that, non functional pituitary adenomas have the highest rate of EGFR expression. This observation partially correlates with our results, due to the fact that we did not ind any statistically signiicant correlation between the separate expression of each pituitary hormone with EGFR overexpression. However, hormones co-expression analysis, correlated with EGFR overexpression demonstrated that, in EGFR positive cases, we had a statistically signiicant correlation regarding GH-PRL co-expression and partial correlations for co-expressions such as PRL-TSH and total correlations for PRL-ACTH. The presence of PRL in all the hormone associations was anticipated and is supported by an article published in 2008 which reported that EGFR determines the increase of PRL secretion in an experimental model and that EGFR inhibition induces the control of tumor growth and the inhibition of PRL secretion, which may constitute a promising therapeutic taget in case of prolactinomas resistent to the dopaminergic treatment or in rare cases of prolactinomas with malignant transformation [32]. Another particular aspect is represented by the PRLACTH-EGFR association, which was completely. Fukuoka and collaborators studied the inhibitory effects of Geitinib Figure 2. Chromogen in situ hybridization for HER2 oncoprotein in pituitary adenomas conirming HER 2 gene ampliication with variable degrees: 1(a), 2(b), 3(c) and 4(d). _____________________________ A.M. Cîmpean et al 37 on ACTH secretory isolated cells from pituitary adenomas that secrete ACTH of human and canine origin and demonstrated that EGFR inhibition determines the decrease of tumor dimensions, corticosteroids level as well as the reduction of the omental adipose mass [33]. Due to all these results, the authors consider that the therapy based on Geitinib may be a welcomed alternative that may reduce surgical interventions carried out for these types of pituitary tumors, of course with an increase in the patient’s comfort. Other studies regarding EGFR expression in pituitary adenomas support our data. Kontogeorgeous and collaborators demonstrated the EGFR overexpression in over 60 % of corticotrophic pituitary adenomas and only in 20 % of the somatotrophic and latotrophic pituitary adenomas [34]. These results sustain the statistically signiicant correlation obtained for the association of GH-PRL and of PRL-ACTH with EGFR in our study. The decrease of the protein p27/Kip1 in the cases of ACTH secreting pituiray adenomas, correlated with the inhibition of the same type of protein by EGFR expression, suggests the fact that EGFR is implicated in the tumorigenesis of ACTH secreting pituitary adenomas by means of a p27/ Kip1 inhibition mechanism [35]. GH-PRL secreting adenomas are characterized by HER2 and EGFR overexpression this inding suggesting a possible existence of different molecular subclasses with a speciic phenotype. Foliculo/stelate cells are important components of pituitary adenomas because of their ability to secrete several growth factors with prognostic or therapeutic role. Presence of distinct pituitary adenomas subclasses is also sustain on the one hand by association between FSH/LH and HER2 overexpression and, on the other hand by EGFR association with ACTH secreting pituitary adenomas with or without PRL secretion. Pituitary adenomas overexpressing growth factors could be targets for new therapies based on humanized monoclonal antibodies against such factors. In this moment, this kind of therapy is not usually applied in pituitary adenomas treatment. Based on different growth factors expression, a reclassiication of pituitary adenomas is required. Together with hormone proile, this new classiication should include growth factors which can deine and identify new molecular classes of pituitary adenomas with different clinical and therapeutic behaviour inluencing recurrences and prognosis. 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