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Journal of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology, Italian Division of the International Academy of Pathology ORIGINAL ARTICLES 425 Cytologic diagnosis of spinal cord ependymoma in cerebrospinal fluid A. Kalogeraki, D.Tamiolakis, V. Sinatkas, A. Xekalou, M. Papadakis, E.N. Stathopoulos 428 Diffuse leptomeningeal glioneuronal tumours: clinico-pathological follow-up M.P. Gardiman, M. Fassan, P. Nozza, E. Orvieto, M.L. Garrè, C. Milanaccio, M. Severino, G. Perilongo, F. Giangaspero

REVIEW

Periodico bimestrale – POSTE ITALIANE SPA - Spedizione in Abbonamento Postale - D.L. 353/2003 conv. in L. 27/02/2004 n° 46 art. 1, comma 1, DCB PISA Aut. Trib. di Genova n. 75 del 22/06/1949

432 Molecular pathology of colorectal carcinoma. A systematic review centred on the new role of the Pathologist A. Remo, M. Pancione, C. Zanella, R. Vendraminelli

CASE REPORTS 442 High-grade primary peritoneal serous papillary carcinoma with a wide differential diagnosis: a diagnostic challenge S. Gilani, R. Tashjian, L. Fathallah 446 Pancreatic heterotopia in the gallbladder neck associated with chronic cholecystitis F. Limaiem, S. Jedidi, F. Hassan, S. Korbi, S. Aloui, A. Lahmar, S. Bouraoui, S. Mzabi 449 Ovarian fibromatous tumours of uncertain biological potential: study of three cases T. Pusiol, M.G. Zorzi, D. Morichetti 452 Subscapular elastofibrolipoma: a new variant type among elastofibromas or lipomas? A case report S. Squillaci 455 A case of Stengel-Wolbach sclerosis: a half century after the last report B.J. Rocca, M.R. Ambrosio, M.A.G.M. Butorano, A. Ambrosio, R. Santopietro, S. Lazzi

458 Memoir of Karl Lennert S.A. Pileri

Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology

Vol. 104 December 2012

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Journal of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology, Italian Division of the International Academy of Pathology

Editor-in-Chief Marco Chilosi, Verona Associate Editor Roberto Fiocca, Genova Managing Editor Roberto Bandelloni, Genova Scientific Board R. Alaggio, Padova G. Angeli, Vercelli M. Barbareschi, Trento C.A. Beltrami, Udine G. Bevilacqua, Pisa M. Bisceglia, S. Giovanni R. A. Bondi, Bologna F. Bonetti, Verona C. Bordi, Parma A.M. Buccoliero, Firenze G.P. Bulfamante, Milano G. Bussolati, Torino A. Cavazza, Reggio Emilia G. Cenacchi, Bologna P. Ceppa, Genova C. Clemente, Milano M. Colecchia, Milano G. Collina, Bologna P. Cossu-Rocca, Sassari P. Dalla Palma, Trento G. De Rosa, Napoli A.P. Dei Tos, Treviso L. Di Bonito, Trieste C. Doglioni, Milano V. Eusebi, Bologna G. Faa, Cagliari F. Facchetti, Brescia G. Fadda, Roma G. Fornaciari, Pisa M.P. Foschini, Bologna F. Fraggetta, Catania E. Fulcheri, Genova P. Gallo, Roma F. Giangaspero, Roma W.F. Grigioni, Bologna

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Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology

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Vol. 104 December 2012

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CONTENTS Original articles Cytologic diagnosis of spinal cord ependymoma in cerebrospinal fluid A. Kalogeraki, D.Tamiolakis, V. Sinatkas, A. Xekalou, M. Papadakis, E.N. Stathopoulos Ependymoma cells are known to rarely exfoliate into cerebrospinal fluid (CSF). However, the frequency of CSF involvement in patients with ependymoma is unclear, and to the author’s knowledge the cytomorphologic features of tumour cells have not been well described to date. In this study, the CSF findings in a patient with ependymoma and the cytopathological features of this tumor are reported. The patient presented at the University Hospital of Heraklion, Crete, suffering from a chest to back pain. Computed tomography, scanning and magnetic resonance imaging (MRI) were performed and a mass of 3x2 cm in the thoracic aspect of the spinal cord was found. A sample of cerebrospinal fluid (CSF) was sent for cytologic examination and a diagnosis of ependymoma was made. A biopsy was performed and histology confirmed the cytologic diagnosis of ependymoma grade II (WHO). Exfoliated cells from ependymomas of spinal cord are rarely recognizable in CSF samples. Except in patients with myxopapillary tumours and anaplastic tumours, cytomorphologic features of ependymoma have been described only in case reports of intraoperative imprinting or fine needle aspiration biopsies (FNABs) and not in CSF cytology. Diffuse leptomeningeal glioneuronal tumours: clinico-pathological follow-up M.P. Gardiman, M. Fassan, P. Nozza, E. Orvieto, M.L. Garrè, C. Milanaccio, M. Severino, G. Perilongo, F. Giangaspero Glioneuronal tumours are a group of primary brain neoplasms of relatively recent acquisition in the World Health Organization (WHO) Classification of the Central Nervous System tumours. In diagnostic practice it is still possible to encounter glioneuronal tumours that cannot be placed into any of the well-defined WHO categories despite a growing list of entities. We have recently published four paediatric cases of diffuse leptomeningeal tumours that cannot be easily classified in the currently used CNS WHO classification, but which have histological and immunohistochemical criteria to be considered as glioneuronal tumours. The clinical, neuroradiological and pathological long-term follow-up of an unusual diffuse leptomeningeal glioneuronal tumour is presented in the article.

Review Molecular pathology of colorectal carcinoma. A systematic review centred on the new role of the Pathologist A. Remo, M. Pancione, C. Zanella, R. Vendraminelli Colorectal carcinoma (CRC) is the second most frequent malignant disease in developed countries. Many aetiological factors have been reported in CRC development including genetic or non-genetic (environmental) elements. Independently of these, three groups of alterations have been implicated: 1) chromosomal instability (CIN); 2) microsatellite instability (MSI); 3) CpG island methylator phenotype (CIMP). A different multistep association between these alterations contributes to determine three distinct developmental pathways: traditional, alternative and serrated. Each genotypic CRC assessment is associated with specific morphologic or clinical features. Pathologists have to consider the morphologic and clinical features of each CRC when study tumours with molecular tests. Chromatin remodelling is extremely dynamic and depends on several DNA-based

processes, such as transcription, DNA repair and replication. The recent results with whole genome sequencing in a vast array of cancers have provided a catalogue of genetic lesions in chromatin modifiers that were previously unappreciated. It has revealed surprising facts about mutations in several SWI/SNF complex members in many malignancies including CRC. The loss of INI1 expression is detected at a low rate in CRC and may be associated with differentiation grade and survival. Accumulating evidence suggests a critical role of the epithelial mesenchymal transition (EMT) in cancer progression. Some results support the existence of crosstalk between EMT and epigenetic modifications in the MSI-CRC group. We have summarized the role of genetic/epigenetic changes in the origin of the multiple CRC pathway, taking into account current knowledge of pathogenesis and feasibility of designing novel therapeutic approaches. .

Case reports High-grade primary peritoneal serous papillary carcinoma with a wide differential diagnosis: a diagnostic challenge S. Gilani, R. Tashjian, L. Fathallah Serous surface papillary carcinoma of the peritoneum is a rare malignant epithelial tumour that is histologically indistinguishable from high-grade serous papillary carcinoma of ovarian origin. We herein report a case of 66-year-old female with a history of breast carcinoma who presented with abdominal distension and shortness of breath. Imaging studies at the time of initial workup revealed multiple masses in abdominal cavity, and suspicion was high for metastatic carcinoma. The patient underwent exploratory laparotomy with total abdominal hysterectomy and bilateral salpingo-oophorectomy, partial colectomy, and omentectomy. Gross inspection of the omentum showed diffuse tumour caking, and frozen section evaluation of the omental nodules revealed adenocarcinoma with papillary features. Histological examination showed a high-grade papillary carcinoma with numerous psammoma bodies predominantly present within the omentum and peritoneum. Eventually, this case was determined to be a high-grade primary peritoneal serous papillary carcinoma involving the omentum, colon, and appendix, as well as the surfaces of the uterus, bilateral ovaries, and bilateral fallopian tubes. Knowledge of the primary organ of origin in cases of advanced papillary serous carcinomas is essential for both prognosis and staging, as well as for initiation of appropriate treatment. Close post-therapy follow up is required.

Pancreatic heterotopia in the gallbladder neck associated with chronic cholecystitis F. Limaiem, S. Jedidi, F. Hassan, S. Korbi, S. Aloui, A. Lahmar, S. Bouraoui, S. Mzabi The gallbladder is an unusual location of pancreatic heterotopia defined as the presence of pancreatic tissue lacking anatomical and vascular continuity with the main body of the gland. A 55-year-old previously healthy male patient presented with repeated attacks of right hypochondriac pain and vomiting. On physical examination, the right upper abdomen was tender to palpation with a positive Murphy’s sign. Abdominal ultrasonographic examination showed multiple gallstones within a thin-walled gallbladder. Laparoscopic cholecystectomy was performed with uneventful recovery. Macroscopic examination of the surgical specimen revealed a yellowish intramural nodule measuring 7 mm close to the neck of the gallbladder. Histological examination revealed chronic cholecystitis and subserosal foci of heterotopic pancreas made up of exocrine acinar and ductal elements without islet cells corresponding to incomplete

heterotopia. Heterotopic pancreas is usually detected as an incidental microscopic finding in a gallbladder specimen removed by cholecystectomy. Pre-operative diagnosis is difficult primarily due to its non-specific clinical features. Ovarian fibromatous tumours of uncertain biological potential: study of three cases T. Pusiol, M.G. Zorzi, D. Morichetti Background. The classification of ovarian fibromatous tumours with high mitotic activity is controversial. Cases report. The first case was an 18x17x10 cm left ovarian fibromatous tumour with 17 mitoses/10 HPF detected in a 44-year-old woman. The second case consisted of a 4 x 2.5 x 2 and a 2.5 x 2.5 x 2 cm fibrmatous tumours found, respectively, in the left and right ovaries of a 67-year-old woman. The mitotic count varied from 4 to 6/10 HPF. Conclusions. Prat&Scully reported that mitotic activity was the most important factor in diagnosing fibrosarcomas, and that cellular pleomorphism was not reliable. Irving et al. suggested that cellular fibromatous neoplasms with bland nuclear features and mitotic count of ≥ 4 MFs/10 HPFs should be considered mitotically-active cellular fibromas rather than fibrosarcomas. We propose the term ‘fibromatous tumours of uncertain biological potential’ when an average mitotic count of 4 or more per 10 HPFs are found and nuclear atypia and necrosis are absent.

Subscapular elastofibrolipoma: a new variant type among elastofibromas or lipomas? A case report S. Squillaci Elastofibroma is a rare soft tissue benign fibrous proliferation that characteristically occurs in periscapular soft tissues of the elderly, particularly in females, with typical morphological features consisting of an admixture of excessive collagen and abnormal elastic fibers displaying a beaded or globular appearance. Here we report an unusual, recently described histological variant with unclear origin, showing the presence of abundant mature fat tissue, named ‘elastofibrolipoma’, which could lead to confusion with other adipose and mesenchymal cell tumour proliferations. The issue as to whether elastofibrolipoma is reactive or neoplastic, variant of elastofibroma or lipoma, remains controversial. Differential diagnostic problems and histogenetic considerations are provided. A case of Stengel-Wolbach sclerosis: a half century after the last report B.J. Rocca, M.R. Ambrosio, M.A.G.M. Butorano, A. Ambrosio, R. Santopietro, S. Lazzi Stengel-Wolbach sclerosis is a rare form of granulomatosis of the lymphoid tissue, with largely predominant involvement of the spleen. Firstly described by Wolbach in 1910, it has disappeared from the literature from the beginning of the last century. Herein, we describe the case of a 29-year-old female with splenomegaly due to multiple nodular lesions. On the basis of morphological and molecular biology findings, a diagnosis of Stengel-Wolbach sclerosis of unknown aetiology was made. The presentation of this case may reopen the discussion on this enigmatic entity.

pathologica 2012;104:425-427

Original article

Cytologic diagnosis of spinal cord ependymoma in cerebrospinal fluid A. KALOGERAKI, D.TAMIOLAKIS, V. SINATKAS, A. XEKALOU, M. PAPADAKIS, E.N. STATHOPOULOS Department of Pathology-Cytopathology, University of Crete, Faculty of Medicine, Heraklion, Crete, Greece

Key words Ependymoma • CSF • Cytology • Histopathology • Immunocytohemistry

Summary Ependymoma cells are known to rarely exfoliate into cerebrospinal fluid (CSF). However, the frequency of CSF involvement in patients with ependymoma is unclear, and to the author’s knowledge the cytomorphologic features of tumour cells have not been well described to date. In this study, the CSF findings in a patient with ependymoma and the cytopathological features of this tumor are reported. The patient presented at the University Hospital of Heraklion, Crete, suffering from a chest to back pain. Computed tomography, scanning and magnetic resonance imaging (MRI) were performed and a mass of 3x2 cm in the thoracic aspect of the

spinal cord was found. A sample of cerebrospinal fluid (CSF) was sent for cytologic examination and a diagnosis of ependymoma was made. A biopsy was performed and histology confirmed the cytologic diagnosis of ependymoma grade II (WHO). Exfoliated cells from ependymomas of spinal cord are rarely recognizable in CSF samples. Except in patients with myxopapillary tumours and anaplastic tumours, cytomorphologic features of ependymoma have been described only in case reports of intraoperative imprinting or fine needle aspiration biopsies (FNABs) and not in CSF cytology.

Introduction

Cytologic examination of CSF remains clinically useful when leptomeningeal involvement by a neoplastic process is suspected  4. Most ependymomas arise in the vicinity of the ventricles and spinal cord, and even normal ependymal cells occasionally exfoliate into CSF. Despite this, leptomeningeal spread of ependymoma is uncommon  5 6. As a result, studies regarding the cytologic diagnosis of exfoliated tumor cells from ependymomas are to our knowledge very limited. Cytomorphologic features of ependymoma variants have been described only in fine needle aspiration (FNA) cytology and not in CSF cytology 7 8. In this report, we diagnosed a case of ependymoma in CSF that was histologically confirmed as ependymoma (WHO grade II).

Ependymal neoplasms are rare tumours that arise from the ependymal lining of the cerebral ventricles and remnants of the central canal of the spinal cord. They is a group of glial tumours that exhibit ependymal differentiation, including subependymoma (WHO grade I), myxopapillary ependymoma (WHO grade I), classic ependymoma (WHO grade II) and anaplastic ependymoma (WHO grade III). Most are sporadic; ependymomas may be seen as part of neurofibromatosis type 2, a hereditary cancer predisposition syndrome with germline mutations in the NFF2/Merlin gene. Recent evidence supports radial glia as the candidate cell of origin for ependymomas  1. Posterior fossa infra-and supra-tentorial ependymomas are more common in children, accounting for approximately 6% to 12% of all paediatric intracranial neoplasms and representing the third most common brain tumour in this age group after astrocytomas and medulloblastoma. Spinal ependymomas occur more often in adults (peak from age 30-40), and constitute approximately 60% of spinal tumours 2 3.

Patient, methods, and results A 53-year-old-male from Crete, Greece presented to our hospital one month prior. He suffered from chest to back pain. His medical history was uneventful. A chest

Correspondence

Alexandra Kalogeraki, Department of Pathology-Cytology University of Crete, Medical Faculty, PO Box1393, Heraklion, 71110 Crete, Greece - Tel. +30 28 10394692 - Fax +30 28 10394694 - E-mail: [email protected]

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X-ray was normal but computed tomography, scanning and magnetic resonance imaging (MRI) revealed a mass of 3x2 cm in the thoracic aspect of the spinal cord. No other relevant findings were found. He had no lymphadenopathy and haematological evaluation was within normal limits. A diagnostic paracentetesis and CSF sample collection was performed. The CSF sample was sent for cytologic evaluation. Cytologic slides were prepared from the fluid after cytocentrifugion for 4 min at 400 rpm. Four slides were fixed in 95% ethanol and stained with Papanicolaou stain, and four were air-dried for Giemsa stain and immunocytochemistry. The observation of smears revealed the presence of neoplastic cells with large and hyperchromatic oval-tospindle shaped nuclei, without nucleoli and the absence of mitoses. The cytoplasm of the neoplastic cells was scanty and basophilic (Fig. 1). An immunocytochemical study was carried out on air-dried smears, which showed that these atypical cells were of glial origin, GFAP and S-100 positive (Fig.  2). The cytologic diagnosis was of ependymoma. Multiple biopsies of the tumour were obtained and histology confirmed the cytologic diagnosis. In biopsy specimens, perivascular pseudorosettes and canals of neoplastic hyperchromatic cells were identified (Figs. 3, 4). Mitotic figures and necrosis were not found. Immunohistochemically the tumour cells were found to be positive for GFAP and S-100 protein. The final histologic diagnosis was ependymoma (WHO grade II).

spinal cord (particularly in adult patients) and the third most common paediatric central nervous system (CNS) tumour, representing up to 30% of intracranial tumours in individuals under 3 years old  9  10. Infratentorial tumours have their peak age of occurrence in the first decade, while spinal tumours tend to peak from 30-40 years. They have an equal gender distribution, though they are nearly twice as frequent in Caucasians compared to African-Americans. Intracranial ependymomas typically result in blockage of CSF pathways, signs and symptoms related to hydrocephalus and increased intracranial pressure. Spinal ependymomas may cause back pain and motor and/or sensory deficits, depending on their specific anatomic involvement. The CSF detection of ependymoma is surprisingly uncommon compared with other primary CNS neoplasms, such as medulloblastomas and malignant astrocytomas. However in other studies  11 12, patients with ependymoma and positive CSF samples constituted only a very small proportion of cases. The distinction between exfoliated spinal ependymoma cells and normal ependymal cells or cells derived from choroids plexus can be difficult, if not impossible, especially in hypocellular and/or poorly preserved specimens. This may render tumour cells unrecognizable as neoplastic, and may be a potential source of false negative results  13. In this case, we report the cytology and immunocytology of a classical spinal ependymoma in a CSF sample, confirmed histologically as ependymoma WHO grade II.

Discussion

Conclusions The value of CSF cytology in the evaluation of patients with ependymoma may be limited and the overall detection rate of ependymoma cells in CSF is low.

Ependymomas are the most common tumour of the

Fig. 1. Neoplastic cells with large and hyperchromatic oval-tospindle shaped nuclei, without nucleoli and mitoses. CSF. Giemsa stain x600.

Fig. 2. Ependymoma cells. CSF. S-100 immunostain x600.

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Cytologic diagnosis of spinal cord ependymoma in cerebrospinal fluid

Fig. 3. Ependymoma. Tissue section. H&E x200.

Fig. 4. Ependymoma. Tissue section. H&E x200.

References

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1

Fullar CH, Narendro S, Tolicica I. Ependymal tumors. In: Adesina AM, Tihan T, Fuller CE, et al., eds. Atlas of Pediatric Brain Tumors. New York: Springer 2010, pp. 47-59.

2

Bouffet E, Perilongo G, Canate A, et al. Intracranial ependymomas in children: a critical review of prognostic factors and a plea for cooperation. Med Pediatr Oncol 1998;30:319-29.

3

Chamberlain Mc. Ependymomas. Curr Neural Neurosci Rep 2003;3:193-9.

4

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pathologica 2012;104:428-431

Original article

Diffuse leptomeningeal glioneuronal tumours: clinico-pathological follow-up M.P. GARDIMAN1, M. FASSAN1, P. NOZZA2, E. ORVIETO1, M.L. GARRÈ3, C. MILANACCIO3, M. SEVERINO4, G. PERILONGO5, F. GIANGASPERO6 1  Department of Medicine (DIMED), University Hospital of Padua, Padua, Italy; 2 Pathology Unit, G. Gaslini Institute, Genoa, Italy; 3  Neuro-Oncology Unit, G. Gaslini Institute, Genoa, Italy; 4 Neuroradiology Unit, G. Gaslini Institute, Genoa, Italy; 5 Department of Woman’s and Child’s Health, SDB, Division of Paediatrics, University Hospital of Padua, Padua, Italy; 6 Department of Pathology, Policlinico Umberto I, University La Sapienza, Rome, Italy

Key words Glioneuronal tumours • Leptomeningeal tumours • Paediatric oncology

Summary Glioneuronal tumours are a group of primary brain neoplasms of relatively recent acquisition in the World Health Organization (WHO) Classification of the Central Nervous System tumours. In diagnostic practice it is still possible to encounter glioneuronal tumours that cannot be placed into any of the well-defined WHO categories despite a growing list of entities. We have recently pub-

lished four paediatric cases of diffuse leptomeningeal tumours that cannot be easily classified in the currently used CNS WHO classification, but which have histological and immunohistochemical criteria to be considered as glioneuronal tumours. The clinical, neuroradiological and pathological long-term follow-up of an unusual diffuse leptomeningeal glioneuronal tumour is presented herein.

Introduction

We previously reported the clinico-pathological features of four cases that did not easily fall into the WHO classification and proposed their possible distinct pathological and clinical entity in the group of glioneuronal tumours: diffuse leptomeningeal glioneuronal tumours 6. In our original series, three patients showed an indolent course of disease and one developed a left intraventricular frontal lesion with anaplastic/de-differentiating features  6. While showing varying degrees of glial and neuronal cell differentiation and clinical aggressiveness, the four cases collectively represented a distinct pathological entity. The complexity of the investigation was determined by the paucity of accessible material: small dural biopsies without follow-up re-sampling in three cases and a specimen from incomplete resection of the intraventricular lesion. We present the clinical, neuroradiological and histological follow-up (67 months after diagnosis) of one of the previous series (case #4), in which systematic follow-up and a new biopsy allowed better definition of histopathological features and clinical behavior.

One of the novel most intriguing research fields in neuropathology is the definition of diagnostic criteria and natural history for glioneuronal tumours  1  2. This heterogeneous group of primary brain neoplasms has been recently expanded and introduced in the last World Health Organization (WHO) classification of tumours of the Central Nervous Systems with three new entities (i.e., papillary glioneuronal tumour, rosetted glioneuronal tumour with neuropillike islands and rosette-forming glioneuronal tumour of the fourth ventricle) 1 2. This classificatory extension is in part the consequence of the constantly increasing availability of novel immunostains, which have enabled pathologists to more readily identify neuronal differentiation in tumours that morphologically resemble glial neoplasms. However, despite a growing list of new entities, in routine diagnostic practice it is still possible to encounter glioneuronal tumours that cannot be placed into any of the welldefined WHO categories. Moreover, due to the relative rarity of these neoplasms and lack of long-term follow-up studies, further clinical investigations are warranted 3-5.

Correspondence

Marina Paola Gardiman, Surgical Pathology & Cytopathology Unit, Department of Medical Diagnostic Sciences and Special Therapies, University Hospital of Padua, via Giustiniani 2, 35128 Padua, Italy - Tel. +39 049 821 1316 - Fax +39 049 827 2277 E-mail: [email protected]

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Clinical history and neuroimaging In 2007, a 13-year-old boy, with an unremarkable medical history, was admitted to our institution because of sudden awakening at night, confusional state, dysarthria and dyslalia. MRI demonstrated few non-enhancing cyst structures scattered over the cerebellar surface, and postgadolinium images revealed diffuse leptomeningeal thickening and enhancement particularly in the cerebral sulci, cerebellar vermis, basal prepontine and ambient cisterns, as well as within the internal auditory canals with no evidence of intraparenchymal involvement. A first small leptomeningeal biopsy was collected and a final diagnosis of diffuse leptomeningeal glioneuronal tumor was achieved  6. In the 22 months after first presentation and biopsy, the patient has been systematically monitored without treatment. During this follow-up period, at age 14, the boy presented slight clinical worsening consisting in the appearance of seizures, character changes (hyperactivity, mood imbalance) and one episode of partial epilepsy; neurological examination remained normal without focal deficits. Systematic MRI follow-up reviewed by a panel of neuroradiologists at another hospital (Giannina Gaslini Hospital) revealed a considerable worsening of all lesions in the brain and spinal cord. In fact, MRI showed a thick leptomeningeal enhancement of the spinal cord and cauda equina, with secondary nodular infiltration of the conus medullaris and large neoplastic deposits within the dural sac at the level of S2 (Fig. 1A-D). Intracranially, there were multiple cysts scattered over the surface of cerebellum, medial thalami and lateral ventricles. Diffuse leptomeningeal enhancement was present, especially involving the cerebellum, brainstem, basal and chiasmatic/suprachiasmatic cisterns, supra-pineal and optic recesses, and internal auditory canals. Because the first histological diagnosis, was not considered sufficient for a clear classification of histological features and for treatment purpose (i.e., unclear grading of the neoplasm) a new biopsy was performed at the level of the spinal site of involvement (S1-S2 level). The neoplastic tissue within the sacral dural sac was partially removed and an adequate biopsy sampling was obtained. Based on the final diagnosis of a low-grade glio-neuronal tumour with clinical and pathological features of diffuse leptomenigeal glioneuronal tumour, and according to the SIOP Low Grade protocol, a chemotherapeutic regimen with vincristine and carboplatin was adopted. The patient presented unusual severe neuro-toxicity to vincristine and had prolonged myelosuppression. Treatment was shifted to oral temozolamide and then to valproic acid (15 mg/kg twice a day; aiming to maintain a blood level between 100 and 150). Valproic acid was administered for its antiepileptic and behavioural effects, but also for its potential differentiation effect on glial cells 7. At 3 months since initiation of valproic acid, neurological deficits and the leptomeningeal involvement visible at MRI significantly improved. The patient is currently at 67 months since first diagnosis and 45

Fig. 1. Craniospinal MRI findings of the present case at 54 months after diagnosis. (A) Spinal sagittal contrast-enhanced T1-weighted image shows diffuse leptomeningeal thickening and enhancement (white arrows) infiltrating the conus medullaris with a nodular pattern (white arrowheads) and involving the cauda equina (black arrowheads). There is bulky neoplastic tissue (black arrow) within the dural sac at S2 level (site of biopsy). Notice the abnormal enhancement of cerebrospinal fluid signal and the vertebral scalloping of S1-S2 (empty arrows). (B) Cranial axial T2-weighted image reveals multiple hyperintense cysts within the cerebellar folia (arrowheads). Axial (C) and sagittal (D) contrast-enhanced T1weighted images demonstrate leptomeningeal enhancement on the cerebellar and pontine surface (empty arrows), IV ventricle (arrows) and within enlarged internal auditory canals (arrowheads). Nodular foci of leptomeningeal thickening are evident in the suprapineal recess and chiasmatic/suprachiasmatic cisterns (black arrows).

months from the second biopsy. His neurological status is improving. Valproic acid therapy is ongoing with no apparent side effects. Steroids were tapered and interrupted 3 years ago. The boy is enjoying school and has a nearly regular life.

Histopathology The surgical specimens consisted of several fragments ranging in size from 2 mm to 8 mm. The neoplastic tissue showed a predominant pseudopapillary architectural pattern, characterized by perivascular pseudorosettelike structures in which thickened and hyalinized wall vessels were surrounded by cells featuring clear cytoplasm, monomorphic round nuclei with finely granular dispersed chromatin and occasional small nucleoli. Interspersed between these pseudopapillary structures there were collections of neurocytic cells resembling oligodendroglial cells or occasionally multinucleated cells reminiscent of dysplastic ganglion cells (Fig.  2). In one of the samples, the tumour focally infiltrated the

430

Fig. 2. Representative histopathological and immunohistochemical features of the described tumour. Tumour samples were composed of a monotonous population of round cells (A) arranged in pseudopapillary structures (B). Tumour cells showed diffuse immunoreactivity for synaptophysin (C) and GFAP (D). (Original magnifications 20x and 40x).

adjacent meninges. No areas of necrosis, any Rosenthal fibre or vascular endothelial proliferation were present; rare mitoses were found and cell proliferation, as evaluated with MIB-1 labelling index, was less than 3%. Immunohistochemical analysis was performed using the standard avidin-biotin-peroxidase method, as previously described 6 8. Tumour cells showed diffuse immunoreactivity for GFAP, synaptophysin and S100, patchy reactivity for Neu-N and were negative for both neurofilaments and epithelial membrane antigen (Fig.  2). Fluorescence in situ hybridization (FISH) was performed on formalin-fixed, paraffin-embedded tissues and revealed no cytogenetically detectable alterations concerning regions 1p and 19q.

Discussion Glioneuronal tumours are considered a heterogeneous group of primary brain neoplasms characterized by a biphasic neurocytic and glial population: the neuronal component consists of synaptophysin-positive neurocytes with round nuclei and clear cytoplasm occasionally intermingled with neurons and intermediate-sized “ganglioid” cells, whereas the glial component exhibits features of GFAP positive astrocytes  1 2. The histogenesis of these tumours is unclear, but an origin from multipotent precursors capable of divergent differentiation has been suggested 9. Despite the growing list of new nosological entities, in diagnostic practice it is still possible to encounter glioneuronal tumours that cannot be placed into any of the welldefined WHO categories. In this regard, we previously

M.P. GARDIMAN et al.

reported the unique clinico-pathological features of four cases and proposed their possible distinct pathological and clinical entity in the group of glioneuronal tumours, namely diffuse leptomeningeal glioneuronal tumours 6. Leptomeningeal dissemination in glioneuronal tumours is considered to be very rare; however, it has been reported more frequently than in the past, probably because of the more widespread use of magnetic resonance imaging (MRI) in tumour staging and follow-up  10. In fact, the incidence of leptomeningeal dissemination in low-grade gliomas (a name for a wide variety of neoplasms of glial or mixed glial-neuronal origin) is estimated to be 5% at diagnosis and 7-10% at subsequent tumour progression  11. Well-established examples of glioneuronal tumours with leptomeningeal dissemination include ganglioglioma and pleomorphic xanthoastrocytoma  12. The existence of isolated groups of glioneuronal progenitor cells entrapped in the context of the leptomeninges during primitive migration could be a possible explanation for the origin of these diffuse leptomeningeal tumours. Theoretically, these embryonic cells could be capable of divergent differentiation with neuronal, oligodendroglial and astrocytic features  13-15, supporting the existence of a new “superfamily” of tumours with oligodendroglial and neurocytic potential in which this case of diffuse leptomeningeal glioneuronal tumours could be included. The satisfactory sampling of the lesion in the present case allowed adequate histological analysis, demonstrating the glioneuronal commitment of tumour cells. Small biopsy specimens (and the subsequent unfeasibility to perform immuno-histochemical-molecular studies) could be a reason for misdiagnosis as diffuse leptomeningeal oligodendroglioma in similar cases. Actually, we believe that similar cases have already been published in the literature under the name of diffuse leptomeningeal oligodendrogliomas  10  16-23 or neurocytomas/gangliocytomas  24. From a histological point of view, the present case was composed of cells characterized by round to oval nuclei with finely granular dispersed chromatin and inconspicuous nucleoli with clear oligodendrocyte-like cytoplasm. In some of the similar previously described cases, these histological findings might have favoured the diagnosis of oligodendrogliomas and oligodendrogliomatosis 10 16-23. In oligodendrogliomas, synaptophysin immunoreactivity is usually caused by residual parenchyma and is frequently seen at the infiltrating tumour borders. Surprisingly, recent data have demonstrated a paranuclear dot-like pattern of immunoreactivity in morphologically classic oligodendroglioma containing 1p and 19q co-deletions  25. In our cases, the concurrent presence of diffuse synaptophysin and nuclear NeuN immunoreactivities strongly support a glioneuronal commitment of the neoplasms. Interestingly, in the other similar cases presented in literature, considered as diffuse leptomeningeal oligodendrogliomas, the immunohistochemical profiles are quite variable and sometimes inconsistent. This could be related to the glioneuronal nature of the described neoplasms and further underlines the difficulty in classifying these tumors.

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In contrast to the aggressive transformation observed in one of the previously described cases (case #3)  6, the present patient developed an indolent course of the disease that correlated well with pathological diagnosis. In fact, the two different behaviours of the tumours are consistent with the morphological features observed within the follow up period: in the first case, the tumour showed a biphasic architecture with a de-differentiated anaplastic area characterized by increased cellularity and a diffuse honeycomb pattern of growth; on the other hand, in the present case, the dural

sac lesion presented a monotonous differentiated cell population arranged in a predominant pseudopapillary architectural pattern. This case underlines that, from a clinical and pathological point of view, the spectrum of glioneuronal tumors is still not completely explored. The next step would be the multicentre collection and subsequent stratification of ‘diffuse leptomenigneal glioneuronal tumours’ from patients in different stages of clinical aggressiveness to theoretically define patient-specific clinico-therapeutic follow-up strategies.

References

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pathologica 2012;104:432-441

Review

Molecular pathology of colorectal carcinoma. A systematic review centred on the new role of the Pathologist A. REMO, M. PANCIONE*, C. ZANELLA, R. VENDRAMINELLI Department of Pathology, “Mater Salutis” Hospital, ULSS21 Legnago, Verona, Italy; * Department of Biological, Geological and Environmental Sciences, University of Sannio, Benevento, Italy

Key words Molecular biology • Colorectal carcinoma • Pathologist • CIN • MSI • CIMP

Summary Colorectal carcinoma (CRC) is the second most frequent malignant disease in developed countries. Many aetiological factors have been reported in CRC development including genetic or non-genetic (environmental) elements. Independently of these, three groups of alterations have been implicated: 1) chromosomal instability (CIN); 2) microsatellite instability (MSI); 3) CpG island methylator phenotype (CIMP). A different multistep association between these alterations contributes to determine three distinct developmental pathways: traditional, alternative and serrated. Each genotypic CRC assessment is associated with specific morphologic or clinical features. Pathologists have to consider the morphologic and clinical features of each CRC when study tumours with molecular tests. Chromatin remodelling is extremely dynamic and depends on several DNA-based processes, such as transcription, DNA repair and replication.

The recent results with whole genome sequencing in a vast array of cancers have provided a catalogue of genetic lesions in chromatin modifiers that were previously unappreciated. It has revealed surprising facts about mutations in several SWI/ SNF complex members in many malignancies including CRC. The loss of INI1 expression is detected at a low rate in CRC and may be associated with differentiation grade and survival. Accumulating evidence suggests a critical role of the epithelial mesenchymal transition (EMT) in cancer progression. Some results support the existence of crosstalk between EMT and epigenetic modifications in the MSI-CRC group. We have summarized the role of genetic/epigenetic changes in the origin of the multiple CRC pathway, taking into account current knowledge of pathogenesis and feasibility of designing novel therapeutic approaches.

Introduction

largely reflects detection and removal of precancerous lesions through colorectal cancer screening 6. Many aetiological factors have been reported in CRC development including genetic or non-genetic (environmental) elements (Tab. I). Genomic instability is emerging as a fundamental process in colorectal tumourigenesis 7, as highlighted by a number of inherited syndromes such as familial adenomatous polyposis (FAP), MYH associated polyposis (MAP) and hereditary non-polyposis colon cancer (HNPCC). They are caused, in fact, by germline mutations in cancer-related genes involved in DNA duplication or repair, respectively 8 9. The main environmental risk factors consist of the “Western” diet combined with a sedentary lifestyle in addition to behavioural factors (smoking, alcohol consumption and occu-

CRC is the second most frequent malignant disease in developed countries. The incidence of CRC is generally higher for men, and the risk of the disease increases with age, as the majority of cases are diagnosed in patients more than 50 years of age  1 2. Most CRC are located in the sigmoid colon rectum, but the proportion of carcinomas that are more proximally located increases with age 3. Intriguingly, during the last decade, the incidence of CRC in the Asian population has increased from two- to four-fold, whereas it progressively diminished in Western countries, implying yet undefined gene-environment interactions 4 5. The decrease in the incidence rate in Western countries in the most recent time period

Correspondence

Andrea Remo, Department of Pathology, “Mater Salutis” Hospital, ULSS21 Legnago, via Gianella 1 37045 Verona Italy - Tel. +39 0442 622313 - Fax +39 0442 622323 - E-mail: [email protected] aulsslegnago.it

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Tab. I. Many aetiological factors have been reported for CRC development including genetic or non-genetic (environmental) elements.

Genetic (Inherited Syndrome) Autosomal dominant inheritable colorectal carcinoma (gene involved)

Non-genetic

• HNPCC or Lynch Syndrome (MLH1, MSH2, MSH6, PMS2) • Familial adenomatous polyposis (APC) • Peutz Jeghers syndrome (LKB1/STK11) • Juvenile polyposis syndrome (SMAD4) • Hereditary haemmorrhagic teleangectasia syndrome (ENG, ACVRL1) • Hyperplastic polyposis syndrome (MUTYH) • PTEN hamartoma syndrome (Cowden syndrome/ BannayanRuvalcaba-Riley syndromes) (PTEN) • Birt-Hogg-Dube syndrome (FLCN)

• Dietary factors • Alcohol consumption and smoking • Occupational factors • Inflammatory bowel disease • Infectious (Schistosoma japonicum, Schistosoma Mansoni) • Iatrogenic causes: + Radiation (Pelvic irradiation) + Surgery (colecistectomy, Ureterosigmoidostomy, Ileal conduits, ileostomy and anastomoses)

Autosomal recessive inheritable colorectal carcinoma • MUTYH-associated polyposis (MUTYH) Prevention (Role of Pathologist) • Identification of CRC caused by inherited genetic syndrome in order to enrol asymptomatic relatives in a specific screening program 73 74 • Identification of patients with polyps or precancerous lesions determined by inherited genetic syndrome in order to activate surveillance to prevent potential future CRC 84

pational factors). Other non-genetic aetiological factors are chronic inflammatory bowel disease such as ulcerative colitis, Crohn disease and Schistosoma mansoni/ japonicum infection  10. Iatrogenic etiological causes including therapeutic pelvic irradiation11 and ureterosigmoidostomy 12. Independently of aetiological factors, three groups of alterations have been implicated in the development of these tumours: 1) chromosomal instability (CIN); 2) microsatellite instability (MSI); 3) CpG island methylator phenotype (CIMP) 13. The evidence has revealed that CRC arise through a multistep process in which genetic and epigenetic alterations accumulate in a sequential order. In 1990, Vogelstein and colleagues proposed a model whereby CRC proceeds through a series of morphological steps due to specific genetic alterations 14. The model emphasizes the central role of the adenomatous polyp as the precursor lesion and provides evidence that in the majority of CRCs the primary event is the aberrant activation of the APC/β-catenin pathway, followed by RAS/RAF mutations and loss of p53 function at later stages  14. Ten years later, only 7% of CRCs have been shown to bear mutations in all three genes, implying that multiple pathways may be involved in the tumourigenic process  15. Recently, the development of colorectal cancer has been considered from a different point of view  16-19. Genetic alterations are, in fact, only a part of a more complex puzzle  20; epigenetic variations in cancer-related genes and noncoding RNAs also play a role in contributing to ma-

• Involvement in a screening programme to diagnose precancerous lesions or CRC • Involvement in routine to diagnose precancerous lesions or CRC associated with other pathologies (IBD, infectious etc.)

lignant status 21 22. The timing and combinations of genetic and epigenetic events, rather than the mere accumulation of genetic disorders, appear to confer cancer cells a selective advantage resulting in the activation of distinct pathways  18 21 23. Current evidence indicates that only one type of genomic instability predominates, providing the main “genetic or epigenetic signature” to the development of a specific CRC  24. Although the molecular basis of genomic instability remains elusive, a different multistep association between the three groups of pathogenetic alterations contribute to determine three distinct developmental pathways: traditional, alternative and serrated  17 19. This suggests that not a single but multiple pathogenetic mechanisms account for colorectal tumourigenesis. One attractive aspect is to establish the relative contributions of each of these pathways to tumour development and understand the effects they exert on phenotype and clinical behaviour. In this review, we will examine recent achievements in the understanding of CRC pathogenesis.

Pathogenetic alterations and pathways Three different developmental pathways have been implicated in the CRC: 1) CIN is the most common type of genomic instability occurring in 60% of CRCs, mainly in tumours proficient in DNA mismatch repair  25. CIN positive tumours are characterized by frequent loss of heterozygosity (LOH)

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Fig. 1. Multiple pathways in colorectal cancer pathogenesis. A) A least three different pathogenetic pathways have been implicated in the colon cancer pathogenesis: traditional, alternative, and serrated. They show clinical morphologic differences, especially when referred to the site of origin of the tumor. The serrated pathway tends to be localized to the proximal (right) colon in older patients; the traditional to the distal (left) colon and the alternative is common in both sites. B) Colorectal cancers (CRCs) arise through a multistep process in which genetic and epigenetic alterations accumulate in a sequential order. The prevalence and the best known genetic/epigenetic alterations occurring in each pathway are simplified, along with the characteristic precursor lesions (adenomas) and distinctive molecular features of the corresponding carcinomas. The traditional and serrated pathways are more homogeneous and clearly distinguishable; the alternative is more heterogeneous. Epithelial mesenchymal transition (EMT) tumor and crosstalk between cancer cells and neoplastic stroma (tumor microenvironment) are common features and critical for metastatic dissemination of primary tumors.

A

B

Abbreviations: APC, adenomatous polyposis coli; MMR, mismatch-repair or MSI, micro satellite instability; CIMP, CpG island methylator phenotype; CIMPL, CIMP-low; CIN, chromosomal instability; MSS, microsatellite stable; WNT, wingless pathway; EMT, epithelial mesenchymal transition; TGFβ, transforming growth factor-beta; LOH, loss-of-heterozigosity.

at tumour suppressor gene loci, chromosomal rearrangements and numerical abnormalities “manifested as aneuploidy” (Fig. 1A). Mutations frequently occur in APC, KRAS, PIK3CA, SMAD4 and TP53, among other genes 26. The presence of CIN is associated with poorer prognosis, regardless of stage and therapy 27. 2) CRC development has also been associated with frequent mutations at simple sequence repeats or microsatellites, generating what has been termed microsatellite instability (MSI). MSI accounts, instead, for approximately 15-20% of sporadic CRCs with a well-defined phenotype resulting from loss of DNA mismatch repair functions. The characteristic signature of MSI is the deletion of one element in a repetitive region of DNA that generates a frameshift in the coding sequence and hence gene inactivation, generally at tumour suppressor genes loci  28. Interestingly, most MSI-CRCs are apparently diploid or near-diploid (Fig. 1A). Recently, it has been hypothesized that point mutation instability (PIN), a process that increases spontaneous mutations in random nucleotide sequences, may contribute to both CIN and MSI 29 30. 3) During the last decade, epigenetic events, or heritable changes in gene expression not accompanied by changes in the DNA sequence, have been recognized to play a crucial role in CRC development  31. A novel type of instability has been suggested to influence CRC pathogenesis, which consists of DNA hypermethylation at specific regulatory sites enriched in CpG motifs (CpG islands) in the promoter regions of tumour suppressor genes  22  32  33. In 1999, Toyota et al. proposed the term CIMP to describe a subset of CRCs that consistently show widespread CpG island hypermethylation at seven

different loci defined methylated in tumours (MINT) 32. Subsequently, methylation of at least three MINT loci has strongly been correlated with CDKN2A (p16) and hMLH1 methylation constituting the so-called “classic panel”, providing a simplified approach to the definition of CIMP  24  31-33. Using these markers (called CIMP1), CIMP positive tumours are more frequently associated with MSI-CRCs than the MSS (Microsatellite-Stable) counterparts and localized to the right colon (up to 40%) than the left colon and rectum (3-12%). Curtin et al. proposed alternative markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) to the classic list of genes 34. Based on this new panel (called CIMP2), CIMP positive tumours do not have any relationship with KRAS mutations but strongly correlate with the BRAF V600Emutation, and are also referred as CIMP-high CRCs 31 34. The CIMP phenotype has recently been validated as an independent pathway using a whole methylome sequencing approach. According to these studies, four epigenotypes or DNA methylation–based subgroups of CRC have been proposed: CIMP-high, -low and non-CIMP. These latter are separated into two distinct clusters. One nonCIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumours that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations, and are significantly enriched for rectal tumours 35. Based on these group of alterations, CRC development has been classified in at least three distinct multiple pathways, each of which is based on different molecular mechanisms and variable prognosis  13  36. These pathways are illustrated in Figure 1B which takes into consideration

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current understanding. Every genotypic CRC assessment is associated with specific morphologic or clinical features (Fig. 1A). It is our opinion that Pathologists should consider both morphologic and clinical features of each individual CRC when studying tumours with molecular tests to understand the pathway. a) The alternative pathway is the most heterogeneous as it originates mainly from villous but also from serrated adenomas (Fig.  1B). It is characterized by a CIMP-low phenotype, predominant KRAS and occasional BRAF mutations, with no CIN. It is associated with poorer prognosis 16 17 36. b) The traditional pathway is based on APC and KRAS mutations (by CIN alterations) and epigenomic instability is not present (CIMP negative). Usually, they originate from tubular adenoma and are located in the left colon. c) The serrated pathway is characterized by BRAF mutations and epigenomic instability (CIMP-high). It originates from serrated adenoma and is located in the right colon. This pathway includes two kind of tumours: CRC-MSI morphologically including mucinous, medullary and tumours with intraepithelial lymphocytes (MSI-morphology), and another group consisting of CRC-MSS with a serrated morphology. It is still unclear whether these two groups constitute two different pathways or rather are two face of the same coin. This group has the highest rate of CRC that could benefit from targeted therapy with antiEGFR agents (KRAS “wild type”). We have recently proposed that the specific identity of these pathways may be established at an early evolutionary stage and fully enforced within precancerous lesions through an “epigenetic memory” that might operate differentially in colon crypt compartments, predisposing to the epigenetic characteristics of the adult cancer cells  13. The grade of “epigenetic memory” is variable within the colon crypt and may be dependent upon the expression of the PcG polycomb repressive complexes (PRCs). This condition may predispose the adult cancer cell to distinct degrees of epigenetic abnormalities, explaining the differences between the serrated, alternative and traditional pathways and CIMP positive versus negative CRC. Tumour development through the traditional pathway is, in fact, relatively slow (5-20 years), probably due to the fact that the initial events occur in the differentiated cells of the colonic crypt  13. APC mutations, generally, are detected in the cells of the upper crypt compartment according to the top-down morphogenetic model  37. The causal events underlying the serrated pathway, instead, may take place in the cells of the lower crypt compartment, whose functions are finely regulated by epigenetic mechanisms carried out by the components of the PcG polycomb repressive complexes (PRCs)  20  3841 . Albeit speculative, the present model integrates both morphological and molecular evidence and may explain why the precursor lesions of the traditional pathway originate in the upper part of the crypt tend

to grow upward, while those of the serrated pathway grow downward or laterally, are rapidly progressive and prone to CIMP  13  19  36  42. The early appearance of CIMP and higher CpG island hypermethylation present in the serrated or alternative pathway could be related to the specific chromatin organization program of the cell of origin. Among the genetic alterations associated with these events, BRAF mutations have been proposed as the earliest genetic event in the serrated pathway, followed by inactivation of p16/INK4a  42 43. Whether this is the seminal genetic lesion and what the subsequent ones are in serrated adenomas remains to be established (Fig. 1B). Moreover, the features of the tumour-initiating cells (TICs) from which the different pathogenetic pathways originate are not yet known, although they remain the focus of intense research 40 44. TICs form a malignant stem-cell compartment with a hierarchical organization and a specialized microenvironment, the so-called “niche,” resembling the normal stem-cell system at the bottom of the crypt. Recently, three different subtypes of TICs have been described, and only one defined long-term TICs (LT-TICs) is able to initiate tumour formation, maintain self-renewal and promote distant metastasis  44. Genetic heterogeneity does not significantly contribute to the functional differences between distinct types of TICs. Next generation sequencing has highlighted that the methylation profile of hypermethylated regions in CIMP tumours appears to be very similar to normal stem cells, as it is enriched in repressive marks of the PRC family and in specific transcription factors. The relative extent of CpG island hyper- and hypomethylation in tumours may reflect different features of the TIC subclones of origin (i.e., long-term stem cells versus transient amplifying precursors or differentiated cells). The “hierarchy model” hypothesizes that cancer stem cells initiate the malignant process and provide a continuous source of transformed cells that expand the tumour mass and tumour heterogeneity. Moreover, they display an increased ability to survive genotoxic stress and injury, suggesting that they are responsible for chemo- and radioresistance, for metastasis and, ultimately, patient demise 44. The discovery of different types of TICs suggests a distinct contribution of the stem-cell-like population to tumour formation and progression; however, the molecular link between the “cell of origin” and the specific pathogenetic pathway remains to be demonstrated 20 36 40 44.

Cromatin remodelling and epigenetic abnormalities in colon carcinogenesis Chromatin contains the heritable material of eukaryotic cells which provides the scaffold for the packaging of the entire genome. The functional unit of chromatin is the nucleosome, which is composed of a macromolecular complex of DNA and histone proteins (H2A, H2B, H3, H4). In general terms, chromatin can be subdivided

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Fig. 2. Cromatin Remodelling in Colon Carcinogenesis. A) The Chromatin “macromolecular complex of DNA and histone proteins” can be subdivided into two major regions: (1) heterochromatin, which is highly condensed and primarily contains inactive genes; and (2) euchromatin, which is relatively open and contains most of the active genes. Several processes regulate the chromatin dynamic: DNA methylation, histone modifications, and nucleosome remodeling ATP dependent. B) Only few components from the switching defective/sucrose nonfermenting (SWI/SNF) chromatin-remodeling family are known to be mutated or altered in several cancers, raising the possibility that they may be bone fide tumor suppressor. Their role in CRC are only just beginning to emerge. Our recent data on sporadic CRC suggest that loss of SNF5/INI1 is common to the poorly differentiated cases and correlates with poor patients’ prognosis.

A

Abbreviations: SWI: switching mating-type; SNF: sucrose nonfermenting; BRM: brahma; BRG1: brahma-related gene-1; ARID1A, AT-rich interactive domain-containing protein 1A; SNF5/INI1, sucrose nonfermenting, yeast, homolog-like 5/Integrase interactor 1 protein: Note: representative SWI/ SNF components are shown; at least 15 subunits have been described in mammals so far.

into two levels of organization “major regions”: 1) heterochromatin, which is highly condensed and mainly contains inactive genes; 2) euchromatin, which is relatively open and contains DNA coding genes that are actively transcribed (Fig. 2A). The structural changes of chromatin are extremely dynamic and finely controlled, and depend on several DNA based processes, such as transcription, DNA repair and replication. Consequently, abnormal expression patterns or genomic alterations in chromatin regulators can lead to the induction and maintenance of cancers. Globally, at least four

processes regulate the chromatin dynamic: DNA methylation, histone modifications, non-coding RNA-mediate targeting and nucleosome remodelling (Fig. 2A). The identification of the CIMP phenotype and, more recently, the discovery of various classes of epigenetic regulators, has significantly modified our knowledge of carcinogenesis  45-47. Over the last few years, the advent of several global proteomic and genomic technologies has allowed for appreciation of the contribution of epigenetic complexity and plasticity to tumour development. Most of the complexity surrounding the

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epigenome comes from the modification pathways that have been identified, and in particular cellular enzymes that chemically modify histone (“chromatin writers”) and specialized proteins with unique domains that specifically interpret these modifications (“chromatin readers”). The recent results from International Cancer Genome Consortium (ICGC) on whole genome sequencing in a vast array of cancers has provided a catalogue of genetic lesions in chromatin modifiers and global alterations in epigenetic landscape previously unappreciated  35 48. The myriad of covalent modifications on the nucleosome often provides the scaffold and context for dynamic ATP-dependent chromatin remodelling. Emerging evidence has linked the perturbation of complexes that remodel the structure of chromatin with oncogenic transformation. Nucleosome remodelling complexes (NRCs) are relevant in determining the accessibility of transcription factors to their target DNA sequences altering the interaction between DNA and histone proteins or relocating nucleosomes in an ATP-dependent manner (Fig.  2A). They are grouped into four subfamilies based upon their associated ATPase; 1) The mating-type switching and sucrose non-fermenting SWI/SNF family; 2) the imitation SWI (ISWI) family; 3) the nucleosome remodelling and deacetylation (NuRD)/Mi-2/chromodomain helicase DNA binding (CHD) family; 4) and the inositol requiring 80 (INO80) family. The SWI/SNF subfamily has received particular attention in cancer research  46. The complex contains two subunits with ATPase activity, Brahma-(Brm), Brahma-related gene-1 (BRG1) and several Brahma associated factors (BAFs)(Fig. 2B)  4951 . Loss of function of several members of SWI/SNF such as hSNF5/INI1, BRG1, BRM and MTA1 has been implicated in human cancers, raising the possibility that they may be bone fide tumour suppressors. Next generation sequencing (NGS) of cancer genomes has revealed surprising facts about high-frequency mutations in several SWI/SNF complex members in a range of haematological and solid malignancies, including CRC (Fig.  2B). SNF5 (also called SMARCB1, INI1, BAF47) is a core subunit of the SWI/SNF encoded at the 22q11.2 chromosome region. It is the most extensively studied subunit of the SWI/SNF complex due to its critical role in the development of undifferentiated sarcomas with rhabdoid features (Fig. 2B) 51 52. In fact, SNF5 is inactivated either at the germline or somatic level in malignant rhabdoid tumours (MRTs), a paediatric and highly lethal neoplasm of the kidney and brain  52-55. Despite their extremely aggressive behaviour, most of these tumours display a normal karyotype and the so-called rhabdoid cells. MRTs have also been described in extrarenal organs including colorectal, giving rise to rhabdoid colorectal tumours (RCTs), which is a rare, highly aggressive neoplasm frequently observed at the right colon of elderly patients. Variable loss of SNF5 immunohistochemical staining has been identified in other malignant neoplasms including renal medullary carcinoma, epithelioid sarcoma,

epithelioid malignant peripheral nerve sheath tumour (MPNST), myoepithelial carcinoma and extraskeletal myxoid chondrosarcoma (EMCS). Loss of SNF5 in other tumours is considered extremely rare. However, our studies on 90 sporadic CRC revealed that loss of INI1 expression is detected in about 11% of cases, and this subgroup is associated with a poor grade of differentiation (P