© Copyright by PZWL Wydawnictwo Lekarskie, Warszawa 2020

All rights reserved.

Reprinting and reproduction in any form of the whole or part of the book without the written consent of the publisher is prohibited.

The authors and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with current recommendations and practice. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug.

Publisher: Sylwia Chrabałowska, Jolanta Jedlińska

Leading Editor: Barbara Nowak-Pacholczak

Substantive Editor: Weronika Janowska

Producer: Monika Dąbrowska

Cover and title pages design: Sabina Bicz

Histopathological photographs courtesy of Jolanta Orłowska-Heitzman, MD, Department of Pathomorphology of the Jagiellonian University Medical College.

This book has been prepared on paper edition from 2020 (First edition)

Warsaw 2020

ISBN 978-83-200-5955-7

PZWL Wydawnictwo Lekarskie

02-460 Warszawa, ul. Gottlieba Daimlera 2

ph. 22 695 43 21

www.pzwl.pl

Online bookshop:

ph. 42 680 44 88; helpline: 801 33 33 88

e-mail: wysylkowa@pzwl.pl

Conversion for PZWL Wydawnictwo Lekarskie: Monika Lipiec / Woblink

AUTHORS

Dariusz Adamek, MD, DSc, Professor

Chair of Pathomophology, Department of Neuropathology, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland

Department of Pathomorphology, University Hospital, Krakow, Poland

Jinfeng Cao

Department of Ophthalmology, LUMC, Leiden, The Netherlands

Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, China

Sarah E. Coupland, MBBS, PhD, FRCPath, Professor

Royal Liverpool University Hospital, Liverpool, UK

George Holt Chair of Academic Pathology, University of Liverpool, Liverpool, UK

Bertil Damato, MD, PhD, FRCOphth

Nuffield Department of Clinical Neurosciences, Medical Sciences Division, University of Oxford, UK

Martyna Elas, PhD, DSc

Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

Bita Esmaeli, MD, FACS

Orbital Oncology and Ophthalmic Plastic Surgery, Department of Plastic Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

Joshua Ford, MD

Orbital Oncology and Ophthalmic Plastic Surgery, Department of Plastic Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

Martine J. Jager, MD, Professor

Department of Ophthalmology, LUMC, Leiden, The Netherlands

Barbara Jakubowska

Department of Ophthalmology and Ocular Oncology, University Hospital, Krakow, Poland

Chair of Ophthalmology, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland

Department of Ophthalmology and Ocular Oncology, University Hospital, Krakow, Poland

Jakub Khzouz, MBBS

Department of Pathology and Laboratory Medicine, King Hussein Cancer Center, Amman, Jordan

Joanna Kobylarz, MD

Department of Ophthalmology and Ocular Oncology, University Hospital, Krakow, Poland

Agnieszka Kubicka-Trząska, MD, DSc

Chair of Ophthalmology, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland

Department of Ophthalmology and Ocular Oncology, University Hospital, Krakow, Poland

Jolanta Orłowska-Heitzman

Chair of Pathomophology, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland

Bożena Romanowska-Dixon, MD, DSc, Professor

Chair of Ophthalmology, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland

Department of Ophthalmology and Ocular Oncology, University Hospital, Krakow, Poland

Beata Sas-Korczyńska, MD, DSc, Professor

Institute of Medical Sciences, Medical College of Rzeszow University, Rzeszow, Poland

Department of Ophthalmology and Ocular Oncology, Jagiellonian University Collegium Medicum, Krakow, Poland

Carol L. Shields, MD, Professor

Ocular Oncology Service, Wills Eye Hospital, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

Jacek Składzień, MD, DSc, Professor

Department of Otolaryngology, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland

Department of Otolaryngology, University Hospital, Krakow, Poland

Christina Stathopoulos, MD

Pediatric Ocular Oncology Service, Jules-Gonin Eye Hospital, Lausanne, Switzerland

PREFACE

Dear Readers and Colleagues

The book that you hold in your hands presents the most common problems in ophthalmological oncology. Authors of this publication specialize in various fields, and have dedicated most of their professional lives to oncological patients. They have extensive knowledge and experience which they are kind enough to share.

The book is based on materials collected over many year of professional activity. It offers a review of oncological diseases, and discusses diagnostic and therapeutic methods currently applied by all centers which specialize in ocular and orbital tumors. It also discusses the immunological, genetic and pathomorphological aspects of ocular neoplasms as well as looks into the complex interdependencies between genetic, chromosomal and immunological disorders that predispose patients to the development of cancer. Additionally, it analyses disease risk factors and patient survival rates.

An accurate diagnosis and a sufficiently early treatment is crucial in terms of prognosis (both health and survival outcomes) in primary ophthalmological neoplasms and metastatic tumors alike.

We point out the significance of different complementary imaging techniques in ophthalmological diagnostics, which make it possible to offer correct diagnosis. We also pay special attention to morphological features revealed in individual studies, juxtaposing them with the clinical findings. We make use of color photographs, red-free photography and autofluorescence, ultrasound tests (10 MHz US, 35–50 MHz US), SS-OCT, A-OCT, and in selected conditions also fluorescein and indocyanine angiography. Moreover, we present interesting cases with the differential diagnostics involved. All of the figures included in the publication come from the authors’ resources.

We do hope that our experience backed by the available literature will be useful in your daily clinical practice.

Bożena Romanowska-Dixon, MD, DSc, Professor

FOREWORD

The University Eye Clinic of Krakow was one of the first ophtalmological institutions in Europe to use circumscribed irradiation with radioactive applicators for the treatment of intraocular tumors. Successful results obtained in Poland after the treatment of uveal melanomas with Cobalt 60 plaques had a positive contribution for the global acceptance by the international ophtalmological community of the conservative management of intraocular tumors.

Since 1968, the University Eye Clinic of Krakow is a national and European reference Center in ocular oncology. This allowed a cumulation of large experience and enlargement of therapeutic armamentarium including recently the creation of a facility for proton beam irradiation of uveal melanomas.

The present textbook on ocular oncology, edited by professor Romanowska-Dixon, on the occasion of the 50th Anniversary of brachytherapy in Krakow, reflects major clinical experience which was progressively cumulated in her department.

This perfectly illustrated volume summarizes a complete overview of benign and malignant ocular tumors. The diagnostic modalities as well as the therapeutic options are clearly described.

This textbook, edited in English and Polish, has a high didactic value and will become soon a major theoretical support for the management of intraocular tumors.

Professor Leonidas Zografos

Honorary Professor at the Faculty of Biology and Medicine of the University of Lausanne, Switzerland

RECOMMENDATION

Contemporary oncology advanced considerably in the recent years due to new insights from tumor immunology, tumor-host interactions and significant breakthroughs in cancer detection and treatment. Accurate diagnosis based on the proper diagnostic process, and then choosing the appropriate treatment is crucial for therapeutic success. Taking this into consideration, ocular oncology is one of the most demanding areas of ophthalmology.

‘Ocular Oncology’ reflects the wide-scope range expertise, based on the many years of work of the Authors. The book reviews the eye neoplasms, as well as recent diagnostic and therapeutic modalities used in world-renowned centers of ophthalmic oncology.

The present compendium is a comprehensive summary of all ocular oncology aspects, including the description of eye tumors and their epidemiological, morphological, immunohistochemical, genetic and immune characteristics. Tumor treatment methods are widely described, including malignant tumor biology. The reader will also find history of ocular oncology in Poland. The book outlines the standard care for tumors of the eyelid, conjunctiva, uvea, retina as well as intraocular lymphoma and orbital tumors. The principles of radiation therapy for ocular and orbital tumors are also discussed. The preparation of the material for histopathological evaluation and experimental models of uveal melanoma metastases, and metastases to the eye are presented.

It is worth pointing out that all the Authors are eminent and internationally recognized specialists in ophthalmic oncology, who shared their experience with the readers. What is more, they all come from prominent ophthalmology centers, with large clinical material. The aim of the book is to provide ophthalmologists and specialists in other branches of medical science with the latest updates in ocular oncology. The book is clearly written with rich graphics, photographs and figures.

It is an honor for me to recommend this remarkable publication. I would like to congratulate all the Authors for creating this book, which will serve both the ophthalmologists and their patients. Special thanks should be directed to Professor Bożena Romanowska-Dixon, whose endless commitment and enthusiasm for ocular oncology came to fruition in this book. I firmly believe that the Authors’ experience together with recent literature review will prove useful in your everyday practice.

Professor Ewa Mrukwa-Kominek, MD, PhD

Departament of Ophthalmology, Medical University of Silesia, Katowice, Poland; Prof. Gibinski's University Clinical Center of the Medical University of Silesia in Katowice

TABLE OF CONTENTS

Abbreviations

1. History of ocular tumor radiotherapy in Krakow – Bożena Romanowska-Dixon, Barbara Jakubowska

2. Genetic aspects of ocular tumours - Jakub Khzouz, Sarah E. Coupland

Introduction

Eyelid cancers

Basal cell carcinoma

Sebaceous Carcinoma

Merkel cell carcinoma

Conjunctival cancers

Conjunctival squamous carcinoma and pre-invasive lesions

Conjunctival melanoma

Conjunctival lymphomas

Intraocular cancers

Medulloepithelioma

Retinoblastoma

Uveal melanoma

Vitreoretinal Lymphoma

Summary

3. Immunology of ocular tumors - Martine J. Jager , Jinfeng Cao

Introduction

Immune checkpoint inhibitors

Uveal melanoma: effect of checkpoint inhibitors

Other therapies

Presence of inflammation in UM

Phenotype-genotype comparisons

Comparison to other tumors

Conjunctival melanoma

Side effects of treatment

Conclusions

4. Eyelid tumors - Joshua Ford, Bita Esmaeli

Introduction

Overview of eyelid malignancies

Basal Cell Carcinoma (BCC)

Squamous cell carcinoma

Sebaceous carcinoma (SebC)

Periocular sweat gland carcinoma

Merkel cell carcinoma

Malignant hair follicle carcinoma

Eyelid Melanoma

American Joint Committee on Cancer Staging (TNM) of eyelid malignancies

Surgical management

Basal cell carcinoma

Squamous cell and sebaceous cell carcinoma

Eyelid melanoma

Merkel cell carcinoma

Sentinel lymph node biopsy (SLNB)

Adjuvant therapies

Topical therapies – Imiquimod (Aldara)

Targeted therapies

BRAF targeted therapy (Vemurafenib, sorafenib, debrafenib)

Immune checkpoint inhibitors (Pembrolizumab, ipilimumab, nivolumab, avelumab)

Other therapies (Cryotherapy)

Summary

5. Conjunctival tumors – Bożena Romanowska-Dixon, Barbara Jakubowska

Benign conjunctival tumors

Vascular lesions of the conjunctiva

Pigmented lesions

Congenital and acquired melanosis

Malignant conjunctival tumors

Conjunctival Squamous Intraepithelial Neoplasia (CIN; also termed C-SIN)

Invasive Conjunctival squamous cell carcinoma (SCC)

Conjunctival melanoma

Lymphoid tumors

Metastatic tumors

Secondary conjunctival tumors

Differential diagnosis of conjunctival tumors

Conclusion

6. Uveal tumors - Bożena Romanowska-Dixon, Barbara Jakubowska, Izabella Karska-Basta, Agnieszka Kubicka-Trząska, Bertil Damato

Iris cyst – Bożena Romanowska-Dixon, Barbara Jakubowska

Uveal nevi and melanoma – Bożena Romanowska-Dixon, Barbara Jakubowska

Iris nevus

Iris melanoma

Iris metastases

Ciliary body tumors – Bożena Romanowska-Dixon, Barbara Jakubowska

Ciliary body cyst

Ciliary body and peripheral choroidAL melanoma

Ciliary body metastases

Ciliary body adenoma and ciliary body adenocarcinoma

Differential diagnosis of ciliary body tumors

Choroidal tumors

Choroidal hemangioma - Bożena Romanowska-Dixon, Barbara Jakubowska

Choroidal osteoma - Bożena Romanowska-Dixon, Barbara Jakubowska

Sclero-choroidal calcifications - Bożena Romanowska-Dixon, Barbara Jakubowska

Choroidal pigmented tumors - Bożena Romanowska-Dixon, Barbara Jakubowska, Izabella Karska-Basta

Management of ciliary bodyand/or choroidal melanoma

Surgical treatment – Bożena Romanowska-Dixon, Agnieszka Kubicka-Trząska

Endoresection of choroidal melanoma – Agnieszka Kubicka-Trząska

Exoresection - Bożena Romanowska-Dixon

Enucleation - Bożena Romanowska-Dixon, Barbara Jakubowska

Radiotherapy - Bożena Romanowska-Dixon, Barbara Jakubowska

Prediction of metastatic disease in patients with choroidal melanoma - Bertil Damato

Ciliary body tumors – Bożena Romanowska-Dixon, Barbara Jakubowska

Introduction

Diagnosis

Treatment

Examples of Intraocular metastases

Leukemia – Bożena Romanowska-Dixon, Barbara Jakubowska

7. Differential diagnosis of intraocular tumors – Barbara Jakubowska,Bożena Romanowska-Dixon, Izabella Karska-Basta

Use of optical coherence tomography in a differential diagnosis of uveal tumors

Lesions masquerading as uveal melanoma

Use of indocyanine angiography in a differential diagnosis of uveal tumors

8. Retinal tumors – Bożena Romanowska-Dixon, Barbara Jakubowska, Joanna Kobylarz

Retinal tumors – Bożena Romanowska-Dixon, Barbara Jakubowska

Retinal glioma – Bożena Romanowska-Dixon, Barbara Jakubowska

Retinoblastoma – Joanna Kobylarz, Bożena Romanowska-Dixon

Introduction

Pathogenesis

Symptoms

Diagnosis

Clinical manifestation

Classification

Treatment

Post-treatment management

Prophylaxis

Summary

Intra-arterial Chemotherapy for Retinoblastoma - Christina Stathopoulos, Carol L. Shields

Congenital hypertrophy of RPE (CHRPE) – Bożena Romanowska-Dixon,Barbara Jakubowska

9. Intraocular lymphomas – Agnieszka Kubicka-Trząska

Epidemiology, classification, and clinical characteristics

Diagnosis

Imaging in intraocular lymphoma

Treatment

Prognosis

10. Retrobulbar Orbital Tumors – Jacek Składzień

Orbital anatomy

Diagnosing orbital tumors

Diagnosing and treatment orbital tumors

Imaging

Medical history

Selected orbital malignancies in the retrobulbar region

Dermoid cyst, epidermoid cyst and developmental cyst of the orbit

Cavernous hemangioma of the orbit

Capillary hemangioma

Lymphatic hemangioma of the orbit (venous-lymphatic malformation, vascular hamartoma)

Idiopathic orbital inflammation (IOI)(orbital ‘pseudotumorIgG-4’)

Astrocytoma (glioma)

Optic nerve meningioma

Lacrimal apparatus disorders

Metastases of orbital tumors

Surgical treatment

Summary – basic techniques for accessing tumors

Orbital exenteration

Rehabilitation after surgery

11. Radiotherapy of ocular and orbital tumors – Beata Sas-Korczyńska

Radiotherapy – characteristics

Interaction of ionizing radiation with matter

Energy and dose

Biological effects

Dose fractionation

External Beam Radiation Therapy (EBRT)

Brachytherapy

Radiotherapy of ocular and orbital tumors

Radiotherapy purpose and techniques

Radiation dose

Side effects in the radiotherapy of ocular and orbital tumors

Eyelid dermatitis

Conjunctivitis

Dry eye syndrome

Side effects in the cornea (keratopathy)

Cataract

Side effects on the iris

Side effects in the retina (retinopathy)

Optic neuropathy

Side effects in the sclera

Conclusion

12. Preparation of orbit material for histopathological examination – Jolanta Orłowska-Heitzman

13. Pathology of orbital tumors – Dariusz Adamek

Introduction

Non-cancerous orbital lesions

Most common types of orbital tumors and tumor-like lesions

Tumors of the optic nerve and its sheaths

Peripheral nerve tumors and other neurogenic tumors

Other neurogenic tumors

Tumors arising from the muscular or adipose tissue, fibrohistiocytic tumors, and other mesenchymal tumors

Osseous and chondral tumors and tumor-like lesions

Tumors and other proliferative lesions of the lymphatic, hematopoietic and histiocytic systems

Malignant secondary and metastatic orbital tumors

14. Experimental models to study metastasis in uveal melanoma - Martyna Elas

Cell culture and tumoroids

Animal models

CAM

Zebrafish

Rat, mouse and hamster

Rabbit

Transgenic models of UM

Conclusions

Notes

ABBREVIATIONS

3D-CRT – three-dimensional conformal radiation therapy

5-FU – 5-fluorouracyl

ACS – American Cancer Society

AF – autofluorescence

AJCC – American Joint Committee on Cancer

angio-OCT – optical coherence tomography angiography

ARVO – Association for Research in Vision and Ophthalmology

BAP1 – BRCA1 associated protein-1

BCC – basal cell carcinoma

Bq – bekerel

BRAF – a human gene that encodes a protein called B-Raf

BRCA – human tumor suppressor genes, which when mutated may lead to hereditary breast-ovarian cancer

BT – brachytherapy

CHRPE – congenital hypertrophy of retinal pigment epithelium

CM – conjunctival melanoma

CNS – central nervous system

CNV – choroidal neovascularisation

CT – computed tomography

CTLA4 – cytotoxic T cell antigen 4

DNA – deoxyribonucleic acid

DRI-OCT – deep range imaging OCT

EBRT – external beam radiation therapy

EGFR – epidermal growth factor receptor

ERM – epiretinal membrane

FA – fluorescein angiography

FAP – familiar adenomatous polyposis

FAZ – foveal avascular zone

Gy – grej

Gy (RBE) – unit of radiation dose, that includes relative biological effectiveness of the applied radiation; may be used for example in proton beam radiation

HIV – human immunodeficiency virus

HPV – human papilloma virus

IAC – intraarterial chemiotherapy

ICGA – indocyanine green angiography

IGRT – image-guided radiotherapy

IMRT – intensity-modulated radiation therapy

LET – linear energy transfer

MBD4 – Methyl-CpG-binding domain protein 4

MCC – Merkel cell carcinoma

MLC – multileaf collimator

MMC – mitomycyn C

MMS – Mohs micrographic surgery

MRI – magnetic resonance imaging

NRAS – gene encoding protein N-ras (neuroblastoma RAS viral oncogene homolog)

OARs – organs at risk

PAM – primary acquired melanosis

PBS – pencil beam scanning

PCPTCL – primary cutaneous peripheral T-cell lymphoma

PD-1 – programmed death receptor 1

PD-L1 – programmed death ligand 1

PDT – photodynamic therapy

PPV – pars plana vitrectomy

PSP – passive scattering protons

PT – proton beam radiotherapy

PVRL – primary vitreoretinal lymphoma

QUANTEC – quantative analysis of normal tissue effects in the clinic

RBE – relative biological effectiveness

RION – radiation-induced optic neuropathy

RON – radiation optic neuropathy

RPE – retinal pigment epithelium

RR – radiation retinopathy

SCC – squamous cell carcinoma

SebC – sebaceous carcinoma

SHH – sonic hedgehog

SLN – sentinel lymph node

SLNB – sentinel lymph node biopsy

SRS – stereotactic radiosurgery

SRT – stereotactic radiotherapy

S-OCT – spectral OCT

SS-OCT – swept source OCT

TCA – triamcinolon

TD – tolerance dose

TFSOM-UHHD – To Find Small Ocular Melanoma Using Helpful Hints Daily

T-NHL – T-cell non-Hodgkin lymphoma

TNM – Tumor Node Metastasis

TNMH – tumor, node, metastasis, heritable trait – retinoblastoma classification system according to American Joint Committee on Cancer Staging

TTT – transpupillary thermotherapy

UBM – ultrabiomicroscopy

UICC – Union for International Cancer Control

USG – ultrasonography

VEGF – vascular endothelial growth factor

1
HISTORY OF OCULAR TUMOR RADIOTHERAPY IN KRAKOW
BOŻENA ROMANOWSKA-DIXON
BARBARA JAKUBOWSKA

In October 1850, the Ophthalmology Department was established as a part of the Medical Faculty of the Jagiellonian University in Krakow. The professors of the newly created Ophthalmology Department and heads of the Ophthalmology Clinic of the Jagiellonian University were as follows: Antoni Sławikowski (1851–1870), Lucjan Rydel (1870–1895), Wiktor Bolesław Wicherkiewicz (1895–1915), Jan Lauber, Kazimierz Wincenty Majewski (1939 i 1945), Jan Lauber (1940–1944), Marian Wilczek (1945–1967), Helena Żygulska-Mach (1967–1996), Maria Starzycka (1996–2004), Bożena Romanowska-Dixon (since 2004 to the present time).

1898 saw the opening of a new building of the Ophthalmology Clinic, which was then modernized at the beginning of the 21st century.

a

b

c

d

e

f

g

h

Figure 1.1. Heads of the Ophthalmology Department and Clinic of the Jagiellonian University: a – Antoni Sławikowski; b – Lucjan Rydel; c – Wiktor Bolesław Wicherkiewicz; d – Kazimierz Wincenty Majewski; e – Marian Wilczek; f – Helena Żygulska-Mach; g – Maria Starzycka; h – Bożena Romanowska-Dixon.

In the 1960s, ophthalmic oncology made significant progress all over the globe. The Clinic in Krakow was no exception in this respect. With Professor Marian Wilczek as Head, the Clinic introduced new diagnostic methods, techniques of conjunctival and eyelid tumor management, as well as reconstructive surgery.

At the beginning of the 1960s, the Clinic purchased the Meyer-Schwickerath xenon arc lamp which was used, amongst other things, for photocoagulation therapy in retinal detachment as well as treatment of other vascular disorders of the retina, primarily diabetic retinopathy, and intraocular neoplasms in adults (melanoma) and children (retinoblastoma).

1.2

Figure 1.2. The building of the Clinic in the 19th century.

1.3

Figure 1.3. Interior rooms of the Clinic in the 19th century.

1.4

Figure 1.4. A team of doctors with Professor Marian Wilczek in the 1960s.

1.5

Figure 1.5. Xenon arc lamp.

Thanks to the efforts of Professor Marian Wilczek, the Clinic made preparations for brachytherapy with cobalt (Co-60), and in 1967 several cobalt plaques (applicators) were purchased and then used for treating uveal melanoma patients. That happened in 1968, only after Professor Wilczek had passed away. The Krakow Ophthalmology Clinic was the second center worldwide, which introduced cobalt applicators (Co-60) for the treatment of intraocular neoplasms [1].

Professor Helena Żygulska-Mach started brachytherapy with cobalt applicators, which was initially used in the treatment of retinoblastoma and choroidal melanoma, and later expanded to other intraocular tumors. There were 3 types of plaques used, varying by size, namely with a 10 mm (CKA1), 15 mm (CKA2) and 20 mm (CKA3) diameter. They were periodically reactivated at the National Center for Nuclear Research in Świerk (Warsaw). Large melanomas and tumors located close to the optic disc were treated with brachytherapy with Co-60 in combination with xenon arc photocoagulation of the top of the tumor. Brachytherapy was also used after removal of conjunctival melanoma, and for irradiation of the orbit after enucleation in large melanomas with extraocular extension.

In addition, strontium applicators (Sr-90) were used to treat neoplasms on the surface of the eye. Patients from all over Poland have been (and still are) referred to the Clinic in Krakow. Reports from treatment have been published in national and international journals. The first publications of treatment outcomes at the Clinic appeared in 1972.

1.6

Figure 1.6. An excerpt from an article on brachytherapy with cobalt, describing the technique of placing cobalt applicators with the use of marker fittings (shown in the picture), published in the Medical Journal of the Medical Academy in 1969.

1.7

Figure 1.7. A drawing of a patient’s fundus with a choroidal melanoma. All hand drawings come from Ophthalmology Clinic documentation.

1.8

a

b

c

Figure 1.8. Drawings of a patient’s fundus with a choroidal melanoma: a – prior to treatment; b – 8 months after brachytherapy with Co-60; c – almost 3 years after brachytherapy with Co-60.

1.9

a

b

Figure 1.9. Drawings of a patient’s fundus with a choroidal melanoma: a – prior to treatment; b – 2 years after brachytherapy with Co-60.

1.10

a

b

Figure 1.10. Drawings of a child’s fundus with retinoblastoma: a – prior to treatment; b – 1 year after brachytherapy with Co-60.

1.11

Figure 1.11. A publication on radiotherapy with cobalt and a photograph of Co-60 applicators.

Use of brachytherapy to treat extraocular tumors has gradually gained proponents and currently this method relies on a number of different radioactive elements. Until recently, the Krakow Ophthalmology Clinic was the only center in Poland and one of the few centers in Europe to use brachytherapy for treatment of ocular tumors, and therefore has accumulated extensive clinical data. Brachytherapy was successful in salvaging the globe, often with a satisfactory visual acuity, without compromising the patients’ survival rates. The probability of 5- and 10-year survival was, respectively, 0.86 and 0.67. In order to maximize damage to the tumor, brachytherapy was used in combination with xenon arc photocoagulation in most cases. Photocoagulation was also used successfully as a stand-alone therapy for small tumors, achieving a 5-year survival rate of 0.98 for patients treated with the xenon arc lamp.

In 1995, four plaques containing a radioactive isotope of ruthenium (Ru-106) were acquired by the Clinic. In that year, the first pediatric and adult patients received brachytherapy with ruthenium. Ru-106 plaques need to be replaced once a year due to the element’s half-life and the applicator’s active time. We used Ru-106 plaques of different sizes (with diameters ranging from 10 to 25 mm) and shapes (round, with a notch for the cornea, and with a notch for the optic nerve). We usually hold 3 to 4 active ruthenium plaques, which are in regular use (with sterilization in each case). In 1997, three additional plaques containing a radioactive isotope of iodine (I-125) were purchased [2].

At that time, we discontinued the use of Co-60 plaques. As per the standard of care, Ru-106 plaques are used for tumors up to 5 mm thick, and iodine applicators for tumors with a greater thickness. We hold three types of iodine applicators: round (3 large ones with a 20 mm diameter and 1 small one with a 10 mm diameter) as well as one applicator with a notch for the optic nerve (20 mm in diameter). Iodine seeds in the applicators need to be replaced every 2–3 months due to the element’s half-life and the plaque’s active period. Until October 2018, radiotherapy with ruthenium was administered to 2,596 patients and with radioactive iodine to 1,951 patients. Most of them suffered from uveal melanoma while some had choroidal metastases and the treatment was also provided to children with retinoblastoma. Only in few cases, brachytherapy with ruthenium is used after removal of conjunctival melanoma.

Since 1995 we have used transpupillary thermotherapy (TTT) with a diode laser in addition to brachytherapy (in the so-called ‘sandwich therapy’), particularly in case of tumors located near the disc, as adjuvant therapy following brachytherapy, or as a stand-alone therapy in case of small tumors, metastatic choroidal tumors, retinoblastoma, choroidal hemangioma and other.

In 1990s, new surgical methods were introduced for treatment of conjunctival melanoma, iris melanoma and ciliary body melanoma, namely iridocyclectomy and iridocyclochoroidectomy. Since 2010, we have performed endoresection of choroidal melanoma after radiation by pars plana vitrectomy (PPV) to prevent development of the toxic tumor syndrome.

Small, symptomatic choroidal hemangiomas are treated using photodynamic therapy (PDT) while larger tumors are managed by transpupillary thermotherapy (TTT) or brachytherapy.

Patients treated with brachytherapy need to remain on dedicated hospital wards.

Patients who undergo brachytherapy with iodine need to wear eye shields to shield the radiation. Healthcare professionals providing brachytherapy and caring for patients wear protective clothing and carry ring and film badge dosimeters. Each year ca. 170 patients receive brachytherapy with I-125 applicators, roughly 200 with Ru-106 applicators.

The operating suite houses a radiological laboratory and 2 sterilizers – one using steam and the other using gas (to sterilize the applicators). The applicators are kept in a safe box, and the movement of radiation sources is closely monitored and recorded (in accordance with the recommendations and approval issued by the National Atomic Energy Agency). The wards for irradiated patients are in close proximity to the operating suite, separated from the regular wards.

In 1988–1989, the Ophthalmology Clinic in Krakow joined the international Retinoblastoma International Clinical Study (RICS). The study was carried out in 14 countries all over the world, and our Clinic contributed the third largest clinical database.

1.12

Figure 1.12. Ru-106 applicators.

1.13

Figure 1.13. I-125 applicators.

1.14

Figure 1.14. Proton therapy facility of the University Hospital in Krakow located at the Institute of Nuclear Physics of the Polish Academy of Sciences.

The Ophthalmology Clinic in Krakow also collaborates with the Genetic Outpatient Clinic of the Clinical Genetics Department and the Immunology Clinic of the Polish-American Children’s University Hospital in a study on families of children with retinoblastoma. The Clinic also studied genetic markers in various ocular disorders, including retinoblastoma, in collaboration with the Department of Forensic Medicine of the Jagiellonian University.

Following a rapid development in the area of ocular oncology, our Clinic was renamed as the Ophthalmology Department, Ophthalmology and Ocular Oncology Clinic of the Jagiellonian University Hospital.

In January 2011, we started treating patients with choroidal melanoma using a proton beam (proton therapy) in cooperation with the Institute of Nuclear Physics of the Polish Academy of Sciences (PAN) in Krakow.

The therapy is managed by a team of physicians (ophthalmologists, radiotherapists and an oncologist), medical physicists, radiology technicians and nursing staff from the Ophthalmology and Ocular Oncology Clinic of the Jagiellonian University in Krakow while the Institute of Nuclear Physics of PAN in Krakow contributes the proton beam. Every year around 50 patients receive proton therapy.

Everyday the outpatient cancer center of the Ophthalmology and Ocular Oncology Clinic sees between 30 and 40 patients referred from all regions of Poland as well as a few patients from abroad (Lithuania, Latvia, Ukraine and the UK). Our operations were enabled by collaboration with other institutions, including the Anatomical Pathology Department, Otorhinolaryngology Clinic and the Pathomorphology Department of the Jagiellonian University and, with respect to experimental research, the Faculty of Biochemistry, Biophysics and Biotechnology [3, 4].

We have presented our research in diagnosing and treating ocular oncology patients at national and international scientific symposiums, and published papers in Polish and international scientific journals. Irradiation of tumors enabled us not only to salvage the eye and, frequently, retain a satisfactory visual acuity, but also improve patient survival rates. A review of brachytherapy treatment outcomes showed that secondary glaucoma proves to be the most destructive complication. Other complications included: radiation retinopathy, radiation neuropathy and vitreous hemorrhage. Secondary cataract was the most frequently reported complication.

We also engage in extensive international scientific cooperation. In 2016, the Ophthalmology Clinic of Krakow joined the UMCURE 2020 program (grant agreement 667787; https://www.umcure2020.org/en/). It is one of 4 centers in Europe involved in developing new therapies of uveal melanoma, with a particular focus on treating metastatic uveal melanoma.

References

1. Żygulska-Mach H., Maciejewski Z., Link E: Conservative treatment of choroidal melanomas. Combined use of cobalt plaques and photocoagulation. [In:] Intraocular Tumors. Fortschritte der Onkologie. Band 9 (red. P.K. Lommatzsch, F.C. Blodi). Akademie-Verlag, Berlin 1983: 417–423. 2. Romanowska B., Żygulska-Mach H., Księżyk M.: Survival rate of young patients with malignant choroidal melanoma after conservative treatment. Klinika Oczna 1998; 100(4): 211–216.3. Romanowska-Dixon B., Elas M., Swakoń J. et al.: Metastasis inhibition after proton beam, β- and γ-irradiation of melanoma growing in the hamster eye. Acta Biochimica Polonica 2013; 60(3): 307–311.4. Romanowska B., Kukiełczak J., Bryk B. et al.: New technique for implanting Bomirski melanoma into the anterior chamber of Syrian hamster eyes. Klinika Oczna 1995, 97(11–12): 324–327.

2
GENETIC ASPECTS OF OCULAR TUMOURS
JAKUB KHZOUZ
SARAH E. COUPLAND*

INTRODUCTION

Cancers of the eye and orbit include a wide range of neoplasms, varying from carcinomas within the ocular adnexal structures and those arising from the differing coats of the eye, to a range of mesenchymal tumours of differing infiltrative and metastatic potential. They can occur in both children and adults, and may or may not be associated with particular underlying syndromes with germline mutations. In this chapter, we will provide an overview of the underlying genetic alterations of the most common ocular adnexal and intraocular malignancies. In a systematic manner, we will commence with the eyelid tumours, and then progress posteriorly to the back of the eye. Later chapters in this book deal with more intricate details of the clinical management of these lesions.

EYELID CANCERS

BASAL CELL CARCINOMA

Basal cell carcinoma (BCC) of the eyelid is a primary malignant epithelial tumor that originates by neoplastic transformation of the basal cells of the epidermis, which proliferate and invade into the underlying dermis. BCC is the most common cancer worldwide, occurring 80% in the head and neck region, of which 20% on the eyelids and 90% among malignant eyelid tumours. The incidence of BCC is higher in more equatorial latitudes. A high incidence of BCC and a larger sized tumour are two features that are associated with patients living in areas of socioeconomic deprivation.

BCC is strongly associated with chronic exposure to ultraviolet (UV) radiation. In addition, other risk factors for developing BCC include immunosuppression, exposure to arsenic compounds and ionizing radiation. Several tumour suppressor genes and proto-oncogenes have been linked in BCC oncogenesis. Basal cell nevus syndrome or ‘Gorlin-Goltz’ syndrome is a hereditary autosomal dominant condition in which patients develop multiple BCCs at young age and develop different tumours including medulloblastomas, keratocystic odontogenic tumours, ovarian and cardiac fibromas, in addition to developmental anomalies.

The mutant gene is PATCHED1 (PTCH1) gene and is mapped to chromosome 9q22.3. It encodes a transmembrane protein, which is part of the Hedgehog (HH) signalling pathway [1]. The HH pathway (fig. 2.1) is crucial for organogenesis, tissue repair and regeneration and stem cell maintenance. Aberrant HH activation represents the most significant event in BCC. More than 90% of BCCs show loss of PTCH1 function by inactivating mutations of PTCH1 gene or aberrant SMO mutation (fig. 2.1 and 2.2). Sporadic BCCs also show inactivating point mutations of LOH in PTCH1 with evidence of a UV-signature.

Mutations in other components of HH pathway are reported in sporadic BCC including activating SMO mutations and SUFU loss of function mutation. Smaller number of sporadic BCCs were found to also have PTCH2 mutations [2]. Other tumour suppressor genes and proto-oncogenes have been implicated in the pathogenesis of BCC, including the TP53 tumour suppressor gene and members of the RAS proto-oncogene family. TP53 gene mutation in BCC is mostly due to UV- induced damage [3].

Additional mutations in other cancer-related genes might be considered as a new driver in BCC development, such as MYC-N, PPP6C & STK19. Inactivating mutations of the LATS1 & PTPN14 genes (Components of the Hippo-YAP axis) also have been recently identified [4].

The histological types of BCC are solid, nodular (fig. 2.3), nodular-ulcerative, morpheaform, infiltrative, cystic, nodular-cystic, basosquamous, and plexiform (adenoid pattern). The nodular and superficial types of BCC are the most common in the eyelid.

a     b

Figure 2.1. Normal Hedgehog (HH) signalling: a – in the absence of HH ligands (this case Shh), PTCH1 blocks SMO function and keeps GLI transcription factors sequestered in the cytoplasm by suppressor of fused ‘SUFU’; b – the Sonic hedgehog (Shh) binds to the transmembrane receptor protein ‘PTCH1’, thereby releasing the transmembrane G protein coupled receptor-like protein ‘SMO’, allowing its migration to the tip of the primary cilium. GLI transcription factors (GLI1, GLI2 & GLI3) are activated through multistep process including the release of suppression by ‘SUFU’. GLI factors translocate into the nucleus and drive cell proliferation and growth. GLI1 also establishes a feedback loop that auto-regulates this signalling pathway through PTCH1 modulation.

2.2

Figure 2.2. Abnormal HH signalling. In BCC, loss of function of PTCH1 or activating mutations of SMO induces HH pathway in the absence of HH ligands.

2.3

Figure 2.3. Basal cell carcinoma of the eyelid. Dermal infiltration by nodules and nests of basaloid tumour cells with peripheral palisading and peritumoural clefting. Actinic elastosis due to chronic sun exposure is seen in the papillary dermis (Stain: Haematoxylin and Eosin [H & E]).

SEBACEOUS CARCINOMA

Sebaceous carcinoma of the eyelid is generally considered the second most common neoplasm of this anatomical region after BCC. Sebaceous carcinoma usually originates from the tarsus of the upper eyelid and less commonly from the lower lid, the Zeiss gland, or the sebaceous glands of the caruncle with secondary involvement of the conjunctival epithelium by pagetoid invasion (fig. 2.4).

The incidence of sebaceous carcinoma is about 5% of all malignant eyelid tumours, with a mean age at diagnosis ranging from 57–72 years. Risk factors include advanced age, Asian race, and female gender.

Figure 2.4. Invasive sebaceous carcinoma with associated in situ disease. Nests of tumour cells showing nuclear pleomorphism, distinct cell borders and foamy/vacuolated cytoplasm. Mitotic activity is prominent. This tumour shows pagetoid spread (in-situ component) in the overlying conjunctiva (Stain: H & E).

Muir-Torre syndrome (MTS), a form of the rare Lynch syndrome, is associated with sebaceous carcinomas. MTS is an autosomal dominant syndrome with high penetrance and variable expression; some sporadic cases have been also reported. It is associated with mutations involving the mismatch repair proteins (MLH1 and MSH2) [5]. It is characterised by the occurrence of sebaceous neoplasms and various visceral malignancies. The sebaceous neoplasms associated with MTS range from benign sebaceous adenomas and sebaceoma, invasive sebaceous carcinoma as well as variants of BCC with sebaceous differentiation. Visceral malignancies associated with MTS include colorectal-, genitourinary-, endometrial-, breast and gastric carcinomas, and, more rarely, haematological malignancies [6]. However, it was reported that the association with MTS and sebaceous carcinomas is more common in extra-orbital locations [7].

TP53 mutations are also found in a high percentage of eyelid sebaceous carcinomas and may be involved in the early stages of carcinogenesis. These mutations are UV-light independent [8, 9]. The role of HPV was also investigated in few studies since it promotes the degradation of p53 protein but showed variable results [5].

Amplification of the ERBB2/HER2 receptor, detected by immunohistochemistry or in-situ hybridization, has been reported in sebaceous carcinoma. Activation of this transmembrane receptor protein results in activation of signalling pathways including the MAPK and STAT pathways resulting in cell proliferation and inhibit apoptosis. Dysregulation of both of these pathways was identified in sebaceous carcinoma of Meibomian gland, and may facilitate identification of targeted therapy in the future [10].

MERKEL CELL CARCINOMA

Merkel cell carcinoma (MCC) (fig. 2.5) is a rare aggressive neuroendocrine tumour of the skin. MCC mostly affect the head and neck region, including the eyelid, followed by the trunk and the limbs. Interestingly eyelid MCC mostly occurs in the upper lid. Its incidence varies: it is higher in Australia (1.6 per 100,000) than in the USA (0.44 per 100,000). Immunocompromised patients with T-cell dysfunction are more likely to be affected by MCC. The median age at presentation is 77 years (range, 58–91 years), with a slight predominance in women.

Risk factors for MCC include fair skin, chronic sun exposure, chronic immune suppression, and advanced age. MCC development has been linked to Merkel cell polyomavirus (MCPyV) and chronic UV radiation.

MCPyV belongs to the family Polyomaviridae comprised of non-enveloped, double-stranded circular DNA viruses. They are considered part of normal skin flora. Immunocompromised patients (solid organ transplant and AIDS patients) are at increased risk of developing MCC [11].

Viral genome integration into the host-genome results in mutation in the Large T (LT) antigen that is required for Viral DNA replication. This results in expression of MCPyV small T antigen (ST) and truncated large T antigen (LT) which eventually leads to uncontrolled cellular proliferation (fig. 2.6). MCPyV+ MCC are predominantly diploid and show minimal number of somatic alterations. They have wild type Tp53 and RB1 and no UV mutational signatures.

Figure 2.5. Merkel cell carcinoma. Dermal infiltration by sheets of monotonous round cells with scant cytoplasm and vesicular nuclei. Frequent mitotic and apoptotic figures are seen. MCC is positive for CK20 and neuroendocrine markers such as CD56, Synaptophysin and chromogranin, while it is negative for CK7 (Stain: H & E).

In contrast, UV-induced MCCs are aneuploidy and MCPyV negative. They do not express the ST or LT antigens. They show high frequency of UV damage induced mutations with inactivating mutations involving the RB1 and the TP53 in addition to inactivating mutations involving genes involved in different repair pathways [12].

Figure 2.6. Merkel cell carcinoma polyomavirus effect: a – normally, p53 inhibits phosphorylation of pRb through a negative effect of p21 on the cyclinD1 & the Cdk4/6 complex. Hence, E2F transcription factor is not released (inactive); b – with MCPyV+: MCC binding of LT antigen to p53 results in a negative regulation of p21 with consecutive upregulation of cyclinD1 & Cdk4/6 complex, RB phosphorylation, and E2F activation. Binding of LT antigen to pRb also results in the release of E2F and cell cycle transition from G1 to S phase.

CONJUNCTIVAL CANCERS

CONJUNCTIVAL SQUAMOUS CARCINOMA AND PRE-INVASIVE LESIONS

Squamous cell carcinoma (SCC) of the conjunctiva and caruncle is a malignant epithelial tumour with squamous differentiation and can be keratinising or non-keratinising. There is significant geographical variation in the incidence of conjunctival SCC. Worldwide, the mean age-standardised incidence is 0.18 per year per 100,000 for males and 0.08 per year per 100,000 for females. Worldwide, a high incidence has been demonstrated in HIV-positive and in other immunosuppressed populations. A higher incidence is also seen in xeroderma pigmentosum (XP).

UV radiation remains the strongest risk factor. It is the only risk factor where a dose-response relationship has been established. Impaired immune surveillance clearly plays a role in the development of conjunctival SCC, and it unites risk factors such as UV (which suppresses cell-mediated immunity) and immunosuppression (HIV, post-transplant). Reduced immunity potentiates the effect of oncogenic viruses such as HPV. In Africa, the combination of low latitude, HIV and/or HPV infection explains the relatively high incidence of conjunctival SCC. Multiple oncogenic viruses (HPV, KSHV and EBV) were also identified in HIV+ patients with SCC, which may play a role in tumour oncogenesis in those patients [13].

DNA damage is the key step in SCC development: it affects the genome stability leading to cancer. DNA damage can be genetic or epigenetic. UVB radiation causes direct DNA damage by crosslinking adjacent bases to form Cyclobutane Pyrimidine Dimer which distorts DNA’s structure and blocks DNA synthesis. CPDs formation results in specific p53 tumour-suppressor gene mutation, which has been observed in actinic keratosis and what is termed 'ocular squamous surface neoplasia (OSSN), a broad term given to conjunctival dysplasia, carcinoma in situ and invasive carcinoma. While UVA radiation causes indirect DNA damage through reactive oxygen species leading to DNA strands break. DNA damage is monitored normally through three check points in different phases of cell cycle (fig. 2.7). There are different molecular ways to repair damaged DNA. The Nucleotide Excision Repair (NER) pathways is the one involved in repairing Cyclobutane Pyrimidine Dimer in DNA. The Base excision repair (BER) is responsible for repairing DNA damage cause by reactive oxygen species, ionizing radiation and alkylating agents [14].

P53 is a tumour suppression gene located on short arm of chromosome 17 (17p13.1). Its activation results in binding of its protein to DNA with resultant cell cycle arrest at G1-S checkpoint or apoptosis. P53 also has an additional secondary effect by regulating other gene products involved in preventing DNA damage or helping in DNA repair.

Xeroderma pigmentosa (XP) is a rare autosomal recessive genetic disorder in which there are mutations in genes resulting in a deficiency of the enzymes responsible for repairing DNA damage caused by UV radiation by a process called Nucleotide excision repair. There are eight genes (XPA–XPG and XPV) located on different chromosomal locations. Defects in XP-V gene does not affect the nucleotide excision repair mechanism, rather it results in XP variant where they have problems replicating UV damaged DNA. Males and females are affected equally with an incidence in the USA of 1 in 250,000. It is higher in Japan, Middle East and North Africa [15]. Patient’s presents with increased skin sensitivity to sunlight, lentigines, dry scaly skin and pigmentation, photophobia, and severe keratitis. In addition, the patients have increased risk of cutaneous neoplasia and ocular neoplasia at an early onset of life. Neurological problems and increased risk of Central Nervous System (CNS) neoplasms are also manifestations of XP. Conjunctival SCC occurring in background of XP usually presents earlier in life and tend to be more aggressive with bilateral involvement seen frequently [16].

Figure 2.7. The G1/S checkpoint prevents cells with damaged DNA from entering the S phase by inhibiting the initiation of replication. The intra-S-phase checkpoint deals with DNA damage that may occur during S-phase or unrepaired damage that escaped the G1/S checkpoint. The G2/M checkpoint prevents cells with damaged DNA from undergoing mitosis.

CONJUNCTIVAL MELANOMA

Conjunctival melanoma is a malignant tumour arising from conjunctival melanocytes. It represents 5% of all ocular melanomas. The annual incidence ranges from 0.1 to 0.7 per million population. Geographic variation follows racial composition of populations. Increasing incidence has been shown in several populations over the last 50 years. Roughly 10% of conjunctival melanomas arise in or involve the caruncle. The average age at diagnosis is 50–60 years. They are rare in children and there is no gender predilection.

Evidence linking chronic UV light exposure to conjunctival melanoma is accumulating, but is less robust than for cutaneous melanoma. No other environmental cause is established.

Conjunctiva melanomas may arise from melanocytic intraepithelial neoplasia (about 65%), pre-existing nevus, or de novo (fig. 2.8). These may be present adjacent to the main invasive lesion. Invasive conjunctival melanomas consist of varying combinations of atypical spindle cells, polyhedral cells, and epithelioid cells. These are usually contained within ill-defined nests within the conjunctival stroma (fig. 2.8).

a

b

c

d

Figure 2.8. Conjunctival melanoma in situ, and invasive conjunctival melanoma: a, b – conjunctival melanoma in situ, demonstrating the atypical melanocytes within the conjunctival epithelium, replacing the normal epithelial layers and goblet cells. This is highlighted in the image on the right with the MelanA stain; c, d – invasive conjunctival melanoma composed of predominantly epithelioid cells arranged in nests. These nests are surrounded by collagenous stroma and scattered lymphocytes. Infiltration of the tumour cells into lymph vessels and capillaries should be noted in histomorphology reports.

There is no known genetic susceptibility for conjunctival melanoma. It is genetically different from uveal melanoma (see below). Between 30–50% of conjunctival melanomas have BRAF mutations, and nearly 20% have NRAS mutations, similar to the proportions found with cutaneous melanoma. Other less common mutations involve TERT and KIT. CDK1NA and RUNX2 amplification were detected in 52% and 75% respectively. Less common mutations include TERT and KIT mutations [17, 18].

CONJUNCTIVAL LYMPHOMAS

Conjunctival lymphomas are mostly of the B-cell non-Hodgkin type. Extra nodal marginal zone lymphoma is the most common (70–80%) followed by Follicular lymphoma (10–15%) [19].

Several chromosomal aberrations are identified in ocular adnexal neoplasms. In EMZL (fig. 2.9) the most common translocation is t(11;18)(q21;q21) involving API2 and MALT1, t(14;18)(q32;q21) involving IGH and MALT1, t(3;14)(p14;q32) involving FOXP1 and IGH, and t(1;14)(p22;q32) involving Bcl-10 and IGH. The t(11;18)(q21;q21) translocation is present in approximately 20% of conjunctival EMZLs and is related to oxidative damage. The t(14;18)(q32;q21) translocation is found in around 15–20% of ocular adnexal EMZLs, specifically in the conjunctiva [20].

Trisomy 18 is very common in conjunctival EMZL and is found in 67% of patients [21] and is found to be associated with t(14;18) while trisomy 3 is associated with t(3;14) [22].

Most of the follicular lymphomas including the conjunctival ones are associated with the t(14;18)(q32.3;q21.3) translocation. This translocation involves BCL2 and IGH genes and leads to overexpression of the anti-apoptotic protein BCL2.

Most cases of MCLs are associate with t(11;14)(q13;q32) translocation which has also been found in conjunctival MCL. It involves both cyclin D-1 (CCND1) and IGH and results in the overexpression of the cyclin D-1 gene, leading to dysregulation of the cell cycle and enhanced proliferation. Table 2.1 summarises chromosomal translocations in conjunctival lymphomas.

a

b

Figure 2.9. Conjunctival extranodal marginal zone lymphoma (EMZL): a – monotonous proliferation of centrocyte-like B cells with involvement of the overlying epithelium; b – positivity of the tumour cells for the B-cell antigen CD20, and with evidence of the surface.

Table 2.1. Summarises chromosomal translocations in conjunctival lymphomas

Lymphoma

Translocations

Genes involved

Extranodal marginal zone lymphoma

t(11;18)(q21;q21)

API2 and MALT1

t(14;18)(q32;q21)

IGH and MALT1

t(3;14)(p14;q32)

FOXP1 and IGH

t(1;14)(p22;q32)

Bcl-10 and IGH

Follicular lymphoma

t(14;18)(q32.3;q21.3)

BCL2 and IGH

t(3;14)(q27;q32)

BCL6 and IGH

Mantle cell lymphoma

t(11;14)(q13;q32)

Cyclin D-1 (CCND1) and IGH

INTRAOCULAR CANCERS

MEDULLOEPITHELIOMA

Medulloepithelioma (fig. 2.10) is a non-hereditary congenital neoplasm of the non-pigmented ciliary epithelium arising from the primitive medullary epithelium located at the inner layer of the optic cup. It typically presents in the first decade of life.

Cytogenetic abnormalities described include der(16) t(1;16) chromosome, del(6q), and monosomy 15 [23]. Recently few cases of medulloepithelioma were described in patients having DICER-1 related pleuropulmonary blastoma [24]. DICER-1 mutation increases the risk of other cancers including cancers of the brain, female genital tract, thyroid, lung and testes. It is inherited as autosomal dominant trait and localised on the long arm of chromosome 14 (12q32.13) [25]. DICER1 is essential for the production of miRNAs which regulates gene expression during embryologic and early developmental stages through blocking mRNA action or degrading mRNA targets.

Figure 2.10. Medulloepithelioma: tubules of multiple layers of polarized neuroepithelium resembling embryonic medullary epithelium surrounded by loose stroma. This tumour shows malignant features in form of increased mitotic activity and pleomorphism. Some tumour can be teratoid containing cartilage, rhabdomyoblasts or brain like tissue (not shown here).

LIN28A encodes a protein LIN28A that binds a miRNA and function as post-transcriptional negative regulators of the let-7 family of miRNAs, which serve together as a tumour suppressor. Increased expression of LIN28A inhibits differentiation and as a result there will be accumulation of MYC(N), RAS, CDK6 and HMGA2 pro-oncogens. LIN28A is expressed in primitive neuronal tumours and primarily identified in the cytoplasm. LIN28A is not seen in any adult cell. LIN28A is being detected using immunohistochemichal stains in a subset of primitive CNS tumours namely Embryonal tumours with multi-layered rosettes (ETMR) and intra-ocular medullopeithelioma. The intensity of staining is suggested to predict an aggressive behaviour of the intra-ocular tumour [26].

RETINOBLASTOMA

Retinoblastoma (RB) is a malignant neoplasm of the neurosensory retina, affecting young children. Hereditary cases are typically bilateral with multiple tumour foci in one or two eyes and, more rarely, intracranially at midline.

Figure 2.11. Retinoblastoma: arising from the retina and showing early retina differentiation in the form of Flexner-Wintersteiner rosettes (cuboidal cells surrounding a central lumen which corresponds to subretinal space). Homer Wright rosettes lacks central lumen in contrast to Flexner-Wintersteiner rosettes.

RB is the most common primary intraocular malignant tumour in infants and young children, and is thought to be the most common primary malignant intraocular neoplasm overall. It accounts for approximately 3% of all cancers occurring in young children. The incidence of RB is reported as 1 in 16,000–18,000 live births with approximately 7000 to 8000 new cases worldwide yearly. The incidence of retinoblastoma appears to be higher in Africa, India, and among children of Native American descent.

Histopathologically, retinoblastoma is a ‘small round blue cell tumour’, composed of primitive neuroblastic cells with basophilic nuclei and scant cytoplasm (fig. 2.11).

RB can be either hereditary or non-hereditary. Hereditary form results from germline mutations involving the Rb1 gene and is inherited in an autosomal- dominant fashion. Tumours resulting from germline mutations usually presents with bilateral multifocal tumours (25% of retinoblastoma cases) or may present as unilateral or unifocal form (10%). The remaining 65% of cases are sporadic, and present as unilateral and usually unifocal tumour.

In 25% of hereditary form the Rb1 gene is inherited from an affected survivor; however, 75% results from new germline mutation. Genetic screening for Rb1 gene mutation is essential especially for the 10% unilateral cases without prior family history [27].

In 1971 Knudson proposed his two-hit hypothesis, postulating that two rate-limiting mutational events in the retinoblastoma susceptibility gene (RB1) were required for tumour formation in retinoblastoma [28] (fig. 2.12).

2.12

Figure 2.12. Two-hit hypothesis (Knudson hypothesis).

2.13

Figure 2.13. In the normal cell (a) – pRb binds and inhibits the E2F transcription factor in G0/G1 phase. Then during proliferation pRb is phosphorylated by Cyclin D and Cdk4/6 (which are under the inhibitory control of p16). E2F is released and is required for G1/S transition and cell proliferation. In the tumour cell (b) – E2F transcription factor is released and is not in an interaction with pRb, so the cell is proliferating. Possible causes include: Rb gene mutations, pRb binding to viral oncoproteins (HPV E7), or inappropriate phosphorylation of pRb, either by loss of p16 inhibition or over expression/mutations of Cdk4/6 or Cyclin D1.

The Rb1 gene is located on chromosome 13q14, spans 180kb and has 27 exons. Its product ‘pRb’ is a tumour suppressor gene. Its absence or inactivation results in deregulated cell proliferation (fig. 2.13). Most retinoblastoma cases are caused by a biallelic loss of RB1. Only 1% of children who carry an RB1 mutation remain unaffected.

In the familial hereditary form of retinoblastoma, the first mutational hit occurs in the germline, generating an RB1+/− genotype, and the second hit occurs sporadically in the somatic cells. In the non-familial, sporadic retinoblastoma, two sporadic mutational hits occur in the somatic cells.

The mutant RB1 allele is identifiable in blood samples of 95% of bilaterally affected patients. Low-level mosaicism can account for the remaining 5% of blood samples in which no mutation is found [29].

A recent study described the distribution of RB1 mutations in 1173 patients and classified them as; nonsense (37%), frameshift (20%), splice (21%), large indel (9%), missense (5%), chromosomal deletions (7%), and promoter (1%) [30].

Inactivation of RB1 gene also cause chromosomal instability leading to formation of further mutations. Additional genetic abnormalities were also detected in retinoblastoma. Two-thirds of tumour has MDM2/MDM4 amplification leading to p53 inactivation.

A small proportion of unilateral tumours with normal RB1 gene has been describe in the literature. Most of those cases have shown N-MYC oncogene amplification and expressed functional RB1 protein. They have distinct histological features with only a few of the genomic copy-number seen in retinoblastoma and are diagnosed at a very early age [31].

UVEAL MELANOMA

Uveal melanoma (UM) is a malignant tumour occurring predominantly in the choroid (90%) with remainder being confined to the ciliary body and the iris. The overall incidence of UM is 6–7 per million per year. It increases with age from about 2.5 per million between the ages of 15 and 44 years to 25 per million after the age of 65 years. The tumour is extremely rare in children. There is no significant sex difference.

UM is much more common in Caucasians than in Africans or Asians. Epidemiological studies suggest that UM is 2–3 times more common in blue/grey than in brown eyes. The aetiology of UM is unknown; however, there are known associations with UM. These include: ocular melanocytosis (increased population of melanocytes within the uvea and episclera), oculodermal melanocytosis (naevus of Ota, which also involves the periocular skin and meninges), simple and dysplastic cutaneous naevi and cutaneous melanomas, and neurofibromatosis type 1.

Rare reports of families with an excess of UM cases have been published. UM is part of the autosomal dominantly inherited ‘BAP1 (BRCA1-associated protein-1) familial cancer syndrome’. Affected patients, who have germline mutations in BAP1 develop mesotheliomas and unusual benign atypical melanocytic skin tumours as well as UM.

UM can be either dome- or mushroom shaped lesions, the latter due to perforation of Bruch’s membrane. They consist predominantly of tumour cells with some admixed reactive cells usually representing a smaller proportion of the tumour’s cellular content. According to the modified Callender classification, UM consist of spindle B cells and/or of epithelioid cells (fig. 2.14). The tumour cells stain for melanocytic markers, such as MelanA, HMB45, MITF and S-100P. Immunohistochemical staining is routinely performed using BAP1, which aids determination of the status of the BAP1 gene and indirectly, chromosome 3 status [32, 33] (see below).

It has been known for almost 20 years that UM show specific chromosomal alterations, which are quite distinct from melanomas at other sites, particularly those of the skin [34, 35]. The most striking abnormality in UM is the complete or partial loss of chromosome 3. Other common genetic abnormalities of UM include loss on 1p, 6q, 8, and 9p as well as gain on 1q, 6p, and 8q. These alterations were initially identified by standard karyotypic analyses. They have subsequently been confirmed by several groups using differing technologies, including: fluorescence in situ hybridization (FISH); comparative genomic hybridisation (CGH); spectral karyotyping; microsatellite analysis (MSA); Multiplex ligation-dependent probe amplification (MLPA), and single nucleotide polymorphisms (SNPs).

Recent advances in the molecular characterisation of UM using multiple and complex molecular techniques have identified additional molecular changes that contribute to the development and progression of this disease. These include alterations in the following genes with decreasing frequency: BAP1, SF3B1, EIFAX1, PLCB4, CYSLTR2, TP53BP1, CSMD1, TTC28, DLK2, and KTN1. The biological significance of all of these findings is not yet clear. Other genetic changes of relevance to metastatic progression include the amplification of DDEF1 and PTP4A3 on chromosome 8q.

The above-mentioned chromosomal alterations in primary UM are clinically relevant because of their correlation with the risk of metastatic death. Chromosome 3 loss is associated with a reduction of the 5-year survival probability from approximately 100% to 50%. Similarly, chromosome 8 gains and loss of chromosome 1 significantly correlate with reduced survival. Both chromosome 3 loss and polysomy 8q are also associated with other poor prognostic factors, including increasing tumour basal diameter, ciliary body involvement, presence of epithelioid cells, high mitotic count, and closed connective tissue loops. Conversely, gains in chromosome 6p correlate with a good prognosis, suggesting this aberration may have a functionally protective effect.

A PCR-based 12-gene assay based on gene expression profiling (GEP), divides UM into two ‘classes’ on the basis of an mRNA expression signature: class 1 and class 2 [36]. Class 1 UM often show 6p and 8q gain. Class 2 UM tend to show more aneuploidy with 1p loss, 3 loss, 8p loss, and 8q gain. Class 2 UM are also strongly associated with inactivating mutations of BAP1, located at 3p21. Increasingly prognostic testing is being performed on small intraocular biopsies. Essential for precise prognostication is:

a

b

c

d

Figure 2.14. Uveal melanoma morphology and BAP immunohistochemistry: a, b – UM of plump spindle morphology with clear nuclear positivity of the tumour cells for BAP1; c, d – UM composed of large spindle cells with connective tissue loops and with loss of nuclear expression for BAP1 protein. Loss of nuclear BAP1 protein expression as demonstrated by immunohistochemistry is associated with a high risk of metastatic spread.

a) morphological examination of the biopsy;

b) the integration of relevant clinical, histological and genetic factors, resulting in an individualised curve allowing for improved downstream personalised care.

VITREORETINAL LYMPHOMA

Vitreoretinal Lymphoma (VRL) is an aggressive B-cell lymphoma, usually demonstrating diffuse large B cell lymphoma morphology and immune profile (fig. 2.15). It is closely associated and related to primary CNS lymphoma. Most of the VRL cases are of the activated B-cell (ABC) subtype of DLBCL and are derived from a late stage of germinal centre B cell differentiation [37]. VRL expresses monoclonal immunoglobulins heavy chains and light chains rearrangement, which demonstrate a high number of somatic hypermutations (SHM).

Genetic studies for VRL showed a subset of tumours with a high frequency of IgH/BCL2 re-arrangement by PCR due to t(14:18) which is seen also in Follicular lymphoma and some DLBCL [38].

BCL6 translocations has been demonstrated in up to 47% PCNSL. Also, there is a high frequency of MYD88 mutations has been identified in PCNSL and ~70% of PVRL [39, 40].

a

b

Figure 2.15. Vitreoretinal lymphoma: a – a May-Grunewald Giemsa stained vitreous biopsy revealing numerous lymphatic blasts of medium-to-large size, with a narrow cytoplasm, and with prominent nuclei and nucleoli; b – immunostaining using the CD79a antibody demonstrates that the vast majority of the blasts are of B-cell type.

SUMMARY

Presented was an overview of the range of the most common ocular tumours with a brief description of their epidemiological, morphological, immunohistochemical and genetic characteristics. More details are provided in the later chapters regarding treatment of these tumours. It is clear that whilst significant progress has been made in the underlying biology of these malignancies, there is still considerable room for improvement in their treatment, particularly their metastases. It will be through the application of new technologies to detect tumour spread at an earlier time point and through the application of new targeted medicines as adjuvant therapies (alone or in combination), that breakthroughs will be made and the prognoses improved for the ocular cancer patients.

References

1. Fujii K., Miyashita T.: Gorlin syndrome (nevoid basal cell carcinoma syndrome): update and literature review. Pediatrics International 2014; 56: 667–674. 2. Pellegrini C., Maturo M.G., Di Nardo L. et al.: Understanding the molecular genetics of basal cell carcinoma. International Journal of Molecular Sciences 2017; 18(11): 2485.3. Bolshakov S., Walker C.M., Strom S.S. et al.: p53 mutations in human aggressive and nonaggressive basal and squamous cell carcinomas. Clinical Cancer Research 2003; 9(1): 228–234.4. Bonilla X., Parmentier L., King B. et al.: Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma. Nature Genetics 2016; 48(4): 398–406.5. Stagner A.M., Jakobiec F.A.: Updates on the molecular pathology of selected ocular and ocular adnexal tumors: potential targets for future therapy. Seminars in Ophthalmology 2016; 31(1–2): 188–196.6. Bhaijee F., Brown A.S.: Muir-Torre syndrome. Archives of Pathology and Laboratory Medicine 2014; 138(12): 1685–1689.7. Shalin S.C., Lyle S., Calonje E. et al.: Sebaceous neoplasia and the Muir-Torre syndrome: important connections with clinical implications. Histopathology 2010; 56(1): 133–147.8. Hussain R.M., Matthews J.L., Dubovy S.R. et al.: UV-independent p53 mutations in sebaceous carcinoma of the eyelid. Ophthalmic Plastic and Reconstructive Surgery 2014; 30(5): 392–395.9. Kiyosaki K., Nakada C., Hijiya N. et al.: Analysis of p53 mutations and the expression of p53 and p21WAF1/CIP1 protein in 15 cases of sebaceous carcinoma of the eyelid. Investigative Ophthalmology & Visual Science 2010; 51(1): 7–11.10. Kumar A., Kumar Dorairaj S., Prabhakaran V.C. et al.: Identification of genes associated with tumorigenesis of meibomian cell carcinoma by microarray analysis. Genomics 2007; 90(5): 559–566.

11. Uchi H.: Merkel cell carcinoma: an update and immunotherapy. Frontiers in Oncology 2018; 8: 48. 12. Becker J.C.: Merkel cell carcinoma. Annals of Oncology 2010; 21(Suppl. 7): vii81–vii5.13. Simbiri K.O., Murakami M., Feldman M. et al.: Multiple oncogenic viruses identified in Ocular surface squamous neoplasia in HIV-1 patients. Infectious Agents and Cancer 2010; 5: 6.14. Gichuhi S., Sagoo M.S., Weiss H.A. et al.: Epidemiology of ocular surface squamous neoplasia in Africa. Tropical Medicine and International Health 2013; 18(12): 1424–1443.15. Lehmann A.R., McGibbon D., Stefanini M.: Xeroderma pigmentosum. Orphanet Journal of Rare Diseases 2011; 1: 6–70.16. Gupta N., Sachdev R., Tandon R.: Ocular surface squamous neoplasia in xeroderma pigmentosum: clinical spectrum and outcome. Graefe’s Archive for Clinical and Experimental Ophthalmology 2011; 249(8): 1217–1221.17. Griewank K.G., Westekemper H., Murali R. et al.: Conjunctival melanomas harbor BRAF and NRAS mutations and copy number changes similar to cutaneous and mucosal melanomas. Clinical Cancer Research 2013; 19(12): 3143–3152.18. Lake S.L., Jmor F., Dopierala J. et al.: Multiplex ligation-dependent probe amplification of conjunctival melanoma reveals common BRAF V600E gene mutation and gene copy number changes. Investigative Ophthalmology & Visual Science 2011; 52(8): 5598–5604.19. Kirkegaard M.M., Rasmussen P.K., Coupland S.E. et al.: Conjunctival lymphoma – an international multicenter retrospective study. JAMA Ophthalmology 2016; 134(4): 406–414.20. Cerrone M., Collina F., De Chiara A. et al.: BCL10 expression and localization in ocular adnexa MALT lymphomas: a comparative cytogenetic and immunohistochemical study. Histology and Histopathology 2014; 29(1): 77–87.

21. Ruiz A., Reischl U., Swerdlow S.H. et al.: Extranodal marginal zone B-cell lymphomas of the ocular adnexa: multiparameter analysis of 34 cases including interphase molecular cytogenetics and PCR for Chlamydia psittaci. American Journal of Surgical Pathology 2007; 31(5): 792–802. 22. Streubel B., Vinatzer U., Lamprecht A. et al.: T(3;14)(p14.1;q32) involving IGH and FOXP1 is a novel recurrent chromosomal aberration in MALT lymphoma. Leukemia 2005; 19(4): 652–658.23. Betts D.R., Leibundgut K.E., Niggli F.K.: Cytogenetic analysis in a case of intraocular medulloepithelioma. Cancer Genetics and Cytogenetics 1996; 92(2): 144–146.24. Priest J.R., Williams G.M., Manera R. et al.: Ciliary body medulloepithelioma: four cases associated with pleuropulmonary blastoma – a report from the International Pleuropulmonary Blastoma Registry. British Journal of Ophthalmology 2011; 95(7): 1001–1005.25. Shields J.A., Eagle R.C. Jr, Ferguson K. et al.: Tumors of the nonpigmented epithelium of the ciliary body: the Lorenz E. Zimmerman Tribute Lecture. Retina 2015; 35(5): 957–965.26. Jakobiec F.A., Kool M., Stagner A.M. et al.: Intraocular medulloepitheliomas and embryonal tumors with multilayered rosettes of the brain: comparative roles of LIN28A and C19MC. American Journal of Ophthalmology 2015; 159(6): 1065–1074.27. Rodriguez-Galindo C., Orbach D.B., VanderVeen D.: Retinoblastoma. Pediatric Clinics of North America 2015; 62(1): 201–223.28. Knudson A.G. Jr.: Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Sciences of the United States of America. 1971; 68(4): 820–823.29. Rushlow D., Piovesan B., Zhang K. et al.: Detection of mosaic RB1 mutations in families with retinoblastoma. Human Mutation 2009; 30(5): 842–851.30. Dommering C.J., Mol B.M., Moll A.C. et al.: RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients. Journal of Medical Genetics 2014; 51(6): 366–374.

31. Rushlow D.E., Mol B.M., Kennett J.Y. et al.: Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies. Lancet Oncology 2013; 14(4): 327–334. 32. Farquhar N., Thornton S., Coupland S.E. et al.: Patterns of BAP1 protein expression provide insights into prognostic significance and the biology of uveal melanoma. Journal of Pathology. Clinical Research 2017; 4(1): 26–38.33. Kalirai H., Dodson A., Faqir S. et al.: Lack of BAP1 protein expression in uveal melanoma is associated with increased metastatic risk and has utility in routine prognostic testing. British Journal of Cancer 2014; 111(7): 1373–1380.34. Damato B., Dopierala J.A., Coupland S.E.: Genotypic profiling of 452 choroidal melanomas with multiplex ligation-dependent probe amplification. Clinical Cancer Research 2010; 16(24): 6083–6092.35. Höglund M., Gisselsson D., Hansen G.B. et al.: Dissecting karyotypic patterns in malignant melanomas: temporal clustering of losses and gains in melanoma karyotypic evolution. International Journal of Cancer 2004; 108(1): 57–65.36. Harbour J.W.: The genetics of uveal melanoma: an emerging framework for targeted therapy. Pigment Cell and Melanoma Research 2012; 25(2): 171–181.37. Coupland S.E., Hummel M., Stein H. et al.: Demonstration of identical clonal derivation in a case of 'oculocerebral' lymphoma. The British Journal of Ophthalmology 2005; 89(2): 238–239.38. Wallace D.J., Shen D., Reed G.F. et al.: Detection of the bcl-2 t(14;18) translocation and proto-oncogene expression in primary intraocular lymphoma. Investigative Ophthalmology & Visual Science 2006; 47(7): 2750–2756.39. Bonzheim I., Giese S., Deuter C. et al.: High frequency of MYD88 mutations in vitreoretinal B-cell lymphoma: a valuable tool to improve diagnostic yield of vitreous aspirates. Blood 2015; 126(1): 76–79.40. Araujo I., Coupland S.E.: Primary vitreoretinal lymphoma – a review. Asia-Pacific Journal of Ophthalmology 2017; 6(3): 283–289.

Notes

* Prof. Sarah E. Coupland participates in the project UMCure2020 „New therapies for uveal melanoma”, no 667787 under Horizon 2020.