DISSERTATIONS | MINNA SOPO | EPITHELIAL OVARIAN CANCER AND ANGIOGENESIS , BIOMARKER AND GENE ... | No 639
MINNA SOPO
Epithelial ovarian cancer and angiogenesis
Biomarker and gene therapy study
Dissertations in Health Sciences
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
EPITHELIAL OVARIAN CANCER AND ANGIOGENESIS
BIOMARKER AND GENE THERAPY STUDY
Minna Sopo
EPITHELIAL OVARIAN CANCER AND ANGIOGENESIS
BIOMARKER AND GENE THERAPY STUDY
To be presented by permission of the
Faculty of Health Sciences, University of Eastern Finland
for public examination in Kuopio University Hospital Auditorium 1, Kuopio on Friday 1st October 2021, at 12 o’clock noon
Publications of the University of Eastern Finland Dissertations in Health Sciences
No 639
Department of Obstetrics and Gynecology, Institute of Clinical Medicine, Faculty of Health Sciences,
University of Eastern Finland Kuopio
2021
Series Editors
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Professor Tarja Kvist, Ph.D.
Department of Nursing Science Faculty of Health Sciences Professor Ville Leinonen, M.D., Ph.D.
Institute of Clinical Medicine, Neurosurgery Faculty of Health Sciences
Professor Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D.
School of Pharmacy Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
P.O.Box 1627 FI-70211 Kuopio, Finland
www.uef.fi/kirjasto PunaMusta Oy
Vantaa, 2021
ISBN: 978-952-61-4291-3 (print/nid.) ISBN: 978-952-61-4292-0 (PDF)
ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s address: Department of Obstetrics and Gynecology Kuopio University Hospital
University of Eastern Finland KUOPIO
FINLAND
Doctoral programme: Doctoral programme of Clinical Research
Supervisors: Docent Hanna Sallinen, M.D., Ph.D.
Department of Obstetrics and Gynecology University of Eastern Finland
KUOPIO FINLAND
Docent Maarit Anttila, M.D., Ph.D.
Department of Obstetrics and Gynecology University of Eastern Finland
KUOPIO FINLAND
Professor Seppo Ylä-Herttuala, M.D., Ph.D.
Department of Molecular Medicine University of Eastern Finland KUOPIO
FINLAND
Docent Kirsi Hämäläinen, M.D., Ph.D.
Department of Pathology University of Eastern Finland KUOPIO
FINLAND
Reviewers: Docent Ralf Bützow, M.D., Ph.D.
Department of Pathology University of Helsinki HELSINKI
FINLAND
Docent Anna Kanerva, M.D., Ph.D.
Department of Obstetrics and Gynecology Helsinki University Hospital
HELSINKI FINLAND
Opponent: Docent Johanna Hynninen, M.D., Ph.D.
Department of Obstetrics and Gynecology Turku University Hospital
TURKU FINLAND
7 Sopo, Minna
Epithelial ovarian cancer and angiogenesis, Biomarker and gene therapy study Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 639. 2021, 214 p.
ISBN: 978-952-61-4291-3 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-4292-0 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
So far, the prognosis of epithelial ovarian cancer (EOC) has not significantly improved despite fast-developing treatment strategies. The antiangiogenic
approach has been part of standard treatment in advanced and recurrent ovarian cancer with variable success. The mechanisms behind the resistance to treatment and variable efficacy of the treatment have remained unclear. Biomarkers for optimal patient selection and monitoring the treatment response in addition to new modalities to boost the therapeutic effect are urgently needed. The aim of this study was to explore nine principal angiogenic factors in ovarian carcinoma tissue and to assess their relevance as prognostic biomarkers. Furthermore, the therapeutic efficacy of a new antiangiogenic treatment strategy was investigated in a xenograft model of ovarian cancer.
The clinical study population consisted of 86 patients with epithelial ovarian carcinoma, primarily operated in Kuopio University Hospital between 1999–2007.
In addition, 17 patients with borderline and 36 patients with benign epithelial ovarian tumors were included in study IV. Omental metastatic samples were also explored from 16 patients with serous carcinomas. Immunohistochemical stainings of vascular endothelial growth factor (VEGF) -A, -C, -D, VEGF receptors (-R1, -R2, -R3), angiopoietin-2 (Ang-2), tyrosine kinase receptors Tie-1 and Tie-2 were analysed in all malignant primary and metastatic tumor samples, assessing separately the expression in neoplastic, stromal and endothelial cells.
Furthermore, micro- and lymph vessel parameters, assessed by cluster of
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differentiation (CD) 34, 105 and D2-40, were calculated from all 139 patients with different histologies. Additionally, adenovirus-mediated antiangiogenic gene therapy with soluble (s)VEGFR1, -R2 and -R3 was combined with paclitaxel and compared with single chemotherapy and bevacizumab in an ovarian cancer animal model with athymic nude mice.
We found that tumor epithelial expression of VEGF-A, -D, -R1, Ang-2 and Tie-2 was significantly higher in omental metastatic lesions than in primary high-grade serous carcinoma. In addition, high expression of Tie-2 predicted poor overall survival (OS) in high-grade serous carcinoma. In contrast, low expression of VEGF-A and Ang-2 in primary tumors was associated with poor overall survival.
Microvessel parameters, assessed by CD34 and CD105, were higher in malignant tumors than in borderline and benign ones. Further, small lymph vessels in ovarian carcinoma predicted shorter survival, and a high density of lymph vessels was related to lymph node metastases and cancer recurrence. In the first study, intravenous antiangiogenic gene therapy with sVEGFR1, -R2 and -R3 in
combination with paclitaxel prolonged the overall survival of mice compared to single chemotherapy, bevacizumab and control groups. Tumors of the gene therapy group were also smaller and less vascularized than tumors of other groups.
An increase in angiogenesis is a feature of epithelial ovarian carcinoma.
Accumulation of angiogenic factors in abdominal metastases might reflect more active angiogenesis and capacity to disseminate. According to this study, Tie-2 had the most potential as an angiogenic tissue biomarker, warranting further studies on its prognostic value. The endothelial antibodies, CD34 and CD105, define different properties of microvessels. Neither showed superiority over the other to predict angiogenic activity in ovarian cancer. Instead, the prognostic significance of lymphangiogenesis in ovarian cancer became clarified. Antiangiogenic gene therapy with VEGF receptors seems to have treatment efficacy in a xenograft model and could have potential in clinical studies.
National Library of Medicine Classification: WP 322, QZ 33, QS 523, QZ 365, WB 142, QU 107, QY60.R6, QW165.5.A3, WH 700
Medical Subject Headings: Carcinoma, Ovarian Epithelial, Ovarian Neoplasms, Angiogenesis, Angiogenesis Inhibitors, Immunohistochemistry, Microvessels, Vascular Endothelial Growth Factor, Angiopoietins, Receptors, Lymphangiogenesis, Gene Therapy, Disease Models, Animal
9 Sopo, Minna
Epiteliaalinen munasarjasyöpä ja angiogeneesi, Biomerkkiaine- ja geeniterapiatutkimus
Kuopio: Itä-Suomen yliopisto
Publications of the University of Eastern Finland Dissertations in Health Sciences 639. 2021, 214 s.
ISBN: 978-952-61-4291-3 (nid.) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-4292-0 (PDF) ISSN: 1798-5714 (PDF)
TIIVISTELMÄ
Epiteliaalisen munasarjasyövän ennuste on gynekologisista syövistä heikoin nopeasti kehittyvistä kohdennetuista hoitomuodoista huolimatta. Verisuonten uudismuodostusta estävä lääkehoito on kuulunut osana edenneen ja uusiutuneen munasarjasyövän hoitoon jo usean vuoden ajan. Hoidon haasteiksi ovat
muodostuneet sen rajallinen teho, resistenssin kehittyminen ja sivuvaikutukset.
Hoidon tehon seuraamiseksi ja potilasvalinnan optimoimiseksi ei ole olemassa biomerkkiaineita. Tämän tutkimuksen tavoitteena oli selvittää yhdeksän merkittävän angiogeenisen tekijän esiintymistä munasarjasyöpäkudoksessa ja niiden yhteyttä syövän ennusteeseen. Lisäksi tutkittiin antiangiogeenisen geenihoidon tehoa munasarjasyövän eläinmallissa.
Kliiniseen tutkimukseen osallistui 86 epiteliaalista munasarjasyöpää sairastavaa naista, jotka operoitiin ensivaiheessa Kuopion Yliopistollisessa sairaalassa vuosina 1999–2007. Tämän ohella neljännessä osatyössä tutkittiin näytteet 17
rajalaatuisesta ja 36 hyvänlaatuisesta munasarjakasvaimesta. 16 syöpäpotilaalta analysoitiin lähtökasvaimen lisäksi näytteet vatsapaidan etäispesäkkeestä. Kaikista syöpäkasvainnäytteistä tehtiin immunohistokemialliset värjäykset verisuonten endoteelikasvutekijöistä (VEGF-A, -C, -D), niiden reseptoreista (VEGFR1, -2, -3) ja angiopoietiini-2:sta (Ang-2) sekä Tie-1 ja Tie-2 reseptoreista. Näytteistä analysoitiin neoplastisten ja strooman solujen ekspressio erikseen. Veri- ja imusuonivärjäykset CD34, CD105 ja D2-40 endoteelimerkkiaineilla tehtiin kaikista 139
munasarjakasvainnäytteestä. Lisäksi adenovirusvälitteisen antiangiogeenisen
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geenihoidon tehoa liukoisilla VEGF-reseptoreilla yhdistettynä paklitakseliin verrattiin kemoterapiaan ja monoklonaaliseen VEGF-vasta-aineeseen munasarjasyövän eläinmallissa immuunipuutteisilla nude hiirillä.
Tutkimuksen perusteella verisuonten uudismuodostus oli aktiivisempaa syöpäkasvaimissa verrattuna raja- ja hyvänlaatuisiin kasvaimiin. Lisäksi etäispesäkkeissä verisuonikasvutekijöiden (VEGF-A, -D, -R1, Ang-2, Tie-2) ilmentyminen oli runsaampaa verrattuna lähtökasvaimeen viitaten vilkkaaseen angiogeneesiin. Korkea Tie-2 ilmentyminen lähtökasvainten epiteelisoluissa oli yhteydessä lyhyeen elinaikaennusteeseen korkea-asteisessa seroosissa munasarjasyövässä. Toisaalta VEGF-A ja Ang-2 ilmentyivät heikommin huono- ennusteisten potilaiden primaarisyöpäkasvaimissa. Kasvainten pienikokoiset imusuonet ennustivat lyhyttä elinaikaa ja korkea imusuonitiheys oli yhteydessä imusolmukelevinneisyyteen ja syövän uusiutumiseen. Antiangiogeeninen
geenihoito liukoisilla VEGF-reseptoreilla yhdistettynä paklitakseliin pidensi hiirten kokonaiselinaikaa pelkkään kemoterapiaan, bevasitsumabiin tai kontrolliryhmään verrattuna. Lisäksi geenihoitoryhmän kasvaimet olivat pienempiä ja vähemmän verisuonitettuja kuin vertailuryhmien.
Lisääntynyt angiogeneesi on pahanlaatuisten munasarjakasvainten ominaisuus ja angiogeenisten tekijöiden kumuloituminen etäispesäkkeisiin voi viitata
kasvaimen aggressiivisempaan kykyyn metastasoida. Tämän tutkimuksen perusteella Tie-2 oli ennusteellisesti potentiaalein angiogeeninen tekijä
kasvainkudoksessa. Endoteelivasta-aineet CD34 ja CD105 kuvastivat verisuonten eri ominaisuuksia eikä eroa niiden ennusteellisuudessa pystytty osoittamaan. Sen sijaan imusuonten uudismuodostuksen merkitys munasarjasyövän ennusteeseen ilmeni tässä tutkimuksessa. Antiangiogeenisen geenihoidon teho liukoisilla VEGF reseptoreilla tuli osoitettua ja potentiaali edetä kliinisiin tutkimuksiin vahvistui.
Luokitus: WP 322, QZ 33, QS 523, QZ 365, WB 142, QU 107, QY60.R6, QW165.5.A3, WH 700
Yleinen suomalainen ontologia: Munasarjasyöpä, angiogeneesi,
immunohistokemia, verisuonet, endoteeli, kasvutekijät, angiopoietiinit, reseptorit, imusuonet, eläinkokeet, geeniterapia, adenovirukset, hoitomenetelmät
11 To Marko, Aleksi, Annika and Elias
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ACKNOWLEDGEMENTS
This study was carried out at the Department of Gynecology and Obstetrics and Department of Pathology, Kuopio University Hospital and University of Eastern Finland during 2016-20. The first study was conducted in the Department of Molecular Medicine, A.I. Virtanen Institute, University of Eastern Finland in 2009- 2012.
First of all, I owe my deepest gratitude to my principle supervisors, Docent Hanna Sallinen and Docent Maarit Anttila. You two have made this project possible. In the beginning, you, Hanna, introduced me to the world of
experimental studies with animals and ovarian cancer. And from thereon You have encouraged, supported, taught and concretely helped me not only with your warm, human and gentle spirit, but also with your uncompromising expertise and precision. I greatly appreciate our friendship and sharing also other aspects of life.
Maarit, your soul is in gyn oncology and research, and you are a role model for me as for many others. I am extremely grateful for your support, encouragement and belief you have poured on me also during tough times. In some way, you always find the time, (which you don`t have), to discuss and go through
problematic steps to find the solutions. Your persistent strength, knowledge and experience of the field is irresistible and astonishing.
I was honored to have Professor Seppo Ylä-Herttuala as my supervisor in A.I.
Virtanen Institute. I owe my greatest gratitude to Professor Ylä-Herttuala, whose enormous expertise, enthusiasm, optimism and endless ideas were extremely inspiring and encouraging for a novice reasearcher. Professor Ylä-Hettuala also provided me a possibility to work in a top-class research team of gene therapy field, which I highly appreciate and which was an invaluable experience to me.
I am very grateful to Professor Leea Keski-Nisula for your genuine interest, enthusiasm and supportive attitude towards my work. I want to thank Professor Seppo Heinonen for guiding me in my first steps of my study.
I express my thanks to Docent Kirsi Hämäläinen for kindly offering your highly professional view and patiently teaching me to analyse immunohistochemical samples. I wish to thank Professor Veli-Matti Kosma for providing the
circumstances for this kind of research project.
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I am honored to have Docent Johanna Hynninen from the University of Turku as my opponent. I want to express my sincere gratitude to the official reviewers of this thesis, Docent Anna Kanerva and Docent Ralf Bützow. I am grateful to Docent David Laaksonen for the careful linguistic revision of the previously unpublished parts of this thesis.
I wish to thank my research team in A.I. Virtanen Institute and fellow co-authors for proficient and highly professional collaboration: PhD Annukka Kivelä, PhD Anniina Laurema, Svetlana Laidinen, PhD Pasi Tuunanen, PhD Jonna Koponen, PhD Laura Tuppurainen, Docent Petra Korpisalo and Oona-Tuuli Muukkonen. I want to thank Professor Kari Alitalo from University of Helsinki for collaboration. My deepest thanks belong to invaluable technical assistance from Mrs Helena Kemiläinen and Mrs Seija Sahrio. I also want to thank Tuomas Selander for kind assistance with statistical questions.
I want to express my warm gratitude to my present and former colleagues in the Department of Gynecology and Obstetrics. Special thanks belong to PhD Maijakaisa Harju for friendship, support and sharing the ”ups and downs” of life and PhD Maija-Riitta Orden for your encouraging attitude and believing in me. I want to thank Professor Marjo Tuppurainen and PhD Marja Komulainen for your supportive attitude towards my research. I owe my heart-felt gratitude to Marja- Liisa Eloranta for your kind and patient support and teaching me in gyn oncologic surgery and working together. I also want to thank the whole gyn oncology team for having me as a part of the team.
I wish to thank all friends along my lifetime, especially my trusty beloved ”non- biological sister” Tanja Nuotio. We have grown together, been soulmates from childhood until today and gone through heaps of unmemorable moments together. I want also express my special thanks to our group of ”lovely ladies” or
”thirties” with Helena, Terhi, Pia, Riikka, Kati and Sirkku for our hilarious meetings and upkeep of mental health with laugh and joy.
My loving thanks belong to my parents, Tuula and Esa Leinonen, for always caring and believing in me. My dear dad, wish you could be sharing this moment with us. I know you would be proud. I am thankful for my little brother Timo and his spouse Merja for being part of my life. I want to express my respect and gratitude to my aunt Seija Niskanen for inspiring discussions and deeper lifetime support. I also wish to thank my parents-in-law Pirkko and Raimo Sopo for helping our family during these years.
15 Finally, I owe my deepest, loving feelings to my own family. The greatest
achievements of my life, my precious children, Aleksi, Annika and Elias, you have kept me in real life and arranged numerous unmemorable happy and joyful moments to my life. I am very proud of you all. And lastly, Marko, you just have made this all possible! Without your everlasting loving support and total
commitment to our family, this thesis would not have been completed. Words are not enough to describe the appreciation for your patience, understanding, gentle caring and optimistic spirit towards me and this project. Thank you for being there, I love you.
Kuopio, November 2020 Updated May 2021 Minna Sopo
This study has been financially supported by Kuopio University hospital EVO- and VTR grants, Kuopio University Hospital Research Foundation, Kuopio University Foundation, the Finnish Medical Foundation, the Finnish Cultural Foundation of Northern Savo, the Emil Aaltonen Foundation, the Finnish Cancer Society of Northern Savo, the Foundation of Gynecology and Obstetrics, the Paavo Koistinen Foundation, the Ida Montin Foundation
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LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications:
I Sopo M, Anttila M, Sallinen H, Tuppurainen L, Laurema A, Laidinen S, Hämäläinen K, Tuunanen P, Koponen J, Kosma VM, Heinonen S, Alitalo K, Ylä-Herttuala S: Antiangiogenic gene therapy with soluble VEGF receptors - 1, -2 and -3 together with paclitaxel prolongs survival of mice with human ovarian carcinoma; Int J Cancer 2012 Nov 15; 131(10): 2394–401.
II Sopo M, Anttila M, Hämäläinen K, Kivelä A, Ylä-Herttuala S, Kosma VM, Keski-Nisula L, Sallinen H: Expression profiles of VEGF-A, VEGF-D and VEGFR1 are higher in distant metastases than in matched primary high grade epithelial ovarian cancer; BMC Cancer 2019 June 14; 19(1): 584.
III Sopo M, Sallinen H, Hämäläinen K, Kivelä A, Ylä-Herttuala S, Kosma VM, Keski-Nisula L, Anttila M: High expression of Tie-2 predicts poor prognosis in high grade serous ovarian cancer; PlosOne 2020 Nov, 15(11): e0241484.
IV Sopo M, Anttila M, Muukkonen OT, Ylä-Herttuala S, Kosma VM, Keski-Nisula L, Sallinen H: Microvessels in epithelial ovarian tumors; high microvessel density is a significant feature of malignant ovarian tumors; Anticancer Research 2020 Dec; 40(12):6923–6931.
The publications were adapted with the permission of the copyright owners.
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CONTENTS
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ... 13
1 INTRODUCTION ... 29
2 REVIEW OF THE LITERATURE ... 31
2.1 Ovarian Cancer ... 31
2.1.1 Epidemiology and risk factors ... 31
2.1.2 Classification and pathogenesis ... 32
2.1.3 Genetic features of ovarian cancer ... 35
2.1.4 Prognostic factors ... 37
2.1.5 Diagnostics and current standard of treatment ... 40
2.1.6 Targeted therapies for ovarian cancer ... 42
2.2 Angiogenesis And Biomarkers ... 47
2.2.1 Physiological angiogenesis ... 47
2.2.2 Mechanisms of tumor angiogenesis and metastasis ... 48
2.2.3 Angiogenic pathways ... 51
2.2.4 Biomarkers of angiogenesis ... 58
2.3 Antiangiogenic Treatment And Gene Therapy ... 74
2.2.5 Studies of antiangiogenic treatment in ovarian cancer ... 74
2.2.6 Anti-angiogenic gene therapy for ovarian cancer ... 75
3 AIMS OF THE STUDY ... 85
4 ANTIANGIOGENIC GENE THERAPY WITH SOLUBLE VEGF-RECEPTORS -1, -2 AND -3 TOGETHER WITH PACLITAXEL PROLONGS SURVIVAL OF MICE WITH HUMAN OVARIAN CARCINOMA ... 87
4.1 Abstract... 87
4.2 Introduction ... 88
4.3 Materials And Methods ... 89
4.3.1 Cell line ... 89
4.3.2 Chemotherapy and anti-VEGF antibody ... 89
4.3.3 Viral vectors ... 90
4.3.4 Animal model ... 90
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4.3.5 Histology, immunohistochemistry, microvessel measurements and real-time quantitative PCR ... 92 4.3.6 Magnetic resonance imaging ... 93 4.3.7 Clinical chemistry ... 93 4.3.8 Statistical analyses... 94 4.4 Results ... 94 4.4.1 Transgene expression ... 94 4.4.2 Intraperitoneal tumor growth ... 94 4.4.3 Microvessel measurements ... 98 4.4.4 Survival and safety ... 98 4.5 Discussion ... 101 5 EXPRESSION PROFILES OF VEGF-A, VEGF-D AND VEGFR1 ARE HIGHER IN
DISTANT METASTASES THAN IN MATCHED PRIMARY HIGH GRADE
EPITHELIAL OVARIAN CANCER ... 105 5.1 Abstract... 105 5.2 Background ... 106 5.3 Methods... 108 5.3.1 Patients ... 108 5.3.2 Immunohistochemistry ... 110 5.3.3 Quantitative real-time polymerase chain reaction (qRT-PCR)
analysis ... 110 5.3.4 Statistical analysis ... 111 5.4 Results ... 111
5.4.1 Immunohistochemical analyses of VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2 and VEGFR3 in primary high-grade serous and endometrioid ovarian tumors ... 111 5.4.2 Immunohistochemical analyses of VEGF-A, VEGF-C, VEGF-D,
VEGFR1, VEGFR2 and VEGFR3 in metastatic lesions of high-grade serous ovarian cancer ... 116 5.4.3 Angiogenic factor expression in primary high-grade tumors
compared to the related metastases ... 116 5.4.4 qRT-PCR levels and correlations with immunohistochemical
expression ... 117
21 5.4.5 Relation of clinicopathological data to the expression of VEGFs and
VEGF receptors ... 119 5.4.6 Overall survival and progression-free survival of ovarian cancer
patients ... 121 5.5 Discussion ... 123 5.6 Conclusions ... 127 6 HIGH EXPRESSION OF TIE-2 PREDICTS POOR PROGNOSIS IN PRIMARY
HIGH GRADE SEROUS OVARIAN CANCER ... 129 6.1 Abstract... 129 6.2 Introduction ... 130 6.3 Materials And Methods ... 132 6.3.1 Patients and data collection ... 132 6.3.2 Immunohistochemistry ... 134 6.3.3 Quantitative real-time polymerase chain reaction (qRT-PCR)
analysis ... 135 6.3.4 Statistical analysis ... 135 6.4 Results ... 136
6.4.1 Immunohistochemical analyses of Angiopoietin-2, Tie-1 and Tie-2 in primary ovarian tumors and in metastatic lesions ... 136 6.4.2 Expression of angiogenic factors in primary high-grade serous
tumors as compared to related metastases... 137 6.4.3 Correlation of qRT-PCR results with immunohistochemical
staining ... 140 6.4.4 Relation of clinicopathological data to the expression of Ang-2,
Tie-1 and Tie-2 receptors ... 140 6.4.5 Overall survival and progression-free survival of ovarian cancer
patients ... 141 6.5 Discussion ... 144 7 MICROVESSELS IN EPITHELIAL OVARIAN TUMORS; HIGH MICROVESSEL
DENSITY IS A SIGNIFICANT FEATURE OF MALIGNANT OVARIAN
TUMORS ... 153 7.1 Abstract... 153 7.2 Introduction ... 154 7.3 Patients And Methods ... 156
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7.3.1 Patients and data collection ... 156 7.3.2 Immunohistochemistry ... 157 7.3.3 Statistical analysis ... 158 7.4 Results ... 160 7.4.1 Association of microvessel staining with ovarian tumor type ... 160 7.4.2 Relation of clinicopathological data to the microvessels in
ovarian cancer... 164 7.4.3 Progression-free survival and overall survival of the ovarian
cancer patients ... 165 7.4.4 Correlation between density, percentage, size and number of
microvessels, with VEGF and VEGF receptor expression in
ovarian tumors ... 166 7.4.5 Correlation of density and number of microvessels between
CD34 and endoglin staining ... 166 7.5 Discussion ... 167 8 GENERAL DISCUSSION ... 169 8.1 Principle Results ... 169 8.2 Results In Relation To Other Studies ... 170 8.3 Validity And Limitations Of The Study ... 173 8.4 Clinical Significance And Future Prospects ... 175 9 CONCLUSIONS ... 179 REFERENCES ... 181
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ABBREVIATIONS
AAV Adeno-associated virus
ACTH Adrenocorticotropin
Ad Adenovirus
AGO Arbeitsgemeinschaft Gynäkologische Onkologie Alt Alanineamino
transferase Ang Angiopoietin ARID1A AT-Rich Interaction
Domain 1A
ATM Ataxia-Telangiectasia AUC Area under the curve bFGF basic fibroblast growth
factor
BM Basement
membrane
BOT Borderline ovarian tumor
BRAF Serine/threonine- protein kinase BRCA Breast cancer-
associated gene BRIP1 BRCA1-interacting
protein 1 Bv8 Prokineticin-2 Ca12-5 Cancer antigen 125
CAR coxsackie-virus and adenovirus receptor
CAR-T Chimeric antigen receptor-T-cell
CD Cluster of
differentiation CDK Cyclin-dependent
kinase CEC Circulating
endothelial cells CIAS Computer image
analysing system c-kit Tyrosine-protein
kinase KIT
CMV Cytomegalo-virus
CR Complete response
Crea Creatinine
CT Computer tomo-
graphy CTLA-4 Cytotoxic T-
lymphocyte
associated protein 4 CTNNB1 Cadherin-Associated
protein beta 1
DDP cis-
diamminedichloroplati num
Dll4 Delta-like 4
DNA Deoxyribonucleic acid EC Endothelial cell ECM Extracellular matrix ECOG Eastern Cooperative
Oncology Group Performance Status
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EGF/EGFR Epidermal growth factor/receptor EMA European Medicines
Agency EMT Epithelial
mesenchymal transition
EOC/ EOT Epithelial ovarian cancer/ tumor ER Estrogen receptor ErbB Erythroblastic
leukemia viral oncogene homolog ESGO European Society of
Gynecological Oncology FIGO International
Federation of Gynecology and Obstetrics
Flk-1 Fetal liver kinase-1/
murine VEGFR2
i-NOS Inducible form of nitric oxide
synthase
Flt-1 fms-like tyrosine kinase-1/ VEGFR1 Flt-4 fms-like tyrosine kinase-4/ VEGFR3 FVIII-ab Factor VIII antibody gBRCA germline breast
cancer- associated gene
GI Gastrointestinal
GMP good manufacturing practice
GOG Gynecologic Oncology Group
GT Gene transfer
HE4 Human epididymis
secretory protein HGSC/ LGSC High-/ Low-grade serous carcinoma HIF-1α Hypoxia inducible
factor 1α
HIV Human
immunodeficiency virus
HNPCC Hereditary non- polyposis colorectal cancer
HRD Homologous
recombination deficiency
HSV-tk Herpes simplex virus thymidine kinase hTERT human Telomerase
reverse transcriptase i.m. intramuscular
i.p. intraperitoneal i.v. intravenous IGF-1 Insulin-like growth
factor-1
IgG Immunoglobulin-G
IHC Immunohisto-
chemistry Il-6/ -12/ -1α/ -17 Interleukin
IO Immuno-oncology
25 IOTA The International
Ovarian Tumor Analysis
IRS Immunoreactive score JAG1 Jagged Canonical Notch
Ligand
kDA kilodalton
KDR kinase domain
region/ human VEGFR2 KRAS Kirsten rat sarcoma
viral oncogene homolog gene LacZ β-galactosidase
(marker gene) lpm low potential
malignancy
LVD lymph vessel density LYVE-1 lymphatic vessel
hyaluronan receptor-1
MAPK/ ERK Mitogen activated protein kinase/
Extracellular signal-
regulated kinase MAPK3KI Mitogen activated
protein kinase gene
mc monoclonal
MEK Mitogen activated protein kinase
miR Micro ribonucleic acid
MMP Matrix
metalloproteinase MMRD Mismatch repair
deficiency
MRI Magnetic resonance imaging
mRNA messenger ribonucleic acid MSH 2/6 DNA mismatch repair gene mutS homolog 2/6 MV-CEA Measles virus-
human
carcinoembryonic antigen
MVD Microvascular density NaCl Natriumclorid NCI National Cancer
Institute
nm not mentioned
Nras Neuroblastoma rat sarcoma
NRP 1/2 Neuropilin 1/2 ONYX-015 E1B-deleted
adenovirus against TP- 53 deficient
human tumor cells OS Overall survival Ovca Ovarian cancer/
carcinoma
PALB2 Partner and localizer of BRCA2
PARP Poly(ADP-ribose) polymerase
pc polyclonal
PD-1 Programmed cell death -1
PDGF-β Platelet derived growth factor -β
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PD-L1/2 Programmed cell death ligand 1/2
PDPN Gene encoding podoplanin PFS Progression free
survival
pfu plaque-forming unit PHIL-12/PPC Il-12 plasmid
formulated with PEG- PEI-cholesterol lipopolymer nano- particle-encased Il-12 DNA plasmid vector PI3K-PKB/Akt Phosphatidylinositol-3-
kinase-protein kinase B PID Pelvic inflammatory
disease
PIK3CA Phosphatidylinositol-3 kinase, oncogene PKC Protein kinase C PlGF Placental growth
factor
PP Percentage of positive stained cells PPP2R1A Protein phosphatase 2
Scaffold Subunit Alpha PR Partial response PROX-1 Prospero homeobox
protein 1
PTEN Phosphatase and tensin homolog gene qRT-PCR Quantitative Reverse
Transcription Polymerase chain reaction
∆24-RGD Oncolytic adenovirus against aberrant retinoblastoma protein pathway RAD51D/C human homolog of E.
Coli RecA, gene of homologous recombination s.c. Subcutaneus SD Stable disease SI Staining intensity SKOV-3 human ovarian
adenocarcinoma cell line
SPK Sphingosine kinase SPSS Statistical Product and
Service Solutions
ST Stage
STIC/ STIL Serous tubal intra- epithelial
carcinoma/lesion TEM Tie-2 expressing
macrophages
TGF-α/ -β Transforming growth factor
Tie-1/2 Tyrosine kinase with immunoglobulin and EGF homology domains
TNFR1-FAS Tumor necrosis factor receptor 1- FAS ligand inducing apoptosis
27 TNM Tumor, nodes,
metastases staging classification
TP53 Nuclear phophoprotein 53, tumor
suppressor gene
TR Terminal repeat
TSH Thyroid-stimulating hormone
TSP 1/4 Thrombospondin 1/4 TVA Total vascular area VE-Cadherin Vascular endothelial
Cadherin
VEGF Vascular endothelial growth factor VEGFR Vascular endothelial
growth factor receptor WHO World Health
Organization
VPF Vascular permeability factor
28
29
1 INTRODUCTION
The mortality of epithelial ovarian cancer (EOC) has remained high in spite of extensive research on new therapies. The silent nature of early-stage disease and lack of screening possibilities contribute to the delay of diagnosis until
disseminated, higher stages of carcinoma. Another main factor worsening the prognosis is the frequent development of resistance to standard chemotherapies despite early chemosensitivity. New targeted treatments, including those that take advantage of the deficency in homologic gene repair system and cause synthetic lethality in deficient cells, might be changing the nature of EOC from uncurable to a more chronic disease. Combinations of several different therapy targets might even enhance the treatment effect further in near future. However, the
heterogenic genetic and pathogenic background of the cancer set major challenges also in upcoming times.
Antiangiogenic monoclonal vascular endothelial growth factor (VEGF) -antibody, the first clinically used targeted treatment in ovarian cancer, was accepted as part of first-line therapy of advanced EOC in Europe in 2011. The survival advantage of therapy was limited to advanced stages and unresectable tumors in primary carcinoma and high-risk cases in the recurrent setting. Since then, despite of multiple trials, new antiangiogenic treatments have not come into clinical use. In recent studies, the combination of antiangiogenic therapy and poly(ADP-ribose) polymerase (PARP) -inhibition have improved treatment efficacy compared to single therapies. The fact, that angiogenesis is crucial for the survival and dissemination of tumors, may justify an antiangiogenic approach as a part of treatment combination.
Biomarkers for patient selection and monitoring are essential to maximize the treatment efficacy and to avoid adverse effects. Angiogenic factors have been studied in tissue, plasma or serum and at the ribonucleic acid (RNA) level with varying results, and no consistent prognostic or predictive value. The
understanding of the mechanisms and information on the distribution of angiogenic proteins in malignant tissue could help in targeting biomarker investigation.
This thesis was intended to expand the comprehension of the distribution and the roles of different angiogenic factors, and endothelial and lymph vessel
30
parameters in primary and metastatic lesions of EOC in relation to the
clinicopathological and prognostic aspects. Microvessel parameters were also compared between malignant, borderline and benign ovarian tumors and the correlations between immunohistochemical results and messenger (m)RNA levels of factors as assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) were evaluated. Furthermore, the therapeutic efficacy of antiangiogenic gene therapy with soluble (s)VEGF receptors (VEGFR1, -R2 and R3) in combination with chemotherapy was compared to anti-VEGF antibody and chemotherapy in an ovarian cancer xenograft model.
31
2 REVIEW OF THE LITERATURE
2.1 OVARIAN CANCER
2.1.1 Epidemiology and risk factors
Epithelial ovarian cancer (EOC) is the leading cause of death among gynecological malignancies. In Finland, 540 women were diagnosed with ovarian cancer during 2019, while 385 women died of the same disease over the same period. The corresponding statistics globally were 295 414 newly diagnosed EOCs and 184 799 deaths from EOC in 2018. The five-year survival of the ovarian cancer was 45 % in Finland by 2019. Despite this, EOC is a rare disease. The incidence of EOC in Finland was 17.28/ 100 000, and the cumulative risk to have EOC by the age of 70 years was 0.8 %, which correlates the global statistics. The incidence and prognosis of EOC have not changed significantly over the last years in highly developed countries despite of the advanced diagnostic tools and treatment strategies.
(Finnish Cancer Registry, 2021, Bray et al., 2018)
Family history and age are the most important known risk factors of EOC. The risk of EOC rises with age, and is highest in the 60–69-year age group. The risk is 3.6 times higher among women whose first-degree relative has been diagnosed with EOC, and 2.9-fold for second-degree relatives (Al Bakir, Gabra, 2014), with increasing risk when the diagnosis is made under 50 years of age. A personal history of breast cancer has been associated with a three-fold increased risk of high-grade serous ovarian cancer (HGSC) (Stewart et al., 2018). It is likely that some women with a family history of EOC or personal history of breast cancer carry mutations that are associated with an increased risk of breast and ovarian carcinoma.
Other known risk factors are related to the ovulatory frequency: early onset of menarche, nulliparity, infertility and late menopause refer to the increased risk of epithelial injury to the ovary. Endometriosis is in turn associated with an elevated risk of endometrioid and clear cell cancers. Furthermore, women who have ever used hormone replacement therapy have a 20% higher risk of EOC. With current users and those who have stopped within 5 years, the risk of serous and
endometrioid cancer can be even 40% higher (Collaborative Group on
32
Epidemiological Studies of Ovarian Cancer, 2015). Pelvic inflammatory disease (PID) and chlamydia trachomatis seropositivity has also been associated with an elevated risk of EOC, since inflammation of tubal epithelium can induce mutations and promote carcinogenesis (Fortner et al., 2019). On the other hand, factors related to a high standard of living, have only a modest influence on the risk of EOC, although the incidence of EOC is higher in more developed countries (La Vecchia, 2017, Wentzensen et al., 2016).
Pregnancies, longer duration of breast feeding and the use of hormonal contraceptives reduce the risk of ovarian cancer through changes in hormonal milieu and anovulation. In addition, tubal ligation prevents exposure to
inflammatory and carcinogenic agents, and along with salpingo-oophorectomy, diminish the possibility of EOC. (Gockley, Elias, 2018)
2.1.2 Classification and pathogenesis
According to the World Health Organization (WHO) histogenic classification of ovarian tumors 90-95 % of ovarian malignancies are epithelial originating from coelomic epithelium-derived cells, whereas 5-10 % originate from sex cords of the developing gonads (e.g. adult granulosa cell and Sertoli-Leydig cell tumor) or stroma— and 3-5 % from germ cells (e.g. dysgerminoma, yolk sac tumor, immature teratoma) (Chen, W. V. et al., 2003).
The updated classification in 2020 categorized epithelial ovarian tumors (EOT) into five main histological subtypes and generally subdivided tumors into benign, borderline and malignant tumors (Figure 1). Serous EOCs resembling fallopian tube epithelium comprise under 80 % of the EOCs, while 10 % are endometrioid carcinomas, which mimic morphologically normal endometrium. Clear cell carcinomas form 10 % of EOCs whereas mucinous carcinomas resembling gastrointestinal epithelium represent 3 %. The most recent diagnostic group, named seromucinous tumors, are reminiscent of the endocervical epithelium. The remaining EOCs include so-called Brenner tumors, featured by transitional or urothelial epithelium and undifferentiated carcinomas, made up of monotonous cells without differentiation. (McCluggage, 2011, Kuhn, Ayhan, 2017)
Since the WHO Classification of Tumors of Female Reproductive Organs published in 2014, serous carcinoma has been divided into high-grade (70 % of EOCs) and low-grade (5 % of EOCs) tumors instead of a three-category division (Duska, Kohn, 2017). That emphasizes the distinct molecular pathogenesis, genetic
33 variability and clinically diverse progression of the two different tumor types. For endometrioid cancer the traditional three-grade system is clinically used as in endometrial cancer. Mucinous and clear cell tumors are not graded. (Ayhan et al., 2009)
Figure 1. HE-stainings and genetic mutations behind the main types of EOC (Hirst et al., 2018)
The existing theory of the evolution of the serous carcinoma supports serous tubal intraepithelial carcinoma (STIC) lesions as being precursors of HGSC.
However, some of the HGSCs can develop from the peritoneal endosalpingiosis and cortical inclusion cysts of ovary through malignant transformation influenced by several cytokines, hormones and reactive oxygen species. Many advanced HGSCs, that are not accompanied by malignant precursors in the tubes, are thought to originate from early serous proliferations of tubal epithelium containing tumor suppressor gene (TP)53 mutations. These serous tubal
intraepithelial lesions (STILs) become detached and exfoliate into the peritoneal
34
cavity and some undergo malignant transformation (Soong et al., 2019) (Figure 2).
Nevertheless, the spread of distal tubal lesions to the peritoneal surface or implants to the epithelium of the ovary is thought to explain the majority of HGSCs.
Figure 2. Evolution of HGSC (Soong et al., 2019)
Low-grade serous carcinoma (LGSC) developes from the gradual progression of benign serous cystadenomas to serous borderline tumors, including
micropapillary variant and finally to invasive LGSC.
The majority of endometrioid and clear cell carcinomas are associated with endometriosis, and they often show shared mutations, suggesting that
endometriosis or endometriomas are precursor lesions of these carcinomas.
These facts support the traditional theory of mutagenesis to atypical endometriomas due to inflammation and iron overload of cystic fluid and
35 malignant transformation to endometrioid and clear cell borderline tumors and finally to invasive carcinomas. (Kuhn, Ayhan, 2017)
The histogenesis of mucinous tumors has remained uncertain. Mucinous cancer is categorized histologically into expansile and more aggressive infiltrative subtypes, which have an influence on both the prognosis and treatment protocol.
(Colombo et al., 2019)
2.1.3 Genetic features of ovarian cancer
The genetic background behind EOC is heterogenous, indicating different origins, histopathologic types and clinical courses of the cancer (Table 1). A somatic mutation of TP53 can be found in 96 % of high-grade serous ovarian carcinomas (Ahmed et al., 2010, Vang et al., 2016). Defects in genes encoding proteins for the homologous recombination (HR) gene repair system are found commonly (24- 50%) in high-grade serous ovarian cancer either as somatic or germline mutations.
The most well-known mutations and methylation of breast cancer-associated (BRCA)1 and 2 `caretaker` genes affect approximately 14 % of patients with HGSC (Table 2), although in Finnish HGSC population screened since 2016 the
percentage has been lower: germline mutations in approximately 5 % and a total percentage of germline and somatic mutations around 13 % in Kuopio University Hospital data. There are over 200 different known BRCA mutations including a Finnish founder mutation. Inactivation of the gene and the type of autosomally dominantly inherited mutation influences the individual cancer risk (Table 2).
Microsatellite instability (MSI) can be found either in sporadic cancer or in relation to hereditary Lynch syndrome (HNPCC). Endometrioid and clear cell ovarian carcinomas may occur in Lynch syndrome, which also has an elevated risk of colorectal cancer 43-48 % and endometrial cancer 40-62 % (Aarnio et al., 1999).
Mutations in the DNA mismatch repair (MMR) genes MSH6 and MSH2 are
particularly associated with endometrioid and clear cell ovarian carcinoma (Table 2).
Patients with germline BRCA1/2 mutations or a suspicion of Lynch syndrome are sent to genetic counseling. Prophylactic surgery for these patients is
recommended: salpingo-oophorectomy for BRCA1/2 carriers by 40 and 45 years of age and hysterectomy with salpingo-oophorectomy for Lynch syndrome patients after the age of 45.
36
Table 1. Pathogenic models of EOC (Prat et al., 2018)
Variable Type I Type II
Histology LGSC, LGEC, Clear cell, Mucinous, Brenner
HGSC, HGEC, Carcinosarcoma, Undifferentiated, Mixed tumors
Stage I-II III-IV
Incidence/
Mortality
25 %/ 10 % 75 %/ 90 %
Mutations (somatic)
ARID1A, BRAF, CTNNB1, KRAS, PIK3CA, PPP2R1A, PTEN, RNF43, hTERT, ERBB2
TP53, BRCA 1/2 /BRCA1 methylation, HNPCC (MMRD), HRD
Genetic Stable Unstable
Pathogenesis Stepwise from adenoma –
borderline Tubal fimbria, STIC/ STIL precursor
lesions
H/LGEC=high-/low-grade endometrioid cancer, HNPCC=hereditary non-polypotic colon cancer, MMRD=mismatch repair defect, HRD=homologous recombination defect
Table 2. High- and moderate-risk germline mutations of ovarian cancer patients (Suszynska et al., 2019, Kohlmann, Gruber, 2020)
Variable Incidence in ovarian cancer Risk of ovarian
cancer Risk of breast
cancer
BRCA1 8 %,
1/500 in general population
40–45 % (OR 35)
60–80 % (OR 9.3)
BRCA2 6 %,
1/500 in general population 10–30 %
(OR 11.9) 40–60 %
(OR 5.7)
RAD51D 0.6 % OR 7.3 not increased risk
PTEN 1/200000 in general
population, Cowden syndrome OR 5.5 50–85 %
OR 5.4 TP53 0.8% among breast cancer
patients, LiFraumeni syndrome
OR 5.0 OR 4.4
BRIP1 1 % OR 4.9 OR 1.5
MSH6
MSH2 0.4 %
1/500–1/1000 in general population
22 % (OR 4)
4–20 % OR 1.5
5–18 %
RAD51C 0.6 %, 1.5–2 % OR 4.2, x6 not increased risk
PALB2 0.7 % OR 2.1 OR 4.9
ATM 0.7 % OR 2.0 OR 2.4
OR= odds ratio
37 2.1.4 Prognostic factors
The prognostic markers for ovarian cancer are the tumor stage, the residual tumor after the primary surgery, the primary response to platinum-based chemotherapy, tumor grade, histological type, patient`s age and comorbidities (Wimberger et al., 2007).
Due to the unspecific symptoms in the beginning of the disease, HGSC is often diagnosed at an advanced stage, 80 % at stage (ST) III-IV, which significantly worsens the prognosis (Table 3). The five-year survival of ST I-II EOC varies around 70-95 %, whereas only 26 % of ST IV patients are alive five years after diagnosis (Torre et al., 2018).
38 Table 3. FIGO and TNM staging of ovarian, fallopian tube and peritoneal cancer (Bhatla, Denny, 2018). I Tumor confined to ovaries or fallopian tube(s)T1 IATumor limited to one ovary or tubes (capsule intact), no tumor on surface, negative washings/ ascites T1aN0M0 IBTumor limited to both ovaries or tubes (capsules intact), no tumor on surface, negative washings/ascites T1bN0M0 ICTumor limited to one or both ovaries or tubes, with any of the following IC1: Surgical spill IC2: Capsule rupture before surgery or tumor on the ovarian or tubal surface IC3: Malignant cells in the ascites or peritoneal washings
T1cN0M0 T1c1N0M0 T1c2N0M0 T1c3N0M0 II Tumor involved below pelvic brim or primary peritoneal cancer T2 IIAExtension and/or implants on the uterus and/or fallopian tubesT2aN0M0 IIBExtension to other intraperitoneal pelvic tissuesT2bN0M0 IIIConfirmed spread outside the pelvis and/or metastasis to the retroperitoneal lymph nodes IIIA IIIA1 Positive retroperitoneal lymph nodes only (cytologically or histologically proven) IIIA1(i) Metastasis up to 10mm IIIA1(ii) Metastasis more than 10mm in greatest dimension IIIA2 Microscopic extrapelvic (above the pelvic brim) peritoneal involvement with or without retroperitoneal lymph node metastasis
T1/2N1M0 T3a2N0/1M0 IIIB Macroscopic peritoneal metastasis beyond the pelvis up to 2cm in greatest dimensions, with or without the metastasis to the retroperitoneal lymph nodesT3bN0/1M0 IIIC Macroscopic peritoneal metastasis beyond the pelvis more than 2cm in greatest dimensions, with or without retroperitoneal lymph node metastasis (includes extension of tumor to the capsule of liver and spleen without parenchymal involvement of either organ)T3cN0/1M0 IVDistal metastasis excluding peritoneal metastasis IVA Pleural effusion with positive cytology IVB Parenchymal metastasis and metastasis to extra-abdominal organs (including inguinal lymph nodes and lymph nodes outside of the abdominal cavity) T1/2/3N0/1M1 TNM= Tumor, nodes, metastases staging classification
39 The type of histology is related to the prognosis of EOC. Eighty percent of mucinous tumors are diagnosed in ST I due to the rapidly growing, symptomatic adnexal tumors. Endometrioid tumors are also more often confined to the pelvis.
Clear cell histology and a carcinosarcomatoid component are in turn unfavorable prognostic indicators.
Cytomorphologic differentiation of the tumor epithelial cells, based on nuclear atypia and mitotic rate, is a clear characterizer of prognosis. The five-year survival has been 75 % for low-grade serous cancer (LGSC), but only 40 % for HGSC, including all stages. Furthermore, the progression-free survival (PFS) for HGSC has been shorter (Kaldawy et al., 2016). Also grade three endometrioid and mucinous infiltrative tumors exhibit poorer survival than analogous low-grade and expansile tumors. (Colombo et al., 2019)
Residual tumor after primary surgery has been shown to be an independent prognostic factor for EOC. Complete surgical tumor resection with no residual tumor improved median OS by 66 and 72 months compared to the groups with residual tumor less than 1cm and with residual more than 1cm (du Bois, Andreas et al., 2009). Incomplete cytoreduction usually follows high tumor volume and metastatic disease.
Patients experiencing the recurrency of cancer during the first-line
chemotherapy with platinum or in the following months thereafter are linked to the unfavorable prognosis. A platinum-free interval more than 12 months is associated with better PFS and OS (Pignata et al., 2017).
Patient-related factors: such as high age, gerasteny or frailty, malnutrition, low albumin, and chronic diseases are significant prognostic factors. The five-year survival rate of patients over 75 years old is 18 % compared to 68 % for patients under 55 years of age in the Finnish population during 2014–2016 (Finnish Cancer Registry, 2019).
Carriers of BRCA1 and -2 mutation have greater chemosensitivity to platinum due to the deficient repair system of the DNA double strand breaks, which contributes to the DNA damage and, cross-links between strands caused by platinum agents (Xu et al., 2017). That explains the different course of progression and better prognosis of mutation carriers.
Serum cancer antigen Ca12-5 has been used as a diagnostic and post-
treatment biomarker particularly in HGSC. A high Ca12-5 value after surgery and failure to achieve a normal Ca12-5 level (< 35) after three cycles of chemotherapy predicts poor prognosis (Ayhan et al., 2017).
40
2.1.5 Diagnostics and current standard of treatment
The diagnosis and the treatment strategy of EOC is based both on the radiologic imaging and histopathologic evaluation of the tumor. Transvaginal two-
dimensional ultrasound with doppler technology is the `golden standard` for differential diagnosis of adnexal masses (the International Ovarian Tumor Analysis, IOTA, rules) (Sayasneh et al., 2013). Pre-operative evaluation of primary staging is made by computer tomography of the thorax and abdomen. Computer- based tomography (CT) is also used in the follow-up of ovarian cancer. Magnetic resonance imaging (MRI) and diffusion-weighted MRI have their place in the differential diagnosis of pelvic tumors, on suspicion of recurrence or in primary staging if CT is contraindicated (Forstner et al., 2016). Positron emission
tomography can be used as a complementary modality for suspected recurrence.
As a diagnostic biomarker, human epididymis protein 4, HE4 has shown higher sensitivity (90 % vs 83,3 %) and specificity (95 % vs 85 %) in EOC than Ca12-5, which can be elevated in several benign conditions (Hamed et al., 2013).
Debulking surgery and platinum-based chemotherapy have been the
cornerstones of treatment for years. The goal of primary cytoreductive surgery is to completely remove all visible tumor tissue. Since in upto 60 % of patients, the residual tumor load is more than 1cm after primary surgery, neoadjuvant
chemotherapy would be more beneficial for that group of patients (Vergote et al., 2018, Coleridge et al., 2021).
Different selection criteria for patients suitable for primary debulking surgery have been established. Radiologic and external validation algorithms failed to predict resectability, whereas laparoscopy-based scoring of carcinosis (Fagotti score) was associated with better accuracy and is used with patient-related factors like Eastern Cooperative Oncology Group (ECOG) performance status, nutritional factors: such as serum albumin count, comorbidities and age. In the Fagotti score, the presence of omental cake, diaphragmatic and mesenterial carcinosis, bowel and/or stomach infiltration and liver metastases are each assigned an index value of two. A predictive score of at least eight identified patients undergoing
suboptimal surgery. (Fagotti et al., 2006). The advantage of laparoscopy is also proper histological sampling for diagnosis. In the setting of recurrence, good patient performance status, complete cytoreduction at primary surgery and absence of ascites more than 500ml in the AGO score, are related to the probability of complete resection of the recurrent tumor (Harter et al., 2011).
41 Primary debulking or staging surgery, made via laparotomy with midline
incision, is both therapeutic and defines the tumor extension at its primary
presentation. In ST I EOC the staging can be made laparoscopically including pelvic and para-aortic lymphadenectomy (except LGSC and mucinous expansile type), as 10-15 % of ST I patients have lymph node metastasis (Bogani et al., 2017). In advanced ovarian cancer, the systematic pelvic and para-aortic lymphadenectomy, in case of normal lymph nodes in pre- and intraoperative evaluation does not show OS benefit (Harter et al., 2019). Adequate operation procedures for staging include, besides total hysterectomy and bilateral salpingo-oophorectomy, total omentectomy, peritoneal biopsies of diaphgram, paracolic recesses, cul-de-sac, bladder reflection, adhesiones and all suspicious lesions, washings or cytology of ascites and appendectomy for mucinous tumors. In addition, for optimal results more than one bowel resection, complete peritoneal and diaphragmatic stripping, splenectomy, cholecystectomy and hepatic or pancreatic resection can be
required, making the operation ultraradical (Rodriguez et al., 2013).
Neoadjuvant chemotherapy with a combination of carboplatin area under curve (AUC)5 and paclitaxel 175mg/m2 every three weeks is initiated for patients not filling the criteria of operability. Interval debulking surgery should be proposed to patients fit for surgery who have a response or stable disease compatible with complete resection usually after three cycles of chemotherapy (European Society of Gynecological Oncology, ESGO, guidelines). Selected patients with recurrent ovarian cancer sensitive to platinum might benefit from undergoing complete resection (Pignata et al., 2017).
Combined chemotherapy with carboplatin and paclitaxel is offered as an adjuvant therapy after surgery to all EOC patients except those with stage IA low- grade serous, ST IA, grade one endometrioid and ST IA mucinous expansile type of tumors. A minimum of four, but usually six cycles at three-week intervals are given. HGSC is often primarily chemosensitive unlike its low-grade counterpart and mucinous carcinoma. Unfortunately, however, 70 % of patients relapse within the first three years (Ledermann et al., 2013). Combinations of liposomal doxorubisin (PFS 11.3 months vs 9.4 months with pacli+carboplatin) (Wagner et al., 2012), gemsitabin (Berg et al., 2019) or taxanes (pacli- and docetaxel) with platinum are used in platinum-sensitive and partially sensitive relapses (6-12 months after previous platinum therapy).
In platinum-refractory or resistant cases weekly dosed paclitaxel 80mg/m2 is usually the first choice (Gynecologic Oncology Group et al., 2006). Single-agent
