Mammographic breast density, tumour characteristics and the expression of hyaluronan as prognostic surrogate markers for breast cancer

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DISSERTATIONS | AMRO MASARWAH | MAMMOGRAPHIC BREAST DENSITY, TUMOUR CHARACTERISTICS... | No 351

uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2110-9 ISSN 1798-5706

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

AMRO MASARWAH

MAMMOGRAPHIC BREAST DENSITY, TUMOUR CHARACTERISTICS AND THE EXPRESSION OF HYALURONAN AS PROGNOSTIC SURROGATE MARKERS FOR BREAST CANCER

Mammographic breast density (MBD) is a topic of broad and current interest both in the

literature and the lay press. While most focus is usually placed on MBD’s effect on screening

sensitivity and its role as a breast cancer risk factor, this thesis looks at MBD from a different

angle, its role as a breast cancer prognostic factor. This study concluded that very low MBD is indeed an independent prognostic

factor for breast cancer, and has a strong reciprocal relationship with high expressions

of Hyaluronan and its synthesizing enzymes.

AMRO MASARWAH

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Mammographic Breast Density, Tumour Characteristics and the Expression of Hyaluronan as Prognostic Surrogate

Markers for Breast Cancer

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AMRO MASARWAH

Mammographic Breast Density, Tumour Characteristics and the Expression of Hyaluronan as Prognostic Surrogate

Markers for Breast Cancer

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Kuopio University Hospital Auditorium 1,

On Friday, June 17th 2016, at 10 am Publications of the University of Eastern Finland

Dissertations in Health Sciences Number 351

Departments of Clinical Radiology and Oncology, Institute of Clinical Medicine, School of Medicine, Faculty of Health Sciences,

University of Eastern Finland Kuopio

2016

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Grano Oy Jyväskylä, 2016

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Professor Hannele Turunen, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophtalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm. Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy).

School of Pharmacy Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-2110-9

ISBN (pdf): 978-952-61-2111-6 ISSN (print): 1798-5706

ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

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Grano Oy Jyväskylä, 2016

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Professor Hannele Turunen, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophtalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm. Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy).

School of Pharmacy Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-2110-9

ISBN (pdf): 978-952-61-2111-6 ISSN (print): 1798-5706

ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

III Author’s address: Kuopio University Hospital

Department of Clinical Radiology

School of Medicine, Faculty of Health Sciences University of Eastern Finland

KUOPIO FINLAND

Supervisors: Professor Ritva Vanninen, M.D., Ph.D.

Department of Clinical Radiology Institute of Clinical Medicine

School of Medicine, Faculty of Health Sciences University of Eastern Finland

KUOPIO FINLAND

Professor Päivi Auvinen, M.D., Ph.D.

Kuopio University Hospital Department of Oncology KUOPIO

FINLAND

Reviewers: Docent Tapani Tikkakoski, M.D., Ph.D.

Keski-Pohjanmaa Central Hospital Department of Radiology

KOKKOLA FINLAND

Docent Pasi Hirvikoski, M.D., Ph.D.

Oulu University Hospital Department of Pathology OULU

FINLAND

Opponent: Professor Peter B. Dean, M.D., Med.Sc.D.

Department of Diagnostic Radiology Institute of Clinical Medicine Faculty of Medicine

University of Turku TURKU

FINLAND

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V Masarwah, Amro

Mammographic Breast Density, Tumour Characteristics and the Expression of Hyaluronan as Prognostic Surrogate Markers for Breast Cancer

University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences 351. 2016. 93p.

ISBN (print): 978-952-61-2110-9 ISBN (pdf): 978-952-61-2111-6 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

ABSTRACT

Breast cancer constitutes a major worldwide health issue; it is the most common cancer and leading cause of cancer related deaths in women all around the world. Nowadays, tumour size, lymph node status, hormone receptor status, tumour grade and HER2 status are considered as the strongest and most important prognostic factors correlating with patient outcomes. Finding newer and potentially more accurate prognostic factors could help to better understand breast cancer as a disease and successfully personalize and tailor future treatment regimens.

Mammographic breast density (MBD) is an established risk factor for the development of breast cancer and women with breast density in the upper quartile have a 4 - 6 fold increased risk for developing breast cancer compared to those within the lower quartile. Hyaluronan (HA) is a glycosaminoglycan widely found in different tissues; it is one of the molecular markers and effectors with a strong influence on the progression of breast cancer and by itself is considered as a prognostic factor.

The aim of this thesis was to investigate MBD in terms of its prognostic value as opposed to its more usual and better recognized role as a risk factor, the secondary aim was to assess the potential association between mammographic density and mammographic tumour features with hyaluronan and its synthesis. For the purpose of this study, 139 consecutive HER2 positive invasive breast cancer patients who were operated in Kuopio University Hospital during the years 2002–2008, were matched with an equal number of HER2 negative patients, i.e. there was a total of 278 patients examined in these analyses. The first study was intended to examine the prognostic value of mammographic breast density and mammographic tumour features and their relationship with the established prognostic factors. The second study aimed to assess the potential association between HA and its synthetizing enzymes with that of MBD and other mammographic and tumour characteristics in an attempt to uncover any potential underlying relationship that would help connect those two previously separated and not previously evaluated aspects of breast cancer biology. The third study examined the possible additional value of very low mammographic breast density (VLD), HER2, ER and PR statuses in a patient group within the matched Nottingham Prognostic Index (NPI) categories and whether the addition of any of those factors could serve to improve its prognostic capabilities.

The first study concluded that very low MBD is indeed an independent prognostic factor for breast cancer, and is associated with higher tumour grades and predicted worse survival, even after correcting for all possible confounders. The second study further elicited the value of low MD and discovered the existence of a strong reciprocal relationship between low breast density and high expressions of HA and its synthesizing enzymes.

Moreover, a dramatic reduction in patient survival was found when HA abundance was combined with low breast density. In the third study, VLD and HER2 positivity were found to be prognostic factors for breast cancer, independent of the NPI. Furthermore, it was possible to incorporate those factors into a newly coined prognostic index called the Kuopio-Nottingham Prognostic Index (KNPI), which has a higher predictive power than the original NPI.

National Library of Medicine Classification: WP 870, WP 815, WB 142, QU 83

Medical Subject Headings: Breast cancer; Mammography; Density; prognosis; Radiology; Oncology;

Hyaluronic acid.

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VII Masarwah, Amro

Rintojen tiiviys mammografiassa, tuumorin ominaisuudet ja Hyaluronaanin ennusteellinen merkitys invasiivisessa rintasyövässä.

Itä-Suomen yliopisto, terveystieteiden tiedekunta

Publications of the University of Eastern Finland. Dissertations in Health Sciences 351. 2016. 93p.

ISBN (print): 978-952-61-2110-9 ISBN (pdf): 978-952-61-2111-6 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

TIIVISTELMÄ

Rintasyöpä on merkittävä terveysongelma, sillä se on naisten yleisin syöpä ja johtava syy naisten syöpäkuolemiin maailmanlaajuisesti. Tärkeimmät rintasyövän ennustetekijät ovat kasvaimen koko ja erilaistumisaste, leviäminen kainaloimusolmukkeisiin, kasvaimen hormonireseptoristatus ja HER2- positiivisuus. Uusien, aiempaa tarkempien ennusteellisten tekijöiden kehittäminen olisi merkityksellistä, sillä tuloksilla saattaa olla tulevaisuudessa merkitystä rintasyöpäpotilaiden yksilöllisen hoidon suunnittelussa.

Rinnan tiiviyden yhteys rintasyövän riskiin on hyvin tiedossa. Naisilla, joilla on erittäin tiivis rauhaskudosrakenne on 4-6 -kertainen riski sairastua rintasyöpään. Hyaluronaani on soluväliaineen sokerimolekyyli, joka esiintyy normaalisti lähes kaikissa kudoksissa, ja sen aineenvaihdunta häiriintyy syövässä edistäen syövän etenemistä.

Tutkimuksen tarkoituksena oli arvioida rintakudostiiviyden ja hyaluronaanipitoisuuden sekä niiden yhdistelmän ennusteellista merkitystä rintasyövässä. Tutkimuksessa oli 278 rintasyöpätapausta vuosilta 2002- 2008, ja heistä puolet oli HER2-positiivisia. Rintakudoksen tiiviyden osuutta rinnan pinta-alasta tarkasteltiin mammografiakuvista, joista syöpä oli todettu. Ensimmäisessä osatyössä rintarauhaskudoksen tiiviyden ja muiden tekijöiden vaikutusta taudin ennusteeseen tarkasteltiin runsaan kuuden vuoden seurannassa. Toisessa osatyössä selvitettiin rintarauhaskudoksen tiiviyden, mammografiassa näkyvien tuumorin morfologisten piirteiden ja hyaluronaanin välistä yhteyttä. Seuranta-aika oli kahdeksan vuotta. Kolmannessa osatyössä arvioitiin yleisimmin käytössä olevan ennustemallin, Nottinghamin prognostisen indeksin, käyttökelpoisuutta kun sen laskentakaavaan lisättiin uusia prognostisia tekijöitä kuten hormonireseptorit, HER2 ja rinnan matala tiiviys.

Tulokset osoittivat, että hyvin matala rintarauhaskudoksen tiiviys oli itsenäinen rintasyövän ennustetta huonontava tekijä (sekä tautivapaa että kokonaiselinaika), ja matalaan tiiviyteen liittyi myös kasvainten suurempi aggressiivisuus. Tilastollinen merkitsevyys säilyi myös monimuuttuja-analyysissa, jossa huomioitiin mm. potilaiden ikä, menopausaalinen status ja obesiteetti. Toisessa osajulkaisussa osoitettiin, että jos rinnan tiiviys oli <25 %, hyaluronaanin korkea pitoisuus sekä syöpäsoluissa että stroomassa korreloi tilastollisesti rintasyövän huonoon ennusteeseen ja oli itsenäinen riskitekijä riippumatta HER2-statuksesta. Huomioitavaa on, että hyaluronaanin korkea konsentraatio ja rintakudoksen matala tiiviys liittyivät toisiinsa niin, että potilailla, joilla oli sekä matala rinnan tiiviys että runsaasti hyaluronaania rintasyöpäkudoksessa, oli erityisen huono ennuste. Kolmannessa osajulkaisussa rintojen matala tiiviys oli itsenäinen ennustekijä, ja se yhdessä HER2-positiivisuuden kanssa sisällytettiin uuteen ennustemalliin (Kuopio-Nottinghamin prognostinen indeksi) jonka prediktiivinen ennusteellinen kyky osoittautui paremmaksi kuin alkuperäisessä mallissa.

Yleinen Suomalainen asiasanasto: Rintasyöpä; Mammografia; Tiiviys; ennusteet; radiologia;

syöpätaudit; hyaluronaani.

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"I almost wish I hadn’t gone down that rabbit hole — and yet

— and yet — it’s rather curious, you know, this sort of life!"

-Alice in Wonderland

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Acknowledgements

This study is part of the Cancer Imaging project and based on research carried out in the Departments of Clinical Radiology and Oncology, University of Eastern Finland and Kuopio University Hospital, between the years of 2013-2016.

To commence, it is my radiant sentiment to place on record my best regards, deepest sense of respect and gratitude to my principle supervisor Professor Ritva Vanninen. There aren’t enough words in the English vocabulary to describe the joy and pride I feel for having had you as my main supervisor. Your brilliance, work-ethic, enthusiasm, guidance and humility have been an utter inspiration. Since day one, you welcomed me with open arms and made me feel straight at home. I very much appreciate what you have done for me both professionally and personally. Everything from the mentoring and the countless enriching scientific and medical discussions and even to the now “infamous” skiing weekends we

“enjoyed” at your winter cottage. The joy you have for your work and for research is contagious, and none of this would have been possible without you. You have and will continue to be a great influence on me.

I would also like to extend my gratitude to my second supervisor Professor Päivi Auvinen.

It has been a personal honour of mine to have been able to work with you for the past 3 years.

You have introduced me to new fields of medicine and research that were unknown to me, and you have done this ever so patiently. Your humour, smile, positive attitude and encouraging words have been a driving force for me especially during the tough times of this project. I will always appreciate all the times you took out of your schedule to go through every article and every review word by word with me, your scientific expertise and encouragement in every step really made the tough times go un-noticed.

I would also like to dedicate a special regard to Dr. Mazen Sudah. You are many things to many people, but to me, you’ve been a colleague, teacher, mentor, big brother and best friend.

You have been a true source of inspiration for me, not just during this journey, but throughout my lifetime as well. The extent of your knowledge and passion for the field is staggering to say the least. I always feel privileged whenever we sit down to discuss any science-related topics, for the knowledge and critical eye you possess is certainly unmatched.

I owe a lot to you, unfortunately if I started to write about it, I would need a whole new thesis book altogether. But for now, I will say that you were the one who inspired me to go into radiology, the one who encouraged me to start my research career and gave me all the pointers in the right direction. Without your constructive, sometimes challenging, but yet ever so brilliant input, I wouldn’t be where I am today.

I would also like to thank my fellow co-authors; Anna Sutela, Suvi Rautiainen and Outi Pelkonen, you are great scientists and it has been a joy to work with, thank you for all the hours we spent analyzing those mammograms in the department’s “trenches”. Sanna Oikari,

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you were always more than happy to offer your expert advice and help in any possible way.

To Professors Markku Tammi and Raija Tammi, who in spite of being extraordinarily busy, they took time to hear me and guide me through my second article. I was honored by your contribution and critical review and it has been an absolute pleasure working with both of you. To Professor Veli-Matti Kosma, your professional input and your expert help with this thesis is deeply appreciated.

I wish to express my warm gratitude to the Head of the Department of Clinical Radiology, Professor Hannu Manninen for the opportunity to conduct my research in such an outstanding department. I would also like to thank the official reviewers of this thesis, Docent Tapani Tikkakoski from the Keski-Pohjanmaa Central Hospital and Dr.Pasi Hirvikoski from the Oulu University Hospital, your valuable comments and revisions are truly appreciated.

I would also like to thank all of my researcher colleagues with whom I have shared many beautiful memories. Mikko, Sanna, Antti and Otso - it has been a privilege to get to know you, thank you for your friendship and support during all those years. Otso, apart from being a valuable co-author, you have also been my friend and travelling companion during all those conferences, I will always treasure those memories.

My thanks also extend to our department secretaries and research assistants. Starting with my next door neighbour Tuula Bruun, I have always cherished our conversations which have grown to become a part of my daily routine. Thank you for forcing me to speak Finnish at all times and ignoring each and every attempt that I made to communicate in English or even in desperate times, sign language. I wouldn’t have passed my exams if it wasn’t for your belief and your support, and for that I will be eternally grateful. Taina, you welcomed me on the first day I arrived in Kuopio as an exchange student with a gleaming smile, and to this day your smile has not waned for a second. Thank you for all the help you provided along the way, and for always answering swiftly to each and every one of my silly requests. Marika and Maire for taking care of me and tolerating me during all those conference and travel arrangements, you have saved me on countless occasions. I would also like to thank the brilliant Tuomas Selander for his invaluable statistical advice, and Dr. Ewen MacDonald for his prompt linguistic revision of this thesis.

Last but not least, I would like to thank my family for all their love and encouragement. To Vaiva, thank you for your everlasting support during all the tough times, thank you for believing in me, even when I lost belief myself, thank you for being there for me and most importantly, thank you for being you. You are my rock, my safe place and the light of my life. To Mazen, Leena, Rami and Christiina, you have aided and encouraged me throughout this endeavor, helping me out in every step of the way. You've been a constant source of love, concern, support and strength and to me you're worth more than words could ever describe.

To my parents, Nazih and Muna, to my brothers, Tareq and Sa’ad, thank you for showing me love in its rarest form, showing me what it feels like, and how it can push you to overcome life’s greatest challenges. One couldn’t have wished for a better family and for a better upbringing, all that I have accomplished and any success that I may enjoy in the future will be built on the support and belief that you have installed in me, this thesis is for you.

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This work was supported by grants from the Kuopio University Hospital-VTR funds, EVO funding (grant nos. 5063525, 5063532), University of Eastern Finland, the Cancer Center of the University of Eastern Finland, the University of Kuopio Foundation, Mauri and Sirkka Wiljasalo Grant, the Instrumentarium Foundation, the Radiological Society of Finland, the Finnish Oncological Society, Inkeri and Mauri Vänskä Foundation, Ida Montin Foundation, Paavo Koistinen Foundation, the Finnish Medical Foundation (Gust. Rud. Idman Fund), Northern Savo Cancer Association and the Cancer Society of Finland.

Amro Masarwah Kuopio, Finland May 2016

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List of the original publications

This dissertation is based on the following original publications:

I Masarwah A, Auvinen P, Sudah M, Rautiainen S, Sutela A, Pelkonen O, Oikari S, Kosma VM, Vanninen R. Very low mammographic breast density predicts poorer outcome in patients with invasive breast cancer. Eur Radiol. 2015 Jul;25(7):1875-82.

II Masarwah A, Tammi M, Sudah M, Sutela A, Oikari S, Kosma VM, Tammi R, Vanninen R, Auvinen P. The reciprocal association between mammographic breast density, hyaluronan synthesis and patient outcome. Breast Cancer Res Treat. 2015 Oct;153(3):625- 34.

III Masarwah A, Auvinen P, Sudah M, Dabravolskaitė V, Arponen O, Sutela A, Oikari S, Kosma VM, Vanninen R. Prognostic contribution of mammographic breast density and HER2 overexpression to the Nottingham Prognostic Index in patients with invasive breast cancer. (Submitted)

The publications were adapted with the permission of the copyright owners.

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Abbreviations

ADC apparent diffusion coefficient BCDDP Breast Cancer Detection

Demonstration Project Bhabc biotinylated HA-binding

complex

BI-RADS Breast Imaging Reporting and Data System

BMI body mass index

CEUS contrast-enhanced ultrasound CI confidence interval

c-index concordance index CISH chromogenic in situ

hybridization CMF cyclophosphamide,

methotrexate, 5-fluorouracil DBT digital breast tomosynthesis DCIS ductal carcinoma in situ DFS disease Free Survival ECM extra-cellular matrix

EGFR epidermal growth factor receptor ER estrogen receptor

EUSOMA The European Society of Breast Cancer Specialists

GLcA glucuronic acid GlcNAc N-acetyl-glucosamine HA hyaluronan

HAS hyaluronan synthase

HER2 human epidermal growth factor receptor 2

HR hazard rate

ICC intraclass correlation coefficient K-NPI Kuopio-Nottingham prognostic

index LOD low density

MBD mammographic breast density MID mixed density

MRI magnetic resonance imaging NPI Nottingham prognostic index NSAID non-steroidal anti-inflammatory

drug

PACS picture archiving and communication system PR progesterone receptor

SEER Surveillance Epidemiology and End Results

tdROC time-dependent receiver operating characteristic curve TNM Classification of Malignant

Tumours (tumour, lymph nodes, Metastasis)

US ultrasound VLD very low density

WHO World Health Organization

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1 Introduction

Mammographic breast density (MBD) refers to the appearance and relative amount of radio-opaque breast parenchyma in a mammogram; it is a topic of interest in both the medical literature and the lay press. Historically, Wolfe et al. (1) were the first to describe mammographic density and classify it into different patterns. Furthermore, Wolfe (2) was the first to describe an association between mammographic breast patterns and the risk of breast cancer. Since then, numerous newer methods have been proposed for assessing mammographic density and several studies have been published, not only validating those earlier findings but also trying to provide a biological explanation. Nowadays, increased mammographic breast density is universally accepted to be a strong independent risk factor for the development of breast cancer (3, 4). Nonetheless, a surprisingly small number of studies have focused on MBD from a prognostic point of view, with those who did so, delivering conflicting and often inconclusive results.

Hyaluronan (HA) is a large glycosaminoglycan widely found in different tissues; it possesses several roles e.g. as a molecular marker and as an effector on breast cancer progression (5). Three isoenzymes called hyaluronan synthases (HAS1-3) in the plasma membrane are responsible for the synthesis of HA (6). In human breast cancer, a high level of HA is associated with epidermal growth factor receptor 2 (HER2) positivity and CD44 (the principal transmembrane cell surface receptor for HA) positive stromal cells, and it is considered as an independent factor for a poor prognosis, along with hyaluronan synthases which contribute to the accumulation of HA in breast cancer (7, 8).

This doctoral dissertation focuses on mammographic breast density as a prognostic factor for patient outcome in patients with newly diagnosed breast cancer. For the first time, the relationship between mammographic characteristics and density are compared with hyaluronan and its synthetizing enzymes in a bid to reveal underlying biological mechanisms and thus enhance our understanding of cancer as a disease. Furthermore, this dissertation incorporates MBD into the well-known Nottingham Prognostic Index (NPI) to form a new prognostic system in an effort to provide a gateway for these results to be applied in clinical practice.

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2 Review of the Literature

2.1 BREAST CANCER SUBTYPES AND PREVALENCE 2.1.1 History of breast cancer

Due to its visible and progressive symptoms, breast cancer was amongst the first cancers ever to be described in history. The earliest recorded cases of breast cancer can be traced as far back as 1600 BC in ancient Egypt. The ancient Egyptian “Edwin Smith” papyrus described 8 cases of “untreatable” breast fungating ulcers; and this represents the first historical attempt to actually look at cancer in a scientific manner. Mastectomy as a surgical treatment for breast cancer has been performed as early as 548 AD, but the linking of axillary surgery to original breast surgery was not done until the 17^th century when the first successful radical mastectomy was performed. Nowadays, breast cancer is considered as the most common cancer and the commonest cause of cancer related deaths amongst women all around the world (9, 10).

2.1.2 Breast cancer statistics

According to the latest international cancer statistics report, 1,676,600 new cases were diagnosed in the year 2012 and 521,900 women died of breast cancer in the same year (10).

Breast cancer alone accounts for 25% of all cancer cases and 15% of all cancer deaths among females. The more developed countries account for about one-half of all breast cancer cases and 38% of deaths. The international variations in breast cancer incidence rates reﬂect differences in the availability of early detection as well as risk factors.

Over the course of their lifetime, about every eighth woman in the USA will develop invasive breast cancer. An estimated 231,840 new cases of invasive breast cancer are expected to be diagnosed in women in the U.S. during 2015, along with 60,290 new cases of non- invasive (in situ) breast cancer. According to the Nordcan project statistics (11), an average of 4462 women were diagnosed annually in Finland for the years 2009-2013 which means that breast cancer accounted for 30% of all cancers suffered by Finnish women. By the end of the year 2013, there were 62,103 women living with breast cancer in Finland, representing a prevalence of 2241 per 100,000 of the population. According to the Finnish cancer registry, 695 women were diagnosed with breast cancer in the Kuopio region (North, East and South Savo, North Karelia and Central Finland) in 2013, i.e. 29.5% of the 2355 women diagnosed with cancers in that year. This marks a 14.7% increase from the 606 cases of breast cancer diagnosed in the previous year, and is a record high for breast cancer in the Kuopio region.

The vast majority of cancers in Finland (85.6%) are diagnosed in women over the age of 50. The highest incidence rate has been recorded in patients aged between 60 and 65 (18.0%).

About 5-10% of breast cancers can be linked to inherited paternal gene mutations. The remaining 85% of breast cancers occur in women with no close family history of breast cancer. These occur due to genetic mutations resulting from the normal aging process and life in general, rather than heredity.

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4 2.1.3 Breast cancer survival rates

The relative survival of breast cancer varies from region to region. According to the Finnish cancer registry (http://www.cancer.fi/syoparekisteri/en), one- and five-year relative survival rates of breast cancer patients diagnosed in Finland in 2005– 2012 and followed up between 2010–2012 was 97% and 90%, respectively.

According to the American Cancer Society (12), breast cancer five-year survival rate for women aged 15 or older is 89% in the United States, 82% in Switzerland, and 80% in Spain.

Survival rates are generally lower in the developing countries than in Europe and North America, with rates as low as 38.8% in Algeria, 36.6% in Brazil, and 12% in Gambia (13, 14).

The stage at diagnosis is the most important prognostic variable. For instance, according to the Surveillance Epidemiology and End Results SEER cancer statistic (15), the relative 5 year survival for breast cancer was 89%. The rates in table 1 below come from the same SEER database, and show the relative survival of patients by stage.

Table 1. Percentages of 5 year relative survival by disease stage according to the Surveillance Epidemiology and End Results SEER cancer statistics.

Stage 5-year Relative Survival Rate

0 100%

I 100%

II 93%

III 72%

IV 22%

2.1.4 Breast cancer subtypes

The majority of breast malignancies have epithelial origins and are therefore categorized as carcinomas. Although breast carcinomas are often discussed as a single homogenous disease, they are actually a diverse group of lesions that differ in both their microscopic appearance and biologic behaviour.

Breast cancers can be grouped into either in situ carcinomas or invasive carcinomas according to their ability to metastasize and infiltrate adjacent tissues. Moreover, based on histopathologic properties, both of the aforementioned types can be divided into subtypes (16). The in situ carcinomas of the breast could be either ductal or lobular (the latter is included in lobular neoplasia) while invasive breast carcinomas are more complex and consist of several histologic subtypes.

The estimated percentages according to the SEER database of the National Cancer Institute are as follows (15):

• Infiltrating ductal – 76%

• Invasive lobular - 8%

• Ductal/lobular – 7%

• Mucinous (colloid) - 2.4%

• Tubular - 1.5%

• Medullary - 1.2%

• Papillary – 1%

• Other subtypes account for fewer than 5% of cases

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5 2.1.4.1 Ductal carcinoma in situ (DCIS)

DCIS is a neoplastic intraductal lesion; it is characterized by an increase in cellular atypia, epithelial proliferation, and a strong predilection to progress to invasive breast carcinoma (17). The terminal ductal lobular unit (TDLU) is the site where DCIS originates, and it also the site from where it extends into the lobular glands’ epithelium and spreads inside the ductal structures. Since the start of the widespread screening programs, there has been a considerable increase in DCIS detection. An estimated 10-30% of all detected malignancies in current screening programs are DCIS (18, 19).

2.1.4.2 Invasive carcinoma of no special type

With 70-80% of all invasive lesions, infiltrating ductal carcinoma is the most common type of invasive breast cancer. According to the World Health Organization (WHO) classification, it is a “heterogenous group of tumours that fails to exhibit sufficient characteristics to achieve classification as a specific histological type” and “it comprises the largest group of malignant breast tumours” (17, 20). The 5-year relative survival of the patients with ductal carcinoma is 79% (20).

2.1.4.3 Invasive lobular carcinoma

Infiltrating lobular carcinomas are the second most common type of invasive breast cancer, accounting for about 5 to 10 percent of invasive lesions, and often presents as bilateral or multifocal lesions (21, 22). Invasive lobular carcinoma is difficult to define in both its macroscopic and mammographic form, when viewed in the microscope, its growing pattern exhibits strands of tumour cells that are thread-like and loosely dispersed throughout the fibrous stroma (16, 23).

2.1.4.4 Other types of invasive carcinoma

Several other types of invasive carcinomas such as invasive cribriform, tubular and mucinous carcinomas are usually well-differentiated histopathologically and are detected by mammography as either spiculated masses (tubular and cribriform carcinomas) (24, 25) or well delineated masses (mucinous carcinoma) (17, 26). These tumours have favourable 10- year outcomes in a range between 90-100% (27, 28). Carcinomas with medullary features are an overlapping group of tumours with a medullary appearance, in the WHO classification of breast tumour 4^th edition, the use of older names such as medullary carcinomas, atypical medullary carcinomas and invasive carcinomas of no special type are discouraged. They are often well-defined both clinically and in imaging studies (17). Inflammatory carcinoma is a rare and particularly aggressive form of breast carcinoma with a 5-year survival ranging between 18% to 41% (20, 29, 30).

2.2 TRADITIONAL RISK AND PROGNOSTIC FACTORS FOR BREAST CANCER

2.2.1 Risk factors

Breast cancer occurs 100 times more frequently in women than in men, in the United States approximately 2000 cases of male breast cancer are diagnosed annually while 200,000 are diagnosed in women (9, 10, 31). Moreover, a positive family history of breast cancer increases the risk according to the type of the relative affected, age at which the relative developed the disease and the number of relatives affected (32), the risk is increased to about twofold for women with one affected first-degree relative and threefold in women with two such relatives (33). Age is also one of the strongest risk factors associated with cancer development, with the risk increasing with increasing age. The probability of a woman developing breast cancer in the USA according to age is as follows: (34)

(30)

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• Birth to age 39 – 0.49% (1 in 203 women)

• age 40 to 59 – 3.76% (1 in 27 women)

• age 60 to 69 – 3.53% (1 in 28 women)

• age 70 and older – 6.58% (1 in 15 women)

• Birth to death – 12.29% (1 in 8 women)

Obesity (body mass index, BMI ≥30 kg/m²) is associated with an overall increase in both breast cancer morbidity and mortality. However, this risk of breast cancer that is related to BMI seems to be dependent on the patient’s menopausal status. A higher body mass index (BMI) and/or perimenopausal weight gain have been consistently associated with a higher risk of breast cancer among postmenopausal women (35, 36). On the other hand, increased BMI is associated with a lower risk in premenopausal women (37).

Only 5-10% of breast cancers are directly caused by inherited breast cancer susceptibility genes such as BRCA1, BRCA2, TP53, CDH1, LKB1, CHEK2, BRIP1, PTEN, ATM, and PALB2 i.e. it is rare that one encounters a specific genetic mutation predisposing to breast cancer (17, 38).

The most common mutations implicated in breast cancer are in the BRCA1 and BRCA2 genes. On average, women with a BRCA1 mutation have a 55-65% lifetime risk of developing breast cancer. For women with a BRCA2 mutation, the risk is 45%. Breast cancer that is positive for the BRCA1 or BRCA2 mutations tends to develop more often in younger women.

An increased ovarian cancer risk is also associated with these genetic mutations. In men, BRCA2 mutations are associated with a lifetime breast cancer risk of about 6.8%; BRCA1 mutations are a less frequent cause of breast cancer in men. PALB2 mutations are an important cause of hereditary breast cancer, and it is suggested that PALB2 mutations overlap with BRCA2 mutations in terms of breast cancer risk (39).

Several reproductive factors are associated with an increased breast cancer risk. Early age at menarche is associated with a higher risk of breast cancer (40). Women with menarche at or after 15 years of age were less likely to develop estrogen receptor/progesterone receptor positive breast cancer compared with women who experienced menarche before the age of 13 years (40). Women with menarche at or after age 15 years also had a 16% decreased risk of hormone negative breast cancer. Nulliparity also increases the relative risk of breast cancer by 1.2 to 1.7, and it is postulated to increase the risk synergistically with increased BMI (31, 41). Advanced age at the time of the first pregnancy is also associated with an increased risk (42). Infertility has a controversial association with breast cancer risk, some epidemiological data link anovulatory disorder infertility with a decreased risk while others observed no association or a slight increase in risk (43-45). Increased levels of endogenous estrogen also increase the risk of breast cancer (46) and elevated androgen levels are linked with an increased risk of postmenopausal and premenopausal breast cancer (47).

Women with mammographically dense breast tissue in the upper quartile (≥75 percent density), have a four to six times the risk of developing breast cancer compared with women of similar age in the lowest quartile (fatty breasts, density <25%) (3, 4, 48, 49).

The risk of developing invasive breast cancer in the contralateral breast is increased by a personal history of ductal carcinoma in situ (DCIS) or invasive breast cancer (50). The risk associated with a positive family history of breast cancer is strongly affected by the number of female first-degree relatives with cancer and with the age at which they were diagnosed.

The risk is increased almost twofold if a woman had one affected first-degree relative and threefold if she had two affected first-degree relatives, while the risk is threefold higher if the relative was diagnosed before the age of 30 and 1.5 fold if diagnosed after the age of 60 (33).

Several lifestyle factors such as alcohol consumption (51), smoking (52), diet (53) and radiation exposure are also associated with an increased relative risk of breast cancer.

On the one hand, the literature generally shows no association between previous or current oral contraceptive use and the risk of breast cancer later in life (54, 55). On the other hand, there is evidence that the risk of developing breast cancer is increased in

(31)

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postmenopausal women using hormonal replacement therapy and increases with the increasing duration of use (56, 57).

2.2.2 Protective factors

Factors that might protect from breast cancer, independent from those mentioned in the previous section have also been described. For instance, breastfeeding’s protective effect has been described in several case-control and cohort studies, the magnitude of its impact depends on several factors including the duration of breast feeding and the confounding effect of parity (58-60). One hypothesis for the mechanism of this effect is that breastfeeding delays the re-establishment of ovulatory cycles, and it has been estimated that a 4.3%

reduction in relative risk occurs with every 12 months of breastfeeding (59).

Exercise and physical activity are also linked with a decrease in relative risk (61). A review of nine prospective studies that included 11,656 women found that the postmenopausal breast cancer risk was decreased by 12% for each 5 ng/mL increase in vitamin D (25(OH)D) levels between 27 and <35 ng/mL, with no further reduction for 25(OH)D levels greater than 35 ng/mL (62). Moreover, non-steroidal anti-inflammatory drug (NSAID) use has been linked with an average 12% reduction in breast cancer relative risk, with ibuprofen (21%) showing the greatest protective effect (63).

2.2.3 Prognostic and predictive factors

Although closely related and often mistaken for each other, it is important to make a distinction between what is considered a prognostic and what is considered a predictive factor. By definition, a prognostic factor has the ability to provide information on the clinical outcome independent of therapy at the time of diagnosis. Such factors are often indicators of invasion, growth, and metastatic likelihood. In contrast, a predictive factor is the one that is able to provide insights into the likelihood of a positive response to a specific modality of treatment (64).

For women with newly diagnosed early breast cancer, several factors are utilized to help determine prognosis. Both young and old age (<35 and >65) are associated with a worse prognosis, with differing impacts amongst the differing breast cancer subtypes (65).

Tumour size is recognized as an important prognostic factor and is defined as the largest diameter of the primary tumour (66, 67). The five-year breast cancer survival rates in the SEER (Survival, Epidemiology and End Results) program ranged from 91 percent for size

=<2 cm to 80 percent for sizes of >2 to 5 cm and 63 percent for size >5 cm (66).

Tumour size is correlated with nodal involvement but the prognostic value of the two factors is independent. For patients who are without metastasis, the five-year survival rates for those who present with localized versus regional disease (i.e., pathologic node involvement) are 99% and 84%, respectively (68). In the SEER program which involved 25,000 cases, the five-year relative survival was 96%, 86% and 66% percent for patients who were pathologically node-negative, had one to three nodes involved, or greater than four nodes involved, respectively (66).

With regards to pathological factors, tumour stage by itself is considered to be a prognostic factor. The tumour node metastasis (TNM) staging system for breast cancer (Table 2) is an internationally accepted system used to determine the disease stage (69). Disease stage is a measure of the extent of the disease (Table 3), which is used to determine prognosis and guide management.

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Table 2. Tumour-Node-Metastasis (TNM) classification of breast tumours according to the seventh edition (2010) of the TNM staging system.

Category Description

pTX Primary tumour cannot be assessed pT0 No evidence of primary tumour Tis Carcinoma in situ

pT1mi Tumour ≤ 1 mm in greatest dimension pT1 Tumour 2 cm or less in greatest dimension

pT2 Tumour more than 2 cm but not more than 5 cm in greatest dimension pT3 Tumour more than 5 cm in greatest dimension

pT4 Tumour of any size with direct extension to chest wall or skin only pNX Regional lymph nodes cannot be assessed

pN0 No regional lymph node metastases

pN1mi Micrometastases (> 0.2 mm and/or > 200 cells, but none > 2.0 mm)

pN1 metastases in 1-3 axillary lymph nodes and/or in clinically negative internal mammary nodes

pN2 Metastases in 4-9 axillary lymph nodes or in clinically detected‡ internal mammary lymph nodes in the absence of axillary lymph node metastases pN3 Metastases in ≥ 10 axillary lymph nodes; or in infraclavicular lymph nodes; or in

clinically detected ipsilateral internal mammary lymph nodes in the presence of ≥ 1 positive level I, II axillary lymph nodes; or in > 3 axillary lymph nodes and in internal mammary lymph nodes, with micrometastases or macrometastases not clinically detected; or in ipsilateral supraclavicular lymph nodes

MX Distant metastasis cannot be assessed M0 No distant metastasis

M1 Distant metastasis

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The relationship between the expressions of the estrogen receptor (ER) and progesterone receptor (PR) and prognosis is unfortunately not a straightforward one, and has been a matter of debate for years. Multiple studies have shown that the expression of estrogen and progesterone receptors in the breast cancer tissue correlates with a good outcome with hormonal therapy in the adjuvant setting and even metastatic diseases (70-72). On the other hand, beyond the first five years, the hazard rate (HR) for recurrence is nearly zero percent per year for ER-negative cancers while it remains above zero (of the order of one-half to two percent per year) for those patients with ER-positive disease. Therefore, the prognostic value based on hormone receptor status tends to recede with time (70, 73).

PR appears to have its own prognostic capabilities and acts independently of ER (74, 75).

In a multivariate analysis of a large population based cohort study, PR was shown to be independently predictive of disease free survival (HR 1.94) and breast cancer-specific survival (HR 2.12). In contrast to ER, the prognostic significance of PR appeared to increase beyond the sixth year of follow-up.

The human epidermal growth factor receptor 2 (HER2) oncogene encodes for a 185 KD transmembrane glycoprotein receptor with intracellular tyrosine kinase activity (76). It contains an epidermal growth factor receptor (EGFR) which plays an important role in the activation of subcellular signal transduction pathways that control epithelial cell differentiation, growth, and angiogenesis (77-79). HER2 is expressed in a wide range of normal adult and fetal epithelia, playing an important role in growth and development (80).

HER2 amplifications or its protein product over-expression are observed in 18-20% of human breast tumours (81, 82).

The recommendations for HER2 testing have been updated by the American Society of Clinical Oncology and the College of American Pathologists (83), and these recommendations have also been adapted and evaluated in other countries (84-86).

• HER2 status should be determined in all patients with invasive breast cancer on the basis of one or more test results.

• HER2-positive status is indicated by evidence of protein overexpression or gene amplification.

• When results are equivocal, reflex testing should be performed with an alternative assay, and repeat testing should be considered if results are discordant with other findings.

Table 3. Stage according to the Union for International Cancer Control (UICC) classification.

Stage T N M

0 Tis N0 M0

I T1 N0 M0

IIA T0-1 N1 M0

T2 N0 M0

IIB T2 N1 M0

T3 N0 M0

IIIA T0-2 N2 M0

T3 N1-2 M0

IIIB T4 N0-2 M0

IIIC T0-4 N3 M0

IV T0-4 N0-3 M1

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• Laboratories should be able to show high concordance with a validated HER2 test on a large and representative set of specimens, and testing must be performed in an accredited laboratory.

• Providers should recommend HER2-targeted therapies if the patient's HER2 test result is positive and such treatment is clinically appropriate. Most experts do not recommend HER2-targeted therapy if the HER2 test result is negative and there is no histopathological discordance with HER2 testing. It is recommend to delay the decision to initiate HER2-targeted therapy if the HER2 test result is equivocal. Reflex testing should be done on the same specimen.

• Several methods are used to measure the human epidermal growth factor receptor 2 (HER2) oncogene’s activity; there is no consensus about which is the best method, both in terms of the type of assay used and the optimal way to perform each type of assay, is controversial. The available assays are as follows:

o HER2 gene amplification by in situ hybridization (ISH) – Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), silver- enhanced in situ hybridization (SISH), or differential polymerase chain reaction (PCR).

o Overexpression of the HER2 protein product – Western blotting, enzyme- linked immunosorbent assay (ELISA), or immunohistochemistry (IHC).

o Overexpression of HER2 RNA – Northern blotting or reverse transcription PCR (RT-PCR).

The assay for HER2 amplification is nowadays considered as a routine part of the diagnostic work-up of all primary breast cancers (87, 88). HER2 overexpression forecasts an unfavourable prognosis, particularly if patients do not receive HER2-directed agents with chemotherapy (87). Apart from its prognostic role, the main benefit of HER2 testing is its predictive ability in selecting who should receive HER2-directed agents. In the absence of systemic therapy, HER2 overexpression is a marker of poor prognosis in patients with pathologically node-positive (89) and node-negative breast cancer (90). Prognosis vastly improves with trastuzumab treatment (91). In addition, there are data suggesting that HER2 retains prognostic value even in the presence of small tumours of ≤1 cm.

Gene expression studies have managed to identify several distinct breast cancer subtypes that differ markedly in terms of prognosis and in the therapeutic targets they express (92-98).

The intrinsic list is the name given to the list of genes that differentiates these subtypes and it is made up of several clusters of genes relating to ER expression (the luminal cluster), HER2 expression, proliferation, and a unique cluster of genes called the basal cluster. Others are being identified as investigators continue to study the genomic data derived from breast cancer specimens.

The intrinsic subtypes segregate into two groups that correspond to expression of hormone receptor-related genes. This segregation is consistent with both the literature and clinical experience showing that ER-positive and ER-negative cancers define biologically distinct phenotypes that may derive from different progenitor cells (95).

Luminal subtypes — luminal A and luminal B express genes associated with the luminal epithelial cells of normal breast tissue and overlap with ER-positive breast (99).

The name "luminal" refers to the similarity between these tumours and the luminal epithelium of the breast. They form the majority of ER-positive breast cancer, and are characterized by the expression of ER, PR, and some other genes associated with ER activation. Despite comprising the majority of ER-positive breast cancers, the luminal A and luminal B subtypes exhibit some important molecular and prognostic differences. The clinico-pathological surrogate definitions of subtypes as adopted by the expert panel of the Saint Gallen international breast cancer conference (100) are summarized in Table 4.

Triple-negative breast cancer is a term that refers to cancers that are low in expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor

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receptor 2 (HER2). They account for about 15-20% of all breast cancers and are usually diagnosed in younger women (<40 years) (101, 102). They are usually of a high grade (103) and in comparison to other types of breast cancer, they usually tend to behave more aggressively (104).

Mammographic breast density, tumour characteristics and the expression of hyaluronan as prognostic surrogate markers for breast cancer

uef.fi

Dissertations in Health Sciences

AMRO MASARWAH

MAMMOGRAPHIC BREAST DENSITY, TUMOUR CHARACTERISTICS AND THE EXPRESSION OF HYALURONAN AS PROGNOSTIC SURROGATE MARKERS FOR BREAST CANCER

AMRO MASARWAH

Mammographic Breast Density, Tumour Characteristics and the Expression of Hyaluronan as Prognostic Surrogate

Markers for Breast Cancer

Mammographic Breast Density, Tumour Characteristics and the Expression of Hyaluronan as Prognostic Surrogate

Markers for Breast Cancer

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Review of the Literature