Musculoskeletal side effects

of aromatase inhibitors

treatment in women with

breast cancer

Author: Maria Rodríguez Sanz

DOCTORAL THESIS UPF YEAR 2016

Supervisors: Dr. Natalia Garcia Giralt and Dr. Xavier Nogués Solán

Tutor: Dr. Josep Mª Antó Boqué

Musculoskeletal research group

Inflammatory and Cardiovascular Research Program

Hospital del Mar Medical Research Institute (IMIM)

iii

To my family and friends,

v

ACKNOWLEDGMENTS

First of all, I woul” like to a“knowle”ge my PhD supervisors, Dr. Natalia

Gar“ia Giralt an” Dr. Xavier Nogués Solan for giving me the opportunity to

join their laboratory an” for their ex“ellent gui”an“e. I woul” also like to

thank my “olleagues at the URFOA: Laura De Ugarte, Guy Yoskovitz, Sergi

Ariño, Laura Tio, Anna Martín, Marta Pine”a, Enrique Caro, Ivan Valle”or,

Selene Luna, Anabela Areias...

Gra“ias a Laura ”e Ugarte Corbalán (The Prin“ess), por “ompartir “a”a

minuto ”el “amino, ”es”e el primer hasta el último ”ía. Contigo las

”ifi“ulta”es han si”o menos ”ifí“iles, aunque las “ajas ”e kleenex se a“aben

antes. Las ané“”otas que “ompartimos son innumerables y jamás las

olvi”aré. Deseo que se “umplan to”as tus metas profesionales para el

futuro: ingeniera ”e biomateriales, ”iseña”ora ”e mo”a, “oreβgrafa,

”ete“tive priva”o por las no“hes, mo”elo, fisioterapeuta ”e juga”ores ”e

alto ren”imiento, jui“y girl en Hawaï... (seguro que alguna se me olvi”a). Eso

sí, por favor, ¡bajo ningún “on“epto te hagas pastelera, ni ”ejes que yo lo

sea!. Creo que la etapa que ahora llega será maravillosa, pero hasta nueva

or”en, ya sabes: ¡uñitas, me“hitas y más uñitas!.

Gra“ias a Guy Yoskovitz (alias Dios) por enseñarnos tantas “osas “on tu

pa“ien“ia infinita. Gra“ias por haberme he“ho, no sβlo “reer en otras formas

”e “on“ebir la familia, sino a”emás presumir ”e haber teni”o “omo

“ompañero a un pa”re maravilloso e inmejorable. Gra“ias in“luso por tus

listas ”e análisis esta”ísti“os pen”ientes que pare“ían repro”u“irse por

biparti“iβn. ¡Nene, nene! Te e“hamos mu“hísimo ”e menos.

Gra“ias a Sergi Ariño (“atalán ”e origen pero “ubano ”e alma), por tanta

risa. Pare“e que los enanitos nos están ”an”o un respiro a ambos ¿no?.

Finalmente el Papa Noel ”e la esquina “umpliβ “on su trabajo. Bueno, o eso, o

están pillan”o “arrerilla para la venganza más gran”e ”e la historia XD. EL

humor ”e URFOA se que”β “ojo “uan”o te fuiste, pero me alegro mu“ho ”e

que vayas ha“ién”ote un hue“o en la farán”ula.

Gra“ias a Anna Martín, ¡por esas inye““iones ”e energía fresquísima!. Por tu

alegría ”esbor”ante “a”a vez que entras por la puerta. ¡Hello!. No sé si en tu

trajín ”e ir y venir te habrás lleva”o mu“ho ”e URFOA, pero si estoy segura

”e que has apren”i”o algo muy importante: en el siglo XV antes ”e Cristo ya

había gente en el mun”o. Espero seguir vién”ote mu“ho tiempo más.

Gra“ias a las más re“ientes aunque fuga“es in“orpora“iones: Enrique Caro,

Marta Pine”a e Iván Valle”or. Aunque hay personas a las que “uesta mu“ho

reemplazar , to”os vosotros habéis supera”o las expe“tativas. Marta

Pine”a, mi su“esora, te ”eseo mu“ha suerte en tu nueva etapa. A ti también

Enrique Caro. A”miro tu toleran“ia y tus ganas ”e lu“har por un mun”o

mejor. También te ”oy las gra“ias por ha“erme ver que existen personas

igual (¡¡¡¡¡ o mu“hísimo más!!! XD) ”e ”espista”as que yo (jeje). A ti, Iván

Valle”or, gra“ias por to”as las “ari“aturas en papel y por to”os los buenos

vii

momentos ”e telenovela. Re“uer”a: Respon”e al nombre ”e Soraya .

Gra“ias a Aina, que está lejos, pero apren”ien”o mu“hísimo. Me alegro ”e

que estés vivien”o miles ”e experien“ias. Espero que tú también nos e“hes

mu“ho ”e menos a nosotros y al “ajβn ”e Guy. ¡¡¡¡Vuelve algún ”ia!!!

Gra“ias a Lara ”e Llobet por reapare“er, y por juntarte “on to”os nosotros.

Eres una más ”e nuestra pequeña familia.

Gra“ias a mis amigos fuera ”e URFOA: Carmen, Virginia, Moki, Quique, Osqui,

Canu, Esti, Alain, Simone, Ali“ia, Naty... A to”os: gra“ias por formar parte ”e

mi vi”a. Mis logros también tienen una parte ”e “a”a uno ”e vosotros. Sé

que nos que”an mu“hísimas aventuras juntos, así que... ¡Nos vemos el fin ”e

semana! :D

Gra“ias a Jor”an, por millones ”e “osas que no “abrían en estas páginas. Has

si”o uno ”e los pilares más importantes ”urante to”o este pro“eso. Espero

que sigamos apren”ien”o el uno ”el otro por mu“hísimo más tiempo.

Y por último y más importante, gra“ias a mis pa”res, que me han brin”a”o

to”o el apoyo físi“o y mental ne“esario para llevar a”elante esta exigente

etapa ”e mi vi”a. Sin ellos, ver”a”eramente no habría si”o posible.

ix

ABSTRACT

The intro”u“tion of aromatase inhibitors (AI) therapy for the treatment of

postmenopausal women with hormone re“eptor-positive breast “an“er has

le” to a signifi“ant improvement of patient survival. However, AI-asso“iate”

si”e effe“ts, whose etiology is still unexplaine”, “ompromise quality of life

an” lea” to non-“omplian“e, representing a signifi“ant bur”en. In this thesis,

we fo“us on mus“uloskeletal symptoms, essentially joint pain an” bone

mineral ”ensity loss with the asso“iate” in“rease in fra“ture risk. In a “ontext

of every”ay “lini“al pra“ti“e, we ”es“ribe the evolution of these out“omes

”uring AI therapy. A signifi“ant in“rease in the intensity of joint pain has

been ”ete“te”. Patients also experien“e an a““elerate” bone mineral ”ensity

loss whi“h is ”imme” by oral bisphosphonates. Our resear“h lea”s us to state

that the geneti“ ba“kgroun” of the in”ivi”ual is essential in this setting. Thus,

polymorphi“ variants in genes involve” in steroi” an” vitamin D metaboli“

pathways may “ontribute to the onset an”/or intensity of these un”esirable

symptoms. Furthermore, bone is raise” for the first time as a potential

steroi”ogeni“ tissue. The results are promising an” reveal new insights into

the fun“tioning of these mus“uloskeletal events.

xi

RESUMEN

La terapia “on inhibi”ores ”e aromatasa (IA) para el tratamiento ”el “án“er

”e mama positivo para re“eptor ”e estrβgenos en mujeres

postmenopáusi“as ha pro”u“i”o un aumento signifi“ativo ”e la

superviven“ia. No obstante, los efe“tos se“un”arios aso“ia”os a este

tratamiento, “uya etiología es to”avía ”es“ono“i”a, “omprometen la “ali”a”

”e vi”a ”e las pa“ientes y ”isminuyen la a”heren“ia al tratamiento,

representan”o un gran in“onveniente. Esta tesis se enfo“a en los síntomas

mus“uloesqueléti“os, espe“ífi“amente el ”olor arti“ular y la pér”i”a ”e

”ensi”a” mineral βsea “on el “onse“uente aumento ”el riesgo ”e fra“tura. Se

”es“ribe la evolu“iβn ”e estos síntomas ”urante la terapia “on IA en la

prá“ti“a “líni“a habitual. Se ha ”ete“ta”o un aumento en la intensi”a” ”el

”olor arti“ular. A”emás las pa“ientes experimentan una pér”i”a a“elera”a ”e

la ”ensi”a” mineral βsea, que pue”e ser atenua”a “on bifosfonatos orales.

Nuestros estu”ios nos permiten afirmar que el “omponente genéti“o ”el

in”ivi”uo es esen“ial en este “ontexto. Así, variantes polimβrfi“as en genes

involu“ra”os en las vías metabβli“as ”e los estrβgenos y la vitamina D pue”en

“ontribuir a la apari“iβn y/o intensi”a” ”e estos efe“tos a”versos. A”emás, el

hueso se plantea por primera vez “omo un teji”o esteroi”ogéni“o poten“ial.

Los resulta”os son promete”ores y revelan nuevos “ono“imientos a“er“a ”el

fun“ionamiento ”e estas manifesta“iones mus“uloesqueléti“as.

xiii

PREFACE

The work presente” in this ”o“toral thesis was “arrie” out in the

Inflammatory an” Car”iovas“ular Disor”ers Program of Hospital ”el Mar

Me”i“al Resear“h Institute (IMIM) in Bar“elona, Spain, un”er the supervision of

Dr. Natalia Gar“ia Giralt an” Dr. Xavier Nogués Solán.

The “ontent of this thesis provi”es novel insights on the “ausal me“hanisms

un”erlying the si”e effe“ts asso“iate” to AI treatment in postmenopausal

women with breast “an“er. The results presente” here illustrate an important

role for the geneti“ ba“kgroun” in the onset an”/or intensity of joint pain

an” bone mineral ”ensity loss asso“iate” to this me”i“ation.

xv

CONTENTS

V ACKNOWLEDGEMENTS

IX ABSTRACT

XI RESUMEN

XIII PREFACE

XV GLOSSARY

1 CHAPTER 1- INTRODUCTION 3 1. BREAST CANCER

4 1.1. BREAST CANCER EPIDEMIOLOGY

6 2. BREAST CANCER CLASSIFICATION

6 2.1. RECEPTOR STATUS

6 2.1.1 Human epi”ermal growth fa“tor re“eptor-2 (HER2)

7 2.2.1. Hormonal re“eptors

9 3. RISK FACTORS FOR BREAST CANCER

9 3.1. GENETIC RISK FACTORS

9 3.1.1. High penetran“e genes

9 3.1.2. Low penetran“e genes

10 3.2. HORMONAL RISK FACTORS

10 3.2.1. Repro”u“tive fa“tors

11 3.2.2. Other hormonal fa“tors

xvi

12 3.3. NON-HORMONAL RISK FACTORS

12 3.3.1. Age

12 3.3.2. Geographi“al variation an” Ethni“ity

13 3.3.3. Ra”iation

13 3.3.4. Previous benign ”isease

14 3.3.5. Lifestyle an” ”ietary fa“tors

15 4. ESTROGENS AND BREAST CANCER

15 4.1. STEROID BIOSYNTHESIS

15 4.1.1. Cholesterol transport

16 4.1.2. Cyto“hrome P450 an” HSDs

17 4.1.3. Steroi”ogenesis

19 4.2. STEROIDOGENIC TISSUES AND REGULATION

22 4.3. STEROIDS IN POSTMENOPAUSAL WOMEN

24 4.4. STEROID MECHANISMS OF BREAST CARCINOGENESIS

24 4.4.1. ER signalling.

25 4.4.2. Pro”u“ts of estrogen metabolism

27 5. BREAST CANCER THERAPIES

28 5.1. SURGERY

28 5.2. RADIOTHERAPY

28 5.3. CHEMOTHERAPY

29 5.4. BIOLOGICAL THERAPY

29 5.5. ENDOCRINE THERAPY

29 5.5.1. Surgi“al Oophore“tomy

30 5.5.2. LH-RH (Luteinizing Hormone-Releasing Hormone) Analogues

30 5.5.3. Sele“tive estrogen re“eptor mo”ulators (SERMs) an”

”ownregulators (SERDs)

32 5.5.4. Aromatase Inhibitors (AI)

36 6. SECONDARY EFFECTS OF AI THERAPY

36 6.1. CARDIOVASCULAR RISK AND LIPID METABOLISM

37 6.2. MUSCULOESKELETAL EFFECTS

38 6.2.2. Etiology of AIA

40 6.2.3. AIA signifi“an“e: a”heren“e, treatment effi“a“y an” mortality

41 6.2.4. AIA management

xvii

42 6.3. OSTEOPOROSIS AND RISK OF FRACTURE

44 6.3.1. Bone stru“ture

46 6.3.2. Bone matrix

47 6.3.3. Osteoblasts

47 6.3.4. Osteo“ytes

48 6.3.5. Osteo“lasts

48 6.3.6. Bone remo”elling

50 6.3.7. Mole“ular effe“ts of estrogen ”efi“ien“y on remo”elling

52 6.3.8. Aromatase inhibitor in”u“e” bone loss (AIBL) an” fra“ture risk.

54 7. AIBL MANAGEMENT

54 7.1. LIFESTYLE INTERVENTIONS

54 7.2. BONE LOSS SCREENING AND MONITORING

56 7.3. PHARMACOLOGICAL THERAPY: BISPHOSPHONATES (BP)

56 7.3.1. Me“hanism of a“tion

58 7.3.2. Clini“al role of BP

58 7.4. FARMACOLOGICAL THERAPY: DENOSUMAB

59 7.5. THE ROLE OF BONE MICROARCHITECTURE IN FRACTURE RISK:

TRABECULAR BONE SCORE (TBS)

63 8. VITAMIN D AND AI ADVERSE EFFECTS

63 8.1. SYNTHESIS OF VITAMIN D

66 8.2. EFFECTS OF VITAMIN D ON AIA

67 8.3. EFFECTS OF VITAMIN D ON AIBL

69 9. GENETIC STUDY OF COMPLEX TRAITS

70 9.1. GENETIC BASIS OF AIA

71 9.2. GENETIC BASIS OF AIBL

73 CHAPTER 2- RESULTS 75 Manuscript 1. Geneti“ ”eterminants of aromatase inhibitor-relate”

arthralgia: the B-ABLE “ohort stu”y

89 Manuscript 2. AI-relate” BMD variation in a“tual pra“ti“e “on”itions: a

prospe“tive “ohort stu”y

xviii

101 Manuscript 3. CYP11A1 expression in bone is asso“iate” with aromatase

inhibitor-relate” bone loss

115 Manuscript 4. Evolu“iβn ”e la DMO ”urante el tratamiento “on inhibi”ores ”e

aromatasa y su rela“iβn “on el gen CYP11A1: estu”io prospe“tivo ”e la

“ohorte B-ABLE

125 Manuscript 5. TBS an” BMD at the en” of AI-therapy: a prospe“tive stu”y of

the B-ABLE “ohort

135 CHAPTER 3- DISCUSSION 138 JOINT PAIN IN THE B-ABLE COHORT

141 BMD EVOLUTION IN THE B-ABLE COHORT

146 EFFECTS OF BP ON BMD EVOLUTION

148 BONE MICROARCHITECTURE: A POSSIBLE ROLE FOR TBS

150 HIGH VARIABILITY IN THE STUDIED OUTCOMES

155 INTERINDIVIDUAL DIFFERENCES IN SEX STEROID LEVELS: A POSSIBLE

UNDERLYING MECHANISM FOR VARIATION IN AI SECONDARY EFFECTS

158 THE ROLE OF LOCAL ESTROGEN SYNTHESIS

159 P450SCC IN BONE: FURTHER UNKNOWN FUNCTIONS?

160 VITAMIN D ACTIVATION AND AI SECONDARY EFFECTS

161 THE CHALLENGES AHEAD OF THE STUDY

162 I”entifi“ation of “ausal sequen“e variants

163 Sex steroi” hormone levels in AI-treate” women

164 Bio“hemi“al assessment of bone remo”elling an” “artilage ”egra”ation

”uring AI treatment

167 CHAPTER 4- CONCLUSSIONS

169 CONCLUSSIONS

171 BIBLIOGRAPHY

xix

GLOSSARY

Abbreviation Meaning

17 HSDs 17 -hy”roxysteroi” ”ehy”rogenases enzymes

3 HSD 3 -hy”roxysteroi” ”ehy”rogenase

ACTH A”reno“orti“otropi“ hormone

AI Aromatase inhibitors

AIA AI-relate” arthralgia

AIBL Aromatase inhibitor in”u“e” bone loss

ASCO Ameri“an So“iety of Clini“al On“ology

ATAC Arimi”ex, Tamoxifen, Alone or in Combination trial

BMD Bone mineral ”ensity

BMI Bo”y mass in”ex

BMU Basi“ multi“ellular unit

BP Bisphosphonates

BTM Bone turnover markers

COMT Cate“hol-O-methyltransferase enzyme

CV Coeffi“ient of variation

CYPs Cyto“hrome P450s

DBP Vitamin D bin”ing protein

DHC Dehy”ro“holesterol

DHEA Dehy”roepian”rosterone

DHEA-S Dehy”roepian”rosterone-sulphate

DHT Dihy”rotestosterone

DXA Dual-energy X-ray absorptiometry

xx

ECM Extra“ellular matrix

ER Estrogen re“eptor

FGF Fibroblast growth fa“tor

FN Femoral ne“k

FSH Folli“ular stimulating hormone

GnRH Gona”otrophin releasing hormone

GWAS Genome wi”e asso“iation stu”y

HER-2 Human epi”ermal growth fa“tor re“eptor-2

HRT Hormone repla“ement therapy

HSDs Hy”roxysteroi” ”ehy”rogenases

IGF Insulin growth fa“tor

IL Interleukin

INF-ɣ Interferon gamma

IOM Institute of me”i“ine

LBD Ligan” bin”ing ”omain

LH Luteinizing hormone

LH-RH Luteinizing hormone-Releasing Hormone

LRP5 Low-”ensity lipoprotein re“eptor-relate” protein 5 gene

LS Lumbar spine

M-CSF Ma“rophage “olony stimulating fa“tor

NAD Ni“otinami”e a”enine ”inu“leoti”e phosphate

NADPH Ni“otinami”e a”enine ”inu“leoti”e phosphate

NCI National Can“er Institute

OPG Osteoprotegerin

P450“17 17 -hy”roxylase/17,20-Lyase

P450s““ Cholesterol si”e-“hain “leavage enzyme

PPI Inorgani“ pyrophosphate

PR Progesterone re“eptor

PTH Parathyroi” hormone

RANK-L Re“eptor a“tivator of nu“lear fa“tor-κ

RCT Ran”omize” “ontrol trial

xxi

SEER Surveillan“e, Epi”emiology, an” En” Results

SERDs Sele“tive estrogen re“eptor ”ownregulators

SERMs Sele“tive estrogen re“eptor mo”ulators

SNPs Single nu“leoti”e polymorphisms

SRD5 5 -re”u“tase enzymes

StAR Steroi”ogeni“ a“ute regulatory protein

SULT Sulfotransferase enzymes

TBS Trabe“ular bone s“ore

TCL1A T-“ell leukaemia 1A gene

TGF- Transforming growth fa“tor beta

TNF- Tumour ne“rosis fa“tor alpha

VAS Visual analogi“ s“ale

VDR Vitamin D re“eptor

VitD2 Vitamin D2

VitD3 Vitamin D3

1

Chapter1

Dios no juega a los ”a”os “on el Universo

Albert Einstein

INTRODUCTION

3

INTRODUCTION

1. BREAST CANCER

The human mammary glan” is mainly “ompose” of glan”ular stromal

tissues. The stromal tissue in“lu”es a”ipose an” fibrous material

surroun”ing an” supporting the lobules an” ”u“ts of the glan”ular tissue,

responsible for milk synthesis an” transport to the nipple, respe“tively. The

breast also “ontains lymphati“ an” bloo” vessels (Fig. 1).

Figure 1. The anatomy of the breast. Sagittal se“tion of the human mammary glan”.

A”apte” from http://www.a”amon”eman”.“om/anatomy-an”-physiology-of-

the-breast/#.VrxpTrR97Gh

Breast “an“er is a malignant tumour that starts in the “ells of the breast

tissue, primarily in the ”u“ts or lobules, although a small number “an

originate in other tissues1.

4

INTRODUCTION

1.1. BREAST CANCER EPIDEMIOLOGY

Breast “an“er is the most frequently ”iagnose” “an“er in women. It is

estimate” that 1.7 million new “ases worl”wi”e were ”iagnose” in 2012,

a““ounting for 25% of all new “an“er “ases in women. These ”ata are goo”

representative for the Spanish population (Fig. 2), in an interme”iate

situation at European level2. It is estimate” that 1 in 8 women will ”evelop

breast “an“er in their lifetime3.

Figure 2. Estimated cancer incidence by tumour type in Spanish women for the year

2012. A”apte” from http://www.seom.org

Breast “an“er is the se“on” lea”ing “ause of “an“er ”eath (following lung

“an“er) among women in ”evelope” “ountries an” the first “ause of “an“er

”eath among women in Spain, with 6,075 ”eaths in 20122. Fortunately, in

“ontrast to the in“rease in the in“i”en“e rate, breast “an“er mortality rate in

5

INTRODUCTION

western “ountries has remaine” stable or ”e“reasing ”uring the past 25

years (Fig. 3).

Figure 3. Trends in incidence (A) and mortality (B) from female breast cancer in

European countries. Age-stan”ar”ise” rate per 100,000. * Regional ”ata. A”apte”

from: http://globo“an.iar“.fr/Default.aspx

A““or”ing to the Surveillan“e, Epi”emiology, an” En” Results (SEER) program

of the National Can“er Institute (NCI), the “urrent 5-year relative survival

for breast “an“er patients, “ompare” with the rest of the population, is 89%.

Even those with metastati“ ”isease have a 23% 5-year survival, on average.

Earlier ”ete“tion, effe“tive s“reening programs an” a”van“es in therapies

are among fa“tors un”erlying this survival improvement4,5.

6

INTRODUCTION

2. BREAST CANCER CLASSIFICATION

Breast “an“er variability in terms of risk fa“tors, “lini“al presentations,

pathologi“al features, response to therapy an” out“omes lea”s to

“onsi”er that it is, in fa“t, a “olle“tion of ”ifferent ”iseases affe“ting the

same anatomi“al organ. Therefore, its optimal ”es“ription must in“lu”e

several gra”ing systems influen“ing prognosis an” treatment

response. Among them, histology6, gra”e7 an” stage8 of the tumour,

proliferation rate8 an” re“eptor status “an be highlighte”.

2.1. RECEPTOR STATUS

Determination of re“eptor status of breast tumours is useful as a

prognosti“ an” pre”i“tive fa“tor an” has be“ome a stan”ar” pra“ti“e in the

management of this neoplasm. Currently, the presen“e of 3 possible

re“eptors is assesse”.

2.1.1. Human epi”ermal growth fa“tor re“eptor-2 (HER2)

HER2 is a transmembrane re“eptor, member of the human epi”ermal

growth fa“tor re“eptor family. Ligan” bin”ing to HER2 results in the

a“tivation of intra“ellular signalling pathways of enhan“e” “ell growth,

survival an” ”ifferentiation. HER2 is involve” in the regulation of normal

breast growth an” ”evelopment9. HER2 overexpression ”isrupts normal

“ontrol me“hanisms, potentially lea”ing to the formation of aggressive

tumour “ells10 an” has been asso“iate” with in“rease” “ell proliferation, “ell

motility, tumour invasiveness, progressive regional an” ”istant metastases,

a““elerate” angiogenesis an” re”u“e” apoptosis11. This re“eptor “an be

7

INTRODUCTION

valuable as an a”verse prognosis in”i“ator12, pre”i“tive fa“tor for response

to therapy an” therapeuti“ target13.

2.1.2. Hormonal re“eptors

The biologi“al effe“ts of estrogens are me”iate” through the estrogen

re“eptor (ER), a member of the nu“lear re“eptor superfamily. Estrogen union

to the re“eptors “an trigger genomi“ an” non-genomi“ signalling pathways,

often “onverging in the regulation of trans“riptional pro“esses14.

Progesterone re“eptors (PR) are ligan”-a“tivate” trans“ription fa“tors also

belonging to nu“lear re“eptor family, me”iating progesterone a“tions.

Estrogens, progesterone, as well as other sex steroi”s are absolutely

essential for the proliferation an” morphogenesis of the mammary glan”15.

However, in the normal a”ult mammary glan”, ER an” PR are relegate” to

7 10% of the epithelial “ell population, whi“h remain in a non-proliferative

state, while normal proliferating “ells are ”evoi” of this kin” of re“eptors16.

By “ontrast, it is estimate” that ER are expresse” in approximately three-

quarters of “ases of breast tumours, rea“hing 80% among postmenopausal

patients17. About 65% of breast “an“ers expressing ER also express PR18.

Breast “an“ers expressing ER an”/or PR are ”esignate” as hormone

re“eptor-positive (or hormone sensitive) breast “an“ers. The ER pre”i“tive

value falls on its ability to i”entify patients who may benefit from en”o“rine

therapy, a pivotal treatment that works by blo“king estrogen pro”u“tion or

by interfering with hormone a“tion19. Thus, with appropriate a”juvant

treatment, patients with re“eptor-positive ”isease have substantially better

prognoses than those with hormone re“eptor-negative ”isease19. For its

part, PR “an be useful to i”entify ER−/PR+ tumours, representing from 3%

8

INTRODUCTION

to 5% of patients, as they may also respon” to hormonal therapy. There are

also some evi”en“es suggesting that, PR status is an in”epen”ent pre”i“tive

fa“tor for benefit from a”juvant en”o“rine therapy with tamoxifen20,

helping in the sele“tion of initial a”juvant therapy21,22. Gui”elines from

Ameri“an So“iety of Clini“al On“ology (ASCO) re“ommen” that both ER an”

PR analysis shoul” be performe” routinely in all invasive breast “an“ers, as a

sele“tion “riteria for patients who shoul” re“eive en”o“rine therapy23.

Unfortunately, the potential of hormone-re“eptors as a prognosti“ fa“tors

”e“lines over time, so that the benefi“ial effe“t of its presen“e is limite” to

the first 5 years after ”iagnosis24,25.

9

INTRODUCTION

3. RISK FACTORS FOR BREAST CANCER

Tremen”ous efforts have been ma”e in or”er to un”erstan” the etiology of

breast “an“er. Resear“h into the “auses of the malignan“y has allowe” to

i”entify several well-establishe” risk fa“tors an” a variety of others are

“urrently un”er stu”y. These s“ientifi“ inquiries agree that breast “an“er is a

“omplex, multifa“torial ”isease where there is a strong interplay between

geneti“ an” environmental elements. Some of the most investigate” risk

fa“tors are highlighte” below.

3.1. GENETIC RISK FACTORS

It is estimate” that the heritable fa“tors a““ount for 27% of all breast

“an“er “ases26. Two “lasses of sus“eptibility genes exist.

3.1.1. High penetran“e genes

Alleli“ variants “onferring high in”ivi”ual risk of breast “an“er belong to this

“ategory. The tumour suppressor genes BRCA1, BRCA2, ATM an” p53 are the

most “ommon high-penetran“e genes27. Nonetheless, ”espite of being

involve” in aroun” half of the familiar “lustering of early onset breast

“an“er27 they only explain 5%-10% of all breast “an“ers28.

3.1.2. Low penetran“e genes

In “ontrast to the high penetran“e genes, low penetran“e genes “ontain

”isease-asso“iate” variant alleles that only “onfer a small to mo”erate risk

to in”ivi”uals. Notwithstan”ing, low penetran“e genes are more prevalent in

the population an” therefore explain a greater proportion of breast “an“ers

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INTRODUCTION

than the highly penetrant genes. Can”i”ate low penetran“e genes that have

been evaluate” to ”ate in“lu”e those en“o”ing for enzymes involve” in DNA

repair29, estrogen an” “ar“inogen metabolism, an” those parti“ipating in the

”etoxifi“ation of rea“tive oxygen spe“ies emerging in these rea“tions30-33.

These tumours are likely to result from the interplay of many geneti“ an”

environmental fa“tors an” have a relatively late age at ”iagnosis.

3.2. HORMONAL RISK FACTORS

A wealth of epi”emiologi“al an” experimental information points to the

involvement of estrogens an” other hormones in human breast “an“er.

3.2.1. Repro”u“tive fa“tors

It has been shown repeate”ly that a woman's risk for breast “an“er is

asso“iate” with her lifetime exposure to estrogen34-36 whi“h is, in turn,

”etermine” by several en”ogenous an” exogenous variables. Repro”u“tive

history is among the most important ”eterminants of breast “an“er risk.

Roughly, all those fa“tors in“reasing the number of menstrual “y“les are

asso“iate” with an in“rease” likelihoo” for ”eveloping breast “an“er.

Ø Age at first live birth: in“reasing maternal age at first live birth is

asso“iate” with in“rease” risk for ”eveloping the ”isease. In women

having the first “hil” at 20, the risk of breast “an“er is about half that

of those having the first “hil” at 3037.

Ø Number of births: the risk of breast “an“er ”e“lines with the number of

“hil”ren borne. Women who have un”ergone 5 or more full-term

pregnan“ies have a 50% re”u“e” risk of ”eveloping the malignan“y

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INTRODUCTION

when “ompare” with those who have not given birth38.

Ø Duration of la“tation: breastfee”ing, regar”less of ”uration or timing,

has also been foun” to be prote“tive against ”eveloping breast “an“er39.

Ø Age at menar“he an” menopause: early menar“he an” late menopause

are known to in“rease women's risk of ”eveloping breast “an“er. The

risk in“reases by almost 3% for ea“h year ol”er at menopause40. Thus,

women who have attaine” menopause at 55 years rather than 45 years,

have approximately 30% higher risk. Breast “an“er risk also in“reases

by 5% for every year younger at menar“he31.

3.2.2. Other hormonal fa“tors

Ø Bo”y mass in”ex (BMI): overweight an” obesity have been relate” with

an in“rease” risk of postmenopausal breast “an“er41. When the ovaries

“ease to pro”u“e estrogen, a”ipose tissue be“omes the major sour“e of

estrogen in postmenopausal women. Although the relationship between

BMI an” breast “an“er is not “ompletely establishe”, it may result, at

least in part, from the in“rease” levels of estrogens in obese women42.

Ø Exogenous estrogens: the asso“iation of exogenous hormones,

primarily hormonal “ontra“eptives an” hormone repla“ement therapy

(HRT), with breast “an“er has been wi”ely stu”ie”. The ”iffi“ulty to

rea“h a “onsensus may be attribute” in part to the variability of the

exposures. Changes in patterns of use, re”u“tions in hormone ”ose an”

temporal “onsi”erations all “ontribute to the ”iffi“ulty to “ompare the

many stu”ies. Despite the “onfli“ting results obtaine”, a “ombine”

analysis of 54 stu”ies “on“lu”e” that “urrent use of oral “ontra“eptives

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INTRODUCTION

poses a slight (24%) in“rease in the risk, whi“h ”isappears 10 years

after the “essation of use43. The use of HRT as a risk fa“tor for

”eveloping breast “an“er has also been “ontroversial. Overall, some

strong evi”en“es suggest risk in“reases with long-term use44,45.

Ø An”rogens: Normal an” malignant mammary epithelial “ells also have

an”rogen re“eptors (AR), in”i“ating spe“ifi“ responsiveness to

an”rogens. Besi”es estrogens, elevate” en”ogenous an”rogens have

also long been impli“ate” as a potential risk fa“tors for breast “an“er46.

3.3. NON-HORMONAL RISK FACTORS

A number of non-hormonal risk fa“tors have been asso“iate” with the

”evelopment of breast “an“er; however, some of them may also be

in”ire“tly tie” to mo”ulation of estrogen exposure.

3.3.1. Age

One of the best-”o“umente” risk fa“tors for breast “an“er is age, whi“h is

“onsi”ere” to be a surrogate for DNA ”amage a““umulate” ”uring life. The

”isease is less “ommon in women younger than 30 years, after whi“h it

in“reases sharply until the age of 80. At that point the in“i”en“e rates

flatten out an” then start to ”e“line3.

3.3.2. Geographi“al variation an” ethni“ity

Age-a”juste” in“i”en“e an” mortality for breast “an“er vary by up to a

fa“tor of five between “ountries. The highest breast “an“er in“i”en“e rates

are observe” in high-in“ome nations, in“lu”ing “ountries in North-Ameri“a,

Australia an” Northern an” Western Europe. Conversely, the lowest

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INTRODUCTION

in“i”en“e rates are ”ete“te” in Mi””le an” Eastern Afri“a, Eastern an”

South-Central Asia an” Central Ameri“a47.

Data from SEER1 program of the NCI in”i“ate that white women have the

highest rate of getting breast “an“er, followe” by Afri“an-Ameri“an,

Asian/Pa“ifi“ Islan”er, Hispani“ an” Ameri“an In”ian/Alaska Native women.

However, ”ifferen“es in breast “an“er in“i”en“e rates between most

ra“ial/ethni“ groups “an be largely explaine” by ”ifferen“es in other risk

fa“tors48. Moreover, stu”ies show that migrants assume the rate in the host

“ountry within one or two generations, in”i“ating that environmental

fa“tors are of greater importan“e than geneti“ fa“tors49.

3.3.3. Ra”iation

Mammography has “onvin“ingly ”emonstrate” to result in a “lear re”u“tion

in ”eath from breast “an“er for women over the age of 50. However, some

stu”ies have ”emonstrate” that low-”ose ra”iation from mammographi“

equipment in”ee” in“reases breast “an“er risk, espe“ially in those high-risk

women50. Overall, it seems that the risk-benefit equation is “learly in favour

of su“h s“reening programs51.

3.3.4. Previous benign ”isease

Breast “an“er risk in women with severe atypi“al epithelial hyperplasia is 4

to 5 times higher than in women who ”o not present any benign proliferative

“hanges52. In fa“t, mammographi“ ”ensity, referring to the amount of

ra”iologi“ally ”ense breast-tissue appearing on a mammogram, represents

one of the most important risk fa“tors for breast “an“er53. However, if

breast ”ensity is an in”epen”ent risk fa“tor is still un”er ”ebate. Some

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INTRODUCTION

stu”ies note that it may serve as a marker for other fa“tors, su“h as the

history of estrogen exposure54.

3.3.5. Lifestyle an” ”ietary fa“tors

The International Agen“y for Resear“h on Can“er estimates that 25% of

breast “an“er “ases worl”wi”e are ”ue to overweight/obesity an” a

se”entary lifestyle55. Certain mo”ifiable lifestyle behaviours “an affe“t the

risk of the ”isease.

Ø Al“ohol “onsumption: taken as a whole, the stu”ies suggest that al“ohol

“onsumption at a level of 1 to 2 ”rinks per ”ay mo”estly in“reases

breast-“an“er risk56. The me“hanism un”erlying the “ar“inogeni“ effe“t

asso“iate” with al“ohol is not still un”erstoo”. However, some stu”ies

suggest that al“ohol “onsumption in“reases the exposure to estrogen,

lea”ing to an in”u“tion of ra”i“al oxygen spe“ies pro”u“tion57 an”

a””u“t formation58.

Ø Dietary fa“tors: it is well known that measurement of ”ietary intake is

inexa“t an” prone to mis“lassifi“ation. However, a large number of

fa“tors have been ”es“ribe” to in“rease (fat59, meat60) or re”u“e (fruits

an” vegetables40, soy protein61 an” vitamins A, C, E an” D62) breast

“an“er risk.

Ø Exer“ise: physi“al a“tivity has been propose” to have a benefi“ial effe“t

on breast “an“er risk63. Apparently, physi“al a“tivity “oul” exert its

influen“e on the ”isease through an estrogen-”epen”ent me“hanism,

by ”e“reasing the total number of ovulatory “y“les64.

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INTRODUCTION

4. ESTROGENS AND BREAST CANCER

Alrea”y over 100 years ago when Dr. George Beatson ”es“ribe” the

regression of metastati“ breast “an“er after ovarian ablation65 the

influen“e of hormonal fa“tors in breast “an“er was re“ognize”. Sin“e then, a

wealth of epi”emiologi“ an” experimental information has pointe” to the

involvement of estrogens, an” other repro”u“tive hormones, in human

breast “an“er46,66-68. Person-to-person ”ifferen“es in synthesis, regulation as

well as “onjugation pathways of sex steroi”s may ”efine subpopulations of

women with higher lifetime exposure to hormone-”epen”ent growth

promotion or to “ellular ”amage from parti“ular estrogens an”/or estrogen

metabolites. Su“h variation “oul” explain a portion of the “an“er

sus“eptibility asso“iate” with hormone exposure.

4.1. STEROID BIOSYNTHESIS

Steroi”ogenesis is the “omplex multi-enzyme pro“ess by whi“h “holesterol

is “onverte” to biologi“ally a“tive steroi” hormones. The greatest supply of

“holesterol to steroi”ogenesis “omes from plasma low-”ensity lipoproteins

”erive” from ”ietary “holesterol69.

4.1.1. Cholesterol transport

The first step in steroi”ogenesis takes pla“e within mito“hon”ria. However,

“holesterol nee”s to be transferre” from the outer mito“hon”rial

membrane to the inner membrane. The aqueous phase between these two

membranes “annot be “rosse” by the lipophili“ “holesterol by itself.

Although the me“hanisms by whi“h “holesterol is transporte” within the

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INTRODUCTION

mito“hon”ria are not “ompletely un”erstoo”, it is now known that the

pro“ess is primarily me”iate” by the a“tion of the steroi”ogeni“ a“ute

regulatory protein (StAR).

4.1.2. Cyto“hrome P450 an” HSDs

Biosynthesis of estrogens involves series of enzymati“ steps from

“holesterol to an”rogens an” estrogens. Most enzymes involve” in steroi”

biosynthesis are either “yto“hrome P450s (CYPs) or hy”roxysteroi”

”ehy”rogenases (HSDs).

Cyto“hrome P450 is a generi“ term for a superfamily of oxi”ative enzymes

“ontaining a single heme “ofa“tor70. The term P450 (pigment 450) is ”erive”

from their ability to absorb light at 450 nm in their re”u“e” states

“omplexe” with “arbon monoxi”e. Human CYPs are primarily membrane-

asso“iate” proteins either of the inner membrane of the mito“hon”ria

(type I) or of the en”oplasmi“ reti“ulum (type II). All P450 enzymes a“tivate

mole“ular oxygen using their heme “entre an” a”” ele“trons from the

re”u“e” form of ni“otinami”e a”enine ”inu“leoti”e phosphate (NADPH).

P450 enzymes are asso“iate” with the oxi”ative metabolism of a large

number an” variety of organi“ “ompoun”s, both en”ogenous an”

exogenous71. These rea“tions are me“hanisti“ally an” physiologi“ally

irreversible72. The human genome in“lu”es genes for 57 CYPs ”ivi”e” among

18 families73. Six CYP enzymes are involve” in steroi”ogenesis.

HSDs are enzymes that inter“onvert a“tive an” relatively ina“tive forms of

in”ivi”ual steroi” hormones using ni“otinami”e “ofa“tors NADP+/NADPH

an” NAD+/NADH74. Base” on their a“tivities, the HSDs “an be “lassifie” as

”ehy”rogenases or re”u“tases. These enzymes ten” to fun“tion in one

17

INTRODUCTION

”ire“tion, ”etermine” by the available “ofa“tors74. Unlike P450 enzymes,

ea“h of the rea“tions “atalyse” by HSDs “an be “on”u“te” by at least two

isoenzymes75.

4.1.3. Steroi”ogenesis

The “onversion of “holesterol to pregnenolone by the “holesterol si”e-“hain

“leavage enzyme (P450s““) is the initial step in the biosynthesis of sex-

steroi” hormones (Fig. 4). P450s““ is one of the few CYPs lo“alize” in the

mito“hon”ria an” “atalyses the “onversion in three monooxygenase

rea“tions, with the involving of two other proteins for the ele“tron transfer:

ferre”oxin an” ferre”oxin re”u“tase. Although this rea“tion is “onsi”ere”

the rate-limiting step of steroi”ogenesis, P450s““ is always a“tive. In”ee”,

its a“tivity is limite” by the true rate-limiting step of the pathway: the

supply of “holesterol in the inner membrane by StAR.

On“e pregnenolone has been synthetize” it “an leave the mito“hon”ria to

be “onverte” to progesterone, by 3 -hy”roxysteroi” ”ehy”rogenase

(3 HSD) in the mi“rosomal “ompartment76. An alternative option for

pregnenolone, is to un”ergo the a“tivities of 17 -hy”roxylase/17,20-lyase

(P450“17), thus being first hy”roxylate” to 17-OH-pregnenolone an” then

“onverte” to ”ehy”roepian”rosterone (DHEA).

It shoul” be note” that progesterone “an also be the substrate for

P450“1777, resulting in the pro”u“tion of 17-OH-progesterone77, the

pre“ursor of glu“o“orti“oi”s78. However, the 17,20-lyase a“tivity of P450“17

is not effi“ient for the “onversion 17-OH-progesterone to

an”rostene”ione77, so that un”er normal “ir“umstan“es, progesterone is not

an important pre“ursor for human sex steroi” synthesis.

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INTRODUCTION

Figure 4. Steroidogenic pathway. Arrows in bol” in”i“ate the “lassi“al synthesis

pathway. The existen“e of an alternative pathway (non-bol” arrows) to the a“tive

an”rogen ”ihy”rotestosterone (DHT), has been ”es“ribe”. This ba“k”oor pathway

o““urs in those steroi”ogeni“ tissues expressing both P450“17 an” the 5 -re”u“tase

enzymes (SRD5).

3 HSD, also a“ts upon DHEA to generate an”rostene”ione76. Both DHEA an”

an”rostene”ione are substan“es of weak an”rogeni“ a“tivity whi“h serve

pre”ominantly as a pre“ursors for more potent an”rogens su“h as

testosterone79. The inter“onversion of an”rostene”ione an” testosterone, as

well as multiple other rea“tions, are asso“iate” to the 17 -hy”roxysteroi”

”ehy”rogenases enzymes (17 HSDs). The spe“ifi“ step from an”rostene”ione

to testosterone is “arrie” out by 17 HSD380 an” 17 HSD581, while the opposite

pro“ess, that is, ina“tivation of testosterone, is mainly “arrie” out by

17 HSD282.

Finally, estrogens are pro”u“e” by the aromatization of an”rogens,

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INTRODUCTION

an”rostene”ione an” testosterone, by a “omplex series of rea“tions

“atalyse” by P450“19, also known as aromatase (Fig. 4).

Multiple estrogen metabolites have been i”entifie”. However, in general, only

4 estrogen hormones are “onsi”ere” of interest: estra”iol, estrone, estrone

sulphate an” estriol83, being the latter of the ex“lusively importan“e in

pregnant women. The inter“onversion of estra”iol an” estrone is also

possible with the intervention of 17 HSDs enzymes. Moreover, steroi”

sulphates, as DHEA-sulphate (DHEA-S) an” estrone-sulphate may be

forme” by sulfotransferase enzymes (SULT)84. Thus, while estrone an”

estrone-sulphate are ina“tive by itself, a substantial ”egree of estra”iol may

be synthesize” by re”u“tion of estrone85 (Fig. 4). However, the relative

“ontribution of this pathway to plasma an” tissue estra”iol has not been yet

”isentangle”.

4.2. STEROIDOGENIC TISSUES AND REGULATION

A tissue is sai” to be steroi”ogeni“ if it is able to “onvert “holesterol into

pregnenolone via P450s““. In women, “lassi“ steroi”ogeni“ tissues in“lu”e

the a”renal glan”s an” ovaries86.

The regulation of steroi”ogenesis “an be ”ivi”e” into 3 key events in the

steroi”ogeni“ pathway. Unlike other se“retory tissues, steroi”ogeni“ “ells

store very little amounts of steroi”s75. Thus, a rapi” steroi”ogeni“ response

is nee”e”. A“ute regulation is me”iate” by StAR protein, fa“ilitating the

rapi” influx of “holesterol into mito“hon”ria. Chroni“/quantitative

regulation is prin“ipally at the level of trans“ription of P450s““, whi“h is the

enzymati“ally rate-limiting step. Finally, qualitative regulation, ”etermining

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INTRODUCTION

the “lass of steroi” pro”u“e”, is prin“ipally ”etermine” by P450“17, the key

bran“h point in steroi” hormone synthesis, lea”ing to tissue-spe“ifi“ steroi”

synthesis ”epen”ing on the presen“e/absen“e of its a“tivities75.

Moreover, in ea“h steroi”ogeni“ “ell, the pattern of steroi” pro”u“ts

se“rete” is regulate” by ”ifferent hormonal tissue-spe“ifi“ systems. In fa“t,

”iagrams of steroi”ogeni“ pathway ”iffer in ea“h steroi”ogeni“ “ell type75.

Originally “onsi”ere” to be a major sour“e of “ir“ulating estrogens in

postmenopausal women, the a”renal “ortex is the main sour“e of “ir“ulating

an”rogens (Fig. 5).

Figure 5. Steroid synthesis in adrenal glands. The presen“e/absen“e of some

strategi“ enzymes ”is“usse” above, ”etermine the ability of the 3 ”ifferent a”renal

layers to synthesize steroi” pro”u“ts. The zona glomerulosa pro”u“es al”osterone

un”er regulation by the renin/angiotensin system87. The zona fas“i“ulata mainly

pro”u“es “ortisol an” “orti“osterone un”er the influen“e of the a”reno“orti“otropi“

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INTRODUCTION

hormone (ACTH)75. Finally, the a”renal zona reti“ularis express large amounts of

P450s““, so that DHEA is pro”u“e”, mu“h of whi“h is sulphate” to DHEAS by

SULT2A1. Moreover, small amounts of DHEA are “onverte” to an”rostene”ione, an”

very small amounts of this an”rostene”ione are “onverte” to testosterone75.

Extra“te” from Han et al. (Nature Reviews En”o“rinology, 2014)88.

In “ontrast to the a”renal glan”, the ovary “annot pro”u“e glu“o“orti“oi”s

or mineralo“orti“oi”s be“ause it la“ks the enzymes 21-hy”roxylase an” 11 -

hy”roxylase. In the ovary, the synthesis an” release of estrogens are

“entrally regulate” by the hypothalamus-pituitary-gona”al axis (Fig. 6).

Figure 6. Steroid synthesis by cooperation of cells in the ovarian follicle. Differential

regulation by luteinizing hormone (LH) an” folli“ular stimulating hormone (FSH) of

ovarian estrogen, progesterone an” an”rogen pro”u“tion. In general terms, LH

stimulates the expression of P450s““ in granulosa “ells, in”u“ing pregnenolone an”

also progesterone synthesis. Granulosa “ells ”o not express P450“17 an”

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INTRODUCTION

“onsequently, pregnenolone an” progesterone ”iffuse into a”ja“ent the“a “ells.

On“e there, they “an be “onverte” to an”rostene”ione by P450“17 an” 3 HSD2.

Small amounts of this an”rostene”ione are se“rete” or “onverte” to testosterone,

but most an”rostene”ione returns to the granulosa “ells where it is “onverte” to

estrone an” then to estra”iol, un”er the influen“e of FSH75. Extra“te” from

http://k“ampbell.bio.umb.e”u/MamTox/Presentations/Session3/ Session%203.html

Hypothalamus, se“retes gona”otrophin releasing hormone (GnRH), whi“h in

turn a“ts on pituitary to release LH an” FSH. These two hormones a“t on

gona”s to pro”u“e estrogen in a “y“li“ manner ”uring menstrual “y“le.

Steroi”ogenesis in the ovary is also “omplex be“ause the enzymati“ steps

are partitione” between the granulosa an” the“a “ells (Fig. 6).

Although ”e novo synthesis from “holesterol is restri“te” to a limite”

number of sites, estrogens “an also be synthesize” from their “ir“ulating

an”rogen pre“ursors in a number of extragona”al tissues expressing the

aromatase enzyme, su“h as the mesen“hymal “ells of a”ipose tissue

in“lu”ing that of the breast, osteoblasts an” “hon”ro“ytes of bone, the

vas“ular en”othelium an” aorti“ smooth mus“le “ells, an” numerous sites in

the brain89. The quantitative “ontribution of this synthesis to “ir“ulating

estrogen levels has not been elu“i”ate”.

4.3. STEROIDS IN POSTMENOPAUSAL WOMEN

Menopause is “hara“terize” by a marke” re”u“tion in the amount of

“ir“ulating estrogens. Thus, when the ovaries “ease fun“tioning,

aromatization of an”rogens by peripheral tissues be“omes the responsible

me“hanism for estrogen synthesis. Although the total amount of estrogen

synthesize” by these extragona”al sites may be small, the lo“al tissue

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INTRODUCTION

“on“entrations a“hieve” are probably high an” exert biologi“al influen“e

lo“ally. Un”er these “ir“umstan“es, estra”iol is not just an en”o“rine fa“tor

but a“ts as para“rine or even intra“rine fa“tor90, playing an important

physiologi“al role.

We have gaine” only a limite” un”erstan”ing of whi“h fa“tors ”etermine

plasma an” tissue estrogen “on“entrations in postmenopausal women.

Aromatase is the pro”u“t of CYP19A1, a gene spanning 10 exons. There are at

least 10 variants of exon I that “an be spli“e” into the 5 -untranslate” region

in a tissue-spe“ifi“ fashion91 (Fig. 7). Thus for example, the promoter from

exon I.4 is preferably utilize” for a”ipose tissue an” bone.

Figure 7. Partial structure of human aromatase gene. Human aromatase gene is

lo“ate” on “hromosome 15. The aromatase gene is ~123 kb long an” “ontains nine

“o”ing exons (II-X). Partially tissue spe“ifi“ promoters ”ire“t aromatase gene

trans“ription. Extra“te” from: Khan et al. (Repro”u“tive Biology an” En”o“rinology,

2011)92.

Moreover, a phenomenon of promoter swit“hing ”epen”ing upon ”ifferent

regulatory fa“tors, in“lu”ing hormones, “ytokines an” “ell ”ifferentiation

in”u“ers has been shown in many tissues.

Testosterone an” estra”iol “ir“ulate in the bloo”stream, boun” mostly to

the sex-hormone bin”ing globulin93, whi“h influen“e their bioavailability.

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INTRODUCTION

Therefore, only a very small fra“tion of about 1-2% is unboun”, or "free ,

an” thus biologi“ally a“tive94.

4.4. STEROID MECHANISMS OF BREAST CARCINOGENESIS

Several stu”ies have ”emonstrate” that estrogens are “ar“inogens in various

tissues, in“lu”ing the ki”neys, liver, uterus, an” mammary glan”s95,96. Two

”ifferent but “omplementary pathways are likely “ontributing to the

“ar“inogeni“ity of estrogen.

4.4.1. ER signalling.

Most of the a“tions of estrogens are me”iate” via intra“ellular ERs (Fig. 8).

Figure 8. Estrogen receptor-signalling Pathways associated with increased

proliferation and inhibition of apoptosis. In the “lassi“ me“hanism of ”ire“t a“tion,

estrogens bin” to ERs in the nu“leus, “ausing ”imerization with a“tivation of the

re“eptor trans“riptional ”omain97 an” the subsequent bin”ing to estrogen-response

elements98 of the target genes. The presen“e of ERs has also been “onfirme” in

other “ellular “ompartments, su“h as plasma membrane an” mito“hon”ria99,100.

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INTRODUCTION

Some evi”en“es in”i“ate that estrogens inhibit the early stages of apoptosis

through signalling pathways involving membrane an” mito“hon”rial ERs101. In

a””ition to the genomi“ a“tions of ER, plasma membrane-asso“iate” ERs “an

me”iate the a“tivation of multiple signalling “as“a”es as: phospholipase C/protein

kinase C102, Ras/Raf/MAPK103, phosphati”yl inositol 3 kinase/AKT104, an” “AMP/protein

kinase A105. These non-genomi“ effe“ts fa“ilitate “ross-talk between the membrane

ER-signalling pro“ess an” multiple signal-trans”u“tion pathways. Extra“te” from:

Yager et al. (New Englan” Journal of Me”i“ine, 2006)106.

Two ”istin“t types of signalling “an be me”iate”, often referre” to as

genomi“ an” non-genomi“ pathways. In either “ase, the final result is the

alteration of gene expression emphasizing the effe“ts of in“rease”

proliferation an” inhibition of apoptosis14,107.

4.4.2. Pro”u“ts of estrogen metabolism

ER-me”iate” pro“esses are “onsi”ere” epigeneti“ “ar“inogens: they ”o not

play the “riti“al role in “an“er initiation be“ause the hypotheti“al mutations

obtaine” are ran”om, but instea”, they stimulate abnormal “ell proliferation,

a pro“ess that “an lea” to “ar“inogenesis. However, the ”is“overy that

spe“ifi“ oxi”ative metabolites of estrogens “an rea“t with DNA, supports the

hypothesis that estrogens “an be“ome en”ogenous “ar“inogens by

generating the mutations lea”ing to abnormal proliferations an”, therefore,

to the initiation of “an“er108.

Estrogen metabolism in humans “omprises two phases. Phase I, in whi“h

estrogen an” estrone are oxi”ize” by several CYP enzymes an” Phase II, the

”etoxifi“ation pathways in“lu”ing sulfation, methylation an” rea“tion with

glutathione. Un”er normal “on”itions, these pro“esses are “hara“terize” by

homeostasis, a balan“e” set of a“tivating an” prote“tive enzymes in whi“h

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INTRODUCTION

the “ar“inogeni“ metabolites of estrogens are not available to rea“t with

DNA. However, a variety of en”ogenous an” exogenous fa“tors “an ”isrupt

estrogen homeostasis. The elevation of estrogens by overexpression of

CYP19A1109, an in“rease in 4-OH pathway by overexpression of CYP1B1110 as

well as low levels of COMT a“tivity111 “oul” behin” this ”ysregulation.

27

INTRODUCTION

5. BREAST CANCER THERAPIES

Over the past two ”e“a”es, breast “an“er treatment has evolve” to a more

effe“tive an” targete” ”ire“te” approa“h, “ompletely transforming the

”isease. For many years, the me”i“al treatment of breast “an“er was reliant

solely on “ytotoxi“ “hemotherapy. Currently, a”van“es in un”erstan”ing

tumour biology have le” to the ”evelopment an” approval of many novel

agents that have “hange” the lan”s“ape of therapy. This tren” is expe“te”

to “ontinue, sin“e agents that use novel approa“hes are “onstantly being

teste”. Patient out“omes will improve along with the a”vent of personalize”

me”i“ine.

Breast “an“er treatments “an be “lassifie” ”epen”ing on when the

treatment is a”ministere” as either neoa”juvant (pre-surgery) or a”juvant

(post-surgery). The neoa”juvant approa“h to breast “an“er is use” in the

management of patients with high-risk breast “an“ers, large tumours an”

for lo“ally a”van“e” ”isease. Su“h treatment offers several “lini“al

a”vantages. First, it is able to re”u“e the size of primary tumour in or”er to

in“rease the likelihoo” of breast “onservation rather than maste“tomy112.

Se“on”, neoa”juvant therapy allows for the in vivo assessment of tumour

response to the treatment, thus saving patient exposure to potentially toxi“

therapy. In fa“t, more re“ently, the neoa”juvant strategy has be“ome

re“ognize” as an in vivo potential platform to explore the effi“a“y of new

therapeuti“ agents113.

On the other han”, the goal of a”juvant systemi“ therapy is era”i“ating

mi“rometastasis, that is, “lini“ally o““ult tumours that are present after

28

INTRODUCTION

surgery, with a potential to metastasize. Nearly 30% of women with “an“er

“onfine” to the breast an” 75% of women with no”al involvement will

ultimately relapse114.

5.1. SURGERY

In early breast “an“er, surgery “an remove any ”isease that has been

”ete“te” in or aroun” the breast or regional lymph no”es. In breast

“onserving surgery, only the part of the breast “ontaining the tumour is

remove”. By “ontrast, ”uring maste“tomy the entire breast is remove”.

Lymph no”es “an also be remove” either on the same surgery or as a

separate operation.

5.2. RADIOTHERAPY

Ra”iotherapy is a highly targete” an” effe“tive treatment base” on high

”oses of ionizing ra”iation. After surgery, ra”iotherapy is the most effe“tive

“urative treatment for “an“er. Given as a”juvant treatment after breast

“onservative surgery or even after maste“tomy, ra”iotherapy “an pro”u“e a

substantial re”u“tion in the risk of re“urren“e113,115.

5.3. CHEMOTHERAPY

Chemotherapy is a systemi“ treatment that uses alone or “ombine”

“ytotoxi“ “hemi“al substan“es in or”er to prevent “ell ”ivision an” therefore

slowing tumour growth. It “an be a”ministere” orally or intravenously.

Chemotherapy is usually use” as an a”juvant treatment after surgery, but it

also “an be use” before surgery. Chemotherapy has ma”e signifi“ant

progress with several lan”mark stu”ies i”entifying “lear survival benefits116.

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INTRODUCTION

5.4. BIOLOGICAL THERAPY

Biologi“al therapy is the most re“ently ”evelope” treatment strategy,

subsequent to new knowle”ge regar”ing signalling trans”u“tion pathways in

breast “an“er. Some of the most important spe“ifi“-targete” ”rugs for

treating breast “an“er are mono“lonal antibo”ies or tyrosine kinase

inhibitors. The mono“lonal antibo”y trastuzumab targets the extra“ellular

”omain of the HER2 protein, blo“king its ”imerization an” with it the

”ownstream signalling pathways that lea” to “ell growth, survival an” “ell

”ifferentiation117. The ”rug is now regar”e” as one option for stan”ar”

therapy in HER2-overexpressing metastati“ breast “an“ers. Data from

several stu”ies has ”emonstrate” that trastuzumab has “ontribute” to

re”u“e the rates of breast “an“er mortality an” re“urren“e118,119.

5.5. ENDOCRINE THERAPY

The lan”s“ape of breast “an“er “hange” ”ramati“ally with the intro”u“tion

of en”o“rine/hormonal therapies. As the growth of “ertain breast “an“ers

”epen”s on estrogen, it might, therefore, be expe“te” that if the sour“e of

estrogen is remove” or if estrogen is prevente” from bin”ing to its

re“eptors, tumour growth “oul” potentially be prevente”. Thus, en”o“rine

therapy has be“ome a pivotal treatment for women with ER-positive

tumours. Multiple forms of hormone therapy “urrently exist.

5.5.1. Surgi“al Oophore“tomy

Surgi“al oophore“tomy in other wor”s, surgi“al removal of ovaries is

the ol”est form of hormone therapy65. It “auses an imme”iate an”

permanent ”e“rease in estrogen levels. Several stu”ies have shown the

30

INTRODUCTION

benefits of oophore“tomy in terms of ”isease-free survival120. However, the

pro“e”ure has many ”isa”vantages, in“lu”ing the morbi”ity an” mortality121

as well as its irreversibility.

5.5.2. LH-RH (Luteinizing Hormone-Releasing Hormone) Analogues

As with surgi“al oophore“tomy, the use of LH-RH analogues (e.g. goserelin,

leuproli”e, an” buserelin) is “onfine” to pre- or perimenopausal women.

These “ompoun”s a“t on the hypothalami“-pituitary axis, ”e“reasing LH an”

suppressing ovarian fun“tion an” therefore estrogen levels122. However, in

“ontrast to ovarian ablation, this ”e“rease is potentially reversible an”

normal ovarian fun“tion may return when treatment is stoppe”. These

“ompoun”s have also ”emonstrate” to improve long-term survival in breast

“an“er patients123.

5.5.3. Sele“tive estrogen re“eptor mo”ulators (SERMs) an”

”ownregulators (SERDs)

SERMs (also known as anti-estrogens) are a unique “lass of therapeuti“

agents that a“t as “ompetitive inhibitors of estrogen bin”ing to ERs. Upon

bin”ing to hormone, the ligan” bin”ing ”omain (LBD) of the ER un”ergoes a

“onformational “hange, in or”er to fa“ilitate the union of “ofa“tors (“o-

a“tivators or “o-repressors) require” for ER-me”iate” gene regulation.

These “ofa“tors are exquisitely sensitive to LBD “hanges124. SERMs generate

an abnormal re“eptor “onformation, ”isrupting “o-a“tivator bin”ing to the

LBD125. Subsequently “o-repressor mole“ules are re“ruite” to the ER, hol”ing

it in an ina“tive state126.

However, an in”ivi”ual SERM “an behave as an ER agonist in one tissue an”

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INTRODUCTION

as an antagonist in another, generating a “omplex array of tissue-spe“ifi“

effe“ts. Not only that, but they “an have estrogeni“ effe“ts on “ertain

genes, even in tissues in whi“h its pre”ominant a“tivity is anti-estrogeni“.

Although the exa“t me“hanism behin” the mixe” effe“ts of SERMs is not yet

fully un”erstoo”, the variable intera“tion with “ofa“tors is thought to be

involve”. For this reason, they are terme” sele“tive estrogen re“eptor

mo”ulators.

Sin“e its approval by the Foo” an” Drug A”ministration in 1977127, the SERM

tamoxifen has be“ome the most wi”ely en”o“rine treatment for breast

“an“er. Tamoxifen inhibits the expression of estrogen-regulate” genes

involve” in stimulating breast “an“er “ell growth an” progression, in“lu”ing

growth an” angiogeni“ fa“tors se“rete” by the tumour128. The net result is

the tumour regression by a blo“k of the “ell “y“le in the G1 phase129 an”,

perhaps, a slightly in“rease” rate of apoptosis130. Tamoxifen has been

establishe” as an effe“tive therapy for patients with all stages of hormone

re“eptor-positive breast “an“er128, a“hieving re”u“tions in breast “an“er

re“urren“e an” “ontralateral breast “an“er of 40-50%131. Furthermore, it “an

be use” as a breast “an“er preventive, with notable ”e“reases between 16%

an” 70% in breast “an“er in“i”en“e132,133.

An“illary benefits “an also be ”erive” from the partial agonist properties of

tamoxifen. Among the most remarkable ones are the prote“tion against

menopausal bone loss134 an” “ar”iovas“ular ”isease, although the ”ata for

the latter are still “ontroversial135,136. However, this partial agonist a“tivity is

not release” from serious a”verse effe“ts, as for example its “ar“inogeni“

potential. An alarming “ase, is the en”ometrial “an“er, for whi“h in“reases

similar to those reporte” in estrogen repla“ement therapy have been

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INTRODUCTION

observe”137.

Fulvestrant is the only SERD “lini“ally available an” it is “urrently approve” in

the Unite” States for patients with metastati“ breast “an“er whose ”isease

has progresse” on antiestrogen therapy. Fulvestrant bin”s to the ER with

greater affinity than tamoxifen, resulting in marke” ”ownregulation of both

the ER an” PR, not only on breast tissue but also on the en”ometrium an”

bone. Fulvestrant must be given in intramus“ular inje“tions, whi“h may limit

its usage.

5.5.4. Aromatase Inhibitors (AI)

Tamoxifen provi”e” the mainstay of en”o“rine therapy for many years.

However, the ”evelopment of AI has brought an alternative strategy for

first-line managing hormone-positive breast “an“er in postmenopausal

women. Given that the main sour“e of estrogen pro”u“tion in

postmenopausal women “omes from the peripheral “onversion by

aromatase, inhibition of this parti“ular enzyme results in the signifi“ant

further re”u“tion of estrogen. AI a”ministration triggers a “ompensatory

response, with ”ramati“ in“reases in gona”otropin se“retion. In

premenopausal women (still having large amount of aromatase substrate

present in the ovary) gona”otropins stimulate ovarian folli“les138, making AI

less effe“tive in inhibiting ovarian estrogen pro”u“tion. Thus, in

premenopausal women, AI use is restri“te” to spe“ial “ir“umstan“es, su“h as

prior tamoxifen failure. When this is the “ase, these agents must be use” in

“ombination with surgi“al or me”i“al ovarian ablation139.

Nowa”ays, a “onsi”erable number of AI exist, representing several

generations of evolution, ea“h of them a“hieving in“rease” spe“ifi“ity an”

33

INTRODUCTION

greater poten“y. The first AI to be evaluate”, su“h as testola“tone, were

steroi”al inhibitors, analogues of the natural substrate an”rostene”ione140.

They “ompete with the natural substrate for the bin”ing site an” therefore,

high “on“entrations of ”rug were require” to maintain the inhibition.

Moreover, an”rogeni“ effe“ts were usually ”erive” from their steroi”al

nature.

The starting point in the ”evelopment of “ompetitive nonsteroi”al AI was

aminoglutethimi”e, an inhibitor of several steroi”ogeni“ CYPs, in“lu”ing

aromatase. The ”rug ”emonstrate” “lini“al effi“a“y for the treatment of

postmenopausal breast “an“er141. However, the “on“omitant inhibition of

other steroi”ogeni“ enzymes implie” the nee” of “orti“osteroi” substitution.

The se“on”-generation AI in“lu”e formestane an” fa”rozole whi“h were

asso“iate” with fewer si”e effe“ts “ompare” with stan”ar” treatment

regimens at that time. However, their anti-tumour effe“ts were not superior

than those of aminoglutethimi”e or tamoxifen142. Moreover, formestane has

the ”isa”vantage of requiring intramus“ular inje“tion, an” fa”rozole also

“auses al”osterone suppression. None of these “ompoun”s are in “lini“al use

any longer.

Thir” generation AI, with superior toxi“ity profile an” “onvenien“e of

a”ministration (ex“ellent oral bioavailability in on“e a ”ay ”osing), have

superse”e” first an” se“on” generation “ompoun”s in the treatment of

breast “an“er. This group of agents “luster type I an” type II AI. Type I

inhibitors of thir” generation are steroi”al analogues of an”rostene”ione,

but unlike testolola“tone, they bin” irreversibly to aromatase be“ause of its

“onversion by the enzyme to rea“tive alkylating spe“ies143. This pro“ess

34

INTRODUCTION

generates long-term an” spe“ifi“ effe“ts, in”epen”ent of “ontinue”

presen“e of the ”rug, so the ”uration of inhibitory effe“ts is primarily

”epen”ent on the rate of ”e novo synthesis of aromatase. These types of

inhibitors in“lu”e exemestane. Type II Inhibitors, by “ontrast, are non-

steroi”al an” bin” reversibly to the aromatase enzyme. They prevent the

union of an”rogens by saturating the bin”ing site. This parti“ular subtype

“urrently in“lu”es anastrozole an” letrozole144. All of these thir”-generation

AI inhibit aromatase a“tivity by more than 98% an” are more potent than

earlier ”rug entities145,146.

As other en”o“rine treatments, AI “an be applie” as a preventive,

neoa”juvant or a”juvant manner. The fin”ings from major stu”ies

evaluating thir”-generation AI, “an be summarize” in two treatment

approa“hes. The first is to “ompare AI with tamoxifen monotherapies an”

the se“on” is to evaluate the sequential treatment, in whi“h 2-3 years of

tamoxifen are followe” by an AI. The rationale behin” the sequential

treatment was to in“rease the effi“a“y of the two agents, while re”u“ing

toxi“ities an” preventing a“quire” resistan“e. A re“ent meta-analysis

“ombining 9 trials ran”omizing patients between AI an” tamoxifen foun”

re”u“e” re“urren“e rates with AI “ompare” with tamoxifen147. Moreover, the

longer follow-up of this meta-analysis allowe” establishing that both breast

“an“er mortality an” all-“ause mortality are also re”u“e”. It has been

inferre” from the results that 5 years of AI “ompare” with no en”o“rine

therapy, woul” re”u“e breast “an“er re“urren“e by about 2/3 ”uring

treatment an” by about 1/3 ”uring years 5 9, an” woul” re”u“e breast

“an“er mortality rate by aroun” 40% throughout the first ”e“a”e, an”

perhaps beyon”147. It has to be emphasize” that the most signifi“ant

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INTRODUCTION

re“urren“e re”u“tions were observe” in the sequential treatments,

reinfor“ing the hypothesis that AI a“quire greater superiority over

tamoxifen after previous exposure to tamoxifen147.

Therefore, at this time, the panel believes that optimal a”juvant hormonal

therapy for a postmenopausal woman with RE+ breast “an“er shoul”

in“lu”e AI either as initial therapy or after treatment with tamoxifen.

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INTRODUCTION

6. SECONDARY EFFECTS OF AI THERAPY

Estrogen a“tions are require” for normal anatomi“al an” physiologi“al

”evelopment as well as for the maintenan“e of health in both males an”

females, so mu“h so that, aromatase ”efi“ien“y was “onsi”ere”

in“ompatible with life for many years. This ”ogma “hange” with the

”es“ription in the worl” literature of several “ases of aromatase ”efi“ien“y

in humans148. Clini“al “ases in whi“h aromatase is ina“tivate” be“ause of

germline mutations148 as well as the generation of aromatase kno“k-out

animals149 have highlighte” the important role of estrogens.

AI are generally safe ”rugs an” are reasonably well tolerate” by most

patients. They have been stu”ie” in very large a”juvant trials with “areful

”o“umentation of both toxi“ity an” patient “omplian“e. However, the

emerging fiel” of breast “an“er survivorship -estimate” at slightly 3.2

million women in most ”evelope” “ountries in 2012150- an” “lini“al experien“e

is suggesting some a”verse effe“ts that negatively impa“t quality of life an”

even persisten“e with therapy151. In fa“t, it has been postulate” that the

relative toxi“ity of AI versus tamoxifen152,153 may explain the ”iffi“ulties of

fin”ing overall survival benefit of AI vs. tamoxifen in postmenopausal breast

“an“er patients154.

6.1. CARDIOVASCULAR RISK AND LIPID METABOLISM

The simple observation of the ”ifferential ”istribution of bo”y fat between

pre- an” postmenopausal women gives an i”ea of the integral role of

estrogens in lipi” homeostasis an” a”ipose tissue ”istribution155,156. Estrogens

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INTRODUCTION

have been postulate” to in”u“e “ar”ioprote“tive effe“ts, not only via

re“eptors, but also influen“ing nitri”e oxi”e generation an” bioavailability157.

It is har”ly surprising, therefore, that AI have been asso“iate” with

unfavourable lipi” profile in some stu”ies154,158. The real “on“erns about this

issue, fall on the asso“iation of serum lipi”s with “ar”iovas“ular ”iseases159. A

number of meta-analysis147,154,160-162 integrating ”ata from 11 ”ifferent

ran”omize” “ontrol trials (RCT) have foun” that the use of AI is asso“iate”

with an in“rease” risk of “oronary heart ”isease in “omparison with

tamoxifen. However, these stu”ies are often subje“t to multiple

“onfoun”ing variables an” the results are “ontroversial. It is not “lear yet

whether “hanges in serum lipi”s an” “ar”ia“ events are the result of

tamoxifen with”rawal rather than a ”ire“t effe“t of AI-therapy163, sin“e

tamoxifen may re”u“e the risk of “ar”iovas“ular ”isease164. This notion has

yet to be fully elu“i”ate”.

6.2. MUSCULOESKELETAL EFFECTS

A major “on“ern with AI therapy is relate” to the mus“uloskeletal pain.

Numerous patients on AI “omplain of severe mus“uloskeletal pain an” joint

stiffness. These symptoms have been observe” at approximately 2 months

after treatment starting an” to peak at aroun” the 6-months, but they “an

also appear up to 2 years after initiation of therapy165. Generally,

symmetri“al joint pain most “ommonly affe“ting the wrists, han”s an”

knees as well as “arpal tunnel syn”rome an” trigger finger are “ommon

“omplaints asso“iate” with these agents. Other symptoms may in“lu”e

morning stiffness, myalgia an” ”e“rease” grip strength. Dis“omfort may be

most noti“eable on awakening an” often improve with morning a“tivities.

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INTRODUCTION

Patients frequently mention that they feel they have age” abruptly.

Whatever mus“uloskeletal symptoms are, they have on average a mo”est

impa“t on ”ay-to-”ay fun“tion an” a“tivity6-8.

Unfortunately, the absen“e of uniform an” “lear ”efinition of the problem

makes us to use the term arthralgia (”efine” as joint pain) to mean a

range of symptoms that are probably broa”er than arthralgia alone. It is

pre“isely this la“k of a “onsistent ”es“ription whi“h ren”ers impossible

having an a““urate assessment of the in“i”en“e of AI-relate” arthralgia

(AIA). Thus, various RCTs have ”es“ribe” a wi”e range of AIA in“i”en“e, likely

be“ause they are ea“h ”efining AIA ”ifferently. Moreover, the majority of

the stu”ies reporting ”ata on AIA were not “reate” primarily for this

purpose. Hen“e, AIA in“i”en“e ranges from 5% of the Intergroup Exemestane

Stu”y an” 35% of the Arimi”ex, Tamoxifen, Alone or in Combination (ATAC)

trial. The single stu”y spe“ifi“ally ”esigne” to assess the prevalen“e of these

symptoms, announ“e as mu“h as 47% of women ”eveloping AI-relate” joint

pain166. Thus, mus“uloskeletal toxi“ities be“ome the most “ommon si”e

effe“t asso“iate” with AI-therapy.

6.2.1. Etiology of AIA

To ”ate, the etiology of mus“uloskeletal pain un”er AI has yet to be ”efine”.

Despite many fa“tors su“h as obesity, previous “hemotherapy or HRT as

well as the exa“erbation of pre-existing myalgia an” arthralgia may explain

the severity of these symptoms167,168, estrogen ”eprivation is inevitably

involve”. Ample physiologi“al an” pharma“ologi“al evi”en“e len” mu“h

weight to this hypothesis. It is well known that the prevalen“e of joint an”

wi”esprea” pain in“reases progressively with age in women, rea“hing a

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INTRODUCTION

maximum in the group of 50 59-years169,170. Arthralgia following

menopause has been informe” in several stu”ies, being reporte” by more

than half of the women171. The majority of stu”ies ”emonstrate that HRT

alleviates these symptoms, although the size of the effe“t seems to be

small. Moreover, HRT with”rawal in“reases the in“i”en“e of pain an”

stiffness172. Nonetheless, the question of the pathogenesis an” anatomi“

features of AI-in”u“e” arthralgia remains to be solve”.

Pain “an emanate from a variety of arti“ular stru“tures innervate” with

no“i“eptive fibbers, in“lu”ing the joint “apsule, synovium, periosteal bone,

ligaments an” even, periarti“ular stru“tures. ERs have been foun” in human

synovia an” “artilage173,174 an” animal stu”ies have shown that ovarie“tomy

a““elerates “artilage turnover, presumably ”ue to the low-estrogen state175.

Perhaps, this higher rate of “artilage turnover “ontributes to bone pain from

la“k of “ushioning in the joints. Some stu”ies have also i”entifie”

ra”iologi“al features, su“h as tenosynovial “hanges an” joint effusions

asso“iate” with AI-therapy176,177. These ra”iologi“ evi”en“es may have an

inflammatory nature. The effe“ts on inflammation within the joint are not

well known but it is alrea”y ”o“umente” that higher levels of estrogen

suppress inflammatory “ytokine pro”u“tion, an” lower estrogen levels

in“rease their pro”u“tion. Thus, for example, women who are beginning the

menopause have higher levels of inflammatory “ytokines su“h as interleukin

(IL)-1 an” tumour ne“rosis fa“tor- (TNF- ). These “ytokines may

“ontribute to both the postmenopausal syn”rome of arthralgia an” to AIA.

The “onversion of an”rogens into estrogens in synovial “ells, by aromatase,

is a““ompanie” by IL-6 re”u“tion178. Thus, AI are thought to in“rease IL-6,

an” subsequently the pro-inflammatory response. Even more relevant may

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INTRODUCTION

be the antino“i“eptive effe“ts of estrogen. Estrogen has ”ire“t effe“ts on

opioi” pain fibbers in the spinal “or” an” brain, whi“h have been foun” to

express ERs179. In some spe“ies, aromatase has been foun” in ”orsal horn

“ells180, in whi“h the “onversion of an”rogens “oul” provi”e a sour“e of

estrogen in the spinal “or”. During arthralgia, no“i“eptive neurons a“quire a

heightene” sensitivity either at the joint itself or “entrally181. Consequently,

the pro“essing of no“i“eptive input from the joint is amplifie”, lea”ing to

enhan“e” responses to inno“uous stimuli an” a perpetuation of the feeling

of pain. In a””ition, inflammatory episo”es in”u“e” by estrogen ”epletion

“oul” a““ount for enhan“e” no“i“eption182. Re“ently, the transient re“eptor

potential ankyrin 1 “hannel, a polymo”al sensor, has been postulate” to be a

me”iator of the proinflammatory/proalgesi“ a“tions of AI 183.

6.2.2. AIA signifi“an“e: a”heren“e, treatment effi“a“y an” mortality.

La“k of a”heren“e to pres“ribe” me”i“ations is a well-known problem in the

me”i“al literature184. Many patients fail to fill the initial pres“ription (non-

initiation), to take the ”rug on a ”aily basis as pres“ribe” (nona”heren“e), or

to “ontinue long-term with the ”rug (early ”is“ontinuation). Su“h

”epartures from optimal ”rug use, frequently result in treatment failure184.

Several stu”ies have foun” superior non-a”heren“e an” ”is“ontinuation

rates for AI than those for tamoxifen185. It has been estimate” that more

than 25% of patients ”o not a”here to pres“ribe” therapy with AI185-189.

Evaluation of both non-a”heren“e an” ”is“ontinuation showe” that only

50% of patients on AI took a”juvant hormonal therapy for the full ”uration

at the optimal s“he”ule188.

Although a number of risk fa“tors have been asso“iate” with non-

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INTRODUCTION

a”heren“e, su“h as “omorbi”ity, age, prior treatments, pre-existing pain,

al“ohol “onsumption, smoking an” “an“er at a non-“urable stage151,187,188,190,

none is an absolute pre”i“tor. On the other han”, AI-asso“iate” toxi“ities are

a major barrier to the full appli“ation of effe“tive treatment. Spe“ifi“ally,

joint pain is so troubling that it is a“tually responsible for an important part

of with”rawals191.

AIA “arries further signifi“an“e beyon” quality of life an” “omplian“e issues

for breast “an“er survivors; It may also be tie” to re“urren“e risk: women

who ”evelope” arthralgia ”uring AI therapy a“tually ha” a lower risk of

breast “an“er re“urren“e an” survival192-194. One “oul” argue that the

appearan“e of symptoms “oul” lea” to lower a”heren“e an” therefore lower

subsequent effi“a“y195,196. However, these symptoms are believe” to be

relate” to lowere” estrogen “on“entrations: that is, women with arthralgia

“oul” simply have more effe“tive estrogen ”epletion on AI, lea”ing to a lower

risk of re“urren“e. Or, perhaps the arthralgia is me”iate” by a totally

”ifferent me“hanism, whi“h may also have antitumor properties.

6.2.3. AIA management

The “urrent management of AIA shoul” involve patient e”u“ation before

beginning AI therapy that joint pain is a very “ommon si”e-effe“t197. The

most effe“tive management option is AI ”is“ontinuation with prompt

resolution of symptoms. Although there are still no “lear evi”en“es to state

that swit“hing to another AI “an be benefi“ial, one trial showe” that women

who ”i” not tolerate one AI be“ause of arthralgia were able to tolerate

another AI instea”198. Similarly, swit“hing patients to tamoxifen may also

provi”e signifi“ant benefit199. Therapeuti“ options in“lu”e the use of non-

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INTRODUCTION

steroi”al anti-inflammatory ”rugs, “y“lo-oxygenase-2 inhibitors,

anti”epressants an” gabapentin, ea“h asso“iate” with their own unwante”

effe“ts. Some patients may even require surgery200. However, a “ombination

of lifestyle “hanges, su“h as intro”u“ing weight-bearing exer“ise an” yoga,

a“upun“ture, abstaining from smoking an” being mo”erate in al“ohol

“onsumption shoul” also be re“ommen”e”166,201,202.

In any “ase, it is “lear that mus“uloskeletal ”is“omfort in breast “an“er

patients treate” with AI have been un”erestimate” an” that these

symptoms “an be severe, ”ebilitating, an” “an limit “omplian“e.

6.3. OSTEOPOROSIS AND RISK OF FRACTURE

The wor” of osteoporosis literally means porous bone (Fig. 9). Osteoporosis

is a bone ”isease “hara“terize” by a ”e“rease in bone mass an”

mi“roar“hite“tural alterations whi“h results in bone fragility an” in“rease”

risk of fra“ture203.

Figure 9. Electron microscope images of normal (A) and osteoporotic bone (B). (A)

Normal bone ar“hite“ture: strong, inter“onne“te” plates of bone are visible. (B)

Osteoporoti“ ar“hite“ture: the bone is heavily ero”e” in pla“es by the a“tion of

43

INTRODUCTION

osteo“lasts an” “onsists mainly of thin, fragile struts, weakening the me“hani“al

properties of the bone. A”apte” from: Boy”e et al. (En”o“rine, 2002)204.

Bone mass, bone ”ensity or bone mineral ”ensity (BMD) refers to the

amount of mineral in bone tissue. Clini“ally it is measure” by proxy,

a““or”ing to opti“al ”ensity per square “entimetre of bone surfa“e,

”etermine” by ”ual-energy X-ray absorptiometry (DXA)205. The T-s“ore is

the primary out“ome of a ”ensitometry, in”i“ating the number of stan”ar”

”eviations above or below the BMD mean of young healthy a”ults. It

“lassifies patients into three ”iagnosti“ “ategories: normal BMD, low bone

mass (osteopenia), or osteoporosis. A““or”ing to the Worl” Health

Organization, osteoporosis is ”efine” as a T-s“ore below -2.5.

It is “al“ulate” that osteoporosis affe“ts 200 million women worl”wi”e.

However, the publi“ health an” “lini“al importan“e of osteoporosis lies in the

fra“tures asso“iate” with the ”isease. Osteoporoti“ fra“tures (fragility

fra“tures, low-trauma fra“tures) are those o““urring from me“hani“al

for“es, without major trauma. Typi“al fra“tures in patients with

osteoporosis in“lu”e vertebral (spine), proximal femur (hip), wrist an”

proximal humerus206. Osteoporosis is estimate” to “ause more than 8.9

million fra“tures annually, resulting in an osteoporoti“ fra“ture every 3

se“on”s. The lifetime of patients with osteoporoti“ fra“ture ”e“reases

“onsi”erably, potentially rising 40% for white women207. Furthermore, 50%

of women with osteoporoti“ hip fra“tures ”evelop ”isability, with signifi“ant

impa“t on the “apa“ity to live in”epen”ently an”, in most “ases,

institutionalization. This morbi”ity bur”en has “onsi”erable me”i“al, so“ial

an” finan“ial impli“ations.

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INTRODUCTION

Osteoporosis may be either a primary or a se“on”ary form. Primary

osteoporosis is the more “ommon form an” it is asso“iate” with the pro“ess

of normal aging. The skeleton a“quires the maximal bone ”ensity ( peak

bone mass ) at 25-30 years. Thereafter at about 30 years, a negative bone

balan“e sets in, so that on average 1% of bone is lost every year. Moreover,

the rate of bone loss a““elerates ”uring postmenopausal perio” ”ue to the

steroi” ”e“lining. Several risk fa“tors have been asso“iate” to primary

osteoporosis, notably among them; a”van“ing age, low BMI, family history

of osteoporoti“ fra“tures, early menopause, se”entary lifestyle, ex“essive

al“ohol, low “al“ium an”/or vitamin D intake as well as ina”equate sun

exposure.

On the other han”, se“on”ary osteoporosis o““urs as a result of “ertain

me”i“al “on”itions, su“h as en”o“rine an” metabolism ”isor”ers (e.g.,

hypogona”ism, hyper“ortisolism, hyperparathyroi”ism, hyperthyroi”ism,

anorexia), lymphoproliferative ”isor”ers, intestinal malabsorption

“on”itions, rheumatoi” arthritis, renal failure an” “ollagenopathies. Certain

”rugs “an also “ontribute to the ”evelopment of se“on”ary osteoporosis,

su“h as “orti“osteroi”s, sele“tive serotonin reuptake inhibitors,

anti“oagulants, anti”iabeti“ me”i“ations an”, in this parti“ular “ase, AI.

6.3.1. Bone stru“ture

Bone is a porous mineralize” stru“ture ma”e up of “ells, vessels, an” “rystals

of “al“ium “ompoun”s. It provi”es support an” prote“tion of vital internal

organs an” bone marrow as well as the mus“le atta“hment for lo“omotion.

Bone also plays a “entral role by “ontributing to life-supporting metaboli“

ex“hange, serving as a reserve of “al“ium an” phosphate nee”e” for the

45

INTRODUCTION

maintenan“e of serum homeostasis.

The stru“tural “omponents of bone “onsist of extra“ellular matrix an” “ells.

Two types of bone are observe” in the normal, mature human skeleton:

“orti“al an” trabe“ular203. Although ma“ros“opi“ally an” mi“ros“opi“ally

”ifferent (Fig. 10), the two forms are i”enti“al in their “hemi“al “omposition.

Figure 10. Anatomy of cortical and trabecular bone. Extra“te” from:

http://“lassroom.s”mesa.e”u/es“hmi”/Chapter6-–oo145.htm

Corti“al bone, whi“h “omprises 80% of the total bone mass of an a”ult

skeleton, “onstitutes the outer layer of all skeletal stru“tures. The major

part of the “orti“al bone is “al“ifie” an” has a slow turnover rate. It is ”ense

an” “ompa“t, thus having a high resistan“e to ben”ing an” torsion. Its

fun“tion is to provi”e me“hani“al strength an” prote“tion. It “an also

parti“ipate in metaboli“ responses, parti“ularly when there is severe or

prolonge” mineral ”efi“it. Trabe“ular or “an“ellous bone is a network of

46

INTRODUCTION

internal plates an” ro”s forming a 3D bran“hing latti“e intersperse” in the

bone marrow “ompartment. It is foun” prin“ipally at the metaphysis an”

epiphysis of long bones an” in “uboi” bones, su“h as the vertebrae.

It represents 20% of the skeletal mass but has nearly ten times the surfa“e

area of “ompa“t bone. Trabe“ular bone is less ”ense, more elasti“ an” has a

higher turnover rate than “orti“al bone, exhibiting a major metaboli“

fun“tion. It “ontributes to me“hani“al support an” provi”es the initial

supplies of mineral in a“ute ”efi“ien“y states.

6.3.2. Bone matrix

Similar to other “onne“tive tissues, bone “ells are not the primary

“onstituents of bone by weight. Rather, the extra“ellular matrix (ECM)

represents approximately 90% of the organi“ “omposition of the whole

bone tissue. The mineralize” portion of the ECM is “ompose” largely of

“al“ium-phosphate in the form of hy”roxyapatite with small amounts of

“arbonate, magnesium, an” a“i” phosphate.

The organi“ “omponent (osteoi”) “onsists primarily of type-I “ollagen, an”

to a lesser extent of non-“ollagenous proteins, in“lu”ing proteogly“ans,

osteo“al“in, osteopontin, osteone“tin an” bone sialoprotein. Bone ECM

”etermines the me“hani“al properties of the skeleton. The mineralize”

portion of the ECM imparts rigi”ity an” loa”-bearing strength to the

material, while the osteoi” provi”e plasti“ity208. Furthermore, the ”iverse

array of ECM proteins support various biologi“al “ell fun“tions208. This

“apa“ity is largely ”etermine” by their ability to bin” multiple intera“ting

partners su“h as other ECM proteins, growth fa“tors, signal re“eptors an”

a”hesion mole“ules208.

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INTRODUCTION

6.3.3. Osteoblasts

Osteoblasts originate from pluripotent mesen“hymal stem “ells, whi“h have

the “apa“ity to ”ifferentiate into osteoblasts, a”ipo“ytes, “hon”ro“ytes,

myoblasts, or fibroblasts209. Commitment of mesen“hymal stem “ells to the

osteoblast lineage requires the “omplex integration of bone morphogeneti“

proteins, Wnt, Not“h, Hh, an” fibroblast growth fa“tor (FGF) pathways. The

trans“ription fa“tor Runx-2 seems to be a potential fo“al point for signalling

integration210. The primary fun“tion of these “ells is the pro”u“tion of the

bone matrix, “ontributing to expansion of bone volume by laying ”own

osteoi” an” se“reting fa“tors that fa“ilitate mineral ”eposition. Moreover,

osteoblasts regulate the ”ifferentiation of osteo“lasts211 an” bone resorption

a“tivity by ”ifferent me“hanisms212.

Towar” the en” of the matrix-se“reting perio”, 15% of mature osteoblasts

are entrappe” in their own bone matrix an” ”ifferentiate into osteo“ytes.

Alternatively, on a quies“ent bone surfa“e, the osteoblast “an ”evelop into a

flattene” bone-lining “ell of a single layer forming the en”osteum against

the marrow an” un”erlying the periosteum ”ire“tly on mineralize” surfa“es.

6.3.4. Osteo“ytes

Osteo“ytes represent terminally ”ifferentiate” osteoblasts an” a““ount for

about 95% of the whole “ell population in the mature bone tissue. From

ea“h osteo“yte “ell bo”y, an extensive filopo”ia pro“esses originate an”

ra”iate through the mineralize” matrix via spa“es “alle” the “anali“uli. This

filopo”ia ra”iate in ”ifferent ”ire“tions an” form an intri“ate inter“ellular

network “onne“ting them with ea“h other an” with the bone surfa“e lining

“ells an” osteoblasts213. Osteo“ytes are the pivotal “ells or“hestrating the

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INTRODUCTION

biome“hani“al regulation of bone mass an” stru“ture for effi“ient loa”

bearing. They trans”u“e stress signals from ben”ing or stret“hing of bone

into biologi“ a“tivity, lea”ing to a”equate bone mass an” ar“hite“ture214.

6.3.5. Osteo“lasts

Osteo“lasts, the ex“lusive “ells with ability to resorb bone, are

multinu“leate” giant “ells ”erive” from mononu“lear pre“ursors of the

mono“yte-ma“rophage lineage of bone marrow211. Re“eptor a“tivator of

nu“lear fa“tor-κ (RANK-L) an” ma“rophage “olony stimulating fa“tor (M-

CSF) are the two “riti“al an” suffi“ient “ytokines for basal

osteo“lastogenesis. Both RANK-L an” M-CSF are pro”u“e” mainly by “ells of

the osteoblasti“ line, making the presen“e of these “ell types, in”ispensable

for the physiologi“al re“ruitment of osteo“lasts from their pre“ursors211.

RANK-L bin”s to its re“eptor, RANK, on the surfa“e of osteo“lasts pre“ursors

an” it is “riti“al for osteo“last formation215. M-CSF “ontributes to the

proliferation, survival an” ”ifferentiation of osteo“last pre“ursors216.

Osteoprotegerin (OPG), also se“rete” by osteoblasts an” osteogeni“ stromal

stem “ells, prote“ts the skeleton from ex“essive bone resorption by bin”ing

to RANK-L with high affinity an” preventing it from intera“ting with RANK,

an” hen“e favours in“rease” bone mass217.

6.3.6. Bone remo”elling

To maintain a healthy skeleton with optimal me“hani“al integrity, bone

un”ergoes the remo”elling pro“ess throughout life. This remo”elling, in

whi“h aging or ”amage” bone is gra”ually repla“e” by new tissue, is an

integral part of the “al“ium homeostati“ system an” provi”es a “ru“ial

me“hanism for a”aptation to physi“al stress218,219. This is a““omplishe”

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INTRODUCTION

through the “arefully or“hestrate” “ollaboration among several types of

bone “ells, “ombine” into ”efine” anatomi“al spa“es terme” basi“

multi“ellular units (BMUs). A remo”elling “y“le might begin with osteo“ytes

re“ognizing that a spe“ifi“ area of bone nee”s to be repla“e”220 an”

signalling through their “anali“ulae to surfa“e “ells (Fig. 11). The “onsequen“e

is the separation of lining “ells from the un”erlying bone an” the

re“ruitment of osteo“lasts an” osteoblasts to generate a new BMU221. As the

entire BMU moves forwar”, osteo“lasts resorb bone an” ”ie by apoptosis. If

new osteo“lasts are not forme”, resorption “eases.

Figure 11. The bone remodelling process. Ol” bone is resorbe” by osteo“lasts.

Following resorption, ma“rophage-like “ells are foun” at the remo”elling site.

Osteoblast pre“ursors are then re“ruite”, whi“h proliferate an” ”ifferentiate into

mature osteoblasts, before se“reting new osteoi”. The matrix then mineralizes to

generate new bone, thus “ompleting the remo”elling pro“ess. Extra“te” from:

http://www.york.a“.uk/res/bonefrombloo”/ba“kgroun”/boneremo”elling.html

On“e the resorption phase is terminate” an” after a brief reversal phase222, a

team of osteoblasts is re“ruite”, laying ”own bone until the resorption

“avity is “ompletely fille”. During this pro“ess, the osteoblasts be“ome

progressively flatter an” wi”er. At the en” of the bone-forming phase some

50

INTRODUCTION

of them be“ome embe””e” in the bone as osteo“ytes, some ”ie by

apoptosis an” those remaining when the pro“ess is “omplete” be“ome the

lining “ells that “over the new quies“ent surfa“e223. The net result is the

repla“ement of a pa“ket of ol” bone with new bone (Fig. 11). Pathologi“al

imbalan“e in these “ellular pro“esses “an lea” to ”isease “on”itions of bone

loss, in parti“ular osteoporosis.

6.3.7. Mole“ular effe“ts of estrogen ”efi“ien“y on remo”elling

An essential requirement for balan“e” remo”elling in a”ults is that

resorption an” formation are balan“e”, so that ol” bone is “ontinuously

repla“e” at the exa“t amount remove” by resorption. Thus, tight “ontrol of

bone remo”elling at the level of the BMU is essential to maintain stru“tural

integrity. The regulation of bone remo”elling is “omplex, being “ontrolle” by

hormones an” many other proteins both at systemi“ an” lo“al level.

Parathyroi” hormone (PTH), vitamin D, “al“itonin, the growth

hormone/insulin growth fa“tor (IGF)-1224 system an” IGF-2225, thyroi”

hormones226, glu“o“orti“oi”s227, FSH228 an” serotonin229 are among the most

important fa“tors influen“ing systemi“ regulation of bone “ell fun“tions.

Estrogens also play a key role in maintaining both normal bone turnover an”

bone mass. Therefore, long-term estrogen ”eprivation is asso“iate” with the

”evelopment of osteoporosis230 an” in“rease” risk of bone fra“ture. The

me“hanisms whereby loss of estrogen in“rease bone turnover are “omplex

an” multifa“ete”, but broa”ly speaking, new osteo“last formation with

subsequent in“rease of BMUs, is involve”231. Su“h a“tivation expan”s the

remo”elling spa“e, in“reases “orti“al porosity an” enlarges the resorption

area on trabe“ular surfa“es in a “ytokine-”riven pro“ess232 (Fig. 12).

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INTRODUCTION

Figure 12. Schematic representation of the main mechanisms by which estrogen

deficiency leads to bone loss. The bone loss in”u“e” by estrogen ”efi“ien“y is ”ue to

a “omplex interplay of hormones an” “ytokines that “onverge to ”isrupt the pro“ess

of bone remo”elling. A”apte” from: Weitzmann et al. (The Journal of Clini“al

Investigation, 2006)233.

Estrogen ”efi“ien“y lea”s to an in“rease of T-“ell a“tivation, proliferation

an” lifespan by several me“hanisms in“lu”ing, antigen presentation,

transforming growth fa“tor beta (TGF- ) stimulation an” IL-7 in“reasing.

On“e a“tivate”, T-“ells, in turn, “ontribute to osteo“last proliferation an”

a“tivation by means of “ytokine pro”u“tion234. Mole“ules su“h as TNF- ,

interferon gamma (INF-ɣ), IL-1 as well as the RANK-L itself a“t

synergisti“ally in or”er to influen“e osteo“last formation, lifespan an”

a“tivity234-239. Osteo“last apoptosis is also inhibite” un”er estrogen

52

INTRODUCTION

”eprivation238,240. In parallel to this phenomenon, the “oupling pro“ess eli“its

a “ompensatory in“rease in bone formation. However, in spite of stimulate”

osteoblastogenesis, the rate of bone formation is ina”equate to “ompensate

for enhan“e” bone resorption an” “onsequently, in“rease” bone loss is the

net effe“t. Augmentation of osteoblasts an” osteo“yte apoptosis an”

limitation of the a“tivity of mature osteoblasts are among the fa“tors

me”iate” by estrogen ”epletion to limit the magnitu”e of the “ompensatory

response241,242. As we have seen, the relationship among estrogen, the

immune system an” the skeleton is wi”ely ”o“umente”.

6.3.8. Aromatase inhibitor in”u“e” bone loss (AIBL) an” fra“ture risk.

As previously state”, extragona”al synthesis of estrogen plays an important

role in postmenopausal women. In human, one of the extragona”al sites of

estrogen biosynthesis is bone. Aromatase has been foun” in bone “ells an”

its a“tivity is “omparable to that present in a”ipose stromal “ells243. Thus,

lo“al aromatase expression in bone “oul” be the major sour“e of estrogen

responsible for the maintenan“e of mineralization89.

Consi”ering the important role of estrogens in bone, it seems logi“al,

therefore, that the profoun” estrogen suppression in”u“e” by AI-treatment

(ex“ee”ing the gra”ual ”e“rease seen in healthy menopausal women244),

lea”s to some unfavourable effe“ts on this “ompartment; AIBL at the

lumbar spine (LS) an” hip, reporte” in a number of RCTs vary from 1.7% to

5.8% per year245-248. On average, the rate of AIBL is estimate” at 2.6%

annually, surpassing the bone loss of 1% per year observe” in healthy

postmenopausal women. Overall, fra“ture risk is in“rease” a 47% with AI use

“ompare” with tamoxifen therapy154. Fra“tures are in“reasingly re“ognize”

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INTRODUCTION

as important “lini“al issues for breast “an“er patients sin“e they are

asso“iate” with “hroni“ pain, loss of mobility an” even shorter survival. An

un”erstan”ing of AIBL is “riti“al for ”etermining how to assess the risk an”

i”entifying whi“h patients may benefit from preventive therapy.

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INTRODUCTION

7. AIBL MANAGEMENT

Preserving an”/or restoring bone health is an important “omponent of

mo”ern breast “an“er management. All women ”iagnose” with the ”isease

shoul” be en“ourage” to follow the osteoporosis s“reening an” prevention

gui”elines of the general population.

7.1. LIFESTYLE INTERVENTIONS

Lifestyle “hoi“es an” ”ietary habits provi”e the ne“essary framework,

improving not only bone health but overall health as well. Patients shoul” be

a”vise” to limit al“ohol intake, avoi” ex“ess “affeine, quit smoking an”

follow a well-balan“e” ”iet. A”equate ”ietary “al“ium an” vitamin D are also

“riti“al, requiring supplements when appropriate249,250. In a””ition, patients

shoul” also be en“ourage” to parti“ipate in weight-bearing physi“al a“tivity

to in“rease bone ”ensity an” mus“le strength249.

7.2. BONE LOSS SCREENING AND MONITORING

Close monitoring an” “onsi”eration of proa“tive measures, in“lu”ing ”rug

therapy when ne“essary, to preserve bone health are en“ourage” for

postmenopausal patients with breast “an“er on AI treatment251. Thus, in

patients initiating AI, DXA of the hip an” LS at baseline an” annually

thereafter is a”vise”. However, available ”ata suggest that BMD

measurement shoul” not be the sole “riterion for ”etermining fra“ture risk,

but a “ombination of BMD an” “lini“al risk fa“tors, is optimal252 (Fig. 13).

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INTRODUCTION

Figure 13. Recommended algorithm for managing bone health in women receiving AI

therapy for breast cancer. aIf patients experien“e an annual ”e“rease in BMD of

10% (use lowest T-s“ore from three sites) se“on”ary “auses of bone loss su“h as

vitamin D ”efi“ien“y shoul” be evaluate” an” antiresorptive therapy initiate”. bDenosumab may be a potential treatment option for some patients. “Although

osteone“rosis of the jaw is an un“ommon event, regular ”ental “are an” attention to

oral health is a”visable in patients re“eiving bisphosphonates or ”enosumab. A”apte”

from Ha”ji et al. (Annals of On“ology, 2011)249.

Risk fa“tors foun” to in“rease fra“ture risk in women with breast “an“er

in“lu”e AI therapy, T-s“ore < −1.5, age > 65 years, low BMI (<20 kg/m2),

family history of hip fra“ture, personal history of fragility fra“ture after age

50, oral “orti“osteroi” use >6 months an” smoking. For the prevention of

fra“ture in patients taking AI, those women at high risk of fra“ture, in“lu”ing

patients with osteoporosis (or history of fragility fra“ture) an” patients

with osteopenia who have risks for fra“ture other than AI therapy249, shoul”

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INTRODUCTION

be treate” with pharma“ologi“al therapy.

7.3. PHARMACOLOGICAL THERAPY: BISPHOSPHONATES (BP)

Although there is “urrently no approve” treatment or any prevention

therapy for the AIBL, “lini“al trial evi”en“e in”i“ates both BP an” ”enosumab

are effe“tive in maintaining bone ”ensity. BP are spe“ifi“ inhibitors of bone

resorption253 an” they are “onsi”ere” the first line pharma“ologi“ therapy

for the management of osteoporosis an” other ”iseases of high bone

turnover254. BPs pronoun“e” affinity for bone, relative to other tissues,

enables them to attain a high lo“al “on“entration throughout the entire

skeleton, a“hieving highly spe“ifi“ intera“tion with the relevant “ellular sites

of a“tion. This “riti“al pharma“ologi“al feature makes them the i”eal

“an”i”ates for treatment of skeletal ”isor”ers. ASCO gui”elines for

a””ressing bone health issues in women with breast “an“er251, as well as the

osteoporosis gui”elines255, rely on BMD measurements as the key in”i“ator

for therapy. In general, these gui”elines re“ommen” BP therapy

(alen”ronate, rise”ronate an” zole”roni“ a“i”) when BMD T-s“ores have

”roppe” into (or near) the osteoporoti“ range251,255. However, earlier

intervention may be benefi“ial to minimize the effe“ts on the skeleton an”

preserve patient quality of life an” fun“tional mobility.

7.3.1. Me“hanism of a“tion

Stru“turally, BP are stable analogues of inorgani“ pyrophosphate (PPi), a

naturally o““urring “ompoun” release” as a by-pro”u“t of many

intra“ellular metaboli“ rea“tions. PPi is an en”ogenous regulator of bone

mineralization “apable of inhibiting “al“ifi“ation by bin”ing to

hy”roxyapatite “rystals256. Like their natural analogue PPi, BP also bin” to

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INTRODUCTION

hy”roxyapatite “rystals, but the “arbon atom repla“ing the oxygen atom

ren”ers the mole“ule resistant to biologi“al ”egra”ation.

The nitrogen-“ontaining BP (e.g., pami”ronate, alen”ronate, iban”ronate,

rise”ronate an” zole”ronate) a“t by inhibiting farnesyl pyrophosphate

synthase257, a key regulatory enzyme in the mevalonate pathway (Fig. 14).

Posttranslational prenylation of small GTPases is essential for the regulation

of “ore osteo“last “ellular a“tivities, in“lu”ing stress fibber assembly,

membrane ruffling an” survival258,259.

Figure 14. BP pathway: mechanism of action of nitrogen-containing BP in

osteoclasts. A”apte” from: Gong et al (Pharma“ogeneti“s an” genomi“s, 2011)260.

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INTRODUCTION

Therefore, inhibition of these rea“tions lea”s to osteo“last apoptosis261,262.

Moreover, BP also “ause “hanges in osteo“last morphology263,264, “ulminating

in a ”e“rease” a“tivity265. Furthermore, it has been suggeste” that BP also

fun“tion to limit both osteoblast an” osteo“yte apoptosis266

7.3.2. Clini“al role of BP

BP have emerge” as the lea”ing effe“tive treatment for postmenopausal

an” other forms of osteoporosis. Either oral (eti”ronate267, rise”ronate268,

iban”ronate269 an” alen”ronate270) or intravenous (zole”ronate271) BP are

a““eptable options. All have been approve” as therapies in many “ountries,

to prevent or re”u“e postmenopausal osteoporosis255. They have also

”emonstrate” effi“a“y in preserving BMD in several trials248,250,272-276.

However, evi”en“e to ”ate is “ontroversial about varying risk for fragility

fra“tures270,277,278.

In a””ition, BP treatment has also been asso“iate” with a re”u“tion in the

risk of skeletal morbi”ity in patients with bone metastasis from breast

“an“er279 as well as in breast “an“er re“urren“e280.

7.4. FARMACOLOGICAL THERAPY: DENOSUMAB

Denosumab is a humanize” mono“lonal antibo”y against RANK-L that

re”u“es osteo“lastogenesis. It has been shown to improve BMD in

postmenopausal women281. Denosumab “an be use” as initial therapy in

“ertain patients at high risk of fra“ture, su“h as ol”er patients who have

”iffi“ulty with the ”osing requirements of oral BP or who have marke”ly

impaire” renal fun“tion. In a””ition, ”enosumab is an option for patients

who are intolerant or unresponsive to BP.

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INTRODUCTION

7.5. THE ROLE OF BONE MICROARCHITECTURE IN FRACTURE RISK:

TRABECULAR BONE SCORE (TBS)

The “urrent ”efinition of osteoporosis refle“ts the “hanging perspe“tive of

the ”isease, being no longer “onsi”ere” as a ”isor”er of low bone mass

”ensity alone. Measurement of BMD with DXA is so-“alle” gol” stan”ar” for

”iagnosis of osteoporosis, serving as a surrogate marker for the me“hani“al

“ompeten“e of bone an” fra“ture risk282. However, it is subje“t to several

“onstraints.

First, DXA provi”es a two-”imensional proje“tion of a three-”imensional

stru“ture an” hen“e it “annot “apture bone geometry or mi“roar“hite“ture.

Thus, the obtaine” values ”o not represent the true volumetri“ BMD but

rather a proje“te” areal BMD. This “auses BMD to be “onfoun”e” by bone

size so it “annot ”istinguish between in“rease” BMD values arising from

thi“ker bones (geometri“ “hange) than those arising from in“rease” tissue

mineral ”ensity (material “hange). Therefore, BMD gives no information

about stru“tural properties su“h as bone size, bone geometry an” also

mi“rostru“tural properties su“h as trabe“ular orientation an” “orti“al

porosity. Moreover, the test “an also be ”istorte” by s“an artefa“ts su“h as

aorti“ “al“ifi“ation, soft-tissue “al“ifi“ation an” others.

It is estimate” that <50% of the variation in the whole-bone strength is

attributable to variations in BMD. In fa“t, most in”ivi”uals with a fragility

fra“ture have BMD values in the osteopeni“ or even normal range283.

Furthermore, improvements in spine BMD ”uring treatment with

antiresorptive agents a““ounts for a pre”i“table but small part of the

observe” re”u“tion in the risk of vertebral fra“ture284. Consequently, BMD

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INTRODUCTION

“annot be use” as the sole pre”i“tor of bone strength.

Assessment of skeletal mi“rostru“ture “an be ma”e with some te“hniques

as histomorphometri“ analysis of the transilia“ “rest bone biopsy, high-

resolution peripheral quantitative “ompute” tomography, flat-panel volume

CT an” magneti“ resonan“e imaging. However, these te“hniques are invasive

an”/or not routinely available.

TBS is a textural in”ex that evaluates pixel grey-level variations in the

lumbar spine DXA image an” has been propose” as a “lini“al tool “apable of

assessing trabe“ular mi“roar“hite“ture (Fig. 15). TBS “an be extra“te” from

any available DXA image, even thought it was obtaine” years before, without

further patient in“onvenien“e.

Figure 15. Representation of the TBS principles. The TBS value is ”erive” by an

algorithm that analyses the spatial organization of pixel intensity, whi“h in turn

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INTRODUCTION

“orrespon”s to the ”ifferen“es in the X-ray absorption power of an osteoporoti“

bone versus a normal trabe“ular pattern. Extra“te” from: Silva et al. (Journal of

Bone Mineral Resear“h, 2012)285.

The ”ense trabe“ular mi“rostru“tures proje“te” onto a plane generate an

image “ontaining a large number of pixel value variations of small

amplitu”e. Conversely, a porous trabe“ular stru“ture pro”u“es an image

with a low number of pixel value variations of high amplitu”e. A variogram

of those proje“te” images “an estimate a 3D stru“ture from the existing

variations on the 2D proje“te” images. A high TBS value is asso“iate” with

better bone stru“ture, whereas low TBS values in”i“ate worse bone stru“ture

(Fig. 15).

TBS has been ”emonstrate” to “orrelate with trabe“ular bone “onne“tivity,

trabe“ular number an” thi“kness as well as spa“e between trabe“ulae286,287.

Hen“e, it is “apable of ”ifferentiating between two 3D mi“roar“hite“ture of

“an“ellous bone with the same bone ”ensity but ”ifferent trabe“ular

“hara“teristi“s288. TBS is a strong an” BMD-in”epen”ent pre”i“tor of “urrent

an” future osteoporoti“ fra“tures in a”ults286,289,290 an” provi”es BMD-

in”epen”ent information when monitoring the effe“ts of osteoporosis

treatments. Combining BMD an” TBS measurements provi”es more a““urate

assessment of osteoporoti“ fra“ture risk than ”oes either te“hnique

alone287.

The effe“ts of AI on bone mi“roar“hite“ture have s“ar“ely been explore”.

The un“ertainty about the “apa“ity of BP to re”u“e fra“ture risk in AI-

treate” patients278 triggers the i”ea that AI may in”u“e “hanges in bone

beyon” BMD re”u“tion. Generally speaking, stu”ies on the impa“t of AI on

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INTRODUCTION

TBS report larger ”e“reases in BMD “ompare” with TBS291,292. Moreover, RCTs

evaluating the skeletal preservation of BP in both healthy

postmenopausal293,294 an” AI-treate”295 women ”o not ”es“ribe remarkable

improvements but rather a positive maintenan“e of bone

mi“roar“hite“ture.

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INTRODUCTION

8. VITAMIN D AND AI ADVERSE EFFECTS

Vitamin D is a “omplex nutrient that fun“tions as a hormone. The

asso“iations between vitamin D “on“entrations an” various “on”itions an”

”iseases have been assesse” in a large an” rapi”ly expan”ing literature.

Histori“ally, vitamin D ha” been linke” to “al“ium, phosphorus, bone

metabolism, osteoporosis, fra“tures, mus“le strength an” falls296. However,

in the last two ”e“a”es, growing s“ientifi“ attention turne” to non-skeletal

”iseases su“h as “ar”iovas“ular, infe“tious an” autoimmune ”iseases,

metaboli“ ”isor”ers, as well as “an“er an” mortality297.

8.1. SYNTHESIS OF VITAMIN D

Vitamin D exists in two main forms, vitamin D3 (VitD3) or “hole“al“iferol an”

vitamin D2 (VitD2) or ergo“al“iferol, ”iffering in their si”e “hain stru“ture. In

humans, the majority of VitD3 is pro”u“e” in the skin, from 7-

”ehy”roxy“holesterol (DHC) upon exposure to ultraviolet B ra”iation (Fig.

16). Only a small proportion is obtaine” from animal sour“es su“h as oily fish

an” egg yolk. VitD2 is pre”ominantly obtaine” from plant sour“es.

Commonly, vitamin D refers “olle“tively to VitD2 an” VitD3298.

Whether it is ”erive” from the ”iet or synthesize” in the skin, vitamin D

un”ergoes two su““essive hy”roxylation steps me”iate” by CYPs enzymes.

The first step o““urs in the liver, by 25-hy”roxylase whi“h “onverts vitamin

D into 25-hy”roxy vitamin D (25(OH)D). Another enzyme, the 1 -

hy”roxylase, “onverts 25(OH)D to the biologi“ally a“tive form of vitamin D,

1,25-”ihy”roxy vitamin D (1,25(OH)2D or “al“itriol) in the proximal tubule of

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INTRODUCTION

the ki”neys. This 1 -hy”roxylation is un”er “ontrol by serum PTH an” FGF-

23 in response to serum “al“ium an” phosphate an” represents the rate-

limiting step in the syntheti“ pathway298.

Figure 16. Vitamin D synthesis, gene activity and effects on target.

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INTRODUCTION

Vitamin D is transporte” in the “ir“ulation pre”ominantly boun” to vitamin

D-bin”ing protein (DBP) an” albumin, with <1% in the free form. The a“tive

vitamin D metabolite me”iates its biologi“al effe“ts by bin”ing to the

vitamin D re“eptor (VDR). VDR belongs to the nu“lear re“eptor superfamily

of steroi”/thyroi” hormone re“eptors an” it is expresse” by “ells in most

organs, in“lu”ing the brain, heart, skin, gona”s, prostate an” breast. VDR

a“tivation lea”s to the maintenan“e of “al“ium an” phosphorus levels in the

bloo” an” to the maintenan“e of bone “ontent298.

The “ataboli“ enzyme 24-hy”roxylase is responsible for the “onversion of

both 25(OH)D an” 1,25(OH)2D into ina“tive metabolites an” via a multistep

pathway to the water soluble “al“itroi“ a“i”, whi“h un”ergoes urinary an”

biliary ex“retion. 24-hy”roxylase is in”u“e” by 1,25(OH)2D, whi“h serves as

an important fee”ba“k me“hanism to avoi” vitamin D toxi“ity298.

Unfortunately, optimal levels of vitamin D have been not universally

establishe”. Diversity in measurement metho”s an” referen“e stan”ar”s

provi”e” by ”ifferent manufa“turers of laboratory kits further “ompli“ate

the issue of ”efining “ut-points an” making ju”gments in the in”ivi”ual

patient. Several prominent investigators reviewe” a large number of stu”ies

to arrive at an answer to this question. The US Institute of Me”i“ine (IOM)

re“ommen”ations for vitamin D (IOM 2011)299, base” on a review of the

evi”en“e, “on“lu”e” that:

• serum 25(OH)D < 30 nmol/L is ”efi“ient;

• serum 25(OH)D of 30 to 50 nmol/l may be ina”equate in some people;

• serum 25(OH)D > 50 nmol/l is suffi“ient for almost the whole population.

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INTRODUCTION

A““or”ing to these “riteria, a high prevalen“e of vitamin D ”efi“ien“y or

ina”equate vitamin D status in Europe has been ”es“ribe”. This is espe“ially

true for el”erly populations, lea”ing to severe “onsequen“es in terms of falls,

osteoporosis an” fra“tures. The prevalen“e of vitamin D insuffi“ien“y an”

severe ”efi“ien“y among postmenopausal patients treate” for early breast

“an“er have also reporte” to be very high300. Moreover, it is now re“ognize”

that the target level of 25(OH)D for vitamin D repletion is likely to ”iffer for

”ifferent tissues301.

8.2. EFFECTS OF VITAMIN D ON AIA

One possible fa“tor un”erlying AIA is the high prevalen“e of vitamin D

insuffi“ien“y or ”efi“ien“y in women with breast “an“er. Hypovitaminosis D

has been suggeste” as an un”erlying etiology in in”ivi”uals with persistent,

nonspe“ifi“ mus“uloskeletal pain, “omparable with the symptoms of

osteomala“ia302. Both RCTs an” observational stu”ies have reporte”

promising results that vitamin D may play an important role in these

symptoms, suggesting benefi“ial effe“ts on mus“uloskeletal ”is“omfort303-

305.

Vitamin D an” estrogen metabolism are “losely tie”; Estrogen in“reases the

a“tivity of 1 -hy”roxylase306 an” enhan“es the expression of VDR gene307.

Hen“e, the ”rop in estrogen levels “ause” by AI might in”u“e a ”e“rease in

a“tive vitamin D levels lea”ing to a vitamin D ”efi“ient-like arthralgia

syn”rome. In a””ition, AI therapy is believe” to in“rease vitamin D

requirements: vitamin D is ne“essary to in”u“e the expression of CYP3A4, an

essential system for AI ”etoxifi“ation in the liver. Vitamin D, in turn, “an

a“tivate the promoter region of the aromatase gene in a number of

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INTRODUCTION

tissues308. Thus, higher “on“entrations of vitamin D may attenuate lo“al

estrogen ”efi“ien“y309. Moreover, vitamin D has non-skeletal effe“ts on a

number of tissues in“lu”ing synovium, mus“le an” “artilage, with putative

roles in arthritis310. Lastly, it has been hypothesize” that ”e“rease” vitamin

D may also lea” to se“on”ary hyperparathyroi”ism, with the ”eposition of

unmineralize” “ollagen matrix. As a “onsequen“e the hy”ration an”

expansion of the sub-periosteal tissue woul” in”u“e se“on”ary pressure on

sensory pain fibbers, “ausing pain347. Vitamin D is also known to affe“t a

number of inflammatory pathways asso“iate” with the ”evelopment an”

persisten“e of “hroni“ pain. Overall, Vitamin D exerts anatomi“, hormonal,

neurologi“al an” immunologi“al influen“es on pain manifestation, thereby

playing a role in the aetiology an” maintenan“e of “hroni“ pain states311.

8.3. EFFECTS OF VITAMIN D ON AIBL

One of the most important roles of vitamin D is to maintain skeletal “al“ium

an” phosphate balan“e to allow passive mineralization of unmineralize”

bone matrix. Serum 1,25(OH)2D ”oes this primarily by stimulating “al“ium

an” phosphorous absorption from the gut. In “ase of vitamin D ”efi“ien“y,

less “al“ium will be available for bone mineralization an” the PTH level will

in“rease, stimulating both the hy”roxylation of 25(OH)D to 1,25(OH)2D an”

bone turnover, in or”er to restore serum “al“ium levels. In perio”s of severe

vitamin D ”efi“ien“y, the mineral ”efi“it lea”s to osteomala“ia (ris“kets in

“hil”ren), a bone ”isor”er, “hara“terize” by ”e“rease” mineralization of

newly forme” osteoi”. Vitamin D status is relate” to BMD, not only in

vitamin D ”efi“ient subje“ts, but also in vitamin D insuffi“ient subje“ts312-314.

The ”ire“t a“tions of vitamin D on bone are still not well un”erstoo”, ”ue to

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INTRODUCTION

the multitu”e of effe“ts on the homeostati“ me“hanisms an” to the

”ifferen“es by spe“ies, states of ”ifferentiation an” responsiveness315,316.

Whether vitamin D ”efi“ien“y is a pre”i“tor for bone fra“tures is less “lear.

Furthermore, it is ”iffi“ult to ”istinguish the ”ire“t effe“ts of vitamin D on

bone from its benefi“ial effe“ts on mus“le that result in ”e“rease” falls an”

thereby, fra“ture risk317. Anyhow, gui”elines for “an“er treatment-in”u“e”

bone loss in“lu”e supplementation with “al“ium an” vitamin D249,251.

Improve” vitamin D status using supplementation has been asso“iate” with

attenuation of AIBL303,318.

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INTRODUCTION

9. GENETIC STUDY OF COMPLEX TRAITS

In the past two ”e“a”es, many genes that were impli“ate” in simple

(Men”elian) ”iseases have been i”entifie” by using geneti“ linkage approa“h.

Although these metho”s have been remarkably su““essful for the

i”entifi“ation of high risk genes, they have low statisti“al power to ”ete“t

genes with mo”est effe“ts involve” in “omplex ”isor”ers. They are terme”

“omplex be“ause they are ”etermine” by the sum total of multiple geneti“

an” environmental fa“tors. Geneti“ asso“iation stu”ies are a powerful

means of i”entifying the “ommon variants that un”erlie the “omplex traits.

In the “an”i”ate gene approa“h, pre-spe“ifie” genes, usually hypothesize”

to have a role in the ”isease, are “hosen in an attempt to fin” an asso“iation

between the gene an” the ”isease. I”entifie” variants in or near those genes

that might either “ause a “hange in the protein or in its expression, or be

in linkage ”isequilibrium with fun“tional “hanges, are genotype” an” teste”

for a possible “orrelation with the phenotype of interest by statisti“al

metho”s319.These variants use to be single nu“leoti”e polymorphisms (SNPs),

variations at a single position in a DNA sequen“e among in”ivi”uals.

The ”is“overy rate has been a““elerate” by the Human Genome Proje“t an”

by improve” te“hnologies for the genome-wi”e interrogation of variation. A

genome-wi”e asso“iation stu”y (GWAS) is an approa“h that involves

systemati“ DNA s“reening with markers evenly spa“e” throughout the whole

genome, without regar” to their fun“tion or “ontext in a spe“ifi“ gene, with

the aim of fin”ing geneti“ variations asso“iate” with a parti“ular “omplex

trait. Su“h stu”ies are parti“ularly useful in fin”ing geneti“ variations that

“ontribute to “ommon, “omplex ”iseases. However, the magnitu”e of the

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INTRODUCTION

effe“t for a parti“ular polymorphism use to be quite mo”est. Higher levels

of risk may be “onferre” a““or”ing to the “ombine” a““umulation of many

polymorphisms. Consequently, very large “ase-“ontrol stu”ies woul” be

ne“essary to quantify the risk asso“iate” with a parti“ular SNP a““urately.

Hormonal therapy with AI is still fa“ing the “hallenge of interpatient

variability in both therapeuti“ response an” intensity of a”verse effe“ts.

Although AI effi“a“y among breast “an“er patients has been prove”, there is

signifi“ant variability in the frequen“y of response rate an” a”verse effe“ts.

As most human “omplex ”iseases, the out“omes an” toxi“ity of most

me”i“ations “oul” be governe” by, among other fa“tors, geneti“ variability.

9.1. GENETIC BASIS OF AIA

Previous se“tions have highlighte” the “entral role of estrogen in many

physiologi“al pro“esses an”, espe“ially, in the onset an”/or intensity of AI

si”e effe“ts. There is ample evi”en“e in”i“ating that ”eterminants of plasma

levels of sex steroi”s are, at least in part, heritable320. In fa“t, “ommon

geneti“ variants have been relate” in some extent to the effe“tiveness of AI

treatment, parti“ularly those in CYP19A1 gene321-323. All the foregoing

in”i“ates that these an”/or other variants may also be involve” in the onset

an”/or intensity of AI se“on”ary effe“ts. Thus, for example, a

tetranu“leoti”e repeat polymorphism (TTTA)n, in CYP19A1 gene, previously

asso“iate” with “ir“ulating estra”iol an” estrone levels, was alrea”y relate”

to the o““urren“e of AIA324. Park et el. also ”es“ribe” an haplotype of

CYP19A1 to be asso“iate” with bone/joint paint ”ue to the AI-therapy325.

Moreover, another stu”y foun” that an introni“ variant of ESR1, whi“h

en“o”es re“eptor ERa, is asso“iate” with in“rease” risk of AI ”is“ontinuation

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INTRODUCTION

”ue to mus“uloskeletal toxi“ity326. On the other han”, a GWAS stu”y in 2010

also foun” that SNPs in the T-“ell leukaemia 1A gene (TCL1A), whi“h, in turn,

was relate” to the IL-17, were asso“iate” with mus“uloskeletal si”e

effe“ts327.

9.2. GENETIC BASIS OF AIBL

Stu”ies in twins an” families in”i“ate that geneti“ fa“tors play an important

role in the regulation of BMD, estimating its heritability between 50% an”

85%328,329. Osteoporosis is a polygeni“ ”isor”er, ”etermine” by the effe“ts

of several genes, ea“h with relatively mo”est “ontribution to bone mass an”

other ”eterminants of fra“ture risk. Can”i”ate gene stu”ies have reveale”

several genes involve” in the pathogenesis of the ”isease in“lu”ing COL1A,

LRP5, CYP19A1, SOST, TGF 1, ESR, AR, VDR, IL-6, “al“itonin re“eptor, OPG,

RANK an” RANK-L330. GWAS have also i”entifie” several lo“i influen“ing BMD

variation331-336. A genome-wi”e meta-analysis in 2012 i”entifie” 56 lo“i (32

novel) asso“iate” with BMD, of whi“h 14 also yiel”e” signifi“ant asso“iation

with fra“ture risk337. Several of these fa“tors “luster within the RANK-

RANKL-OPG, mesen“hymal-stem-“ell ”ifferentiation, en”o“hon”ral

ossifi“ation an” the WNT signalling pathways. However, lo“i “ontaining

genes not known to play a role in bone biology were also ”is“overe”.

Although these fin”ings helpe” explain a large amount of geneti“

ar“hite“ture un”erlying BMD variation an” fra“ture sus“eptibility, the

un”erstan”ing about geneti“ fa“tors “ontributing to osteoporosis response

is still limite”.

As regar”s the spe“ifi“ “ase of AIBL, whether the alrea”y i”entifie” variants,

involve” in BMD ”etermination or AI response, “ontribute to the

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INTRODUCTION

pathogenesis is still unknown. Population analysis involving larger patient

“ohorts an” examining all fun“tional gene variants simultaneously is

require” to vali”ate the existing fin”ings an” to elu“i”ate the me“hanisms

un”erlying the variability of the se“on”ary effe“ts of AI. Tests that are able

to pre”i“t treatment response might allow us to i”entify upfront patients

who will not tolerate AI, provi”ing valuable information for treatment

planning.

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Chapter2

La “ien“ia está maravillosamente prepara”a para

respon”er a la pregunta ¿Cβmo?,

pero se vuelve terriblemente “onfun”i”a “uan”o se

enfrenta a la pregunta ¿Por qué?

Erwin Chargaff

RESULTS

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GENETICDETERMINANTSOFAROMATASE

INHIBITOR-RELATEDARTHRALGIA:THEB-ABLE

COHORTSTUDY

Garcia-Giralt N, Rodríguez-Sanz M, Prieto-Alhambra D, Servitja S, Torres-Del Pliego E, Balcells S, et al. Genetic determinants of aromatase inhibitor-related arthralgia: the B-ABLE cohort study. Breast Cancer Res Treat. 2013 Jul 19;140(2):385–95. DOI: 10.1007/s10549-013-2638-3

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AI-RELATEDBMDVARIATIONINACTUAL

PRACTICECONDITIONS:APROSPECTIVECOHORT

STUDY

Rodríguez-Sanz M, Prieto-Alhambra D, Servitja S, Garcia-Giralt N, Garrigos L, Rodriguez-Morera J, et al. AI-related BMD variation in actual practice conditions: A prospective cohort study. Endocr Relat Cancer. 2016 Apr;23(4):303–12. DOI: 10.1530/ERC-16-0025

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CYP11A1EXPRESSIONINBONEISASSOCIATED

WITHAROMATASEINHIBITOR-RELATEDBONE

LOSS

Rodríguez-Sanz M, García-Giralt N, Prieto-Alhambra D, Servitja S, Balcells S, Pecorelli R, et al. CYP11A1 expression in bone is associated with aromatase inhibitor-related bone loss. J Mol Endocrinol. 2015 Aug 24;55(1):69–79. DOI: 10.1530/JME-15-0079

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EVOLUCIÓNDELADMODURANTEELTRATAMIENTOCON

INHIBIDORESDEAROMATASAYSURELACIÓNCONELGEN

CYP11A1:ESTUDIOPROSPECTIVODELACOHORTEB-ABLE

Rodríguez-Sanz M, Prieto-Alhambra D, Servitja S, García-Giralt N, Garrigos L, Albanell J, et al. Evolución de la DMO durante el tratamiento con inhibidores de aromatasa y su relación con el gen CYP11A1: estudio prospectivo de la cohorte B-ABLE. Rev Osteoporos y Metab Miner. 2015 Dec;7(4):98–105. DOI: 10.4321/S1889-836X2015000400004

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TBSANDBMDATTHEENDOFAI-THERAPY:A

PROSPECTIVESTUDYOFTHEB-ABLECOHORT

María R-S, Marta P-M, Sonia S, Natalia G-G, Tamara M, Ignasi T, et al. TBS and BMD at the end of AI-therapy: A prospective study of the B-ABLE cohort. Bone. 2016 Nov;92:1–8. DOI: 10.1016/j.bone.2016.08.008

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Chapter3

Este hombre, por una parte, “ree que sabe algo,

mientras que no sabe —na”a].

Por otra parte, yo, que igualmente no sé —na”a],

tampo“o “reo—saber algo].

Apología de Sócrates-Platón-

DISCUSSION

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DISCUSSION

AI are “urrently in”i“ate” as first-line a”juvant en”o“rine therapy in the

treatment of early-stage breast “an“er in postmenopausal women with ER-

positive tumours. Sin“e 2001, the effi“a“y of thir”-generation AI has been

well establishe” an” large “lini“al trials have shown these agents to be

superior to tamoxifen in terms of ”isease-free survival, in“i”en“e of

“ontralateral breast “an“er an” time to re“urren“e147. The effi“a“y of AI has

le” to a “onsi”erable in“rease in the frequen“y of their use. Consequently,

the “ompli“ations arising from AI therapy in this patient population have

long-term effe“ts an” greatly impa“t patient quality of life.

Most of the epi”emiologi“al ”ata “on“erning AI se“on”ary effe“ts originates

from RCTs, whi“h are the most rigorous strategy for ”etermining a “ause-

effe“t relation between an intervention an” an out“ome. RCTs assess the

effe“t of the intervention by “omparing it to a “ontrol “on”ition, stan”ar”

treatment or pla“ebo. Ran”omization of parti“ipants to the test an” “ontrol

arms an” “on“ealment of their allo“ation ensures that allo“ation bias an”

“onfoun”ing of unknown variables are minimize”. Although RCTs are

powerful tools for “ausal inferen“e, their use is not ”is“harge” from

“onstraints. The stri“t an” “ontrolle” “on”itions in whi“h they are

“on”u“te”, often “onfines their appli“ability to i”eal “on”itions an” this

limits their ability to portray what happens in the real-life population338.

Therefore, one of the main purposes of the present work was to ”es“ribe the

prospe“tive evolution of mus“uloskeletal a”verse effe“ts of AI treatment in

the real-life usual “are. In this “ontext, patient “hara“teristi“s, as well as

therapy a”heren“e, may ”iffer from those observe” in RCTs. Observational

stu”ies mimi“ every”ay “lini“al pra“ti“e, provi”ing higher external vali”ity.

For this purpose, the B-ABLE “ohort, a prospe“tive, observational, “lini“al

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DISCUSSION

“ohort stu”y was available. All the postmenopausal women ”iagnose” with

early breast “an“er an” “an”i”ates for AI treatment atten”ing the

outpatient Breast Can“er Unit, Hospital ”el Mar (Bar“elona, Spain), were

“onse“utively invite” to parti“ipate in this stu”y an” re“ruite” after

informe” “onsent. Information on a large number of “lini“al variables was

re“or”e” at the time of enrolment, in“lu”ing the intensity of self-reporte”

joint pain an” BMD at baseline an” repeate” at ea“h follow-up visit.

JOINT PAIN IN THE B-ABLE COHORT

Pain is ”efine” as an unpleasant sensory an” emotional experien“e

asso“iate” with a“tual or potential tissue ”amage, or ”es“ribe” by the

patient in terms of su“h ”amage . Pain pro“esses ”o not begin with the

stimulation of re“eptors. Rather, injury or ”isease pro”u“es neural signals

per“eive” in the “ons“ious brain, whi“h parti“ipate in the sele“tion,

abstra“tion an” synthesis of information from the total sensory input. Thus,

pain is essentially a subje“tive per“eptual experien“e influen“e” by a “omplex

intera“tion of behavioral, environmental, biologi“al an” so“ial fa“tors339.

In the B-ABLE “ohort, pain is measure” by the visual analogi“ s“ale (VAS). The

VAS “onsists of a 10-“m line with the two en”points labelle” as no pain an”

worst pain ever (or similar verbal ”es“riptors) (Fig. 17). The question

asso“iate” to the VAS rea”s as follows: Please, s“ore the intensity of the

pain you feel in your peripheral joints (knee, wrist, fingers/toes, elbow,

shoul”er, et“.), ex“lu”ing spine/ba“k pain an” pain at the operate” area

(translate” from Catalan/Spanish by the authors). Patients are require” to

pla“e a mark on the 10-“m line at a point that “orrespon”s to the level of

pain intensity. The ”istan“e in “m from the low en” of the VAS to the

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DISCUSSION

patient s mark is use” as a numeri“al in”ex of the severity of pain.

Figure 17. VAS pain scale ruler. The s“ale is most “ommonly an“hore” by no pain

(s“ore of 0) an” worst imaginable pain (s“ore of 10). Extra“te” from:

http://www.“ustompromotionalrulers.“om/visual-analog-s“ale-vas-rulers/vas-

pain-s“ale-rulers-0-10-“m-w/sli”er/

The major ”isa”vantage of VAS is the assumption that pain is an

uni”imensional experien“e that “an be measure” with a single-item s“ale of

intensity339. Notwithstan”ing, patient reporte” toxi“ity more

“omprehensively “apture the subje“tive si”e effe“ts of therapies (that is,

pain) on ”aily experien“e an” has higher “on“or”an“e with health-relate”

quality of life than “lini“ian as“ertaine” toxi“ity; therefore, it is more

appropriate for the investigation of AIA340. A””itionally, VAS s ratio s“ale

properties make it appropriate to speak meaningfully about per“entage

”ifferen“es between VAS measurements obtaine” at multiple points in time.

Other a”vantages of the VAS in“lu”e its ease an” brevity of a”ministration

an” s“oring, minimal intrusiveness an” “on“eptual simpli“ity.

VAS is sensitive to pharma“ologi“al an” nonpharma“ologi“al pro“e”ures that

alter the experien“e of pain an” “orrelate highly with pain measure” on

verbal an” numeri“ rating s“ales341. Unfortunately, in our stu”y, ”etails of

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DISCUSSION

non-steroi”al anti-inflammatory ”rugs an” other pharma“euti“al

interventions usually in”i“ate” for pain amelioration were not “olle“te”. This

is an important limitation to “onsi”er, sin“e it may be biasing the results

obtaine” for AIA.

A previous stu”y in the B-ABLE “ohort, foun” that the mean joint pain

in“rease” from VAS s“ore of 3 at baseline to 4.5 at 3 months follow-up305. In

our analysis, about 50% of women reporte” worsening pain both at 3 an” 12

months of AI-treatment342. Moreover, approximately 5% of women in the B-

ABLE “ohort ”is“ontinue” treatment ”ue to severe AIA.

Some stu”ies have ”emonstrate” by sonographi“, ele“trophysiologi“ an”

magneti“ resonan“e imaging measurements that patients with AI-relate”

arthralgia often have “hanges in their affe“te” joints an” ten”ons176,177. We

also aime” to explore this possibility in the B-ABLE “ohort. Thus, ”ynami“

ultrasonographi“ exploration in “ross-se“tional an” longitu”inal areas of 10

joints of the han”s (“arpus, 10 meta“arpophalangeal an” 10 proximal

interphalangeal joints) through ESAOTE Mylab 60 equipment (multifrequen“y

probe of 7-13 Hz equippe” with Doppler) was “on”u“te”. The exploration

metho” an” the ”iagnosti“ “riteria for synovial effusion, synovial

hypertrophy an” tenosynovitis are those ”es“ribe” an” re“ommen”e” by the

working group OMERACT-7285. The presen“e of synovial hypertrophy,

effusion an” the intra-arti“ular power-Doppler signal are being evaluate” by

a semi-quantitative s“ale from 0 to 3. In a””ition, “arpal ten”ons with

synovial sheath an” finger flexors are in“lu”e”. A global inflammation in”ex

resulting from the sum of the s“ores in both han”s has been ”efine”. So far,

no signifi“ant morphologi“ “hanges in the affe“te” joints an” ten”ons have

been foun”.

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DISCUSSION

A pro“ess of “entral sensitization might also be “onsi”ere”. Persistent pain

alters the nervous system, lowering the threshol” for pain generation an”

in“reasing the ”uration, amplitu”e an” spatial ”istribution of pain.

BMD EVOLUTION IN THE B-ABLE COHORT

The gol”-stan”ar” for estimation of BMD is the DXA te“hnique be“ause of

its repro”u“ibility, large normative ”ata, non-invasive nature, little time

requirement for pro“e”ure an” minimal ra”iation exposure. However, it is

essential to be aware of some pitfalls in the interpretation of BMD report.

The pre“ision a“hievable is ”epen”ent both on the ma“hine an” the subje“t

being measure”, an” it is limite” by statisti“al error of the signal, the

a““ura“y of the ”ete“tion of the bone e”ge an” the repro”u“ibility of

subje“t positioning. All the DXA s“ans of the B-ABLE patients have been

performe” on the same ma“hine an” by the same te“hni“ian, minimizing

thus the potential variability asso“iate” with some of these fa“tors.

However, while assessing BMD, s“an artefa“ts must be s“reene” sin“e they

may lea” to overestimation of the BMD. Te“hni“al artefa“ts in“lu”e metalli“

obje“ts like surgi“al “lips, navel rings, barium sulphate, metal from zipper,

“oin, “lip, or other obje“ts. A number of ”iseases may also interfere with the

a““ura“y of the BMD measurement. Thus, ”egenerative ”is“ ”isease with

osteophytes (ra”iographi“ markers of spinal ”egeneration representing

enlargements of the normal bone stru“ture), spon”ylosis, osteoarthritis,

s“oliosis, aorti“ “al“ifi“ation an” vertebral fra“tures “an also be present,

parti“ularly affe“ting patients age” 60 years or more343 (Fig. 18).

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DISCUSSION

Figure 18. Example of spinal degenerative changes in a DXA scan. Extra“te” from

Buehring et al. (Journal of Caquexia, Sar“openia an” Mus“le, 2013)344.

The effe“t of osteophytes “an be ”ramati“: the magnitu”e of in“rease in

BMD ”ue to osteophytes “an vary from 9.5% at L4 to 13.9% at L1345. Thus, it

has been reporte” that the effe“t of osteophytes on BMD is suffi“ient

enough to “ause 25% of women with osteopenia an” 10% of women with

osteoporosis to be mis”iagnose”343. Therefore, material “orre“tion by

removal of artefa“ts, or ex“lusion of the affe“te” vertebra shoul” be a

man”atory requirement. In patients with extensive ”egenerative ”isease, the

spine s“an may be of little ”iagnosti“ value. Inspe“tion of s“an images is

parti“ularly important when interpreting follow-up s“ans an” visual

“omparison shoul” always be ma”e with previous stu”ies. Hip s“ans also

require “areful s“rutiny as there is a wi”e range of anatomi“al variations,

some of whi“h “ause ”iffi“ulties in “orre“tly positioning the hip. However,

stru“tural “hanges an” artefa“ts interfering with DXA at proximal femur are

less often as “ompare” to spine346. In our stu”y, those follow-up s“ans results

showing unexpe“te”ly large rates of “hange were reviewe” to ex“lu”e the

mentione” fa“tors as a “ause. Our results in”i“ate” that LS BMD is

substantially influen“e” by ”egenerative fa“tors: when ex“lu”ing from the

143

DISCUSSION

analysis those patients with s“an artefa“ts, LS BMD re”u“tions were more

pronoun“e” (Fig. 19 A). All this suggests that the spine AIBL ”e“line in the

whole “ohort is partially obs“ure” by ”egenerative “hanges.

Figure 19. Summary of BMD evolution in the B-ABLE cohort patients without BP

treatment. Results are presente” as intra-in”ivi”ual per“ent “hange on LS an”

femoral ne“k (FN) BMD from baseline to the en” of AI therapy.

Nearly all ”ata available in”i“ate that AI are superior to tamoxifen148.

However, the ”e“line of plasma estrogen in”u“e” by AI is ”efinitely

asso“iate” with the re”u“tion of BMD245-248 an” in“rease” risk of bone

fra“ture154. The observe” BMD ”e“reases throughout the entire AI therapy in

B-ABLE patients were, generally, mil”er than those reporte” in previous

RCTs246,247,276,347,348. In”ee”, in some of these trials, women experien“e” mean

FN BMD ”e“reases almost twi“e as mu“h as B-ABLE “ohort patients.

There are a number of fa“tors that “an “ontribute to this phenomenon. First,

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DISCUSSION

some baseline patient “hara“teristi“s “an ”etermine ”ifferen“es in BMD

variation throughout the follow-up. Thus, for example, ”ifferen“es in lifestyle

an” ”ietary fa“tors between the stu”y “ohorts shoul” be kept in min”.

Moreover, the lower baseline BMD values in the B-ABLE “ohort, as “ompare”

with those reporte” in most of the mentione” stu”ies, “an lea” to a

regression to the mean bias. That is, those patients starting with low levels of

BMD are less prone to experien“e greater BMD ”e“reases. A““or”ingly,

signifi“ant asso“iation has been observe” between baseline BMD an” BMD

“hanges in other stu”ies349.

Se“on”, the stri“t monitoring of BMD as well as “al“ium an” vitamin D

supplements may also “ontribute to the less pronoun“e” BMD re”u“tions.

High vitamin D ”efi“ien“y rates at baseline have been ”es“ribe” in the B-ABLE

“ohort (~90% of patients). However, after 3 months of vitamin D

supplementation >70% of women were replete (Fig. 20).

Figure 20. Vitamin D levels in the B-ABLE cohort. Per“entage of women with vitamin

D serum levels below ”ifferent threshol”s (10, 20, 30 an” 40 ng/ml) after 3 months of

supplementation with VitD3 ”aily 800 IU + 16,000 IU every 2 weeks, among patients

145

DISCUSSION

who were vitamin D insuffi“ient an” ”efi“ient at baseline. Extra“te” from Prieto-

Alhambra et al. (Breast Can“er Resear“h an” Treatment, 2011)305.

Both higher vitamin D levels an” vitamin D in“reases at 3 months have

alrea”y been asso“iate” to lower BMD loss in B-ABLE patients350, supporting

the hypothesis that vitamin D repletion “an play a prote“tive role against

AIBL. The “ombine” benefit of bone loss attenuation an” ”e“rease” AI-

asso“iate” arthralgia strengthens the “ase for 40 ng/ml level as the optimal

therapeuti“ target. Thus, an in”ivi”ualise” vitamin D supplementation

regimen ”epen”ing on patient “hara“teristi“s an” ante“e”ents shoul” be

“onsi”ere”.

Thir”, the steroi”al stru“ture an” putative an”rogeni“ a“tivity of exemestane

might have ”ifferent effe“ts on bone “ompare” with the nonsteroi”al AI351.

Some stu”ies have even noti“e” BMD in“reases asso“iate” to this agent,

although not statisti“ally signifi“ant352. In the B-ABLE “ohort, nearly 30% of

women re“eive exemestane. This “onsi”erable proportion might also a““ount

to the lower BMD ”e“reases in the B-ABLE “ohort.

Finally, inherent ”ifferen“es between the stu”y ”esign of B-ABLE

(observational stu”y) an” RCTs “an also lea” to ”ifferen“es in the mentione”

out“ome. A““or”ingly, another observational stu”y on the impa“t of AI on

bone fra“tures foun” similar BMD losses at 3 years of follow-up than those

reporte” in our work353.

Previous tamoxifen treatment also mo”ifies the bone loss rate. Thus at the

en” of AI-treatment, those patients who ha” re“eive” previous tamoxifen

(pTMX-AI) experien“e” equal or even higher BMD re”u“tions in half the time

that patients re“eiving AI only (5y-AI) (Fig. 19).

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DISCUSSION

It is not easy to i”entify the exa“t “ause(s) of this aspe“t. Several stu”ies

have suggeste” that the effe“ts of tamoxifen on bone resemble those of

estrogen, preserving bone even in patients with breast “an“er354-356.

Unfortunately, this prote“tive effe“t “oul” be“ome a ”isa”vantage, lea”ing

to a reboun” effe“t: the early in“rease in BMD with tamoxifen may be

followe” by a steep ”e“rease when swit“hing to an AI. However, ”ifferen“es

in some other “hara“teristi“s (for example, tamoxifen is given to

perimenopausal women) “annot be rule” out as a possible “auses of the

”ifferen“e in BMD loss rates between both groups of our stu”y. In spite of

this, the more a““elerate” BMD loss in pTMX-AI patients ”oes not seem to

have “onsequen“es in absolute terms. That is, BMD values at the en” of

treatment in pTMX-AI an” 5y-AI groups were not statisti“ally ”ifferent.

EFFECTS OF BP ON BMD EVOLUTION

In the B-ABLE “ohort, women with osteoporosis or osteopenia plus 1 major

risk fa“tor or prevalent fragility fra“tures were allo“ate” to oral BP

treatment (BP-treate” patients). This group of patients experien“e”

signifi“ant in“reases in both LS an” FN BMD (Fig. 21).

As B-ABLE is not a RCT, we “annot assume that BP are responsible for the

observe” BMD in“reases: a group of patients with osteoporosis at baseline

but without BP treatment woul” be stri“tly ne“essary to ”raw any

“on“lusions about treatment effi“a“y. In our stu”y, pre-existing baseline

”ifferen“es between BP-treate” an” BP-untreate” groups “oul” influen“e

the stu”y results: both groups ”iffer naturally, at least, in BMD values at

baseline. Thus, for example, those patients with lower BMD at baseline woul”

be expe“te” to experien“e lower BMD rates, merely for a regression to the

147

DISCUSSION

mean effe“t. However, the “lear BMD in“rease observe” ex“lusively in BP-

treate” patients suggests an effe“t attributable to these agents. Neither

“an BMD in“reases be as“ribe” to vitamin D an” “al“ium supplementation,

sin“e the BP-untreate” group is also re“eiving the same ”oses of su“h

supplements. In this sense, reliable evi”en“e has been publishe” about the

effe“ts of BP on BMD. In several ran”omize” trials, BP signifi“antly in“rease”

BMD in postmenopausal women with early breast “an“er re“eiving AI248,272.

Therefore, we “onsi”er that BP therapy is the most likely “ause of the

observe” mean BMD in“rease BP-treate” patients.

Figure 21. Summary of BMD evolution in the B-ABLE cohort patients with BP

treatment. Results are presente” as intrain”ivi”ual per“ent “hange on LS an” FN BMD

from baseline to the en” of AI therapy.

The greatest BMD in“reases took pla“e ”uring the first year of the stu”y. As

for the subsequent time-points, BMD in“reases ”i” not yiel” statisti“al

signifi“an“e. Nevertheless, this effe“t may be “ause” by the substantial

”e“rease in the sample size ”uring the follow-up an” therefore the analyses

148

DISCUSSION

shoul” be repli“ate” when higher sample size is rea“he”.

BP therapy seems to in”u“e greater BMD in“reases in 5y-AI group. In fa“t,

pTMX-AI patients ”i” not a“hieve signifi“ant BMD in“reases with BP therapy.

The reboun” effe“t of tamoxifen “oul” also be “ontributing to this

phenomenon.

It is noteworthy that even though BP-treate” patients experien“e”

signifi“ant BMD in“reases, their absolute BMD values never rea“he” those of

non-BP-treate” patients. Thus, by the en” of treatment, average BMD

values for BP-treate” group still remaine” below those of BP-non-treate”

patients. In fa“t, although non-BP-treate” patients experien“e” substantial

BMD ”e“reases, only a few women be“ame osteoporoti“ ”uring the follow-

up. If we stri“tly a”here to preventing bone loss properties of BP one may

state that they shoul” only be pres“ribe” to osteoporoti“ patients. Some

other stu”ies in”i“ate that AIBL is relatively pre”i“table an”, thus, for women

with normal BMD before starting AI therapy, the risk of ”eveloping

osteoporosis over 3 to 5 years on AI is very low; Lifestyle a”vi“e,

reassuran“e, an” very limite”, if any, follow-up measurements of BMD are all

that is require”247. We nevertheless have to bear in min” that BP “oul”, in

a””ition, provi”e on“ologi“al benefit. Thus, a re“ent meta-analysis has

”emonstrate” that a”juvant BP re”u“e the rate of breast “an“er re“urren“e

in the bone an” improve breast “an“er survival in postmenopausal women357.

Overall, a”juvant BP may be “onsi”ere” in a broa”er range of

postmenopausal women.

BONE MICROARCHITECTURE: A POSSIBLE ROLE FOR TBS.

149

DISCUSSION

The measure of TBS has been propose” as a “lini“al tool “apable of assessing

trabe“ular bone mi“roar“hite“ture. In an attempt to “reate an analogy with

the three BMD “ategories, a working group of TBS users from ”ifferent

“ountries establishe” “ut-off points “reating the following range for TBS

values in postmenopausal women: TBS 1.350 is “onsi”ere” to be normal;

TBS between 1.200 an” 1.350 is “onsi”ere” to be “onsistent with partially

”egra”e” mi“roar“hite“ture; an” TBS 1.200 ”efines ”egra”e”

mi“roar“hite“ture358. At baseline, most patients in the B-ABLE “ohort ha” a

partially ”egra”e” mi“roar“hite“ture an” parti“ularly, the ”egra”e”

mi“rostru“ture “ategory prevaile” in patients allo“ate” to BP treatment.

In our stu”y, TBS experien“e” signifi“ant mean ”e“reases in the non-BP-

treate” patients at the en” of treatment359. Images with ”egenerative

“hanges were not ex“lu”e” in this parti“ular observational assessment. In

this sense, some stu”ies have shown that ”egenerative “hanges have little

effe“t on TBS. However, taken into a““ount the effe“ts of osteophytes on

BMD, it is probably that TBS re”u“tions are less pronoun“e” than the true

BMD ”e“lines.

Tamoxifen has also ”emonstrate” to in”u“e a “ompensation an”/or

stabilization of bone texture parameters291. Similarly to BMD, TBS also

”e“rease” more sharply in pTMX-AI group359. The same reboun” effe“t

”es“ribe” for BMD may also be the un”erlying “ause.

In “ontrast to BMD, TBS ”i” not in“rease in BP-treate” patients an” remaine”

un“hange” from baseline359. As with BMD in“reases, we “annot grant that

the stabilization of TBS is “ause” by BP. However, similar observations were

ma”e in RCTs evaluating the effe“ts of BP on TBS360. Overall, TBS is sensitive

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DISCUSSION

to “hanges in estrogen status an” remains un“hange” when BP therapy is

a””e”. This is not surprising, be“ause one woul” expe“t a greater

improvement on BMD with antiresorptive therapy, resulting from in“rease”

mineralization, than in trabe“ular mi“rostru“ture.

TBS stems from the nee” to “apture those fragility fra“tures o““urring in

the osteopeni“ or even the normal range of BMD. In our stu”y, “hanges in

TBS an” BMD were only weakly “orrelate”, supporting the “ontention that

the two measures refle“t ”ifferent aspe“ts of bone status359. However, it

remains to be stablishe” whether TBS variation exert a “lini“ally relevant

effe“t on fra“ture risk. Moreover, there is some ”ata suggesting that AI

substantially affe“t “orti“al bone “ompare” with trabe“ular bone361. The

in“i”en“e of hip or femoral fra“ture among postmenopausal women

un”ergoing breast “an“er therapy seems to be higher than the in“i”en“e of

vertebral fra“tures. Furthermore, hip fra“tures o““urre” mu“h earlier in

women with breast “an“er than in healthy women362. Until now, TBS has only

been implemente” for spine, so it may overlook some a portion of the risk of

hip fra“ture.

HIGH VARIABILITY IN THE STUDIED OUTCOMES

In the previous se“tions, a”verse effe“ts of AI in the B-ABLE “ohort have

been ”es“ribe” in a “omprehensive manner. There is no question, however,

that one of the most remarkable fin”ings in this work is the great variability

in the magnitu”e of the stu”ie” out“omes.

Although one “an state that me”ian VAS for joint pain in“rease” from

baseline to 3 months of follow-up in the B-ABLE “ohort, it is noteworthy

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DISCUSSION

that only 50% of women with no joint pain at baseline reporte” in“i”ent

joint pain at 3 months. From those with prevalent join paint at baseline,

50% reporte” ha” a worse VAS at 3 months but 25% reporte” no “hange

an” approximately 25% even got better305,342.

As regar”s BMD variation ”uring AI treatment, these ”ifferen“es be“ame

evi”ent when analysing patient ”istribution by BMD “hange “ategories. While

it is true that most of BP-non-treate” patients un”ergone “lini“ally

signifi“ant re”u“tions in BMD, just over a thir” of women in this group

ha”n t yet experien“e” any “hange in LS an”/or FN BMD by the se“on” year

of AI363. Some previous stu”ies have also reporte” su“h BMD variability250.

Similar variation was ”ete“te” in TBS. At the en” of AI-therapy,

approximately 25% of non-BP-treate” patients ”e“rease” by one TBS

“ategory, while the remaining in”ivi”uals persiste” in their baseline “ategory.

Even more variability was ”ete“te” in the BP-treate” group, in whi“h 14% of

women raise” from TBS “ategory359.

Several sour“es of te“hni“al an”/or biologi“al variability may un”erlie these

observations.

Ø S“an artefa“ts “an “ause falsely BMD measurements345. Thus for

example, some BMD in“reases >3% observe” in our stu”y “oul” be

explaine” by these anomalies. However, BMD variability remaine” even

after the ex“lusion from the analysis of those patients with s“an

artefa“ts364. Moreover, FN BMD variation, whi“h is not so sus“eptible to

the presen“e of osteophytes, also presents high inter-patient variability.

Consequently, other relevant fa“tors must be “onsi”ere”363,364.

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DISCUSSION

Ø Coeffi“ient of variation (CV) of DXA: CV is the most “ommonly

presente” measure for BMD s“ans repro”u“ibility an” it ”epen”s on

quality assuran“e fa“tors, in“lu”ing quality “ontrol tests, performan“e

of the ma“hine as well as the experien“e of the operator. The small

unexpe“te” “hanges in BMD observe” in some patients “oul” be

attribute” to the CV, but those “hanges higher than 3% “an har”ly be

explaine” by this phenomenon.

Ø Despite a “umulative loa”ing of 168,000 IU of vitamin D supplements, a

“lini“ally signifi“ant proportion of women faile” to attain a”equate

vitamin D status within 3 months305. As previously mentione”, an

asso“iation between vitamin D serum levels an” AIBL has been ”es“ribe”:

those patients with improve” vitamin D status using supplementation

showe” attenuation of bone loss350. In this sense, vitamin D

supplementation may also help to explain a portion of the observe” BMD

in“reases in those patients who are not re“eiving BP. Furthermore, the

in“rease in joint pain was signifi“antly attenuate” in those that rea“he”

high vitamin D “on“entrations, with a lower risk of in“i”ent arthralgia305.

Thus, the interin”ivi”ual ”ifferen“es in vitamin D improvement/status

may be influen“ing the variability of both AIBL an” AIA.

Ø While stu”ies have foun” that a”juvant hormonal therapy for hormone-

sensitive breast “an“er ”ramati“ally re”u“es re“urren“e an” mortality,

a”heren“e to me”i“ations is suboptimal185-189. Non-a”heren“e may also

“onstitute a plausible justifi“ation for an important part of the

interin”ivi”ual variability in the treatment-asso“iate” se“on”ary

out“omes. In the B-ABLE “ohort, self-reporte” a”heren“e to AI, BP an”

vitamin D supplements is assesse” by physi“ian questionnaire at ea“h

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DISCUSSION

time-point. Self-report is a wi”ely a““epte” an” applie” metho” to

assess me”i“ation a”heren“e, however, this may be less reliable to fully

refle“t true a”heren“e. This, in fa“t, is one of the limitations of our

stu”y. Non-a”heren“e “an either affe“t AI, BP as well as “al“ium an”

vitamin D supplements. Therefore, a “ombination of treatment

effi“a“ies “an be ”erive”, lea”ing to a large variability in the se“on”ary

out“omes. Thus, for example, some authors have alrea”y asso“iate”

BMD loss in BP-treate” patients with poor a”heren“e to BP treatment353.

Ø Emotional state, in”ivi”ual's evaluation of his/her own health “on”ition,

so“ial support, health beliefs, e”u“ation level, psy“hologi“al problems as

well as so“ioe“onomi“ status “an also influen“e pain feeling365. These

variables are, in turn, “losely linke” to treatment “omplian“e299. Thus,

psy“hotherapy in“lu”ing relaxation training, biofee”ba“k, visual imagery,

”istra“tion metho”s an” psy“hiatri“ intervention “oul” effe“tively

alleviate pain-relate” emotional stress an” ”epression366.

Ø Besi”es the reporte” “lini“al fa“tors, geneti“ ba“kgroun” may also be of

importan“e to interin”ivi”ual variability. Hen“e, some geneti“ variants in

CYP19A1324,325, ESR1326 an” TCL1A327 ha” been asso“iate” so far with AIA.

A““or”ingly, in our stu”y, a number of SNPs in CYP17A1, CYP27A1 an” VDR

genes were asso“iate” with both joint pain intensity an” worsening

”uring AI-therapy. Moreover, one SNP in CYP27A1 was also relate” to AI-

therapy ”is“ontinuation ”ue to severe arthralgia342. Subsequent to our

stu”y, Lintermans et al.367 teste” the vali”ation of SNPs in sele“te”

genes, base” on literature review, with AI-relate” mus“uloskeletal

symptoms. They only foun” one SNP in OPG gene yiel”ing signifi“ant

asso“iation. No “orrelation between any other teste” SNPs, in“lu”ing

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DISCUSSION

those in CYP17A1 i”entifie” in our stu”y, was foun”. In a““or”an“e,

another stu”y also foun” OPG an” RANKL368 to be relate” to AI

mus“uloskeletal si”e effe“ts. Differen“es in ”efinitions of out“omes an”

patient “ohorts use” are probably responsible for these in“onsisten“ies.

We have also ”es“ribe” an asso“iation between SNPs in CYP11A1 an”

bone loss364. Other stu”ies foun” a””itional polymorphisms to be

asso“iate” with bone loss among patients taking AI, ”uring the “ourse

of our work. Thus, Napoli et al.369 reporte” one SNP in CYP19A1 gene to

be asso“iate” with AIBL. Another stu”y in 2015 foun” that SNPs in ESR1,

ESR2 an” CYP19A1 were asso“iate” with ”e“rease” bone ”ensity in

letrozole an” exemestane-treate” patients348. Moreover, a GWAS in

2014370 i”entifie” SNPs in or near CTS–-SLMO2-ATP5E, TRAM2-

TMEM14A, an” MAP4K4 genes that were asso“iate” with risk for bone

fra“ture in AI-treate” women. Interestingly, these genes all ”isplaye”

estra”iol (E2)-”epen”ent in”u“tion. The ”is“overy of su“h large number

of asso“iations of SNPs in genes involve” in estrogen synthesis pathway

with AI se“on”ary effe“ts (an”/or effe“tivity) suggests, on“e again,

that the estrogen environment is essential for the tolerability of these

”rugs. In our work, SNPs in CYP17A1 an” CYP11A1 have been asso“iate”

with AI si”e effe“ts. As previously mentione”, steroi”ogenesis

regulation is ”etermine” by CYP11A1 (en“o”ing P450s““ protein) gene

expression an” other ”ownstream enzymes, espe“ially P450“17

(en“o”e” by CYP17A1). These genes, therefore, have a key role in the

lo“al sex steroi” hormone levels, probably influen“ing also some AI

se“on”ary out“omes su“h as AIBL an” AIA.

155

DISCUSSION

INTERINDIVIDUAL DIFFERENCES IN SEX-STEROID LEVELS: A POSSIBLE

UNDERLYING MECHANISM FOR VARIATION IN AI SECONDARY EFFECTS.

Despite the ”iffi“ulties in measuring in vivo estrogen “on“entrations, it is

overall a““epte” that AI re”u“e whole-bo”y aromatization by 90.6%

98.9%. However, subtle ”ifferen“es in the ”egree of hormone suppression

among the 3 thir”-generation AI have been ”es“ribe”145. More interesting,

though, is that some stu”ies have reporte” unexpe“te”ly high plasma

estrogen levels in AI-treate” patients. Thus Kallak et al.371 evaluate”

aromatase in”ex in long-term AI treatment, showing that the pattern of

estrogen levels is more unpre”i“table than the ”es“ribe” in previous RCTs. In

a””ition, a wi”e interin”ivi”ual range of “on“entrations of “ir“ulating

estra”iol an” estrone were reporte”.

We personally agree with the fin”ings in”i“ating su“h a high interin”ivi”ual

variability in estrogen levels ”uring AI therapy. In fa“t, we hypothesize that

this is one of the me“hanisms un”erlying the wi”e range of intensities

asso“iate” to these me”i“ations.

Poor ”rug poten“y an” alternative sour“es of estrogeni“ hormones are only

likely to be the “ause of the interin”ivi”ual variability in estrogen levels

”uring AI therapy in o““asional “ases. Non-a”heren“e an” ”rug-intera“tions

also “onstitute a plausible justifi“ation for an important part of this

interin”ivi”ual variation372. Moreover, a positive relationship has been foun”

between estrogen levels an” BMI ”uring treatment with AI373. A”ipose tissue

is the main sour“e of estrogen biosynthesis in postmenopausal women, so

that in“omplete estrogen ”epletion in women with high BMI “an o““ur, even

lea”ing to re”u“e” effi“a“y of AI in obese women408,409. In spite of this, Kallak

156

DISCUSSION

et al. state” that the observe” interin”ivi”ual ”ifferen“es among AI-treate”

patients in their stu”y “oul” not be attributable to in”ivi”ual ”ifferen“es in

BMI nor other variables as time sin“e menopause or self-reporte”

a”heren“e371. Therefore, other fa“tors may be “ontributing to this

phenomenon.

We hypothesize that geneti“ variants are responsible, at least in part, of the

interin”ivi”ual variability in host sex-steroi” an” vitamin D environment

”uring AI-therapy. This, in turn, translates into the interin”ivi”ual variability

in the se“on”ary effe“ts (an” effe“tivity) of AI (Fig. 22).

Figure 22. Scheme of the hypothetic mechanisms underlying the interindividual

variability in AI effectivity and in their associated side effects.

Some fin”ings woul” un”erpin our hypotheses; First, there is a wi”e range in

157

DISCUSSION

the response rates to AI: per“entages between 35 an” 70% in neoa”juvant

stu”ies have been reporte” an” benefits may be lower in a”van“e”

”isease374. In that “onne“tion, resistan“e to AI has alrea”y been asso“iate”

with SNPs in CYP19A1375. In fa“t, it has been alrea”y ”emonstrate” that

high/raise” levels of aromatase may prevent effe“tive blo“ka”e by

inhibitors. A””itionally, the appearan“e of AI unwante” effe“ts has been

”emonstrate” to be a pre”i“tor of en”o“rine treatment response193,376.

Therefore, one “oul” infer that geneti“ variation ”etermining therapy

effe“tiveness may also influen“e the onset an”/or intensity of a”verse

effe“ts.

Se“on”, the importan“e of the low resi”ual estrogen in breast “an“er

patients has alrea”y been ”emonstrate”. Thus, the ATAC trial ”ete“te” an

inverse “orrelation between baseline estra”iol levels an” BMD “hanges: lower

baseline estra”iol is a pre”i“tive not only of lower baseline BMD but also of

greater BMD losses ”uring AI therapy. Remarkably, no other variables

retaine” signifi“an“e after these “ovariates were in“lu”e” in the mo”el349.

Before the wi”esprea” use of AI in breast “an“er patients, the importan“e of

the low resi”ual estrogen levels in postmenopausal women was alrea”y

”emonstrate”. Thus, for example, the lower levels of serum estra”iol are

asso“iate” with the higher rates of bone loss an” the greater risk of

fra“tures in late postmenopausal women377. The me“hanism by whi“h su“h

low estrogen “on“entration be“omes so important remains unknown.

However, some “lini“al observations suggest that long-term ”eprivation of

estra”iol may “ause a”aptive hypersensitivity to low levels of estra”iol357. In

fa“t, there is some evi”en“e that women starting AI who ”evelop joint

manifestations “an experien“e improvement over time165 sin“e tissues may

158

DISCUSSION

a”just to the low estrogen “on“entrations378.

In short, those women with a geneti“ ba“kgroun” favouring a su““essful

inhibition of estrogen synthesis by AI woul” show high intensity-si”e

effe“ts, in”i“ating, in turn, higher likelihoo” of respon”ing to en”o“rine

treatment. On the “ontrary, a la“k of estra”iol response woul” not trigger

symptoms but, unfortunately, breast “an“er re“urren“e prevention woul” be

lower. If true, new pre”i“tive mo”els for hormone treatment response an”

si”e effe“ts intensity “oul” be a“hieve” by “ombining geneti“ tests with

other “lini“al pre”i“tive fa“tors. In this sense, SNPs CYP11A1, CYP27B1, VDR

an” CYP17A1 are likely goo” “an”i”ates for this purpose.

THE ROLE OF LOCAL ESTROGEN SYNTHESIS

Even if “ir“ulating estrogen levels are often use” to evaluate AI treatment

effi“a“y, its importan“e has been questione”. No “orrelation between lo“al

an” plasmati“ estrogen levels has been ”es“ribe”. This is suppose”ly ”ue to

”ifferential uptake from the “ir“ulation an”/or lo“al estrogen pro”u“tion.

The ability to “onvert “holesterol into pregnenolone via P450s““ “onstitutes

the essential prerequisite for a tissue to be steroi”ogeni“. The presen“e an”

bio“hemi“al a“tivity of P450s““ an” P450“17, ”o“umente” in some tissues as

human skin an” brain, “hanges the para”igm of the steroi”ogeni“ “apa“ity

“onfine” so far to the a”renal glan”, ovaries, an” testis. Bone has long been

regar”e” as just one of those “ompartments with “apa“ity to synthesize

estrogens from serum an”rogen pre“ursors. However, in our stu”y, the

expression of P450s““ an” P450“17 at both mRNA an” protein level has been

also ”ete“te” in bone “ells364. This fin”ing evi”en“es that bone possess all the

enzymati“ ma“hinery for ”e novo estrogen synthesis, opening the possibility

159

DISCUSSION

for this tissue to be “onsi”ere” as a steroi”ogeni“ “ompartment. However,

further work is man”atory in or”er to ”emonstrate the enzyme

fun“tionality in this “ompartment.

The “orroboration of bone as steroi”ogeni“ tissue woul” entail a growing

importan“e of lo“al estrogen synthesis. The “hallenge will “ome in

”etermining the fun“tional signifi“an“e of this pathway, sin“e its

“ontribution to lo“al estrogen levels may have important therapeuti“

impli“ations. The importan“e of this aspe“t lies in the fa“t that the low

“ir“ulating levels of estrogens whi“h are observe” in postmenopausal

women have no bearing on the “on“entrations of estrogen rea“he” in

extragona”al site. The signifi“an“e of this para”igm shift “annot be

un”erestimate”, namely that the estrogen whi“h is responsible for breast

“an“er ”evelopment an” for the maintenan“e of bone mineralization an”

“ognitive fun“tion is not “ir“ulating estrogen but rather that whi“h is

pro”u“e” lo“ally at these spe“ifi“ sites within the breast, bone an” brain.

This raises the possibility of using pharma“ologi“ organ or tissue-spe“ifi“

estrogen ”eprivation as an improve” means of targete” therapy. For

example, sele“tive lo“al in”u“tion of this pathway in bone/joint “an be of

therapeuti“ benefit in a “ontext of estrogen re”u“tion while suppressing

breast estrogen levels.

P450SCC IN BONE: FURTHER UNKNOWN FUNCTIONS?

P450s““ have two known isoforms, the full length 60-kDa (521 aa) protein

an” a unique shorter isoform known as isoform b of 42-kDa (363 aa),

missing aa from 1 to 158 from the “anoni“al sequen“e (Fig. 23). This shorter

160

DISCUSSION

isoform is regulate” by alternative spli“ing an” utilization of an internal

translational start “o”on. Unlike the full-length protein, isoform b is not

“on“entrate” into mito“hon”ria.

Figure 23. Full-length and short isoform transcripts of CYP11A1 gene. A”apte” from:

Teplyuk et al. (Mole“ular En”o“rinology, 2009)379.

In our analysis, both isoforms of P450s““ were ”ete“te” in human primary

osteoblasts an” fresh bone tissue364. In fa“t, Teplyuk et al. ha” alrea”y

”ete“te” the expression of the short isoform in mouse an” human

osteoblasti“ “ell lines379. Although its fun“tion remains unknown, they foun”

that kno“k”own of this isoform b in“rease” osteoblast proliferation,

suggesting that it “ontributes to Runx2-me”iate” attenuation of “ell

growth. There are still outstan”ing tasks in or”er to fin” out the fun“tion of

this isoform b in bone “ells.

VITAMIN D ACTIVATION AND AI SECONDARY EFFECTS

In this stu”y, SNPs in VDR an” CYP27B1 were asso“iate” with AIA, suggesting

an important role of vitamin D in the un”esirable joint symptoms342.

CYP27B1, en“o”es for the key 1-hy”roxylase enzyme, pro”u“ing 1,25(OH)2D.

161

DISCUSSION

VDR, in turn, en“o”es the re“eptor for vitamin D. Both genes a“t ”ownstream

of 25(OH)D.

It is worth mentioning that no signifi“ant ”ifferen“es in 25(OH)D serum

levels were foun” among genotypes for any SNP at 3 or 12 months of

follow-up342. The 1,25(OH)2D, the only biologi“ally a“tive form of vitamin D

has not been assesse” in the B-ABLE “ohort sin“e the thigh regulation

together with its short half-life an” the “ir“a”ian variation levels makes it a

ba” a““urate means to estimate vitamin D status. Thus, when trying to

”iagnose vitamin D ”efi“ien“y or to assess vitamin D stores, 25(OH)D is the

preferre” form380.

In“reasing vitamin D substrate via higher ”oses may in“rease the a“tive

hormone 1,25-”ihy”roxyvitamin D with resultant re”u“tion in joint

symptoms. However, this intervention woul” be insuffi“ient for those

patients with unfavourable genotypes for some SNPs in CYP27B1 an” VDR.

As a result, normal serum “on“entrations of 25(OH)D an” AIA “oul” “oexist

in the same in”ivi”ual.

Su“h unfavourable geneti“ ba“kgroun” “oul” be a potential pre”i“tor for

”eveloping joint ”is“omfort ”uring AI therapy. The ability to anti“ipate these

symptoms may en“ourage patient physi“ian “ommuni“ation, treatment

“omplian“e an” imme”iate ”e“ision-making for the benefit of the patient.

THE CHALLENGES AHEAD OF THE STUDY

This work has “ontribute” to the un”erstan”ing of AI-asso“iate” se“on”ary

effe“ts in postmenopausal women with breast “an“er. However, several

issues remain to be settle”.

162

DISCUSSION

I”entifi“ation of “ausal sequen“e variants

The sample size of the B-ABLE “ohort is still limite” in terms of fin”ing

strong “orrelations of geneti“ variants with mo”est effe“t an” quantitative

traits. Thus, further resear“h is nee”e” to vali”ate the ”es“ribe” SNPs as

pre”i“tors of AI-asso“iate” un”esirable symptoms. Repli“ation of our

”is“overies in larger an” in”epen”ent “ohorts woul” be thus highly valuable.

In a””ition, asso“iation ”oes not mean “ausality. Exten”ing these initial

asso“iative fin”ings to i”entifi“ation of the true “ausal variants involves

sear“hing through DNA regions in the vi“inity of ”isease-asso“iate” SNPs.

Those variants in fun“tional elements in“lu”ing protein “o”ing, regulatory

an” stru“tural sequen“es are of true interest. We have starte” working on

this “hallenging task: as a preliminary result, we have foun” a mi“rosatellite

polymorphism lo“ate” at 528 base pairs upstream of the translation level of

CYP11A1. In this regar”, this polymorphism has been alrea”y asso“iate” to

some other interesting phenotypes su“h as poly“ysti“ ovary syn”rome381.

Another key issue with respe“t to our fin”ings relates to the fra“ture

phenotype. Unfortunately, there are few ”ata ”es“ribing the heritability of

osteoporoti“ fra“ture, mainly be“ause re“ruiting a”equate numbers of

stu”y subje“ts with fra“ture is ”iffi“ult an” expensive. Several stu”ies have

shown that a family history of fra“ture is a risk fa“tor for fra“ture, an”

importantly, this is in”epen”ent of BMD382,383. Some others have even noti“e”

that the genes involve” in fra“ture may be separate to those influen“ing

BMD384. Therefore, fin”ing the genes responsible for BMD variation ”oes not

ne“essarily i”entify genes “ausative for fra“ture. In“i”ent fra“tures

thorough AI therapy are being re“ruite” in the B-ABLE stu”y an” the

163

DISCUSSION

“orrespon”ing analysis will be ”one when an appropriate sample size is

a“hieve”.

Sex steroi” hormone levels in AI-treate” women

AI are “onsi”ere” to “ause estrogen level suppression to un”ete“table levels.

However, reliable evi”en“es point to a higher than expe“te” interin”ivi”ual

variability in the estrogen levels ”uring AI therapy371,373. For the purpose of

exploring this issue, serum samples of B-ABLE “ohort patients are “urrently

being “olle“te”.

Taking a”vantage of the previous goal, we also “onsi”ere” of strong interest

to assess “ir“ulating an”rogen levels in B-ABLE patients. It is likely that

an”rogens are able to pro”u“e potent effe“ts in mus“uloskeletal tissue

without influen“e of the aromatase enzyme385-387. Thus, an”rogens “oul” be

“lini“ally relevant, for instan“e, in the “ourse of treatments that mo”ify

their availability, an” parti“ularly when AI are use”. Therefore, it woul” be

interesting to obtain information on the influen“e of AI on the serum levels

of pre-an”rogens an” an”rogens. Spe“ifi“ally, to “larify if the se“on”ary

effe“ts of AI are affe“te” by an”rogen levels, before an” ”uring AI

treatment.

At present, 50 serum samples have been teste” for estra”iol, estrone, estrone

“onjugates, testosterone an” an”rostene”ione levels at baseline an” at 3

months of AI therapy. In a““or”an“e with our hypothesis, a great variability

in hormone levels is expe“te”. Up“oming analyses will examine potential

“onne“tions between the polymorphi“ variants ”es“ribe” in our stu”y, the

hormone levels an” the intensity of mus“uloskeletal effe“ts of AI therapy.

The ”is“overy of a relationship between these fa“tors may she” light on the

164

DISCUSSION

“ausality of AI se“on”ary effe“ts.

Bio“hemi“al assessment of bone remo”elling an” “artilage ”egra”ation

”uring AI treatment

Bio“hemi“al monitoring of bone metabolism ”epen”s upon measurement of

enzymes an” proteins release” ”uring bone formation an” of ”egra”ation

pro”u“ts pro”u“e” ”uring bone resorption. The fiel” of bone turnover

markers (BTM) has ”evelope” “onsi”erably in the past ”e“a”e an” various

bio“hemi“al markers are now available that allow a spe“ifi“ an” sensitive

assessment of the rate of bone formation an” bone resorption of the

skeleton388. The “ombine” use of BMD an” TBS measurement as well as bone

markers is likely to improve the assessment of the risk of fra“tures389.

In several of the trials analysing AI effe“ts on bone, markers of bone

resorption an” formation were in“rease” ”uring AI therapy246,390,391. By

“ontrast, BTM remaine” “onstant or ”e“rease” with tamoxifen therapy390,391.

In or”er to a””ress this point, B-ABLE also re“ruits information of BTM. Thus,

P1NP (type 1 pro“ollagen), NTX (urine N-telopepti”e), CTX, (C-telopepti”e),

OC (osteo“al“in) an” BALP (serum bone spe“ifi“ alkaline phosphatase) are

being measure” at ea“h follow-up visit. Our preliminary analyses agree with

the previous reporte” results: both resorption an” formation BTM in“rease”

at both 3 an” 12 months of AI therapy. By “ontrast, in the group of patients

re“eiving BP, a ”e“rease of all BTM has been ”ete“te”. These analyses will be

repeate” when more sample size is a“hieve”.

Cartilage ”estru“tion lea”s to an a““umulation of break”own pro”u“ts in the

synovial flui”. These are release” into the “ir“ulation an” ultimately filtere”

an” ex“rete”, or broken ”own in vivo392. Analysis of bo”y flui”s “an provi”e

165

DISCUSSION

information regar”ing the health, or turnover of the “artilage, prior to the

”evelopment of gross pathology, or “an highlight any metaboli“ “hanges

attributable to the treatment being stu”ie”. In this regar”, the low-estrogen

state in ovarie“tomize” animals in”u“es a““eleration of “artilage turnover,

presumably from their low-estrogen state393. Thus, P2NP (type 2

pro“ollagen), C2M an” C3M have been alrea”y measure” in a group of

patients of the B-ABLE “ohort. These ”ata, whi“h are “urrently un”er

evaluation, will explore the possibility that the AI-in”u“e” estrogen

”efi“ien“y is “ontributing to AIA from la“k of “ushioning in the joints.

Assessment of “artilage an”/or bone turnover markers may bring the

possibility for physi“ians to pre”i“t whi“h patients are eligible for therapy

intervention before the manifestation of the pathologies (that is, high

arthralgia intensity, with it asso“iate” risk of therapy ”is“ontinuation an”/or

bone mineral ”ensity loss with its asso“iate fra“ture risk).

167

Chapter4

Si realmente viéramos al Universo, tal vez lo enten”eríamos

Jorge Luis Borges

CONCLUSIONS

169

CONCLU

SIONS

1. In a“tual “lini“al pra“ti“e, AI treatment in women with breast “an“er

lea”s to joint pain, whi“h ultimately affe“ts treatment “omplian“e.

2. In a“tual “lini“al pra“ti“e, AI treatment in women with breast “an“er

lea”s to an a““elerate” bone mineral ”ensity loss an” bone

mi“roar“hite“ture ”eterioration.

3. In a“tual “lini“al pra“ti“e, those patients with breast “an“er on AI

treatment re“eiving oral BP experien“e signifi“ant in“reases in bone

mineral ”ensity an” a positive maintenan“e of bone

mi“roar“hite“ture.

4. Geneti“ variants in genes involve” in estrogen an” vitamin D

metaboli“ pathways play a key role in the risk to suffer AI-

asso“iate” mus“uloskeletal si”e effe“ts:

4.1 Geneti“ variants in the CYP17A1, VDR, an” CYP27B1 genes “an help

to pre”i“t the risk of AIA.

4.2 Geneti“ polymorphisms of the CYP11A1 gene are asso“iate” with

AIBL.

5. The bone possess all the enzymes involve” in the steroi”-synthesis

pathway, raising the possibility that this tissue “an synthesize

an”rogens an” estrogens in”epen”ently of serum steroi” pre“ursors.

6. Monitoring of bone health as well as “al“ium an” vitamin D

supplementation are essential for the “lini“al management of the

”etrimental effe“ts of AI on bone tissue.

171

BIBLIO

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