1

Cáncer de Mama y RE

• 70% son RH+ (Luminal)• Estrógenos /RE –> CM

– Exposición a E riesgo– Uso de antiéstrógenos incidencia CM RH+– Antiestrógenos – Tto eficaz en CM RH+

• CDIS recurrencia• SVLP y SG• Eficaz en CMM RH +

• CM local – 20-30% recurrencias tras HT adyuvante

• CMM – HT: Respuesta inicial Progresión

2

3

Rondon-Lagos M et al IJMS 2016

RE – miembro de la superfamilia de receptores

nucleares

4

ESR1 – Cr 6

ESR2 – Cr 14

• AF1 – lugar de fosforilación (MAPKs…), A y B, activación transcripcionalindependiente de ligando

• DNA – binding domain• Ligand binding Domain (LBD- AF2) activación transcripcional dependiente de

ligando

• La actividad del ERα es la predominante en CM y es el que se expresa mayoritariamente en células de CM

• El papel del Erβ en CM esta menos definida

Mecanismos moleculares de la actividad del RE

5

Mediado por Ligando (Estrógeno)Genomico – nuclear- Clásico: Factor de

transcripción (CoA, CoR)- No clásico: Coregulador de

otros FT

No genómico – (ER membrana, citoplásmico)- Interaccion con otras vias de

señalización

No mediado por ligando- Fosforilacion del ER (RTKs-GFR)

Thomas C NRC 2011; Osborne JCO JCO 2005

ER

Nature Reviews | Cancer

EGFR IGFR ERBB2

Oestrogen

PI3K

PI3K PI3K

SRC

SRC

AKTAKT ERKERK

GP

RAS

Cytoplasm

Nucleus

GF

TF

TFTF

REERE ERE ERE RE

ER

ER

ER

ER ER

CoAHAT

P

P

P

P

mRNA

M

Cell cycle regulators, pro-apoptotic proteins, anti-apoptotic proteins, cell adhesion molecules, and components of the membr ane and cytoplasmic signalling cascad e

P

P

UL

ER ER

CoRHAT UL

ER

CoAor CoR

HAT UL

Or

‘Pure’ anti-oestrogens

Drugs that bind the oestrogen

receptor, thereby blocking

the effect of oestrogen, but

that have no detectable

oestrogen-like effects. Most

have a steroidal structure.

synthesis is repressed by the methylation of ER promot-

ers. Promoter hypermethylation has been significantly

associated with the loss of ERα and ERβ in the major-

ity of cancers and cancer cell lines. DNA demethy lating

agents (such as, 5-AZAC (5-aza-2D-deoxycytidine))

were reported to restore ERβ expression in cancer

cells19,43. A number of ER subtype-specific microRNAs

(miRNAs) can affect ER expression, and an inverse

correlation between specific miRNAs and ER levels

has been detected in various cancers49,50–53. Following

translation, the native, unbound ER subtypes are stable

as they form a complex with the HSPs54. Ligand bind-

ing dissociates the receptors from these chaperones

and increases their proteasome-mediated degradation.

This process requires alteration in the phosphory-

lation status of ERs and the interaction of the receptors

with several proteins, including ubiquitin ligases and

ubiquitin-binding proteins. Modulations in the levels

of endogenous ligands, alterations in the tumour micro-

environment (such as, hypoxia), downregulation of

chaperones and increased expression of ubiquitin ligases

are implicated in the proteasome-mediated degradation

of the ERs (FIG. 3). HSP90 inhibitors, including geldana-

mycin, radicicol, physiological oestrogens and ‘pure’

anti-oestrogens (such as, fulvestrant), but not ‘partial’

anti-oestrogens (such as, tamoxifen), downregulate

ERα by increasing its rate of degradation18. Ubiquitin-

protein ligase E3A (UBE3A; also known as E6AP) and

MDM2 are among the ubiquitin ligases that have been

shown to promote the degradation of ERα55,56 (FIG. 3).

Two recent studies have suggested that CUE domain-

containing protein 2 (CUEDC2), a ubiquitin-binding

motif-containing protein, and lemur tyrosine kinase 3

(LMTK3) regulate the stability of ERα and affect the

endocrine resistance of breast cancer57,58. It remains for

future studies to identify whether the upregulation of

novel ubiquitin ligases is a crucial step in the downregu-

lation of the ERs in cancer. This post-transcriptional

regulation of the ERs questions the validity of studies

that based their clinical assessment of ERs on the detec-

tion of ER mRNA and may account for the discrepancy

between ER mRNA and protein levels.

ERs and cell proliferat ion

ERα. Evidence from cell-based and animal model

studies has established the idea that the ER subtypes

influence cancer biology and therapy. Analysis of

Esr1-knockout mice showed phenotypic changes that

are associated with the gonads, skeletal system and

reproductive tract and revealed that ERα is required

for the normal development of the mammary gland59,60.

Similar effects of ERα in the breast have been observed

in mice with CRE-mediated deletion of Esr1 in the epi-

thelium of the mammary gland61. When Esr1-knockout

mice were crossed with mice that develop tumours

owing to the expression of the mouse mammary tumour

virus (MMTV)–Wnt1 transgene or a mammary-specific

mutant of Erbb2, the onset of tumour development in

the offspring was delayed62,63. Similarly, the incidence

of oestrogen- and DMBA-induced mouse mammary

tumours was reduced by the loss of ERα, suggesting that

ERα can influence mammary carcinogenesis64,65. ERα

has additionally been implicated in prostate tumori-

genesis10. Oestrogens were able to stimulate prolif-

eration and the appearance of multilayered prostatic

epithelial cells, a phenotype known as squamous meta-

plasia (SQM), in wild-type, but not in Esr1-knockout,

mice, indicating the involvement of ERα in the induction

of this pathology66. This ERα-mediated increase in pro-

liferation has been linked to inflammation and tumour

development. Treatment with the synthetic oestrogen,

diethylstilbestrol (DES), during neonatal life stimulated

inflammatory cell infiltration in the prostate of wild-

type, but not of Esr1-knockout, mice67–69. In addition, the

ERα-selective antagonist toremifene decreased the inci-

dence of high-grade prostatic intra epithelial neoplasia

(PIN) and prostate cancer in transgenic adenocarcinoma

Figure 2 | Molecular mechanism of ER action. In the classical mechanism of oestrogen

receptor (ER) action, ligands such as oestrogen bind to ERs and the ligand–ER complexes

dimerize and bind to DNA at sequence-specific response elements that are known as

oestrogen response elements (EREs). At these sites, the ERs interact with co-activator

(CoA) or co-repressor (CoR) multiprotein complexes to activate or to repress gene

transcription, respectively. The core of these multiprotein complexes is the active

(phosphorylated (P)) co-activator or co-repressor that recruits co-regulatory proteins such

as a histone acetyltransferase (HAT) and an ubiquitin ligase (UL). ERs can alternatively

regulate gene expression by interacting with other direct DNA-binding transcription

factors (TFs). According to this model of action, ERs can bind to ERE motifs that are near

the response element (RE) of the interacting transcription factor or can indirectly interact

with chromatin through tethering to the partner transcription factor. Oestrogen-bound

ERs localized in the cytoplasm (ERα can be methylated (M)) or at the cell membrane can

interact with SRC, PI3K and G proteins (GPs) and mediate non-genomic signalling. This

signalling through the activation of protein kinase cascades results in the phosphorylation

and activation of target TFs. TFs can regulate transcription through their cognate sites

(RE sites). Growth factor receptors, such as epidermal growth factor receptor (EGFR),

including ERBB2 (also known as HER2 and neu) and insulin-like growth factor receptor

(IGFR) in response to growth factors (GFs) can activate ERK and AKT serine/threonine

kinases, which can phosphorylate and activate ERs in a ligand-independent manner.

REVIEWS

600 | AUGUST 2011 | VOLUM E 11 www.nature.com/reviews/cancer

© 2011 Macmillan Publishers Limited. All rights reserved

Terapia hormonal – CM- RH+

Bloquear la activación por el Ligando (Estrógeno) al Receptor Estrogénico

• Disminuyendo los niveles de E (supresión ovárica, Inhibidores de Aromatasa)

• Antiestrógenos– SERM (Tamoxifeno): Antagonistas/agonistas

parciales (dependiendo de tejido, coactivadores)

– SERD (Fulvestrant): Antagonista + inducción de la degradación del RE

6

7Johnston & Mitch Dowsett. Nar Rev Cancer 2003

Hormonosensibilidad/Hormonorresistencia

• Sensible: cuando hay beneficio a un tratamiento (aumento de SVG, disminución de las recurrencias, aumento de la SLP, aumento de la RR/Beneficio clínico)

• Resistente– Ausencia/pérdida de beneficio: recurrencia, no respuesta,

progresión.

– Primaria vs Secundaria

– Global vs Farmaco específica

– Absoluta (RH negativos, algunos RH+, vía del RE no relevante) vs “relativa” (la activación de la vía RE persiste)

8

Cuando es un CM Hormonosensible?

• Expresión de receptores hormonales- RE y/o RP

– Único factor predictivo con evidencia suficiente es el RE (RE+ y RP +/-)

– RE-/RP+ : infrecuente. Muchos son error en la determinaciondel RE. Beneficio al tratamiento hormonal

9

Perou et al Nature 406, 747-752Sorlie T et al PNAS 2001

Subtipos intrínsecos – expresión génica –RE + – subtipo Luminal (A y B)

10

Expresión de genes regulados por el RE

Luminal A vs Luminal B – alteraciones genómicas

11TCGA. Nature 2012; Ades F JCO 2014

Grupo intrínseco – IHC – Luminal A vs Luminal B

12

<

Ades F JCO 2014

Luminal B vs A• Menor < expresión de genes

regulados por RE• Menor expresión del RP• Mayor grado tumoral• Mayor expresión de genes

relacionados con la proliferación• Activación de vias GFR (IGF-1,R,

PI3K/AKT/MTOR

ASCO/CAP – HR-IHC

13JCO 2010

Expresión del RE y Magnitud del beneficio

• Relación entre la cantidad de expresión del RE (fmol/mg) y el beneficio del TMX complementario

• Allred score y respuesta a neoHT

• RP – ausencia – menor respuesta a terapia hormonal, pero no predictor de ausencia de respuesta.

14

RE – umbral óptimo?

• ASCO/CAP IHC > 1%• LBA - > 10 fmol/mg prot• 1- 9 % ?

– Baja sens a neo HT (SET index: index of ER-related transcription )– Mayor % no luminales (PAM 50) – Menor % con expresión de ESR1-mRNA (24%)– Peor pronóstico, SV mas parecida a los tumores RH neg

15Iwamoto T. JCO 2012; Yi M Ann Onc 2014

Otros predictores de Hsens en RH+?

• Firmas de expresión génica en RH+: RS (Oncotype DX), Mammaprint, PAM50-ROR, Endopredict– Discriminan grupos pronosticos (necesidad QT)– Predicción de recurrencias tardias (ROR, Endopredict)– Predicción de Beneficio a HT (RS – low risk, ROR- low risk)

• CMM - RH + - 1ª línea HT CMM – Subgrupos intrinsecos –PAM50– Luminal A> B> HER2 enriched y Basal like

• SET index (index of ER-related transcription )–– Beneficio de SV tras tto endocrino adyuvante– No factor pronostico inherente

• Otros• Falta de validación prospectiva como marcadores predictivos

16Kwa M NRCO 2017; Pusztai L. JCO 2010; Prat A JAMA Onc 2016

Definición de resistencia endocrina “clínica”

17LOE – Expert Opinion – 67% consensus

HORMONORESISTENCIA (Hres)

18

2 años

Hormonoterapia adyuvante

> 2 años inicio – 1 año post fin tto

6 meses

Hres 1ria Hres 2ria

Hsensible

Enfermedad avanzada -

CMM de novo + no tto adyuvante previo

Ma C et al Nat Rev Ca 201519

Alterations in key cancer pathway components in luminal breast cancers.

Resistencia primaria – Modelo neoadyuvante

• Ki 67 (proliferación)– descenso a 2-4 s post-tto– Cut- off - sensibilidad: < 10% -

criterio de respuesta; otros. – Resistencia intrinseca – 20%– No útil cuando Ki 67 basal < 10% – RR – RFS– Ki-67 variabilidad

intra/interobservador

• PEPI score – Post neoHT Ki 67, T, N

20

Nature Reviews | Cancer

Ki67>10%: AI resistant

≤10%: AI sensitive

>0: AI resistant

Neoadju

vant A

I

Switch therapy

Continue AI

Surgery

PEPI

=0: AI sensitive

AI alone Additional systematictherapy plus AI

ER+ breastcancer diagnosis

2–4weeks

Surgical factors RFS HR PEPI score

Tumour size

T1/2 – 0

T3/4 2.8 3

Node status

Negative – 0

Positive 3.2 3

Ki67 level

0–2.7% – 0

>2.7–7.3% 1.3 1

>7.3–19.7% 1.7 1

>19.7–53.1% 2.2 2

>53.1% 2.9 3

ER status

Negative 2.8 3

Positive 0 0

Pathological complete

response

(pCR). Commonly defined as

the absence of residual

invasive cancers in the breast

and in the axillary lymph

node following completion of

neoadjuvant systemic

therapy, but other definit ions

exist.

Luminal B

One of the five intrinsic

molecular subtypes of breast

cancer characterized by

higher expression levels of

proliferat ion genes and lower

expression levels of

oestrogen receptor

(ER)-regulated genes

compared with the luminal A

subtype, and is associated

with a poor prognosis.

with a significantly increased risk of relapse, with the

lowest risk associated with a natural logarithm of

the Ki67 value of 1 (2.7%) or less, or in the rare patient

with a pathological complete response (pCR). However,

PEPI scores above zero (either because of a high dis-

ease burden at surgery, or high Ki67 levels, or both)

were associated with an incrementally higher risk of

relapse. The occurrence rate of PEPI-0 tumours in

neoadjuvant AI therapy trials ranges from 17% to 37%.

Patients with PEPI-0 tumours represent a low-risk popu-

lation that could be potentially treated with endocrine

monotherapy; that is, no chemotherapy17,23.

The ongoing Alliance A011106 trial (also known as

the ALTERNATE trial) is testing this concept, and this

investigation will prospectively validate the hypothesis

that PEPI-0 status is associated with a 5-year relapse risk

of less than 5% without the administration of chemo-

therapy (ClinicalTrials.gov identifier: NCT01953588).

The Alliance A011106 trial will also identify more

effective neoadjuvant endocrine therapy regimens by

randomizing patient treatment between fulvestrant,

anastrozole or a combination of both for 24 weeks before

surgery and by assessing the PEPI-0 rate on each arm.

An important aspect of the trial is the identification of

patients with intrinsically resistant tumours, defined by

Ki67 levels of above 10% in tumour biopsy samples at

4 or 12 weeks or by a PEPI score of above 0 at surgery

for chemotherapy or an investigational approach. The

genomic analysis of fulvestrant or AI-resistant tumours

and targeting them on the basis of their mutational

profile is clearly the next step in this therapeutic strategy.

Ongoing genomic analyses are also underway for

endocrine therapy-resistant tumours in the advanced

disease setting to identify progression or resistance

mutations and therapeutic targets. Research to produce

a cure for advanced disease is much more daunting and

the acquisition of tumour samples for analysis is more

difficult. Nonetheless, survival improvement for patients

with advanced disease could translate into better cure

rates in adjuvant trials and new technologies such as

circulating tumour DNA analysis may circumvent the

need for a tissue biopsy for somatic mutation diagnosis24.

Somatic mutat ion patternsTo uncover relationships between somatic mutation pat-

terns and the effectiveness of AI treatment of primary

luminal breast cancer, massively parallel sequencing of

77 pretreatment tumour biopsy samples from patients

treated with neoadjuvant AI therapy was conducted21.

This study identified 18 significantly mutated genes

(SMGs), including PI3K catalytic subunit-α (PIK3CA),

TP53 (which encodes p53), GATA3, E-cadher in

(CDH1), retinoblastoma (RB1), mixed lineage leu-

kaemia 3 (MLL3; also known as KMT2C), MAP3K1,

cyclin-dependent kinase inhibitor 1B (CDKN1B; which

encodes p27), T-box 3 (TBX3), runt-related transcrip-

tion factor 1 (RUNX1), low-density lipoprotein recep-

tor adaptor protein 1 (LDLRAP1), stathmin 2 (STMN2),

myosin heavy chain 9 (MYH9), angiotensin II receptor

type 2 (AGTR2), splicing factor 3b, subunit 1 (SF3B1)

and core-binding factor-β subunit (CBFB)21. In addi-

tion, a variety of structural variations including copy

number alterations, deletions, translocations and inver-

sions were identified. The number of SMGs in ER+

breast cancer was further extended to more than 30

by the work of The Cancer Genome Atlas (TCGA; see

Further information)25. The complexity of the genomic

data presents serious challenges for biostatistical ana-

lysis. The biological insights into the remarkable func-

tional diversity of the luminal breast cancer-associated

SMGs are only slowly emerging because many of

these genes have never previously been linked to the

development of breast cancer.

An important follow-on approach for unravelling the

importance of luminal breast cancer-associated SMGs is

to conduct targeted sequencing studies in a larger num-

ber of samples in which treatment is controlled and long-

term clinical outcomes are well documented. Although

these studies are mostly pending, within the context of

neoadjuvant AI trials, this approach has produced some

preliminary data on the clinical importance of three of the

highest frequency SMGs: TP53, MAP3K1 and GATA3.

TP53 mutations were correlated with the luminal B

subtype and high Ki67 levels before and after treatment,

whereas mutations in MAP3K1 associated with the

Box 1 | Diagnosis of AI resistance in early-stage breast cancer

Intrinsic resistance can be diagnosed at 2–4 weeks following the initiation of neoadjuvant

aromatase inhibitor (AI) therapy if tumour Ki67 levels are over 10%. This strategy (see the

figure) is being used in clinical trials of neoadjuvant AI therapies to identify patients with

resistant tumours for whom treatment with chemotherapy or participation in clinical

trials of novel therapies is recommended.

The preoperative endocrine prognostic index (PEPI) score following 4–6 months of

neoadjuvant AI or other endocrine therapy provides another strategy to identify

endocrine-sensitive versus endocrine-resistant tumours in the early-stage setting. A PEPI

score of 0 (pT1/2, node-negative (N0), Ki67<2.7%, oestrogen receptor-positive (ER+)) is

being investigated prospectively as a surrogate of endocrine therapy-sensitive disease

that does not need chemotherapy, and a PEPI of >0 identifies patients with an increased

risk of relapse. The hazard ratio (HR) of each surgical factor for relapse-free survival (RFS)

and assigned PEPI points based on the data from the P024 trial are shown in the table.

REVIEWS

NATURE REVIEWS | CANCER VOLUM E 15 | M AY 2015 | 263

© 2015 Macmillan Publishers Limited. All rights reservedEllis M JNCI 2008

Resistencia primaria – Modelo neoadyuvante

Patrones de mutaciones somáticas (SMG)• 18 genes sign. mutados (PI3K catalytic subunit-α

(PIK3CA), TP53 (which encodes p53), GATA3, E-cadherin (CDH1), retinoblastoma(RB1), mixed lineage leu- kaemia 3 (MLL3; also known as KMT2C), MAP3K1, cyclin-dependent kinase inhibitor 1B (CDKN1B; which encodes p27), T-box 3 (TBX3), runt-related transcrip- tion factor 1 (RUNX1), low-density lipoprotein recep- tor adaptorprotein 1 (LDLRAP1), stathmin 2 (STMN2), myosin heavy chain 9 (MYH9), angiotensin II receptor type 2 (AGTR2), splicing factor 3b, subunit 1 (SF3B1) and

core-binding factor-β subunit (CBFB) TCGA – hasta 30• TP53: Luminal B, niveles altos pre y

post• MAP3K1: Luminal A, niveles bajos

de Ki 67• GATA3: mayor supresión de Ki 67

tras neoAI (pred de respuesta?)

21

Ma C et al Nat Rev Ca 2015Goldstein T et al Clin. Cancer Res 2013

Perfil genómico tras tto corto con HT –Resistencia endocrina

• Tto corto con supresión de E

• Respuesta - Descenso de Ki 67 -

• NGS – Whole exome seq– PIK3CA y TP53 no rel con resistencia/respuesta a

Letrozol

– Amplificaciones rel con resistencia : WHSC1L1, CCND1, FGFR, S6K, BRCA1

– Genes de fusión

• Alteraciones trascriptómicas: activación de E2F, ciclo celular y cel T

22Giltnane J Sci Tr Med 2017

CA de mama metastásico - Resistencia

• Casos con resistencia primaria

• En líneas sucesivas va disminuyendo de forma progresiva la:– RR

– Duración de la respuesta

– SLP

– aumento de la resistencia

• Resistencia como un mecanismo dinámico y evolutivo -

23

Resistencia a terapia endocrina

Tryfonidis K et al. Ca Treat Rev 2016

Persona- Adherencia- SNP, biodisponibilidd

Microambiente tumoral- Cel estromales- Matriz EC- Inmunidad- Hipoxia

Celula tumoral- RE, coreguladores

- perdida expresión- mutaciones ESR1- Modificaciones regul

RE- Ciclo celular- Vias oncogenicas alterna

tivas 24

Ma C et al Nat Rev Ca 2015

Growth factor receptor signalling, PI3K, MAPK, ER and the p53–RB pathway in ER+ breast cancer

25

ESR1 mutation and ESR1-YAP1 translocation

Ma C et al Nat Rev Ca 2015

Mutaciones - ESR1 (ERα)

26

LBDomainD538GY537SActivación constitutiva de ligando

27

Toy W. Nature Genetics 2013Fribbens C et al. J Clin Oncol 2016

Mutaciones - ESR1 (ERα)Raras en el T 1rio – 1-3%Pacientes tratamiento hormonal previo- Tamoxifeno – raro- Inhibidores de Aromatasa (AI)

- CMM > Adyuvancia- 30 % (11-50%)

Resistencia adquiridaDepleción estrogénica (AI)Mutacion de novo vs Selección clonal

Respuesta a SERMsy SERDs – dosis mas altas (modelos preclinicos)

MutadosSERD > AI (SOFEA, PALOMA3)

Se puede medir en tejido y cfDNA

Ma C et al Nat Rev Ca 2015; Rondon Lagos M IJMS 2016

The hallmarks of AI resistance.

28

SERM y SERD- Mec de resistencia

compartidos- Mutaciones ESR1 –

- Menor relevancia- Desarrollo de

nuevos farmacospara revertir estemec de resistencia

Otros: miRNA, LncRNA…Mec epigenéticos

¿Se puede actuar sobre los mecanismos de resistencia hormonal? – evidencia actual -

• Luminales HER 2+: - la combinación HT- antiHER2 > HT sola (TANDEM, Lapatinib-letrozol)

• Luminales HER2 -: – PI3K-AKT-mTOR

• Inh mTOR Everolimus > HT sola tras fallo a inh aromatasa (Bolero2, TamRad, PrECOG)

• Inh de PI3K – neoHT, CMM – resultados discretos, mutación de PIK3CA (predictor ?)

– Inh de ciclinas + HT > HT sola en 1ª y 2ª línea (Palbociclib, Ribociclib, Abemaciclib)

– Mec epigenéticos: HDAC inh (Dasatinib) – tras fallo a IANE

• En todos ellos: RR y SLP pero aun no demostrado impacto en la SV

29

¿Por qué no hay impacto en la SVG en los estudios HT+ terapias dirigidas?

• Larga historia natural, falta de poder estadístico para detectar SVG

• Selección de pacientes inadecuada?– subperfilesbiológicos heterogéneos

• Ausencia de biomarcadores predictivos/selectivos (uso de T1rio archivado – no representativo del CMM)

• Tratamientos posteriores• Evolución postprogresión mas agresiva?• …..

30

… en Ca de mama Luminal

• Las causas de resistencia a terapia endocrina son heterogéneas

• En muchos casos persiste la actividad de la vía del RE actividad independiente de ligando natural – Alteraciones en el ER (mutaciones, fusiones..)– Activación del ER por la interacción con otras vías – Desregulación de genes regulados por el RE

• Necesidad de biomarcadores predictivos– Elección de la terapia endocrina (AIs vs SERM vs SERD)– Terapia combinada (cuando?,todos desde el inicio?, que combinación)– Combinaciones – doble, triple…– Evaluación dinámica de la evolución de la enfermedad (biopsia

líquida?)

31

32