Breast & Colon Cancer: Molecular Breast & Colon Cancer: Molecular Alterations & Therapeutic TargetsAlterations & Therapeutic TargetsPart 1Part 1

April 8, 2005April 8, 2005Nancy HynesNancy Hynes

In the past 25 yrs cancer research has generated a largeIn the past 25 yrs cancer research has generated a largebody of knowledge on molecular mechanisms controlling allbody of knowledge on molecular mechanisms controlling allaspects of normal cellular physiology/signaling pathways. aspects of normal cellular physiology/signaling pathways.

These studies have also provided knowledge on molecules &These studies have also provided knowledge on molecules &mechanisms underlying cancer development. mechanisms underlying cancer development.

GoalGoal –– to design rational/molecularly targeted therapeutics to design rational/molecularly targeted therapeutics for cancer treatment.for cancer treatment.

Molecular targets for cancer therapyMolecular targets for cancer therapy

Major goal of lecturesMajor goal of lectures

Present studies from the area of breast & colon cancerPresent studies from the area of breast & colon cancerto show how molecular analyses can be used to achieve the to show how molecular analyses can be used to achieve the ultimate goal of providing better therapies for patients. ultimate goal of providing better therapies for patients.

Molecular targets for cancer therapyMolecular targets for cancer therapy

Breast cancerBreast cancer1 in 8 women develop the cancer1 in 8 women develop the cancer30% will die of 30% will die of metastaticmetastatic diseasedisease

therapeutics targeting proteins involved in breast cancer therapeutics targeting proteins involved in breast cancer are in clinical useare in clinical use

Colon cancerColon cancer3rd most common cancer3rd most common cancer

therapeutics targeting proteins involved in colon cancer are intherapeutics targeting proteins involved in colon cancer are inpreclinical testing; clinical data on drugs targeting angiogenespreclinical testing; clinical data on drugs targeting angiogenesisishave recently been published have recently been published

The original description of pathological stages of The original description of pathological stages of colorectal cancer as defined by Dukecolorectal cancer as defined by Duke

Davies et al 2005 Nature Rev Cancer 5:199 Davies et al 2005 Nature Rev Cancer 5:199

The colorectal The colorectal adenomoadenomo--carcinoma sequencecarcinoma sequence

Davies et al 2005 Nature Rev Cancer 5:199 Davies et al 2005 Nature Rev Cancer 5:199

A well defined A well defined molecular/pathologicalmolecular/pathological sequence of colorectal cancersequence of colorectal cancerdevelopment has emerged over the past 15 yrs based on the development has emerged over the past 15 yrs based on the pioneering work of Vogelstein & colleagues pioneering work of Vogelstein & colleagues

Pathological/Histological Classification of Breast CancerPathological/Histological Classification of Breast Cancer

Presence or absence of invasionPresence or absence of invasion

Histological appearance of cells Histological appearance of cells -- ductulductul or lobular or lobular

In situ or infiltrating cancerIn situ or infiltrating cancer

DuctalDuctal carcinoma in situ carcinoma in situ

Lobular carcinoma in situ Lobular carcinoma in situ

Infiltrating Infiltrating ductalductal carcinoma carcinoma –– very common very common

Histology, combined with other clinical features, e.g., tumorHistology, combined with other clinical features, e.g., tumorsize, cancer cells in lymph nodes, is routinely used to size, cancer cells in lymph nodes, is routinely used to choose treatment & to predict clinical outcome of the patient. choose treatment & to predict clinical outcome of the patient.

Gene Expression Patterns & Breast Cancer Gene Expression Patterns & Breast Cancer

Gene expression profiling is a broad method for Gene expression profiling is a broad method for molecularly defining tumor types. molecularly defining tumor types.

Tumor subTumor sub--types with distinct gene expression patternstypes with distinct gene expression patternshave been described.have been described.

prognosisprognosisdiagnosisdiagnosisnew therapeutic targetsnew therapeutic targets

A well defined molecular/pathological sequence of A well defined molecular/pathological sequence of breast cancer is not available. breast cancer is not available.

Gene expression profiling & breast cancerGene expression profiling & breast cancer

Breast cancers fall into 5 clinicallyBreast cancers fall into 5 clinically--relevant subtypes*relevant subtypes*

T. Sorlie et al PNAS 2001 98:10869; ibid 2003 100:8413

Basal-like ErbB2+ Normal Luminal Type B Luminal Type A

*cDNA microarrays on a core set of 8100 genes were carried out, then 427 unique genes formed a basis for classification based upon significantly greater variation in expression between different tumors than between paired samples from same tumor.

Gene expression profiling & breast cancerGene expression profiling & breast cancer

Breast cancers fall into 5 clinicallyBreast cancers fall into 5 clinically--relevant subtypesrelevant subtypes

Disease-free survivalOverall survival

Luminal A

Basal-like

BasalBasal NormalNormal Luminal BLuminal B Luminal ALuminal AErbB2ErbB2++Luminal have best prognosisLuminal have best prognosisBasal have worst prognosisBasal have worst prognosis

What influences Breast Cancer development?What influences Breast Cancer development?

Genetic AlterationsGenetic Alterations

HormonesHormones

What influences breast cancer development?What influences breast cancer development?

Many of these factors are related to estrogen exposureMany of these factors are related to estrogen exposure

Estrogen & Breast CancerEstrogen & Breast Cancer

Estrogens play an essential role in development Estrogens play an essential role in development of the normal breast & in cancer.of the normal breast & in cancer.

The life time exposure to estrogens has a strong The life time exposure to estrogens has a strong influence on cancer development.influence on cancer development.

Early onset of menarche & late menopauseEarly onset of menarche & late menopause

Loss of ovarian function prevents breast cancerLoss of ovarian function prevents breast cancer

Low incidence of male breast cancerLow incidence of male breast cancer

Estrogen & Breast CancerEstrogen & Breast Cancer

George George BeatsonBeatson described that removal of the ovaries described that removal of the ovaries from women with from women with metastaticmetastatic breast cancer sometimesbreast cancer sometimesled to tumor regression. led to tumor regression.

“On the treatment of inoperable cases of carcinoma of the “On the treatment of inoperable cases of carcinoma of the mamma: suggestions for a new method of treatment with mamma: suggestions for a new method of treatment with illustrative cases”.illustrative cases”.

Lancet (1896)Lancet (1896)

Estrogen & Breast CancerEstrogen & Breast Cancer

Jensen & Jacobsen identified the target for estrogen action,Jensen & Jacobsen identified the target for estrogen action,the estrogen receptor (ER) in 1962the estrogen receptor (ER) in 1962

“Basic guides to the mechanism of estrogen action”“Basic guides to the mechanism of estrogen action”

Recent Recent ProgProg HormHorm ResRes, 1962, 1962

Jensen correlated the presence of ER with hormone Jensen correlated the presence of ER with hormone responsiveness of the breast tumor. responsiveness of the breast tumor.

““Estrogen receptors & breast cancer response to Estrogen receptors & breast cancer response to adrenalectomyadrenalectomy””

NatlNatl Cancer Inst Cancer Inst MonogrMonogr, 1971, 1971

Estrogen ReceptorEstrogen Receptor

Member of the steroid/thyroid/retinoid receptor Member of the steroid/thyroid/retinoid receptor superfamilysuperfamilyof transcription factors.of transcription factors.

LigandLigand binding induces DNA binding, recruitment of transcriptionalbinding induces DNA binding, recruitment of transcriptionalcoactivatorscoactivators ((NCoAsNCoAs) & ) & histonehistone acetyltransferasesacetyltransferases ((HATsHATs) leading) leadingtranscription of target genes.transcription of target genes.

Hewitt et al 2005 Science vol 307 1572

Classical & novel modes of estrogen actionClassical & novel modes of estrogen action

There are rapid responses that occur within minutes after estrogen administration that cannot be accounted for by changesin gene expression.

Prossnitz & colleagues recently described a non-ER, transmembraneprotein that also initiates signaling cascades in response to estrogen

(Revankar et al 2005 Science 307 1625)

Membrane ERMembrane ERGPCRGPCR

S.Ali & RC Coombes. Nature Reviews Cancer 2: 101 (2002)

Normal Adult Breast Normal Adult Breast

~ 30 % of the epithelial cells in the ~ 30 % of the epithelial cells in the ducts express estrogen receptor (ER). ducts express estrogen receptor (ER).

ER+ cellsER+ cells

ERER++ cells act as sensors in the normal breast cells act as sensors in the normal breast

ER+ER+epithelial cellepithelial cell

ERER--epithelial cellepithelial cell

dividedivide

The ER+ cells respond to estrogens by releasing peptide factors The ER+ cells respond to estrogens by releasing peptide factors that stimulate proliferation of neighboring ERthat stimulate proliferation of neighboring ER-- cells. cells.

estrogenestrogen

Peptide factors: Peptide factors: EGF or EGF or TGFTGFααEGFR on EREGFR on ER-- cellcell

ER+ cells ER+ cells do not usually dividedo not usually dividein the adult!in the adult!

Epithelial cells proliferate in response to cyclical variation Epithelial cells proliferate in response to cyclical variation of ovarian hormones during the menstrual cycle. of ovarian hormones during the menstrual cycle.

The normal increase in cell number is balanced by normal The normal increase in cell number is balanced by normal cell death. cell death.

S.Ali & RC Coombes. Nature Reviews Cancer 2: 101 (2002)

Structure of the normal and malignant breastStructure of the normal and malignant breast

normalnormal

tumortumor

Tumors have many ER+ cellsTumors have many ER+ cells

In breast cancer ERIn breast cancer ER++ tumor cells proliferate tumor cells proliferate

ER+ER+Tumor cellTumor cell

dividedivide

ER+ tumor cells proliferate in response to estrogen.ER+ tumor cells proliferate in response to estrogen.

estrogenestrogen

The normal balance between proliferation & apoptosis is The normal balance between proliferation & apoptosis is lost in the tumor. lost in the tumor.

Estrogen Receptor Expression in Breast Cancer Estrogen Receptor Expression in Breast Cancer ER expression in invasive breast cancers is variable.ER expression in invasive breast cancers is variable.

Panels aPanels a--f are all invasive breast cancers f are all invasive breast cancers -- ER expression ER expression ranges from 0% (a) to ~100% (f) .ranges from 0% (a) to ~100% (f) .

AllredAllred et al 2004 Breast Cancer Research 6: 240et al 2004 Breast Cancer Research 6: 240

Why is this important?Why is this important?Prognosis & treatment Prognosis & treatment

Gene expression profiling & breast cancerGene expression profiling & breast cancer

Breast cancer patients whose tumors show high ER Breast cancer patients whose tumors show high ER expression have the best prognosis. expression have the best prognosis.

T. Sorlie et al PNAS 2001 98:10869; ibid 2003 100:8413

Basal-like Normal Luminal Type B Luminal Type A

High Estrogen Receptor (ER)Moderate ER

ErbB2+

ER negative

The estrogen receptor as target for therapyThe estrogen receptor as target for therapy

ERER++ tumors depend upon estrogen for proliferation.tumors depend upon estrogen for proliferation.

ER antagonists were some of the first targeted, rationalER antagonists were some of the first targeted, rationaltherapeutics .therapeutics .(proposed in 1936 by (proposed in 1936 by LacassagneLacassagne))

In 1971 Jensen correlated the presence of ER with hormone In 1971 Jensen correlated the presence of ER with hormone responsiveness of the breast tumor. responsiveness of the breast tumor.

ER antagonists bind the receptor but prevent transcriptionER antagonists bind the receptor but prevent transcriptionby interfering with by interfering with coactivatorcoactivator binding. binding.

The estrogen receptor as a therapeutic targetThe estrogen receptor as a therapeutic target

The estrogen receptor as a therapeutic targetThe estrogen receptor as a therapeutic target

The nonThe non--steroidal, antisteroidal, anti--estrogen estrogen tamoxifentamoxifen was the 1was the 1stst

agent approved for clinical use. agent approved for clinical use.

1973 UK; 1977 USA1973 UK; 1977 USA

TJ TJ PowlesPowles 2: 787 (2002) 2: 787 (2002)

Estrogen Receptor & Estrogen Receptor & TamoxifenTamoxifen

TamoxifenTamoxifen interferes with AF2 causing block in transcriptioninterferes with AF2 causing block in transcriptionof genes involved with cancer cell proliferation.of genes involved with cancer cell proliferation.

Two distinct domains of ER (AF1 & 2) mediate transcriptional Two distinct domains of ER (AF1 & 2) mediate transcriptional activation.activation.

TamoxifenTamoxifen

The AntiThe Anti--Estrogen* Estrogen* TamoxifenTamoxifen

TamoxifenTamoxifen has made a substantial contribution to the has made a substantial contribution to the reduced mortality rate that has been reported in severalreduced mortality rate that has been reported in severalcountries since 1990. countries since 1990.

TamoxifenTamoxifen was approved for treatment of advanced was approved for treatment of advanced breast cancer in 1973.breast cancer in 1973.Clinical trial - 22% response rate(Cole MP et al 1971 Br.J Cancer 25:270-275)

George George BeatsonBeatson described that removal of the ovaries described that removal of the ovaries from women with from women with metastaticmetastatic breast cancer sometimesbreast cancer sometimesled to tumor regression. led to tumor regression.

Ovarian ablation removes the major estrogen source,Ovarian ablation removes the major estrogen source,thus blocking tumor cell proliferation.thus blocking tumor cell proliferation.

* SERM = selective estrogen receptor modifiers* SERM = selective estrogen receptor modifiers

Many tumors are ER+Many tumors are ER+

Relapse suggests thatRelapse suggests thattumor cells grow in an tumor cells grow in an estrogen/ER independent estrogen/ER independent manner.manner.

Some patients respond toSome patients respond toother drugs targeting other drugs targeting estrogen/ER. estrogen/ER.

S.Ali & RC Coombes. Vol 2: 101 (2002)

Unfortunately after a few years most breast cancer patientsUnfortunately after a few years most breast cancer patientsrelapse & show metastases during treatment with relapse & show metastases during treatment with tamoxifentamoxifen..

In postmenopausal women, adrenal & ovarian androgens In postmenopausal women, adrenal & ovarian androgens are converted into estrogens, in different peripheral are converted into estrogens, in different peripheral tissues: muscle, skin, normal breast & in breast cancer tissues: muscle, skin, normal breast & in breast cancer cells, by the enzyme cells, by the enzyme aromatasearomatase. .

AromataseAromatase inhibitors & breast cancer inhibitors & breast cancer

AromataseAromatase inhibitors & breast cancer inhibitors & breast cancer

Various Various aromatasearomatase inhibitors are in clinical trials. inhibitors are in clinical trials.

Some have shown efficacy in patients who relapsed on Some have shown efficacy in patients who relapsed on tamoxifentamoxifen therapy. therapy.

Review on aromatase inhibitors - IA Smith, 2004, The Breast 13, S3-S9

AromataseAromatase inhibitors & breast cancer inhibitors & breast cancer

Various Various aromatasearomatase inhibitors are in clinical trials. inhibitors are in clinical trials.

Some have shown efficacy in patients who relapsed on Some have shown efficacy in patients who relapsed on tamoxifentamoxifen therapy. therapy.

These results suggest that tumors still require These results suggest that tumors still require estrogen/ER for proliferation; lowering the level of estrogen/ER for proliferation; lowering the level of estrogen in the tumor & adipose tissue has an impact on estrogen in the tumor & adipose tissue has an impact on disease.disease.

These results are very important for breast cancer These results are very important for breast cancer patients:patients:

1 1 –– after relapse on after relapse on tamoxifentamoxifen there’s another there’s another therapeutic option with proven efficacy therapeutic option with proven efficacy

2 2 –– antianti--estogenestogen therapy is relatively nontherapy is relatively non--toxic toxic

Mechanisms of action of therapeutic agents used in endocrineMechanisms of action of therapeutic agents used in endocrinetherapy therapy

Ali & Ali & CoombesCoombes, , VolVol 2 pg 1012 pg 101

Major mechanisms Major mechanisms -- antagonize ER (antagonize ER (SERMsSERMs) or lower estrogen levels ) or lower estrogen levels ((aromatasearomatase inhibitors)inhibitors)

Analysis of ~10,000 breast cancer patients treated with Analysis of ~10,000 breast cancer patients treated with tamoxifentamoxifen as an adjuvant therapy for breast cancer as an adjuvant therapy for breast cancer revealed that the frequency of new breast cancer in revealed that the frequency of new breast cancer in the the tamoxifentamoxifen--treated group was lower then in the treated group was lower then in the control group. control group.

TamoxifenTamoxifen for breast cancer preventionfor breast cancer prevention

NayfieldNayfield et al (1991) JNCI 83: 1450et al (1991) JNCI 83: 1450

Frequency of new breast cancer (Frequency of new breast cancer (contralateralcontralateral breast) inbreast) inclinical trials of clinical trials of tamoxifentamoxifen therapy. therapy.

8 Clinical Trials8 Clinical TrialsTamTam--treatedtreated ControlControl

NATONATO

ScottishScottishNSABPNSABP

No. patientsNo. patients No. cancersNo. cancers No. patientsNo. patients No. cancersNo. cancers

564564

66166114191419

1515

992323

TotalTotal 49754975 79791.6%1.6%

65165114281428

49714971

12123232

1211212.4%2.4%

567567 1717

etcetc

Significant reduction in breast cancer in the TamSignificant reduction in breast cancer in the Tam--treated grouptreated group

TamoxifenTamoxifen for breast cancer preventionfor breast cancer prevention

TJ TJ PowlesPowles 2: 787 (2002) 2: 787 (2002)

Effects of estrogen & Effects of estrogen & tamoxifentamoxifen on human tissueson human tissues

TamoxifenTamoxifen is an ER antagonist in the breastis an ER antagonist in the breast

TamoxifenTamoxifen increases frequency of uterine cancerincreases frequency of uterine cancer

NayfieldNayfield et al (1991) JNCI 83: 1450et al (1991) JNCI 83: 1450

Tam treated patients (n=4028)Tam treated patients (n=4028) Controls (n=4006)Controls (n=4006)No. cancersNo. cancers No. cancersNo. cancers FrequencyFrequency

1919 0.5%0.5% 44

12120.1%0.1%

0.3%0.3%

FrequencyFrequencyuterusuterusendometrialendometrialovaryovary 66 0.1%0.1%

Endometrial cancer is significantly higher in the TamEndometrial cancer is significantly higher in the Tam--treated grouptreated group

TJ TJ PowlesPowles 2: 787 (2002) 2: 787 (2002)

Effects of estrogen & Effects of estrogen & tamoxifentamoxifen on human tissueson human tissues

TamoxifenTamoxifen is an ER agonist in the uterusis an ER agonist in the uterus

TJ TJ PowlesPowles 2: 787 (2002) 2: 787 (2002)

Estrogen Receptor & Estrogen Receptor & TamoxifenTamoxifen

TamoxifenTamoxifen blocks AF2blocks AF2-- the main activator in the breast.the main activator in the breast.

Two distinct activation domains (AF1 & 2) mediate transcriptionaTwo distinct activation domains (AF1 & 2) mediate transcriptional l activation.activation.

In the uterus, AF1 is more important for ER activity In the uterus, AF1 is more important for ER activity –– thus tamthus tamacts as an agonist in this organ.acts as an agonist in this organ.

TamoxifenTamoxifen & breast cancer prevention& breast cancer prevention

Despite the partial ER agonistic activity in some organs,Despite the partial ER agonistic activity in some organs,the decrease in the decrease in contralateralcontralateral breast cancer incidence breast cancer incidence observed in women treated with observed in women treated with tamoxifentamoxifen for breast for breast cancer therapy, led to the proposal that the drug cancer therapy, led to the proposal that the drug might be useful for breast cancer prevention. might be useful for breast cancer prevention.

In 1992 a randomized clinical trial was begun In 1992 a randomized clinical trial was begun “Does “Does tamoxifentamoxifen prevent breast cancer?”prevent breast cancer?”

TamoxifenTamoxifen & breast cancer prevention& breast cancer prevention

13,388 women were enrolled 13,388 women were enrolled

At an increased risk were considered to be those: At an increased risk were considered to be those: >60 yrs of age>60 yrs of age

3535--59 yrs of age with,e.g., family history 59 yrs of age with,e.g., family history

with a history of lobular carcinoma in situwith a history of lobular carcinoma in situ

Results published 1998 (Fisher et al JNCI 90: 1371) Results published 1998 (Fisher et al JNCI 90: 1371)

““TamoxifenTamoxifen administration reduced the risk of invasive &administration reduced the risk of invasive &noninvasive breast cancers by ~50% in all age groups.”noninvasive breast cancers by ~50% in all age groups.”

The role of estrogens/ER in breast cancerThe role of estrogens/ER in breast cancer

The ER is a perfect example of a rational therapeutic The ER is a perfect example of a rational therapeutic target. target.

Ironically, Ironically, tamoxifentamoxifen was developed before the age was developed before the age of molecular biology, which set the stage for molecularof molecular biology, which set the stage for moleculartarget identification in cancers. target identification in cancers.

AntiAnti--estrogens might generally become important inestrogens might generally become important inbreast cancer prevention.breast cancer prevention.

Important questions for the futureImportant questions for the future

Why do patients initially respond to antiWhy do patients initially respond to anti--estrogen therapyestrogen therapythen relapse?then relapse?

Why do patients who failed one type of antiWhy do patients who failed one type of anti--estrogen therapyestrogen therapyrespond to another one? tam vs. respond to another one? tam vs. aromatasearomatase inhibitorsinhibitors

Despite the open questions, antiDespite the open questions, anti--estrogen strategiesestrogen strategieshave been extremely important for treatment ofhave been extremely important for treatment ofbreast cancer patients.breast cancer patients.

Why are some tumors dependent upon estrogen forWhy are some tumors dependent upon estrogen forProliferation & survival? Stem cell?Proliferation & survival? Stem cell?

Breast cancer, stem cells & the ER Breast cancer, stem cells & the ER

*G. Dontu et al 2004 Trends Endocrinology & Metabolism 15: 193-197

Some models* propose that transformation of differentsub-sets of stem cells leads to the observed diversity in breastcancer phenotypes – including ER status.

Why do patients initially respond to antiWhy do patients initially respond to anti--estrogen therapyestrogen therapythen relapse?then relapse?

Why do patients who failed one type of antiWhy do patients who failed one type of anti--estrogen therapyestrogen therapyrespond to another one? tam vs. respond to another one? tam vs. aromatasearomatase inhibitorsinhibitors

Despite the open questions, antiDespite the open questions, anti--estrogen strategiesestrogen strategieshave been extremely important for treatment ofhave been extremely important for treatment ofbreast cancer patients.breast cancer patients.

Why are some tumors dependent upon estrogen forWhy are some tumors dependent upon estrogen forproliferation? Stem cell?proliferation? Stem cell?

Important questions for the futureImportant questions for the future

What influences breast cancer development?What influences breast cancer development?

Genetic AlterationsGenetic Alterations

HormonesHormones

Specific Molecular Alterations & Therapies Specific Molecular Alterations & Therapies

Development of targeted therapeutics based uponDevelopment of targeted therapeutics based uponcharacterizing specific molecular alterations in tumors.characterizing specific molecular alterations in tumors.e.g., ErbB2 gene amplificatione.g., ErbB2 gene amplification

Tumors develop as a result of multiple Tumors develop as a result of multiple genetic alterationsgenetic alterations

loss of tumor suppressor genesloss of tumor suppressor genes

“activation” of dominant “activation” of dominant oncogenesoncogenes

mutationmutation

altered expression altered expression gene amplificationgene amplificationchromosomal rearrangementchromosomal rearrangementincreased transcriptionincreased transcription

Provides hints about how cancer develops.Provides hints about how cancer develops.

Why is it important to characterize Why is it important to characterize molecular alterations in tumors?molecular alterations in tumors?

Identify candidates for rational targeted therapeutics.Identify candidates for rational targeted therapeutics.

Provide new cancer diagnostic tools.Provide new cancer diagnostic tools.

Tumor suppressor genesTumor suppressor genes

Deletion or mutation of one allele is either inherited orDeletion or mutation of one allele is either inherited oroccurs during the development of the cancer. occurs during the development of the cancer.

When the second WT allele is When the second WT allele is lost in a somatic cell, visualized bylost in a somatic cell, visualized byloss of loss of heterozygousityheterozygousity (LOH), (LOH), these cells have a growth these cells have a growth advantage. advantage.

Tumor suppressor genes & breast cancerTumor suppressor genes & breast cancer

BRCA 1 & 2BRCA 1 & 2

Existence of genes responsible for inherited predispositionExistence of genes responsible for inherited predispositionto breast & ovarian cancer was suggested more than to breast & ovarian cancer was suggested more than 100 yrs ago.100 yrs ago.

Due to foresight of certain human geneticists, Due to foresight of certain human geneticists, G.LeniorG.Lenior &&MC KingMC King, who collected material from “cancer families”, , who collected material from “cancer families”, linkage analyses were used initially to determine chromosomallinkage analyses were used initially to determine chromosomallocation of the genes, this was followed by their positional clolocation of the genes, this was followed by their positional cloning. ning.

Family Pedigrees for Breast CancerFamily Pedigrees for Breast Cancer

Hereditary: Breast cancer in all generations. Hereditary: Breast cancer in all generations.

~ 5% of all breast cancers fall in this group; ~ 5% of all breast cancers fall in this group; the majority have mutations in BRCA genes.the majority have mutations in BRCA genes.

What characterizes a family with What characterizes a family with hereditary breast cancer?hereditary breast cancer?

Women with germline heterozygous mutations in BRCA1 or BRCA2 are at increased risk of developing breast, ovarian & other cancers.

BRCA1 & BRCA2 mutations predispose to breast cancerBRCA1 & BRCA2 mutations predispose to breast cancer

BRCA1 mapped in 1990BRCA1 mapped in 1990

Cloned in 1994Cloned in 1994

BRCA2 mapped in 1994BRCA2 mapped in 1994

Cloned in 1995Cloned in 1995

As predicted for a tumor suppressor, the second WT As predicted for a tumor suppressor, the second WT BRCA allele is found mutated in the breast tumor. BRCA allele is found mutated in the breast tumor.

Domain structure of BRCA1 & BRCA2Domain structure of BRCA1 & BRCA2

The BRCA proteins have been implicated in many The BRCA proteins have been implicated in many different processes including different processes including DNA repairDNA repair & & recombination, cell cycle control & transcription. recombination, cell cycle control & transcription.

BRCA proteins & DNA RepairBRCA proteins & DNA Repair

The BRCA proteins are essential for preserving The BRCA proteins are essential for preserving chromosome structure.chromosome structure.

Cells with gross chromosomal rearrangements (GCR) have Cells with gross chromosomal rearrangements (GCR) have defects in DNA repair or recombination pathways. defects in DNA repair or recombination pathways.

GCRsGCRs in BRCAin BRCA--deficient cells result from inappropriate DSB deficient cells result from inappropriate DSB repair during the S & G2 phases of the cell cycle. repair during the S & G2 phases of the cell cycle.

Mouse cells deficient in BRCA2 show spontaneous Mouse cells deficient in BRCA2 show spontaneous aberrations in chromosome structure.aberrations in chromosome structure.

metaphase spreadmetaphase spread

Aberrations in the usual Aberrations in the usual UU--shaped mouse chromosomes shaped mouse chromosomes are visible & enlarged on the are visible & enlarged on the right.right.

chromatidchromatid breakbreak

triradialtriradial &&quatraradialquatraradialstructuresstructures

VenkitaramanVenkitaraman 2002 Cell 108: 1712002 Cell 108: 171

BRCA proteins & DNA RepairBRCA proteins & DNA Repair

GCRsGCRs in BRCAin BRCA--deficient cells result from inappropriate DSB deficient cells result from inappropriate DSB repair during S & G2. repair during S & G2.

VenkitaramanVenkitaraman 2002 Cell 108: 1712002 Cell 108: 171

The preferred errorThe preferred error--freefreeHR pathway does not HR pathway does not fxnfxn in cellsin cellslacking BRCA proteins.lacking BRCA proteins.

nonhomologousnonhomologous end joiningend joiningsingle strand annealingsingle strand annealing homologous recombinationhomologous recombination

BRCA proteins & DNA RepairBRCA proteins & DNA Repair

Turner et al 2004 Nature Reviews Cancer vol 4

The two BRCA proteins have The two BRCA proteins have different functions in this process.different functions in this process.

BRCA proteins & DNA RepairBRCA proteins & DNA Repair

HR = homologous recombination

BRCA proteins & DNA repair complexesBRCA proteins & DNA repair complexes

BRCA1 has a broad range of BRCA1 has a broad range of functions; e.g., transcription*functions; e.g., transcription*

BRCA2 appears to have a more restricted function BRCA2 appears to have a more restricted function --RAD51 dependent DNA repair.RAD51 dependent DNA repair.

*BRCA1 & transcription: *BRCA1 & transcription: MS Chapman & I MS Chapman & I VermaVerma, 1996 Nature 382:678, 1996 Nature 382:678AN AN MonteiroMonteiro et al 1996 PNAS 93:13595et al 1996 PNAS 93:13595T T OuchiOuchi et al 2000 PNAS 97:5208et al 2000 PNAS 97:5208

Turner et al 2004 Nature Reviews Cancer vol 4

Turner et al 2004 Nature Reviews Cancer vol 4

Tumors with BRCA mutations have distinctive featuresIn order for a cell with unrepaired DNA damage to escape apoptosis cell-cycle check-points need to be lost.

This might explain the higher rate of p53 mutations & MYC amplifications

Turner et al 2004 Nature Reviews Cancer vol 4

Tumors with BRCA1 mutations have a distinctive phenotype

A high % of ER negative tumors A high % of ER negative tumors

Turner et al 2004 Nature Reviews Cancer vol 4

BRCA1 vs. BRCA2 phenotype

Gene expression profiling & breast cancerGene expression profiling & breast cancer

BRCA1 tumors have a basal cancer “expression signature”BRCA1 tumors have a basal cancer “expression signature”

Disease-free survivalOverall survival

Luminal A

Basal-like

BasalBasal NormalNormal Luminal BLuminal B Luminal ALuminal AErbB2ErbB2++

BRCA1 & BRCA2 have not been implicated in BRCA1 & BRCA2 have not been implicated in sporadic breast cancersporadic breast cancer

In contrast to other inherited tumor suppressor genes, In contrast to other inherited tumor suppressor genes, sporadic breast cancers do not carry mutations in BRCA sporadic breast cancers do not carry mutations in BRCA genes. genes.

Hereditary Colon CancerHereditary Colon Cancer

~15% of colorectal cancers (CRC) is due to germ line~15% of colorectal cancers (CRC) is due to germ linetransmission of mutated genes.transmission of mutated genes.

APCAPCFamilial Familial AdenomatousAdenomatous PolyposisPolyposis (FAP)(FAP)

GrodenGroden et al Cell 66; et al Cell 66; KinzlerKinzler et al Science 253; et al Science 253; NishishoNishisho et al Science 253 .et al Science 253 .

In 1991 the In 1991 the APCAPC gene was cloned; gene was cloned; germ line & sporadic mutations were described.germ line & sporadic mutations were described.

BRCA1 transcription is silenced by promoter BRCA1 transcription is silenced by promoter methylationmethylation in sporadic breast & ovarian cancers. in sporadic breast & ovarian cancers.

Epigenetic mechanisms of BRCA1 inactivationEpigenetic mechanisms of BRCA1 inactivation

BRCA1 promoter BRCA1 promoter methylationmethylation is infrequently detected is infrequently detected in lung (NSCLC) & cervical cancers; it is never observed in lung (NSCLC) & cervical cancers; it is never observed in other cancers in other cancers –– colon or colon or leukemiasleukemias. .

Turner et al 2004 Nature Reviews Cancer vol 4

BRCA proteins are partsBRCA proteins are partsof large protein of large protein complexes. complexes.

Other proteins on the BRCA pathway might be mutatedOther proteins on the BRCA pathway might be mutatedin sporadic cancer.in sporadic cancer.

Inactivation of BRCA interacting proteinsInactivation of BRCA interacting proteins

How might you use tumor suppressors How might you use tumor suppressors in cancer therapy?in cancer therapy?

Restore WT function which often blocks Restore WT function which often blocks proliferation. proliferation.

This is feasible but technically very challenging for This is feasible but technically very challenging for many reasons. many reasons.

specific introduction into cancer cellsspecific introduction into cancer cellsefficiencyefficiencyetc etc

Tumors develop as a result of multiple Tumors develop as a result of multiple genetic alterationsgenetic alterations

loss of tumor suppressor genesloss of tumor suppressor genes

“activation” of dominant “activation” of dominant oncogenesoncogenes

mutationmutation

altered expression altered expression gene amplificationgene amplificationchromosomal rearrangementchromosomal rearrangementincreased transcriptionincreased transcription

Proteins aberrantly expressed or activatedProteins aberrantly expressed or activatedin breast cancerin breast cancer

ErbB2 & EGF receptor tyrosine ErbB2 & EGF receptor tyrosine kinaseskinases

CC--mycmyc

Cyclin D1Cyclin D1

There are many examples of proteins that are aberrantlyThere are many examples of proteins that are aberrantlyexpressed in breast cancer. expressed in breast cancer.

Therapeutic targets for breast cancer treatmentTherapeutic targets for breast cancer treatment

Goal of rational cancer therapy has been to use these Goal of rational cancer therapy has been to use these “altered” proteins as therapeutic targets.“altered” proteins as therapeutic targets.

What characterizes a good therapeutic target? What characterizes a good therapeutic target?

Enzymatic activity Enzymatic activity

Surface expression Surface expression

Expressed only in the tumor Expressed only in the tumor

Etc. Etc.

The The superfamilysuperfamily of receptor tyrosine of receptor tyrosine kinaseskinases ((RTKsRTKs))

The The ErbBErbB or EGF related family of or EGF related family of RTKsRTKs

ErbBErbB –– avainavain erythroblastosiserythroblastosis virus virus oncogeneoncogene BB

There are approx. 60 There are approx. 60 RTKsRTKs -- at least 30 of these have at least 30 of these have been found altered in human tumors.been found altered in human tumors.

ErbBErbB RTKsRTKs & Breast Cancer& Breast Cancer

ErbB2 amplification correlates with “poor” prognostic factors ErbB2 amplification correlates with “poor” prognostic factors Tumor gradeTumor gradeLymph node status Lymph node status –– sign of metastasessign of metastasesEstrogen receptor expression Estrogen receptor expression

Gene encoding ErbB2 is amplified in ~ 25% of primary Gene encoding ErbB2 is amplified in ~ 25% of primary human breast tumors. human breast tumors.

ErbB2 amplification correlates with poor patient survival. ErbB2 amplification correlates with poor patient survival.

EGFR is not amplified in breast cancer, but the receptor EGFR is not amplified in breast cancer, but the receptor is activated by is activated by autocrineautocrine production of production of ligandsligands in the tumor. in the tumor.

Gene expression profiling & breast cancerGene expression profiling & breast cancer

Disease-free survivalOverall survival

Luminal A

ErbB2+Basal-like

BasalBasal NormalNormal Luminal BLuminal B Luminal ALuminal AErbB2ErbB2++

ErbB2+ group has a bad prognosisErbB2+ group has a bad prognosis

In a normal cell the In a normal cell the ErbBErbB RTKsRTKs depend upon depend upon ligandligand binding for activationbinding for activation

Response Response

Autocrine Stimulationof ErbB1/EGFR

Ligand-Independent Receptor Activationby Overexpression

of ErbB2

EGF1 1

PP P P P P

P PPP

2 2 2 2 2 23 3

TGFα

Transformation of Cells by ErbB ReceptorsTransformation of Cells by ErbB Receptors

ErbBErbB receptors are constitutively active in tumorsreceptors are constitutively active in tumors

Therapeutic approaches to target Therapeutic approaches to target ErbBErbB receptors receptors

AntibodiesAntibodies

Small molecule tyrosine Small molecule tyrosine kinasekinase inhibitorsinhibitors

Trastuzumab(Herceptin)

Pertuzumab(Omnitarg)

Cetuximab(Erbitux)

Matuzumab

Panitumumab

ErbB2

ErbB2

EGFR

EGFR

EGFR

Genentech/Roche

Genentech

ImClone/Merck KGaABristol-Myers Squibb

Merck KGaA Phase II – NSCLC, gynecologic, pancreatic, Esophageal.

Abgenix

Approved ErbB2-overexpressing breast cancer; trials in combination with various drugs ongoing.

Phase II - ovarian, breast, prostate, NSCLC; inhibits ErbB2 dimerization; trials ongoing in low ErbB2expressors.

Approved CRC; trials in combination with various drugs ongoing- pancreatic, HNSCC & NSCLC.

Pivotal trial ongoing – 3rd

line CRC, other indications-RCC and NSCLC.

ErbBErbB targeted recombinant antibodies*targeted recombinant antibodies*

*humanized or *humanized or chimericchimeric

TargetTarget CompanyCompany Status Status

Hynes & Lane 2005 Nature Rev Cancer May issueHynes & Lane 2005 Nature Rev Cancer May issue

P.Carter P.Carter VolVol 1, p1181, p118

Recombinant antibody structuresRecombinant antibody structures

CDRsCDRs -- murinemurine

EGFR targeted C225:EGFR targeted C225:chimericchimericγγ heavy chainheavy chain

ErbB2 targeted ErbB2 targeted Herceptin: Herceptin: humanized humanized γγ heavy chainheavy chain

Antibody treatment canAntibody treatment caninduce human antiinduce human anti--mousemouseantibodies (HAMA).antibodies (HAMA).

Response Response

Autocrine Stimulationof ErbB1/EGFR

Ligand-Independent Receptor Activationby Overexpression

of ErbB2

EGF1 1

PP P P P P

P PPP

2 2 2 2 2 23 3

TGFα

Transformation of Cells by ErbB ReceptorsTransformation of Cells by ErbB Receptors

MAb225 blocks MAb225 blocks autocrineautocrine activated EGFRactivated EGFR

Mendelsohn & Baselga 2000 Oncogene 19:6550

Xenografts - A431 human cancer cells with active EGFR.

Mice treated with mAb225, Doxorubicen or Cis-platin (CDDP)or cytotoxics in combination with C225.

MAb225 Shows Efficacy in Preclinical ModelsMAb225 Shows Efficacy in Preclinical Models

MAPK

PKB

PI3KPI3K

2 22 22 2 2 3

2 3

BT474 CellsBT474 Cells

4D5/Herceptin causes 4D5/Herceptin causes G1 arrest and G1 arrest and downregulation downregulation of of mitogenic mitogenic pathwayspathways

% Cell Cycle Stage% Cell Cycle Stage

G1G1 SS G2/MG2/M

PBSPBS 6767 1212 2121

FRP5FRP5 6565 1616 2020

4D54D5 9696 11 33

BT474 Cells Treated with 4D5/HerceptinBT474 Cells Treated with 4D5/Herceptin®®

Arrest in G1Arrest in G1

Herceptin has AntiHerceptin has Anti--ProliferativeProliferative Activity on Activity on ErbB2ErbB2--Overexpressing Breast Cancer Cells Overexpressing Breast Cancer Cells

MendelsohnMendelsohn & & BaselgaBaselga 2000 2000 OncogeneOncogene 19:655019:6550

BT474 tumor model

Herceptin/Herceptin/TrastuzumabTrastuzumab has Antihas Anti--ProliferativeProliferative Activity on Activity on ErbB2ErbB2--Overexpressing Breast Cancer Cells Growing asOverexpressing Breast Cancer Cells Growing asTumors in Nude Mice Tumors in Nude Mice

Doses:1-30 mg/kg Herceptin/Trastuzumab twice weekly

Combination Treatment With Herceptin + StandardCombination Treatment With Herceptin + StandardChemotherapeutic AgentsChemotherapeutic Agents

MendelsohnMendelsohn & & BaselgaBaselga 2000 2000 OncogeneOncogene 19:655019:6550

BT474 tumor model

0.3 mg/kg: sub-optimal Herceptin dosetwice weekly

Sub-optimal doses of both drugs showincreased activity in combination withHerceptin.

ErbB2 as a target for breast cancer ErbB2 as a target for breast cancer

19851985 gene encoding ErbB2 cloned gene encoding ErbB2 cloned

1987/88 1987/88 gene amplification & protein gene amplification & protein overexpressionoverexpressionfound in ~ 20found in ~ 20--30% primary breast tumors;30% primary breast tumors;correlation with poor prognosis correlation with poor prognosis

19891989 growth inhibitory ErbB2 specific growth inhibitory ErbB2 specific mAbsmAbs describeddescribed

19911991 Phase I clinical trial with Phase I clinical trial with murinemurine mAbmAb 4D54D5

19921992 Phase I clinical trial with “humanized” 4D5 Phase I clinical trial with “humanized” 4D5 --HerceptinHerceptin

19961996 Phase II trial results publishedPhase II trial results published

19971997 Phase III trialsPhase III trials with publishedwith published

19981998 Herceptin approval in the USA for treatment Herceptin approval in the USA for treatment of ErbB2of ErbB2++++, , metastaticmetastatic breast cancerbreast cancer

MendelsohnMendelsohn & & BaselgaBaselga 2000 2000 OncogeneOncogene 19:655019:6550

Pivotal Phase III Clinical Trial with Pivotal Phase III Clinical Trial with TrastuzumabTrastuzumab/Herceptin/Herceptinin Combination with Chemotherapy in Combination with Chemotherapy

n = 469, n = 469, metastaticmetastatic breast cancer patients who had failed previous therapies.breast cancer patients who had failed previous therapies.

MendelsohnMendelsohn & & BaselgaBaselga 2000 2000 OncogeneOncogene 19:655019:6550

Pivotal Phase III Clinical Trial with Pivotal Phase III Clinical Trial with TrastuzumabTrastuzumab/Herceptin/Herceptinin Combination with Chemotherapy in Combination with Chemotherapy

MendelsohnMendelsohn & & BaselgaBaselga 2000 2000 OncogeneOncogene 19:655019:6550

Pivotal Phase III Clinical Trial with Pivotal Phase III Clinical Trial with TrastuzumabTrastuzumab/Herceptin/Herceptinin Combination with Chemotherapy in Combination with Chemotherapy

MendelsohnMendelsohn & & BaselgaBaselga 2000 2000 OncogeneOncogene 19:655019:6550

Pivotal Phase III Clinical Trial with Pivotal Phase III Clinical Trial with TrastuzumabTrastuzumab/Herceptin/Herceptinin Combination with Chemotherapy in Combination with Chemotherapy

Based upon these results Herceptin was approved for the treatmenBased upon these results Herceptin was approved for the treatment of t of breast cancer patients in 1998. breast cancer patients in 1998.

Potential Mechanisms Contributing to Potential Mechanisms Contributing to Herceptin’sHerceptin’sAntiAnti--Tumor ActivityTumor Activity

Blocks ErbB2’s constitutive Blocks ErbB2’s constitutive kinasekinase activity & activity & downstream signaling pathways. downstream signaling pathways.

Induces loss of ErbB2 from tumor cells, i.e.,Induces loss of ErbB2 from tumor cells, i.e.,downdown--regulation. regulation.

Allows recruitment of immune Allows recruitment of immune effectoreffector cells cells to the tumor. to the tumor.

Antibody ClassesAntibody Classes

IgMIgM IgDIgD IgGIgG IgAIgA IgEIgE

Heavy ChainHeavy Chain µµ δδ γγ αα εε

Light ChainLight Chain κκ or or δδ

Activates Activates ++++++++ -- ++++ -- --ComplementComplement

Binds macrophages Binds macrophages -- -- ++ -- --& & neutrophilsneutrophils

γγ heavy chain can recruit immune heavy chain can recruit immune effectoreffector functionsfunctions

Houghton & Houghton & ScheinbergScheinberg 2000 Nat Med 6: 3732000 Nat Med 6: 373

Potential Mechanisms Contributing to Potential Mechanisms Contributing to Herceptin’sHerceptin’sAntiAnti--Tumor ActivityTumor Activity

ClynesClynes 2000 Nat Med 6: 4432000 Nat Med 6: 443

BT474 breast tumor modelwkswks

Herceptin’sHerceptin’s AntiAnti--tumor Activity is Partiallytumor Activity is PartiallyDependent on Dependent on FcFc Receptor ActivationReceptor Activation

WT nude mice FcRγ −/− nude mice

~40% activity is retained in FcRγ -/- mice

Houghton & Houghton & ScheinbergScheinberg 2000 Nat Med 6: 3732000 Nat Med 6: 373

Potential Mechanisms Contributing to Potential Mechanisms Contributing to Herceptin’sHerceptin’s AntiAnti--tumor Activitytumor Activity

mAbmAb bindingbindingdirectlydirectlyaffects ErbB2affects ErbB2activityactivity

BadacheBadache & Hynes (2004) Cancer Cell 5: 299& Hynes (2004) Cancer Cell 5: 299

PertuzumabPertuzumab & & TrastuzumabTrastuzumab block ErbB2 function inblock ErbB2 function intumors tumors ovexpressingovexpressing the receptor.the receptor.

Therapeutic antibodies targeting ErbB2Therapeutic antibodies targeting ErbB2

Is Herceptin a “Perfect” Drug ? Is Herceptin a “Perfect” Drug ?

Patients treated with Herceptin in combination with AC Patients treated with Herceptin in combination with AC ((doxdox + + cyclophosphamidecyclophosphamide) had an increased rate of ) had an increased rate of cardiac toxicity. cardiac toxicity.

Ablation of ErbB2 in mice by gene KO technology, Ablation of ErbB2 in mice by gene KO technology, revealed that this receptor was essential for normalrevealed that this receptor was essential for normalcardiac development. cardiac development. (KO embryos died at E 10.5). (KO embryos died at E 10.5).

Crone et al 2002 Nature Med 8:459

CardiomyocytesCardiomyocytes from ErbB2 KO mice arefrom ErbB2 KO mice aremore sensitive to more sensitive to adriamycin/doxadriamycin/dox--induced cell death. induced cell death.

Control

ErbB2 KO

Specific deletion of ErbB2 in Specific deletion of ErbB2 in cardiomyocytescardiomyocytes reveals a role for reveals a role for the receptor in stress conditions. the receptor in stress conditions.

Herceptin is a Rationally Designed Drug Herceptin is a Rationally Designed Drug

Development of Herceptin is one of the best examples Development of Herceptin is one of the best examples of a rationally designed therapeutic strategy. of a rationally designed therapeutic strategy.

Clinical data suggested that high levels of ErbB2 in breast cancClinical data suggested that high levels of ErbB2 in breast cancerercorrelated with bad prognosis. correlated with bad prognosis.

Biology of the target protein. Biology of the target protein.

Potential of ErbB2 to cause transformation. Potential of ErbB2 to cause transformation.

Animal models were used to show that inhibiting ErbB2 wouldAnimal models were used to show that inhibiting ErbB2 wouldblock tumor growth. (block tumor growth. (Proof of ConceptProof of Concept) )

Thirteen years elapsed between cloning of gene (1985)Thirteen years elapsed between cloning of gene (1985)& approval of an & approval of an ErbBErbB targeted therapeutic (1998). targeted therapeutic (1998).

Not all patients whose tumors Not all patients whose tumors overexpressoverexpress ErbB2 ErbB2 respond to Herceptin. respond to Herceptin.

We need to understand more about how Herceptin We need to understand more about how Herceptin works in order to better predict who will respond to works in order to better predict who will respond to treatment. treatment.

Herceptin is a Rationally Designed Drug Herceptin is a Rationally Designed Drug

However:However:

P.Carter P.Carter volvol 1, p1181, p118

AntiAnti--Tumor Approaches using Antibodies Tumor Approaches using Antibodies

Structure of Single Chain Antibody Structure of Single Chain Antibody -- scFvscFv

Fv

W.Wels

FRP5 FRP5 -- murinemurine mAbmAb that binds ErbB2’s that binds ErbB2’s extracellularextracellular domaindomain

ErbB2 specific ErbB2 specific mAbmAb FRP5FRP5

Binds ErbB2’s Binds ErbB2’s extracellularextracellular domain with high affinity.domain with high affinity.

HybridomaHybridoma cells producing FRP5 were used to isolate cells producing FRP5 were used to isolate cDNAscDNAsencoding the specific heavy and light chain variable domains. encoding the specific heavy and light chain variable domains.

ChimericChimeric scFvscFv--Toxin Fusion ProteinToxin Fusion Protein

W.Wels

ChimericChimeric scFvscFv--Toxin Fusion ProteinToxin Fusion Protein

W.Wels

scFvscFv--Toxin Binds ErbB2 & Internalizes into Tumor CellsToxin Binds ErbB2 & Internalizes into Tumor Cells

Toxin ReceptorToxin Receptor ErbB2ErbB2

W.Wels

Internalized ErbB2 Internalized ErbB2 enters the enters the endosomesendosomes

W.Wels

scFvscFv--Toxin Is Bacterially Produced & Purified to HomogeneityToxin Is Bacterially Produced & Purified to Homogeneity

The The scFvscFv--Toxin FRP5Toxin FRP5--ETA Specifically Kills ErbB2+ CellsETA Specifically Kills ErbB2+ Cells

W.Wels

W.Wels

Tumor Cells with High ErbB2 are Very Sensitive to FRP5Tumor Cells with High ErbB2 are Very Sensitive to FRP5--ETAETA

W.Wels

ErbB2ErbB2--Overexpressing Tumor Xenografts are Sensitive to FRP5Overexpressing Tumor Xenografts are Sensitive to FRP5--ETA ETA

scFvscFv--FRP5FRP5--ETA is currently in early phases of clinical development. ETA is currently in early phases of clinical development.