Significance and Rational Use of Molecular Markers in ...Breast Cancer and the ErbB receptors • A...

Significance and Rational Use of Molecular Markers in

Cancer Management

Ana Maria Lopez, MD, MPHProfessor of Medicine and Pathology

Arizona Cancer Center

Ripped from the Headlines

Topics: Primer of Cancer Molecular Medicine

• Personalized medicine & cancer treatment

• New technologies for the molecular profiling of cancer

• Hallmarks of cancer – essential alterations for malignant transformation

• Genomic instability in cancer

• Specific genetic alterations seen in cancer

• Cancer therapies targeted to specific genetic alterations

Question 1Mrs. Jones is a 58 year-old woman who presents with

abdominal discomfort, fatigue and early satiety. On physical exam, you note an enlarged spleen. Laboratory results reveal a WBC 24,000 with 47N, 6B, 37L, 7E, 2 Monocytes, 1Basophil. Considering the diagnosis of chronic myelogenous leukemia, you arrange for the patient to see an oncologist and send the patient for:

a. Repeat labs in 3 months as this is not an acute process. b. Her2-neu testing as trastuzumab is a known protective

marker. c. Assess for Philadelphia chromosome, a known target for

treatment in CML. d. Send blood for p53, an adverse marker for CML.

Question #2Mr. John Smith is a 43 yo gentleman who is a BRCA

carrier. On physical exam, you note a 2.5 cm breast mass that is diagnosed as invasive ductal carcinoma. When you refer him to the oncologist, you ask about clinical trials related to:

A. Aromatase inhibitors as his tumor is likely to be ER negative.

B. PARP1 inhibitors that target BRCA tumor suppressor gene.

C.Trastuzumab as this new therapy is most effective in men.

D.Lapatinib as this new therapy target BRCA tumor suppressor gene.

Carcinogenesis

Personalized Medicine in Cancer

Vision for the Transformation of Medicine in the 21st Century

Personalized PreemptivePredictive

“I predict that comprehensive, genomics-based health care will become the norm with individualized preventive medicine and early detection of illnesses.”

- Elias A. Zerhouni, 2006

Participatory

Understanding the Technology

• Gene chip technology• Proteomics• Pharmacogenomics

Gene Chip Technology

Diffuse Large B-Cell Lymphoma• Most common type of aggressive

lymphoma

• Standard therapy cures some of the DLBCL patients

• Remaining patients have a high probability of death within 5 years

• Clinical factors are somewhat predictive of treatment outcome

Key Question: What is the biology underlying the disparate treatment outcome?

Gene Expression Profiling of DLBCL

• 240 patients with Diffuse Large B-cell Lymphoma

• Specimens collected at the time of diagnosis

• All patients received adriamycin-based chemotherapy

• Gene expression was compared to patient outcome

Alizadeh et al., Nature, 2000

Gene Expression Profiling & DLBCL

1) Identified DLBCL subtypes2) Subtypes reflect the tumor biology3) Subtypes add to the predictive power of the clinical features

Proteomics in Cancer Treatment

Goals:• Identify biomarkers of

early disease

• Monitor response to therapy

• Predict likelihood of relapse after therapy

DrugInteractions

Pharmacogenomics

• Study of inherited differences in drug disposition and effects

• Largely focused on genetic polymorphisms in drug metabolizing enzymes

Genetic Factors

Anticancer drugs

EnvironmentalFactors

Variations in drug

response

Transport

DrugTarget Metabolism

Pharmacogenomics & Cancer Treatment

TPMT: thiopurine methyltransferaseHPRT: hypoxanthine phosphoribosyl

transferase

Applications of the New Technologies

• Gene expression profiling– discovery of patterns to predict treatment response

• Proteomics– identify serum markers to monitor treatment response

• Pharmacogenomics– use genetic information to optimize treatment dose

Carcinogenesis – The Vogelgram

Carcinogenesis –The Hallmarks of Cancer

Self-Sufficiency in Growth Signals

• Normal cells require mitogenic signals to proliferate

• Tumor cells generate their own signals

Oncogenes for Self-Sufficiency

Oncogene Cancer Mechanism

ERB B2 Breast cancer Gene amplification

BCR-ABL Chronic myelogenous leukemia

t(9;22)

Ras Multiple types Gene mutation

c-myc Burkitt lymphoma t(8;14)

Insensitivity to Anti-Growth Signals

Signal Cancer Mechanism

G1 checkpoint Retinoblastoma Loss of both Rb alleles

DNA damage Many cancers Loss or mutation of p53

TGFβ Pancreatic (100%)Colon cancer (83%)

Mutations in the signaling pathway

Evasion of Apoptosis

t(14;18) in Follicular Lymphoma

Chr 14 Chr 18Promoter bcl-2

Limitless Replicative Potential

Replicative Senescence

SpeciesLifespan (years)

Cell Divisions

Mouse 2 9.2

Rabbit 13 22.5

Chicken 30 25.0

Horse 46 28.8

Human 110 61.3

Number of Cell Divisions

p53 Loss & Limitless Replicative Potential

Sustained Angiogenesis

• New blood vessel formation is required once tumors grow beyond 1-2 mm

• Tumor strategies for angiogenesis:– production of angiogenic factors (e.g., vascular

endothelial growth factor)– loss of anti-angiogenic factors (e.g., von Hippel-

Lindau protein)

Glioblastoma multiforme: Tumor production of VEGF promotes angiogenesis

HIF1α under normoxia:- hydroxylated- is bound by the Von Hippel-

Lindau protein & degraded- is a short-lived protein

HIF1α under hypoxia:- not hydroxylated- is a stable transcription factor- gene targets include VEGF

Von Hippel-Lindau Disease

• Autosomal dominant disease

• Frequency: 1:30,000-40,000

• Pathology:– capillary hemangioblastomas in

the CNS and retina– cysts in the pancreas, liver and

kidney– predisposition to developing

renal cell carcinoma Hemangioblastoma

Tissue Invasion and Metastasis

The abilities to invade and metastasize are the defining attributes of malignant tumors and the major cause of cancer-related deaths.

Strategies for Tissue Invasion and Metastasis

1. Downregulate adhesion molecules

2. Secrete proteases to degrade the basement membrane and the ECM

3. Alter the type of cell surface attachment molecules (e.g., integrins)

4. Co-opt ‘normal’ stromal cells to aid in invasion

Genomic Instability in Cancer –An Enabling Characteristic of

Cancer CellsNormal Spectral Karyotype

Cancer Cell Spectral Karyotype

Mechanisms for Maintaining Genomic Integrity

• DNA repair mechanisms– mismatch repair – corrects mismatched base pairing

occurring during DNA replication– nucleotide excision repair – removes pyrimidine

dimers and oxidatively damaged nucleotides

• DNA damage sensors/regulators of apoptosis– p53– ATM

Ataxia-Telangiectasia• Autosomal recessive disorder

• Neuronal degeneration leads to an ataxic-dyskinetic syndrome beginning in childhood

• ATM is a kinase involved in the cellular response to DNA damage

• Mutations in ATM increase sensitivity to x-ray-induced DNA damage

• ATM carriers may have an increased risk of breast cancer from screening mammography

MSH2 and Colon Cancer• MSH2 encodes a protein involved in mismatch

repair

• Hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is caused by mutations in MSH2

• Homozygous loss of MSH2 increase gene mutations rates by 1000-fold and leads to genomic instability

• HNPCC patients develop colon cancer at a younger age (<50) than unaffected individuals

Genetics Alterations & The Hallmarks of Cancer

Loss of Rb

Invasion & Metastasis

Evading Apoptosis

Ras mutations ↑ Bcl-2

p53 mutation ↓ TGFβ ↑ Her-2 neu

↑ c-myc BCR-ABL ↑ VEGF

Genetics Alterations & The Hallmarks of Cancer

Loss of Rb

Invasion & Metastasis

Evading Apoptosis

Ras mutations

↑ Bcl-2↑ VEGF

↓ TGFβ

↑ Her-2 neu

↑ c-myc

BCR-ABL

p53 mutationp53 mutation

p53 mutation

Epigenetic Changes

• Alterations in DNA, other than in the primary sequence or in the number of gene loci

• Includes DNA methylation and histone acetylation

• Result in a differences in gene transcription and can contribute to carcinogenesis

Altered Gene Expression With Epigenetic Changes

Maternal Supplements(vitamin B12, folic acid, choline)

Methyltransferases attach methyl groups to DNA

HDACS are recruited & transcription repressed

Methyl groups attach to cytosine

HDACS: histone deacetylases

Clinical Applications of Cancer Genetics

Chronic Myelogenous Leukemia

BCR-ABL in Chronic Myeloid Leukemia

• ABL is a non-receptor tyrosine kinase

• The t(9;22) in CML generates novel fusion proteins, designated BCR-ABL

• The fusion proteins have constitutive tyrosine kinase activity (i.e., normal regulation is lost)

• Imatinib mesylate (Gleevec) was developed to target BCR-ABL

Imatinib:Treatment of CML

• Its development was the start of molecularly targeted therapies for cancer

• Alternate names: ST1571, imatinib mesylate and imatinib

• Recognizes the ATP binding site of ABL

• Inhibits the constitutive tyrosine kinase activity

Brian Druker, M.D.

Imatinib – Mechanism of Action

Fausel. J Manag Care Pharm 12(suppl S-a):S8, 2007

Imatinib - Phase III Clinical Trial Results

• Newly diagnosed patients with chronic-phase CML

• Randomly assigned:– interferon alpha plus low

dose cytarabine (553 patients)

– imatinib (553 patients)

• 318 of the combination therapy patients eventually crossed over to imatinib

O’Brien et al. N Engl J Med 348:994, 2003

Imatinib Clinical Trial – Adverse Events*

Combination Therapy

Gleevec

HematologicAnemia 4.3% 3.1%Neutropenia 25.0% 14.3%Thrombocytopenia 16.5 % 7.8%

OtherFatigue 24.4% 1.1%Depression 12.8% 0.4%

*Numbers indicate the percent of patients with the adverse event, Grade 3 or 4

Targeted Therapy

What are Targeted Therapies?

• Targeted therapies block the growth and spread of cancer by interfering with specific molecules involved in tumor growth and progression

Carcinogenesis

Hallmarks of Cancer

How do targeted therapies work?

• Interfere with cancer cell division—proliferation

• Focus on proteins involved in cell signaling pathways—complex system that governs basic cellular functions and activities e.g. – cell division– cell movement – cell responses to specific external stimuli– cell death

• Directly induce apoptosis• Indirectly stimulate immune system to

recognize/destroy cancer cells

Therefore, need a good…

• Target– A target known to play a role in cancer cell

growth and survival– “rational drug design”

What was the 1st targeted therapy?

• Cellular receptor for estrogen in breast cancer

•E2 binds to the ER•Resulting hormone receptor complex activates expression of specific genes involved in:

- cell growth - proliferation

Breast cancer: E2 targets

• SERMs: binds to ER and prevents E2 binding– Tamoxifen– Toremifine

• Fulvestrant (Faslodex): binds to ER and promotes its destruction—reduces ER levels in cell

Aromatase inhibitors

Non-hormonal breast cancer targets

• Tyrosine kinase signal transduction pathways

• Block tumor angiogenesis• Modulate apoptosis• Inhibit histone deacetylation

Types of Receptors

Breast Cancer and the ErbB receptors

• A subset of breast cancers do not express estrogen receptors

• Are on average more aggressive and less differentiated

• Tumor growth is driven through the actions of the ErbB receptor family

• ErbB2 up regulated by gene amplification

erbB Family

• erbB 1: epidermal growth factor receptor or Her 1

• erbB2: Her2• erbB3: Her 3• erbB4: Her 4

ERB B2

• Gene encoding epidermal growth factor receptor-2

• Also called Her2 or neu

• Gene amplification is seen in ~25% of breast cancers

ERB B2 Breast Cancers: More Aggressive

ERB B2 Negative ERB B2 PositiveLow grade High gradeLow mitotic index High mitotic indexNo necrosis NecrosisNo lymphoid infiltrate

Lymphoid infiltrate

PgR+ PgR-

Trastuzumab• Monoclonal antibody binding to HER2/neu

(erbB2) receptor• Standard treatment (in combination with

chemotherapy) for HER2-positive breast cancer• Reduces the risk of recurrent HER2-positive

disease by ~50%• Cardiotoxicity the most important adverse event

– Trastuzumab + paclitaxel: 13% – Trastuzumab + anthracycline: 27%

Piccart-Gebhart MJ.

Treatment of Breast Cancer with Trastuzumab

Slamon et al. N Engl J Med 344:783, 2001

Addition of Trastuzumab To Other Therapies

Synergy: Radiation therapy, carboplatinum, cisplatinum, docetaxal, cyclophosphamide, etoposide, gemcitabine (low dose), tamoxifen

Additivity: Doxorubicin, epirubicin, methotrexate, paclitaxel

Antagonism: 5-Fluoruracil, gemcitabine (high dose)

Trastuzumab Resistance

• Virtually all HER2+ metastatic breast cancers develop resistance

• Adjuvant trastuzumab reduces the annual hazard rate – 50% benefit– 50% relapse

Sledge GW..

Possible Causes of Trastuzumab Resistance

• Suboptimal drug delivery• Altered target expression• Altered target• Modified target-regulating

proteins• Alternative pathway signaling

Sledge GW. Sledge GW.

Overcoming Trastuzumab Resistance

• Block the HER pathway(s) at other points

• Block other growth factor receptor pathways (HER1, IGF-1R)

• Block angiogenesis

Sledge GW.

Lapatinib

Dual inhibitor targeting both erbB1 (or epidermal growth factor) and erbB2 (or HER2/neu) receptors

Lapatinib+Capecitabine vs Capacitabine in Trastuzumab-Resistant Disease

Time to Tumor Progression

Lapatinib/Capecitabine Capecitabinen = 160 n = 161

Progressed or died 45 (28%) 69 (43%)

Median TTP (wk) 36.9 19.7

Hazard ratio (95% CI) 0.51 (0.35, 0.74)

P-value (log rank, 1-sided) .00016

erbB Conclusions

• erbB Family—trastuzumab and lapatinib—activity in metastatic breast cancer

• Lapatinib is effective in treating trastuzumab-resistant tumors; may be beneficial in treating CNS metastases

Targeted therapy: prominent role in the treatment of breast cancer

Hereditary Breast Cancer• Between 10% and 20% of breast cancer cases

are hereditary in nature.• In hereditary breast cancer the age of onset is

considerably younger (< 30 years of age)• In hereditary breast cancer syndromes tumors

are more likely to occur bilaterally

Features of BRCA Breast Cancers

• Histologically indistinguishable from sporadic breast cancers

• Reduced capacity to repair DNA results in increased rates of mutation

• Loss of BRCA gene renders cells highly sensitive to radiation-induced DNA damage

BRCA1 & BRCA2• 50% of hereditary breast cancers are due to inherited

mutations in BRCA1 or BRCA2– Life-time risk for developing cancer is 40-80%

• Associated tumors may include ovarian, pancreas, bile duct, stomach, colon, and endometrium

• Mutations in the other genes accounts for only 10% of the remaining hereditary breast cancers– 40% of familial cases are caused by mutations in unknown genes.

• BRCA1 and BRCA2 are tumor suppressor genesfunction in the repair of DNA double strand breaks (DSBs)

PARP-1 Inhibitor

Clinical Trials and Targeted Therapies

• Clinical trial design that incorporates molecular markers

• Trial endpoints may include pathway modulation

• New data on cross-talk between molecular targets (ie ER and Her2) need to be incorporated

Where do we go from here?

• Rationale: – Clinical developments– Molecular development– Clinical-molecular interfaces:

facilitate understanding of the treatment of human disease at a molecular level