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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
Cell
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
Limitless Replicative Potential
Telo
mer
e Le
ngth
Long
Short
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)
Sustained Angiogenesis
Glioblastoma multiforme: Tumor production of VEGF promotes angiogenesis
Sustained 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
Self-Sufficiency in Growth Signals
Sustained Angiogenesis
Insensitivity to Anti-Growth Signals
Invasion & Metastasis
Evading Apoptosis
Limitless Replicative Potential
Ras mutations ↑ Bcl-2
p53 mutation ↓ TGFβ ↑ Her-2 neu
↑ c-myc BCR-ABL ↑ VEGF
Genetics Alterations & The Hallmarks of Cancer
Loss of Rb
Self-Sufficiency in Growth Signals
Sustained Angiogenesis
Insensitivity to Anti-Growth Signals
Invasion & Metastasis
Evading Apoptosis
Limitless Replicative Potential
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
Chronic Myelogenous Leukemia
Ph
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?
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
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.
Questions?