1. Understanding Genome-Wide Profiling of Cancer
Erin Milliken L. Michelle Bennett
Understanding Genome-Wide
Profiling of Cancer
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Donna Kerrigan, M.S.
E. Milliken, Ph.D.
L.M. Bennett, Ph.D.
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2. Single Gene Tests: A Locally Focused Search
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
World
Continent
Country
U.S. State
Chemical bases
Genome
Chromosome
Gene A G T C
Single-gene tests focus on a specific, known location in a patient’s genome. Using this approach, scientists have looked for single genes linked to cancer. This research has revealed some important discoveries such as gene changes called mutations located within the BRCA1 or BRCA2 genes that may confer a significantly increased risk of breast and ovarian cancer. And some single-gene tests continue to inform treatment decisions. For example, colon cancer patients may be tested for a K-ras mutation, or breast cancer patients for HER2 gene activity, before treatment is planned.
A geographical analogy helps depict the scale of the search involved in single-gene tests.
Cell
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3. Genome-Wide Profiling: A Globally Focused Search
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
World
Continent
Country
U.S. State
Chemical bases
Genome
Chromosome
Gene A G
But single-gene tests alone are unable to completely unravel the complexity of cancer. They very rarely explain why cancer has developed. Because cancer involves the simultaneous interaction of many different mutated genes and proteins within malignant cells and their surrounding normal tissues, a global approach is needed to capture all the activity. Scientists have developed new methods to cast a genome-wide search and look globally for all the changes in DNA, RNA, or proteins* that contribute to cancer’s existence. These new approaches are collectively called genome-wide profiling.
Again, a geographical analogy shows the global scale of the search involved in genome-wide profiling.
*To learn more basic information about DNA, RNA, and proteins in relation to the molecular diagnosis of cancer, please visit T C
Cell
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4. Starts with Proper Collection and Storage
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Informed consent
Biopsy sample
Add unique identifier
Store
Carefully retrieve and analyze
Carefully refreeze
The specimen is biologically viable
Pre-acquisition
Post-acquisition
Genome-wide profiling requires proper sample collection and storage. This is why biopsy specimens must be collected, stored, tracked, and used according to the highest scientific and ethical standards.
Informed consent for use of cancerous biopsy specimens (or a waiver of this consent) must be in place at the time of collection.
Samples must be given an electronic, unique identifier that links the specimen to a patient, to a treatment protocol, and to the informed consent (or waiver).
Samples must be stored and retrieved following best clinical practices.
When all this is in order, a researcher can use the biopsy sample to get a genome-wide profile of the cancer.
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5. Genome-Wide Profiling: Many Ways
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Genome-wide profiling refers to the collection of large-scale information about the sequence and expression of the genes in normal or tumor cells. Techniques include:
• Comparative Genomic Hybridization and Spectral Karyotyping looking at global changes in chromosome structure when cancer cells are compared to normal tissues from the same patient
• Polymorphism Analysis looking at sets of genetic variations called polymorphisms in individuals and in populations (These specific altered regions or sequences are spread across the entire genome.)
• Gene Expression Profiling capturing total gene activities, both increases and decreases, across a genome as patterns of gene expression
Most of these techniques use high-throughput technologies, which are automated systems that can perform rapid analysis of large numbers of samples in a short period of time. Genomic signatures or genomic profiles that are validated can be used to identify a specific tumor type in new tissue samples.
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6. Genome-Wide Profiling: Comparative Genomic Hybridization
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
= equal binding of labeled normal and tumor probes
= more binding of labeled tumor probes—gain of tumor DNA
= more binding of labeled normal probes—loss of tumor DNA
Label probes for all tumor DNA
Label probes for all normal DNA
Hybridize to normal metaphase chromosomes for 48—72 hours
Metaphase chromosome
Comparative genomic hybridization (CGH) is a method that allows researchers to detect large- scale changes in chromosomes. They can visualize where extra genetic material (repeating copies) has been added or deleted. They can see where large portions of genetic material have been inserted into chromosomes. Many cancers exhibit these types of chromosomal abnormalities.
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7. Genome-Wide Profiling: Karyotyping
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Different color probes for each chromosome
Hybridization for 24—72 hours
Human chromosomes
Sorted, labeled human chromosomes
Microscope camera captures different spectra
Spectral karyotyping is a method that allows researchers to visualize each chromosome using a different color. This technology is powerful because it makes it possible to see where genetic material has been added or exchanged within a cancer genome.
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8. Genome-Wide Profiling: Polymorphism Analysis
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Person 1
Person 2
Common Sequence
Variations
Polymorphism
= Variations in DNA
Another approach is called polymorphism analysis. Polymorphism analysis is used to find variation in genetic sequence across the genome; some tests look for sets of telltale variations in single base pairs (single-nucleotide polymorphisms), whereas others look for tandem repeats (simple sequence repeats) or insertions of several telltale bases (restriction-fragment length polymorphisms).
Once sequence polymorphisms that are potentially associated with cancer are identified, these must be validated through further research in independent populations. Polymorphism data will be used in conjunction with other genetic, genomic, and clinical information.
Deletions
Insertions
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9. Genome-Wide Profiling: Gene Expression Profiles
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
What does my patient’s profile say about the diagnosis?
prognosis?
treatment?
All LN+ breast tissue samples (metastatic)
Lymph node positive
Researchers use gene expression profiling to study the activity of genes in a patient’s tumor sample and compare it to normal tissue of the same type. This approach measures levels of messenger RNA (mRNA), which is a good indicator of the proteins about to be made by the cell.
An individual’s messenger RNA is processed and labeled and then applied to a specially designed chip, often referred to as a microarray, which includes complementary sequences for hundreds of thousands of genes. A chip reader measures levels of the expressed genes in the sample.
All LN- breast tissue samples (non-metastatic)
Lymph node negative
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10. Why Is It Important To Collect Genome-Wide Profiles?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
General population
Is there more cancer in group with suspected SNP?
SNP suspected of link to cancer risk
Genomic profiling provides a way to collect extensive information about individuals, analyze the profiles from many patients and their tumor(s), and use databases and analytical tools (informatics) to discover important differences. Researchers hope to correlate genomic profiles with known clinical factors and outcomes.
Sort out people with telltale SNP
Lack suspected SNP
Have suspected SNP
Analysis team
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11. Can Genome-Wide Profiles Predict Cancer Risk?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Same profile, different environment = different result
Precancer
Cancer
No cancer
Not yet. Cancer is a complex disease that is driven by genetic, lifestyle, and environmental factors. Furthermore, each individual and each tumor are unique. Thus, it has proven difficult to identify a predictive genomic profile that is valid for multiple populations. Claims that a particular genomic profile can predict cancer risk should be interpreted with caution. It is likely that genomic profiles will need to be used in conjunction with other genetic and clinical information to accurately assess a person’s cancer risk.
= same genomic profile
= same polymorphism
= cancer
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12. Can Genome-Wide Profiling Discover Polymorphisms
That May Influence Cancer Risk?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Population
No Prostate Cancer
Prostate Cancer
Possibly. Studies of polymorphisms in individual patients and in populations are yielding interesting hints. Some (but not all) sequence polymorphisms appear to influence cancer risk. Some sequence polymorphisms may not themselves be associated with cancer risk but may serve as "markers" for nearby genes that do exert an influence.
Polymorphisms within protein coding regions may alter the function of the encoded protein, particularly if they change its structure or amino acid sequence. Polymorphisms in regions of the DNA that regulate gene expression may result in altered protein levels, alternative splicing, or other modifications.
Changes in a protein's sequence or its regulation can influence cancer risk by altering how a person responds to carcinogens, drugs, chemicals, or other environmental exposures.
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13. Can Genome-Wide Profiles Help Diagnose Cancer?
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Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Diffuse large B-cell lymphoma under the microscope
Pathology sees one cancer
Patient 1
Patient 2
Diffuse large B-cell lymphoma after genome-wide profiling
Region A
Molecular diagnosis sees two cancers
Yes. Genomic profiles can be used to subclassify certain cancers within pathologically defined groups. These profiles can further differentiate tumors that look similar under the microscope. Gene expression microarrays—the major tool used to measure the genome-wide changes in gene expression—have been used experimentally to subclassify acute myelogenous leukemias and to distinguish Burkitt’s from diffuse large B-cell lymphoma. And work is under way to build a genome-based classification system for glioma, the most common type of brain tumor.
While use of genomic profiling for cancer care is still in the research phase, it is already improving diagnosis for some cancers.
Region B
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14. Can Genome-Wide Profiles Inform Cancer Prognosis?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Patient DNA
21-gene panel linked to breast cancer recurrence
Parafin block
Patient’s
tissue sample
Yes. Certain gene changes have already been associated with cancer prognosis. For breast cancer, researchers have identified a set of 21 cancer-related genes whose expression is associated with risk of breast cancer recurrence among women with estrogen receptor-positive breast cancer treated surgically. The profile yields a “score” that predicts the likelihood of disease recurrence. It is not yet clear how each of the genes functions, whether separately or together, whether as “driver” or “passenger,” in the pathophysiology of breast cancer.
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15. Can Genome-Wide Profiles Inform Cancer Treatment Planning?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Colon cancer patients
Individual genome-wide profiles are sorted
Do not have mutated K-ras gene
Have mutated K-ras gene
Profile A...
give targeted therapy A A E A B C C B A B C E E A E B C C B B C B
Yes, although most attempts to do this are occurring within clinical trials. If therapies are available that target genes, proteins, or pathways that appear, on the basis of genome-wide profiling, to be altered in tumors, these can be used to try to treat the patient. Researchers and clinicians are also analyzing large genomic profile databases to help match patients to the best possible treatments. The genomic profile of a newly diagnosed patient is compared with previous patients’ genomic profiles, which are linked to information about how these patients were treated and responded to their treatment. This approach will allow newly diagnosed patients to be given treatments that were effective against cancers with similar genetic profiles.
Respond to targeted therapy A
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16. Can Genome-Wide Profiles Monitor the Response to Cancer Treatment?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
Not yet. Once a molecular pathway and its activities that support a cancer are known, genomic analysis may eventually be used to monitor whether or not a pathway-specific targeted treatment has effectively disrupted this pathway, but this has not yet been demonstrated in the clinic.
The goal: verify that pathway is destroyed
mTOR↓
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17. Will Genomically Informed Cancer Care Be Better for Patients?
National Cancer Institute
Understanding Cancer and Related Topics
Understanding Genome-Wide Profiling of Cancer
What does this profile mean in terms of my cancer care?
Customized
Care
Yes. Genome-wide profiling—whether it involves comparative genomic hybridization, karyotyping, polymorphism analysis, or gene expression profiling—is an essential element of genomically informed cancer care. The goal of genome-based care is to tailor cancer risk assessment, diagnosis, prognosis, and treatment to each individual and malignancy. Researchers are using genome-wide profiling to lay groundwork that should enable this to become a reality. However, there is still much work to be done before this new approach can be translated into better care for patients.
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