Expression Profiling, Protein
 This grade 3 invasive carcinoma has a high mitotic rate  . Additional information is provided by the use of IHC markers to determine prognosis and guide selection of the most effective therapy. |
 IHC markers classify breast cancers into distinct cancer subsets that demonstrate differences in patient characteristics, prognosis, patterns of recurrence, and response to types of treatments. |
TERMINOLOGY
Definitions
Protein expression profiling is identification of patterns of expression that identify clinically significant subtypes of breast cancer
Breast cancer encompasses a group of very heterogeneous malignancies
Carcinomas show distinct differences in natural history, pathologic features, and biologic behavior
Multiple treatment options are available, including use of targeted therapies
Increasingly, clinical decisions on utility of treatment options and targeted therapy require assessment of underlying tumor biology
Need for clinically useful breast cancer classification scheme to help assess prognosis and aid treatment decisions
Breast Cancer Biology and Classification
Technical advances have made it possible to study underlying biology of breast cancer samples
Tumor tissue can be analyzed for changes at level of genome
DNA copy number changes; genomic gains and losses
Global changes in gene expression (mRNA)
Global changes in protein expression
Each of these approaches can be used to classify breast cancers into different biologic subsets
Biologic classification has potential to provide additional prognostic and predictive information
Potential aid to clinical decision making
Each approach has different specimen requirements
Some of these methodologies require fresh or snap frozen tumor samples
Other methodologies can be used to study formalin-fixed paraffin-embedded breast tumors
CLINICAL IMPLICATIONS
Clinically Relevant Tumor Classification
Classification should help distinguish different prognostic groups among patients with similar features
Classification should help predict response to different therapies
Endocrine therapies and type of endocrine therapy (e.g., tamoxifen or aromatase inhibitors)
Chemotherapy: Many types, doses, and combinations available
Biologic/targeted therapies: HER2-targeted treatment has been very successful for cancers overexpressing the protein
Clinically useful breast cancer classification will aid in optimal patient management
Gene Expression Profiling (GEP), mRNA
4 major groups of “molecular subtypes” of cancers identified
2 subgroups of ER(-) cancers
HER2(+) tumors with low or absent expression of ER-related genes
HER2(-) tumors with increased expression of basal cytokeratins
2 subgroups of ER(+) cancers
Luminal A: High ER expression, low levels of proliferation-related gene expression
Luminal B: Lower levels of ER expression, high levels of proliferation-related gene expression, 1/3-1/2 overexpress HER2
These molecular subtypes have been confirmed as reproducible and statistically robust in independent data sets
Significantly correlated with prognosis, independent of traditional prognostic factors
Associated with different patterns of metastatic recurrence
May help predict likelihood of response to chemotherapy
Limitations of GEP
Clinical applicability of gene expression profiling limited
Requires fresh or frozen tissue
Only applicable for larger carcinomas for which diagnosis is known prior to surgery
Not suitable for small carcinomas < 1 cm
If tissue is harvested for this assay, reduces tumor available for histologic evaluation or other types of assays
Technical complexity and technical feasibility in routine practice setting
Reproducibility
Consistency of results across different laboratories or on repeat specimens has not been extensively examined
Cost
IHC Profiling: Single Antibodies
IHC evaluation of single protein markers has demonstrated clinical utility
ER, PR, HER2, Ki-67 (MIB-1)
Clinically validated as useful for risk stratification and decision for specific adjuvant therapies
ER and PR: Response to tamoxifen, aromatase inhibitors
HER2: Response to trastuzumab, lapatinib, other HER2-directed therapy
Ki-67: High proliferative rate may predict increased benefit from chemotherapy
Selected IHC antibody panels can be used to profile breast tumors
Able to identify breast cancer subsets with differing outcomes
Analytical techniques developed for GEP can be applied to IHC
Examination of multiple prognostic markers by IHC in well-defined cohort using unsupervised hierarchical clustering
Demonstrated ability to identify prognostic relevant groups of breast cancer patients
Determined optimal panel of IHC markers necessary to define these groups
Numerous investigators have attempted to “translate” gene expression data into IHC panels for clinical application in breast cancer
Studies suggest that application of selected antibody panels using IHC can identify breast cancer subsets with differing outcomes
May help predict response to specific therapies
IHC Profiling: Cytokeratin (CK) Classification
Breast cancers can be classified based on differential patterns of expression of cytokeratins by IHC
Basal subtype
Expression of high molecular weight “basal” cytokeratins CK5/6, CK14, CK17
Also frequently expresses luminal cytokeratins
Luminal subtype
Expression of low molecular weight “luminal” cytokeratins CK8, CK18
Usually do not express basal cytokeratins
Expression of basal cytokeratins in breast cancer shows a number of clinical correlations
Absence of ER expression
Aggressive clinical course with poor prognosis and increased incidence of early recurrence
Increased incidence of metastases to lungs and brain
High histological and nuclear grade with pushing borders and marked increase in proliferation
BRCA1-associated tumors and familial breast cancer
Poor responses to standard adjuvant chemotherapy
May be more sensitive to anthracycline-based chemotherapy compared with luminal subtype
Tends to occur in patients under age of 40
More common in premenopausal African-American and Hispanic women
IHC Profiling: Surrogates for Molecular Classification by GEP
Limited panel of IHC markers can identify clinically relevant groups
Panel includes ER, HER2, HER1 (EGFR), Ki-67, and basal cytokeratins (e.g., CK5/6)
Stratify breast cancer samples into subsets similar to molecular subtypes defined by expression profiling
Can be used as surrogate for intrinsic molecular classification of breast cancer
IHC surrogates for molecular subsets demonstrate similar prognostic significance compared with expression profiling
May be predictive for patterns of metastatic recurrence
Luminal A subtype
ER(+), HER2(-), Ki-67 low; usually PR(+)
Usually grade 1 or 2
Approximately 70% of all breast cancers
Metastasizes most commonly to bone; least likely subtype to metastasize to brain, liver, or lung
In general, shows little benefit from addition of chemotherapy to hormonal therapy
Luminal B subtype: HER2(-)
ER(+), HER2(-), Ki-67 high; may be PR(-)
Usually grade 2 or 3
Approximately 10% of all breast cancers
Most commonly metastasizes to bone, followed by liver and lung
In general, benefits from chemotherapy and hormonal therapy
Luminal B subtype: HER2(+)
ER(+), HER2(+), Ki-67 high; may be PR(-)
Usually grade 3
Approximately 10% of all breast cancers
Metastasizes most commonly to bone, brain, liver, and lung
More likely to have multicentric disease, multiple positive nodes, and higher risk of local recurrence
In general, benefit from chemotherapy, HER2-targeted therapy, and hormonal therapy
HER2 subtype
ER(-), HER2(+), Ki-67 high; usually PR(-)
Usually grade 3
Approximately 10% of all breast cancers
More likely to have multicentric disease, multiple positive nodes, and higher risk of local recurrence
Metastasizes most commonly to bone, brain, liver, and lung
In general, benefits from chemotherapy and HER2-targeted therapy but not hormonal therapy
Patients are younger compared to women with luminal A cancers
Basal subtype
ER(-), HER2(-), basal cytokeratin(+), EGFR(+), PR(-)
Identifies basal-like carcinomas defined by gene expression with 76% sensitivity, 100% specificity
Other IHC panels have also been used to define this group
Does not identify ˜ 10% that are ER positive or ˜ 10% that overexpress HER2
Usually poorly differentiated
Approximately 15% of all breast cancers
Less likely to have involved nodes but higher risk for local recurrence
Metastasizes most commonly to brain, lung, and distant nodes
May respond to specific types of chemotherapy
Patients are younger compared to women with luminal A cancers
IHC Profiling: Mammostrat® Assay
Alternative approach to profiling breast cancer patients for prognosis and treatment response utilizing IHC
Developed by Applied Genomics, Inc. (Huntsville, AL) in collaboration with researchers at Stanford University
Attempted to translate diversity revealed by gene expression studies into new IHC tests with potential clinical utility
Utilized gene expression data sets to select hundreds of novel targets for production of new antibodies
Antibodies screened across several thousand formalin-fixed paraffin-embedded (FFPE) tumor samples to identify quality IHC reagents
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