Clinical Utility of Comprehensive Microarray Testing in Early-stage Breast Cancer


might be viewed by the pathologist or clinician as a ‘second opinion’ for IHC results. Figure 2 shows the commercial TargetPrint result received by the physician.


BluePrint The pioneering work of Sorlie and Perou4 indicates that valuable


prognostic and predictive information can be obtained by molecular subtyping of a patient’s breast cancer using a genomic classification system based on microarray analysis of a large number of genes. This classification system was first reported in 2000 and since then has undergone several iterations with the key findings grouping breast cancer into four major subtypes: basal, luminal (A and B), and ERRB2 (HER2) type. Each of these subtypes behave differently clinically, and appear to benefit from differing therapeutic approaches.


Figure 4: TheraPrint Patient Result Form


Agendia has developed and commercialized an 80-gene microarray- based assay that mirrors the Perou classification and which is designed for use in concert with the other Symphony profiles, MammaPrint and TargetPrint, to provide a clinically meaningful classification system. Together they not only provide prognostic information, but can also be used to help guide therapy.5


The schema for development of BluePrint is


shown in Figure 3, samples that were analysed both by TargetPrint and IHC in case of concordant results the samples were used for BluePrint development. By using concordant results we ensure to have mRNA expression and protein expression of the ER, PR, and HER2 markers. Of particular interest is the capability of sub-classifying HER2-positive patients into high- and low-risk groups by MammaPrint, with very different recurrence risks. As the overall response rate to herceptin is ~30 %, treatment assignment as a function of genomic profiling most likely have utility in both improving the response rate and minimizing the cost and toxicity of treatment.16


Molecular subtyping is currently being used in clinical trials to help stratify patients, anticipating that such information will have a beneficial effect on patient outcomes.


TheraPrint


While clinical practice guidelines suggest therapeutic choices for neo-adjuvant, adjuvant, and first-line metastatic disease, advanced recurrent disease may prove to be a therapeutic conundrum for the clinician. Patients are usually extensively pre-treated and criteria for disease progression and drug resistance may vary among practitioners.


TheraPrint is a multigene assay that specifically measures the expression level of 56 genes that are the known factors in controlling the response to endocrine agents, chemotherapy drugs, small molecules and monoclonal antibodies. TheraPrint compares the levels of expression of these genes with those of an untreated, invasive breast cancer pool. The genes present in this panel are listed on a multiple page result form (Figure 4 shows a sample first page) and represent the majority of identified genomic biomarkers associated with a defined therapy, which may be useful for patients with breast cancer. A Registry trial is being developed by Agendia to record how physicians will use TheraPrint assay and if its use can be shown to improve patient outcomes when compared to treatments chosen empirically.


US ONCOLOGY & HEMATOLOGY


Figure 3: Rational Based Method of BluePrint Development


THERAPY GENE ASSAY (Page 1 of 3)


CUSTOMER


Doctor: Account:


Address: City, St., Zip:


TheraPrint Therapy Gene Assay Test Results Tumor Percentage: 65%


Pathology Results


Unigene Symbol2


AKT1


AURKA BCL2 BRAF


BRCA1 BRCA2


UniGene Cluster ID


*The tumor cell percentage as reported relates to the tissue sample received by Agendia and serves as a quality control for the test. The presence of tumor cells as reported by Agendia cannot be used as a diagnosis of invasive disease.


Using DNA microarray technology, TheraPrint™ measures the mRNA level of genes that are of potential interest in the context of cancer therapy. Leveraging the chip capacity, the expression of each gene is assessed in multifold, assuring precise results. Provided is the absolute expression (2


from 0 to 19.3. The relative expression percentile represents the patient’s gene expression compared to a reference population of newly diagnosed untreated breast cancer patients (n=373) and indicates the percentage of reference samples with a lower intensity


For each TheraPrint Gene, the absolute expression is measured and compared to the reference distribution. The relative expression of the patient's gene is given as a percentile score. This percentile score indicates the percentage of reference samples with a lower intensity. For instance, a percentile score of 91 indicates that 91% of the samples in the reference distribution had intensities lower than the intensity found in the patient's sample.


Relative Gene Expression Cadherin 3, type 1, P-cadherin (placental) CRYAB


C11orf30 CCND1 CCNE1 CDH1 CDH3


CSK


CXCL12 CXCL14


ECGF1 EGFR


The reference distribution was established using 373 samples from newly diagnosed breast cancer patients untreated with chemotherapy or hormonal therapy. Each of the samples was hybridized onto the Agilent HD 8-pack array, the 2


Assay Description


The absolute expression level is the 2log of the intensity of each gene as measured on the Agilent microarrays (HD 8-pack). The measurement value ranges from 0 to 19.3 and is proportional to the expression level of the gene. However, the value cannot be directly translated to RNA concentration or copy number as there are no known cutoffs to determine whether a given expression level is truly high / low or activated / inactivated. The gene expression level of a given patient’s results can only be compared to the gene expression level of all other samples in the reference distribution to provide relative interpretation of rather high or rather low.


Absolute Gene Expression (2 DHFR


Reference Distribution ERBB4


ERBB3


indicate the expression distribution of the individual gene, the reference distribution is given as the 5-95% expression range on the 0 to 19.3 2 FANCF


ESR2


FLT1 FLT3 FLT4


Sign Off


Chynel F. Henning, M.D., Ph.D., FASCP, FCAP Pathologist Laboratory Director


FRAP1 GSDML


Hs.525622 Hs.250822 Hs.150749 Hs.550061 Hs.194143 Hs.34012 Hs.352588 Hs.523852 Hs.244723 Hs.461086 Hs.554598 Hs.408767 Hs.77793 Hs.522891 Hs.483444 Hs.83765 Hs.592212 Hs.488293 Hs.118681 Hs.390729 Hs.660607 Hs.651196 Hs.507621 Hs.507590 Hs.646917 Hs.338207


Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) V-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) Estrogen receptor 2 (ER beta)


Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) Fms-related tyrosine kinase 3 Fms-related tyrosine kinase 4


Fanconi anemia, complementation group F References:


1. www.agendia.com/researchgenepanel 2. http://www.ncbi.nlm.nih.gov/sites/entrez


FK506 binding protein 12-rapamycin associated protein 1 Hs.306777 Gasdermin-like


log intensity of the research panel genes was measured and the results compiled. To log intensity scale.


Regulatory Information For In Vitro Diagnostic Use Caution: Federal law restricts this device to sale by or on the order of a physician. Agendia, Inc (05D1089250) is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) as qualified to perform high-complexity clinical testing. Research Gene Panel is an aid to measure the mRNA expression levels of certain genes in patients diagnosed with breast cancer. Decisions regarding care and treatment should not be based on a single test such as this test. Rather, decisions on care and treatment should be based on the independent medical judgment of the treating physician taking into consideration all available information concerning the patient’s condition, including other pathological tests, in accordance with the standard of care in a given community.


10.4 7.3 6.2 8.9 7.7 7.8 7.8 7.8 7.1


This test was performed at Agendia’s Irvine, California laboratory.


8.2 - 11.3 6.0 - 8.0 8.6 - 11.4 6.0 - 8.1 6.0 - 6.5 8.4 - 12.6 7.4 - 9.5 6.0 - 8.0 6.3 - 7.8 7.2 - 8.5 6.4 - 9.4


Chemokine (C-X-C motif) ligand 14 Dihydrofolate reductase


Endothelial cell growth factor 1 (platelet-derived)


9.0 7.8 - 10.6 9.5 6.0


Crystallin, alpha B C-src tyrosine kinase


Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) log intensity)


B-cell CLL/lymphoma 2


V-raf murine sarcoma viral oncogene homolog B1 Breast cancer 1, early onset Breast cancer 2, early onset


Chromosome 11 open reading frame 30 Cyclin D1 Cyclin E1


Cadherin 1, type 1, E-cadherin (epithelial)


10.3 7.5 7.5 6.1 8.0


10.3


12.2 8.2 8.9 7.6 8.6


17.0


log intensity) of the patient’s genes ranging 9.4


9.1 - 11.4 7.5 - 9.8 7.2 - 11.1 7.0 - 8.6 6.6 - 8.3 5.9 - 6.8 7.0 - 8.6 8.5 - 12.4 8.4 - 10.7 9.5 - 13.6 7.7 - 11.7 8.4 - 13.3 7.2 - 8.9 7.2 - 10.8 10.3 - 16.6


RNA Integrity Score: 5 Unigene Name


V-akt murine thymoma viral oncogene homolog 1 Aurora kinase A


Pathology Comments:


The specimen has been reviewed to determine the tumor cell percentage.


Absolute Reference Expression (intensity)


9.8 7.9


Distribution 5% - 95%1


SPECIMEN


Requisition #: Collection Date: Date Received: Report Date: Specimen Type: Customer Ref.:


PATIENT Patient:


DOB: Patient #: Gender: SSN:


22 Morgan | Irvine | CA 92618 | ph: 888.321.2732 | fax: 866.756.7548 customercare@agendia.com | www.agendia.com


AG2011V048USA


Conclusions


Gene expression profiling such as the Agendia Symphony Breast Cancer Decision Suite (TargetPrint, MammaPrint, BluePrint, and TheraPrint) can be used for both prognosis and prediction and are important tools for the medical oncologist to use in both evaluating their patients and in choosing an appropriate neo-adjuvant or adjuvant treatment program. Further, all validated multigene assays should be included in guidelines (i.e. St Gallen, National Comprehensive Cancer Network [NCCN], and ASCO guidelines) and this would lend further credence to their growing importance in current practice. n


109


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