San Antonio Breast Cancer Symposium December 4-8, 2012

Continuous association of a 200-gene prognostic risk score with probability of neoadjuvant chemotherapy response and translation from fresh frozen to FFPE tissue for clinical use.
Ryan van Laar Ph.D., James DiPaola BS, Heather Carbaugh BS, Tami Murphy BS, Angela DiRamio BS, Rachel Flinchum BS, Nathan Brown BS, Wasay Hussain MS, Tony Albino Ph.D. Signal Genetics LLC, New York, NY

Abstract
BreastPRS is a suite of complimentary gene signatures that resolves several of the significant limitations of current gene expression signatures in breast cancer. Notably, BreastPRS can accurately provide prognosis prediction, Left: 1,846 histological grade resolution and molecular patients analyzed subtyping of invasive breast cancer. The 200with BreastPRS gene prognostic component was previously plotted by validated in an independent retrospective series prognosis, grade and of 1,016 previously published fresh-frozen breast subgroup. cancers and has now been applied to data from over 3,000 patients1. In part one of this study, a meta-analysis of 890 previously published neoadjuvant treated breast cancer patients was performed2,3,4. A highly significant association was observed between prognosis risk group (high vs. low), continuous risk score (0-100), and a patients probability of achieving pathological complete response (pCR) with neoadjuvant chemotherapy. Data from both fine-needle-aspirate and biopsy tissue were included in the cohort. Notably, fewer than 10% of patients with the lowest (most favorable) pre-treatment BreastPRS prognosis scores achieve pCR. Probability of pCR was observed to increase steadily to over 40% for individuals with the highest prognosis scores. In part two of the analysis, 50 paired fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tumors were profiled on whole genome microarrays containing all three signatures, in a CLIAcertified high throughput genomics laboratory. An additional previously-published 20-patient series was also analyzed5. Results are presented that highlight the consistency of the prognosis signature in clinically relevant FFPE tissue.

Patients & Methods

Compiling a pre-treatment gene expression database of neoadjuvant-treated breast cancer patients Gene expression profiles from 3 previous publications were downloaded from NCBI GEO. Prognostic indexes were calculated using the 200-gene rank-based algorithm. Patients received 6 months of preoperative (neoadjuvant) chemotherapy including paclitaxel, 5fluorouracil, cyclophosphamide and doxorubicin followed by surgical resection of the cancer2,3,4. Paired gene expression profiles of RNA from freshfrozen and FFPE-preserved breast cancer Passing and Bablok regression was used to compare Prognostic Indexes calculated on whole-genome data generated from 50 fresh-frozen and 50 FFPE specimens of breast cancer from the same patients (BioServe, MD). This is a non-parametric linear regression method which is not sensitive to the distribution of measurement errors or outliers. Robust RNA and GeneChip QC metrics were applied prior to analysis. Illumina data from
Variable Neoadjuvant series Freshfrozen/FFPE series Mean: 60 Stdev: 15 (Unknown: 20 pts) Fresh-frozen: 95 FFPE: 95 Positive 32 Negative 20 Unknown: E-TABM-1081[5]

Passing & Bablok regression of BreastPRS Prognostic Indexes in paired RNA from FF and FFPE specimens. Prognostic indexes for RNAs from tumor specimens preserved in different formats showed no significant deviation from linearity in both Affymetrix (final no. pairs =35, P=0.91) and Illumina series (n=20, P=0.84). The classification threshold hold for FFPE tissue is <33.5 = low risk, >33.5 = high risk.
R2=0.67 ICC=0.90 R2=0.94 ICC=0.98

Age (years) Preservatio n method Estrogen receptor status ArrayExpre ss / GEO Study ID Number of patients Gene expression platform

Mean: 50 Stdev: 10 Fresh-frozen biopsy (610), Fresh-frozen FNA (230) Positive: 443 Negative: 324 Unknown: 123 GSE4779[2] GSE20194[4] GSE25066[3] 890 Affymetrix U3X (102) Affymetrix U133A (786) Neoadjuvant chemotherapy including paclitaxel, 5-fluorouracil, cyclophosphamide and doxorubicin pCR: 204 RD: 686 Low: 359, High: 530

a.

b.
a. Affymetrix 200-gene signature in 37 pairs of FF/FFPE RNA. FFPE score = -0.265 + 1.051 x FF score. b. Illumina 169-gene signature in 20 pairs of FF/FFPE RNA. FFPE score = -0.14 + 1.064 * FF score. c. Affymetrix 169-gene signature in same series of patients as (a). FFPE score = -0.214 + 1.005 * FF score.

190 Affymetrix U133 Plus 2.0 (100) Illumina HumanRef-8 (90)

R2=0.66 ICC=0.89

c.

Treatment

Untreated

Conclusions
n/a Low: 8, High: 27

Response BreastPRS risk group:

Mittempergher et al were analyzed separately5.

Results
BreastPRS prognostic indexes were calculated for all 890 pre-treatment genomic profiles. The relative proportion of pCR to RD in each index decile is shown on right. In fresh-frozen breast cancer specimens a score of 41 or higher corresponds to a high risk of recurrence, however the trend in relative pCR:RD can be seen to continue to the upper range of the assay.
Neoadjuvant chemotherapy response vs. Pre-treatment BreastPRS Prognostic Index
100 90 80 70 60 50 40 30 20 10 0

Our meta-analysis of pre-treatment genomic profiles from 890-patients who received neoadjuvant chemotherapy showed that the percent of patients achieving pCR increased from ~10% to over 40% as prognostic indexes increased from 19 to 89. This range corresponds to a very low to very high risk of disease recurrence, following surgery and/or treatment. It is proposed that determining a patients BreastPRS Prognostic Index prior to administration of neoACT may allow physicians to avoid over-treating individuals with a low chance of benefit because of the underlying molecular characteristics of their disease. Comparing the BreastPRS Prognostic index in two series of paired fresh-frozen and FFPE breast cancer profiles showed significant linearity and a revised low/high risk classification threshold was determined for performing the assay on FFPE tissue. As BreastPRS resolves the significant limitations of current multi-gene signatures, it offers a novel and powerful complimentary method to defining treatment options for breast cancer patients.

Objectives
Predicting Neoadjuvant chemotherapy response: 1. Compile a gene expression database from breast cancer patients prior to administration of neoadjuvant chemotherapy from previously published studies. 2. Evaluate the BreastPRS 200-gene prognostic index for its ability to predict whether a patient will achieve pathological complete response following neoadjuvant chemotherapy.

Percent

Pathological complete response Residual disease

References
1. Van Laar, R. K. "Design and Multiseries Validation of a Web-Based Gene Expression Assay for Predicting Breast Cancer Recurrence and Patient Survival." The Journal of molecular diagnostics : JMD 13(3): 297-304. 2. Farmer, P., H. Bonnefoi, et al. (2009). "A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer." Nat Med 15(1): 68-74. 3. Hatzis C, P. L. V. V. and et al. (2011). "A genomic predictor of response and survival following taxane-anthracycline chemotherapy for invasive breast cancer." JAMA: The Journal of the American Medical Association 305(18): 1873-1881. 4. (2010). "The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models." Nat Biotech 28(8): 827-838. 5. Mittempergher, L., J. J. de Ronde, et al. (2011). "Gene Expression Profiles from Formalin Fixed Paraffin Embedded Breast Cancer Tissue Are Largely Comparable to Fresh Frozen Matched Tissue." PLoS ONE 6(2): e17163.

Translation of assay from fresh-frozen to FFPE tissue:

1. Generate a novel dataset of patient-paired fresh-frozen and FFPE breast cancer RNA. 2. Compare prognostic index between pairs, assess linearity and determine the low/high threshold adjustment necessary for performing BreastPRS on FFPE tissue.

N=890

BreastPRS Prognostic Index

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