1. The Present and Future of Genomics in DCIS
On Demand Post-SABCS 2011 Update
Steve Shak, MD
Chief Medical Officer
GHI10116

2. Agenda Background and Unmet Need
Algorithm Development for the DCIS Score™
Clinical Validation
Clinical Applications
Future Research
Closing Remarks

3. DCIS Background and Unmet Need

4. DCIS Background
Ductal carcinoma in situ (DCIS) is a pre‐invasive form of breast cancer
~45,000 new cases in 2010 in the U.S.
Women diagnosed with DCIS are at risk for local recurrence, which may be either DCIS or an invasive breast cancer
Approximately 70% of women with DCIS are treated with radiation following surgical excision
The addition of radiation for DCIS has been shown in clinical trials to reduce local recurrence risk, but has not been demonstrated to prolong survival
Key Points: Ductal carcinoma in situ (DCIS) is a pre-invasive or non-invasive form of breast cancer. Approximately 45,000 new cases of DCIS were diagnosed in 2010 in the US. The advent of wide-spread mammographic screening in the US has greatly increased the detection of DCIS. While women diagnosed with DCIS have an excellent prognosis, they are at risk for local recurrence, which is typically 50% DCIS and 50% invasive carcinoma.
In terms of current treatment the majority of women (up to 70%) with DCIS are treated with surgical excision followed by radiation. The addition of radiation for DCIS has been shown in clinical trials to reduce local recurrence risk, but has not been demonstrated to prolong survival.
Viani GA et al. Radiat Oncol 200; 2:28.;
Early Breast Cancer Trialists' Collaborative Group (EBCTCG). J Natl Cancer Inst Monogr 2010;
Viani GA et al. Radiat Oncol 2007; 2:28.
Early Breast Cancer Trialists' Collaborative Group (EBCTCG). J Natl Cancer Inst Monogr 2010;

5. Key Radiation Trials in DCIS
Key Points: These are the key radiation therapy trials that have been conducted in DCIS. All of the studies reported that there was a benefit with radiation in any subgroup- i.e. low grade, wide margin, favorable histology, etc. Radiation reduces the risk of a recurrence in the ipsilateral breast by approximately 50%. Furthermore, these studies have reported that the risk of contralateral recurrence ranges from 8-11% at 5 years.
Early Breast Cancer Trialists' Collaborative Group (EBCTCG). J Natl Cancer Inst Monogr 2010;
Viani GA et al. Radiat Oncol 2007; 2:28.
Early Breast Cancer Trialists' Collaborative Group (EBCTCG). J Natl Cancer Inst Monogr 2010;

6. Unmet Need
Reliable methods for making treatment decisions based upon patient specific tumor biology in DCIS have not been previously established
There is a need to quantitatively assess the risk of invasive breast cancer recurrence in newly-diagnosed patients with DCIS
There is a significant unmet need for validated tests that identify:
low risk disease which may be treated with surgery alone, avoiding toxicities and costs associated with radiation
high risk disease for which the addition of radiation may be considered
Key Points:
The traditional measures for assessing risk of recurrence for DCIS are similar to the clinical and pathological measures used to assess risk of recurrence in invasive breast cancer: age, residual tumor/margin width, grade, histology, size and menopausal status. None of these characteristics however provide a quantitative assessment and can often vary based on subjective influences such as area of tumor sectioned for pathologic assessment.
There is general agreement that the majority of patients with DCIS are being over treated. But there is currently no good, quantitative, reproducible way to identify patients that are at very low risk of recurrence that they might consider foregoing radiation. Furthermore, none of the traditional measures provide an estimate of the risk of an invasive recurrence. This is especially important because patients that have an invasive recurrence have a much different prognosis and a potentially life threatening disease. (Allegra CJ et al. J Natl Cancer Inst. 2010; 102(3):161–169.)

7. Algorithm Development: DCIS Score™

8. DCIS Score™: Gene Selection and Algorithm Development
DCIS Score developed from analysis of multiple correlative science studies
Compared gene expression in invasive and DCIS
Recurrence Score® tumor biology of invasive breast cancer and DCIS are similar1
Differences seen in the distribution of proliferation genes1
Analyzed local and distant recurrence risk in published data in invasive breast cancer (NSABP B-14: randomized trial of tamoxifen vs. placebo in ER+ patients2,3 and Kaiser: case-control study in ER+ tamoxifen-treated and untreated and ER- untreated patients4)
Selected genes that predict local recurrence
Selected genes that predict recurrence risk regardless of tamoxifen treatment
Coefficients selected on multiple considerations from prior breast and colon studies
Scaling and category cutpoints based on analysis of DCIS Score in separate cohort of DCIS patients
Key Points: The DCIS algorithm was developed based upon an analysis of multiple correlative science studies and evidence suggesting that there is a biologic relationship between DCIS and invasive carcinoma.
Initially, the Oncotype DX assay was performed and the expression levels of the 21 genes were compared for manually micro-dissected DCIS and invasive carcinoma when both were present within the same fixed paraffin-embedded tumor tissue (FPET). A wide range of single gene results and Recurrence Scores were noted in the DCIS components – all DCIS did not have low Recurrence Scores. A strong correlation between gene expression profiles was found for paired invasive and DCIS components; however, the proliferation group scores were lower for the DCIS components.
Selection of genes and algorithm development for the DCIS Score was based on published results for the Oncotype DX assay for invasive breast cancer, examining correlations with both local recurrence and distant recurrence. Seven cancer-related genes (the five proliferation genes, PR, and GSTM1) and the five reference genes were chosen for their ability to predict recurrence, regardless of adjuvant tamoxifen use. The proliferation group score uses the continuous proliferation score which provides important information for predicting IBE risk. The 12 selected genes are a subset of the 21 genes included in the Oncotype DX Recurrence Score.
After the algorithm was developed, a cohort of pure DCIS samples was analyzed and a similar wide range of gene expression was observed (Genomic Health, Inc. internal data on file). This cohort was used to scale the DCIS Score to range from 0 to 100, with a higher score indicating a greater likelihood of recurrence.
1. Baehner FL et al. San Antonio Breast Cancer Symposium 2008; Abstract Paik S et al. N Engl J Med. 2004;
3. Mamounas EP et al. J Clin Oncol. 2009; Habel LA et al. Breast Cancer Res. 2006; R25.

9. DCIS Score™: Gene Selection Hormone Receptor Group
Proliferation
Hormone Receptor Group
Reference
Ki-67
STK15
Survivin
Cyclin B1
MYBL2 PR
Beta-actin
GAPDH
RPLPO
GUS
TFRC
GSTM1
Key Points: The DCIS Score includes 5 genes in the proliferation group, PR, GSTM1 and 5 reference genes.
Similar to the Recurrence Score®, the DCIS Score is a continuous variable from
The pre-specified DCIS Score risk groups are: Low < 39, Intermediate 39 – 54, High ≥ 55.
The DCIS Score was evaluated as a continuous variable ranging from 0 to 100. It was also evaluated as a categorical variable based on three risk groups that were pre-specified as part of the development phase. These cutoffs were not chosen based on E5194 data.
DCIS Score:
Continuous variable
Number between 0 – 100

10. Clinical Validation for DCIS Score™

11. DCIS Score™ Pre-specified for Validation
All aspects of the study were pre-specified in a final protocol prior to initiation of sample processing for the E5194 clinical validation study. This included:
Pre-analytical and analytical methods
Gene coefficients for DCIS Score
Scaling and centering coefficients
DCIS Score risk groups
Low < 39, Intermediate 39 – 54, High ≥ 55
Key Points: All aspects of the study were pre-specified in a final protocol prior to initiation of sample processing for the E5194 clinical validation study.

12. A QUANTITATIVE MULTIGENE RT-PCR ASSAY FOR PREDICTING RECURRENCE RISK AFTER SURGICAL EXCISION ALONE WITHOUT IRRADIATION FOR DUCTAL CARCINOMA IN SITU (DCIS): A PROSPECTIVE VALIDATION STUDY OF THE DCIS SCORE FROM ECOG E Solin LJ, Gray R, Baehner FL, Butler S, Badve S, Yoshizawa C, Shak S, Hughes L, Sledge G, Davidson N, Perez EA, Ingle J, Sparano J, Wood W Eastern Cooperative Oncology Group (ECOG) North Central Cancer Treatment Group (NCCTG) Genomic Health, Inc (GHI) San Antonio Breast Cancer Symposium

13. ECOG E5194 (PARENT STUDY)
Prospective multicenter study (n = 670) Cohort 1: Low/intermediate grade, size < 2.5 cm Cohort 2: High grade, size < 1 cm Study treatment - Surgical excision - Minimum 3 mm negative margin width - No radiation - Tamoxifen option beginning May Reported outcomes at 5 and 7 years (Hughes, JCO, 2009) - Currently 10-year outcomes
Key Points: The E5194 trial was selected as the cohort for the validation study for the DCIS Score because of its status as a landmark trial.
The parent trial was a prospective, multi-center, single-arm study that enrolled 670 patients. The study was designed to test the hypothesis that a group of patients could be identified using standard clinical and pathologic covariates to have a low enough risk of local recurrence that they could be treated with surgery alone. Results of the parent trial were reported in a publication from Hughes et al in JCO 2009 with 5yr and 7yr follow-up.
Eligibility:
Patients with low/intermediate grade, size < 2.5 cm or high grade, size < 1 cm were
A minimum of 3 mm negative margins,
No radiation
Tamoxifen use was allowed at the investigator’s discretion beginning after May There was no collection of duration of Tam. Approximately 30% of patients received tamoxifen at some point.
For the validation study, the two cohorts were pooled and the combined analysis population was used. The median follow up in the validation study was 8.8 years and we are presenting 10 year outcome data.
Hughes LL et al. J Clin Oncol. 2009;
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

14. PRESPECIFIED STUDY OBJECTIVES
Primary:
To determine whether there is a significant
association between the DCIS Score and the risk of an ipsilateral breast event (IBE)
Secondary:
To determine whether the DCIS Score provides value beyond standard clinical and pathologic factors
Conditional (if DCIS Score validated):
To evaluate the Recurrence Score as a predictor of risk of an ipsilateral breast event (IBE)
Key Points: The primary analysis was pre-specified as part of the statistical analysis plan, which was finalized prior to the merging of clinical and Genomic Health’s laboratory data.
The primary study objective was to determine whether there is a significant association between the DCIS Score and the risk of an ipsilateral breast event (IBE). An IBE was defined as a local recurrence of DCIS or invasive carcinoma.
The E5194 clinical validation study was a prospectively defined analysis using archived tissues from a previously completed clinical trial (E5194). Thus, the design of the study is called a prospective-retrospective clinical validation study.
The primary objective was to demonstrate that the DCIS Score was significantly associated with the risk of an IBE (DCIS or invasive). Analyzed as both a continuous variable and a categorical variable based on the 3 risk groups (low <39, Intermediate and high ≥55.
The secondary objective was to determine whether the DCIS Score provided value beyond standard clinical and pathologic factors
There was also a conditional analysis that would be conducted if the primary endpoint was met, which was to evaluate the Recurrence Score as a predictor of an IBE. This would address the question of whether or not the full 21 genes were as informative or more or less informative than the 12 genes.
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

15. METHODS FOR DCIS SCORE VALIDATION STUDY
Prospective-retrospective study design Pre-specified: Study objectives, population, laboratory assays, endpoints, statistical methods
Oncotype DX assay performed (n = 327; 49%) Standardized methods for 21 gene assay Central pathology review Calculated: DCIS Score and Recurrence Score Study endpoint: Ipsilateral breast events (IBE) 1o Endpoint: Any IBE (DCIS or invasive carcinoma) 2o Endpoints: Invasive IBE DCIS IBE
Key Points: We followed the retrospective-prospective study design as outlined by Simon, Paik and Hayes (JNCI 2009). All aspects of the DCIS Score validation study were prospectively defined prior to initiation of sample processing, including the DCIS Score algorithm, laboratory assays, study protocol and statistical analysis plan.
A total of 640 patients enrolled in parent E5194 trial, however, only 327 patients had tissue available, were able to sign informed consent for the validation study or had sufficient RNA extracted.
The Oncotype DX assay was performed according to standard methods, and the DCIS Score and Recurrence Score were calculated according to the pre-specified formulae.
The primary endpoint was any ipsilateral breast event (IBE). Prespecified secondary endpoints included invasive IBEs, and DCIS IBEs with no invasive component.
All samples in E5194 had been reviewed by both local and central pathologists.
Simon RM, et al. J Natl Cancer Inst. 2009;101:
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

16. PATIENT AND TUMOR CHARACTERISTICS
Key Points: Patient characteristics for the validation cohort were similar to the E5194 parent trial.
The average tumor size in the validation study was slightly smaller than in the parent trial. The median size in the parent trial was 6 mm for low/intermediate and (cohort 1) 5 mm for the high (cohort 2).
A total of 29% of patients received tamoxifen (similar to the parent trial).
97% of patients were ER-positive by RT-PCR.
Hughes LL et al. J Clin Oncol. 2009;
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

17. PRIMARY ANALYSES OF THE RISK FOR AN IPSILATERAL BREAST EVENT (IBE)
Key Points: The validation study met its primary objective. The DCIS Score predicted the likelihood of any ipsilateral breast event with a significant p-value of 0.02 and HR of 2.34.
The hazard ratio is for a 50 point difference and that is similar to the range observed between the high and low risk patients.
To ensure that the use of tamoxifen did not confound the study’s primary objective, tamoxifen use as a covariate was included in the model (i.e. adjusting for tamoxifen use) and it had essentially no impact on the results for the DCIS Score, as can be seen by comparing these results to the unadjusted results on slide 22--where the DCIS Score hazard ratio was 2.41 for a 50 point difference.
As anticipated tamoxifen therapy lowered the risk of IBE, however, it was not statistically significant (p-value of 0.12)
The conditional analysis of the RS showed that the RS did not predict the risk of an IBE.
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

18. DCIS SCORE: 10-YEAR IPSILATERAL BREAST EVENTS (IBE) BY RISK GROUP
ANY IBE
Key Points: This slide shows the Kaplan-Meier estimates of the risk of any IBE (panel A) over time for each of the three DCIS Score groups.
The highest estimated risk is in the high DCIS Score group (red) with an estimated 10 year risk of 27.3%, and the lowest is in the low DCIS Score group (blue) with an estimated 10 year risk of 12.0%.
The log rank p-value of 0.02 means that there was a significant trend in IBE risk across the three DCIS Score groups.
It is important to note that 75% of the patients were in the low DCIS Score group, with an estimated 10 year IBE risk of 12.0% (CI: 8.1% to 17.6%).
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

19. DCIS SCORE: 10-YEAR IPSILATERAL BREAST EVENTS (IBE) BY RISK GROUP
ANY IBE
INVASIVE IBE
Key Points: Panel B shows the Kaplan-Meier estimates of the risk of invasive IBE over time for each of the three DCIS Score groups.
Again, the highest estimated risk of invasive IBE is in the high DCIS Score group (red) with an estimated 10 year risk of 19.1%, and the lowest is in the low DCIS Score group (blue) with an estimated 10 year risk of 5.1%.
The log rank p-value of 0.01 means that there was a significant trend in invasive IBE risk across the three DCIS Score groups.
It is important to note that 75% of the patients were in the low DCIS Score group, with an estimated 10 year invasive IBE risk of 5.1% (CI: 2.8% to 9.5%).
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

20. DCIS SCORE: 10-YEAR RISK OF AN IPSILATERAL BREAST EVENT (IBE)
ANY IBE
Key Points: This figure shows the estimated 10 year risk of any IBE according to the continuous DCIS Score. 95% confidence intervals are also shown. The rug plot at the bottom of the graph shows the individual DCIS Scores for all 327 patients in the study.
There is an increasing estimated risk of IBE with increasing DCIS Score, from approximately 9% at a score of 0, up to 40% at a score of 90 (highest observed in the study). But the confidence intervals become wider for the higher scores, where there were fewer patients.
This is based on a proportional hazards regression model for IBE risk including the continuous DCIS Score (no indicator for tamoxifen use). The estimated hazard ratio associated with a 50 point difference in the DCIS Score was 2.41, and the p-value was Adjusting for tamoxifen use had essentially no impact on the results, as can be seen by comparing these results to the primary analysis, which did include a term for tamoxifen use.
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

21. DCIS SCORE: 10-YEAR RISK OF AN IPSILATERAL BREAST EVENT (IBE)
ANY IBE
INVASIVE IBE
Key Points: This right panel shows the estimated 10 year risk of invasive IBE according to the continuous DCIS Score, with 95% confidence intervals. The rug plot also shows the individual DCIS Scores for all 327 patients in the study.
There is an increasing estimated risk of invasive IBE with increasing DCIS Score, from approximately 3% at a score of 0, up to 29% at a score of 90 (highest observed in the study). But the confidence intervals become wider for the higher scores, where there were fewer patients in the study.
This is based on a proportional hazards regression model for invasive IBE risk including the continuous DCIS Score. The estimated hazard ratio associated with a 50 point difference in the DCIS Score was 3.76, and the p-value was 0.01.
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

22. MULTIVARIABLE MODELS OF RISK FOR IBE
Key Points: The DCIS Score was the strongest predictor of IBE recurrence risk.
In multivariable analysis, excluding the DCIS Score, only tumor size and post-menopausal status were significant predictors for the risk of local recurrence. The traditional clinical and pathology variables, including surgical margins, nuclear grade, comedo necrosis, DCIS pattern (papillary, micropapillary, cribiform and solid) and tamoxifen use, were not statistically significant in this analysis.
The DCIS Score was added to this multivariable model, with the results shown here (in the model “including the DCIS Score”). This shows that the DCIS Score was able to predict IBE risk over and above the traditional factors, tumor size and menopausal status, with an estimated hazard ratio of 2.41 associated with a 50 point difference in the DCIS Score. This hazard ratio is unchanged from the model that does not include any additional factors (see slide 17), showing that the DCIS Score is essentially independent of these factors as a predictor of IBE risk.
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

23. EXPLORATORY ANALYSES OF DCIS SCORE: CONSISTENT ASSOCIATION ACROSS SUBGROUPS
No.
2.4
Overall
Pre Menopausal
Post Menopausal
Lesion Size ≤
Lesion Size >
Negative Margins < 5 mm
Negative Margins ≥ 5 mm
Low/intermediate Grade
High Grade
Key Point:
Exploratory subgroup analyses evaluated the consistency of the DCIS Score in relevant patient groups including menopausal status, lesion size, margin size, grade and tamoxifen use. The DCIS Score consistently predicted the risk of a local recurrence across all subgroups evaluated.
These are the results of subgroup analyses in which the hazard ratio for the DCIS Score as a predictor of IBE risk within each subgroup was estimated. It is important to note that this study was not powered for subgroup analyses, which results in very wide confidence intervals. The objective in performing these subgroup analyses to look for general consistency in direction with the overall hazard ratio of 2.41, always taking into account the level of precision as reflected in the confidence intervals.
For each subgroup the hazard ratios for the DCIS Score as a predictor of IBE risk was estimated, with 95% confidence intervals, using a proportional hazards regression model. The hazard ratios were calculated for a 50 point difference in the continuous DCIS Score.
For each subgroup, the hazard ratio was directionally consistent with the overall hazard ratio, and the 95% confidence intervals included the overall hazard ratio of 2.41.
These subgroup analyses showed that the associations of the DCIS Score with IBE risk had similar trends and were directionally consistent with the overall group of patients, with wide confidence intervals.
Tamoxifen Treated
Not Treated
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

24. Association between the DCIS Score and Clinical and Pathological Characteristics
For each characteristic (including tumor grade, tumor size, margin width, patient age, menopausal status), a wide distribution of DCIS Score values was seen within each subgroup
Although lesion size and menopausal status were, as expected, significantly associated with IBE risk, adjusting for these characteristics in multivariate models did not change the estimated association between the DCIS Score and IBE risk
This demonstrates that the DCIS Score provided additional information on IBE risk beyond these characteristics
Key Points: For each pathological and clinical characteristic (tumor grade, tumor size, margin width, patient age, menopausal status), a wide distribution of DCIS Scores were observed across each subgroup.
Although tumor size and menopausal status were significantly associated with IBE risk, adjusting for these variables in a multivariate model did not change the ability of the DCIS Score to predict IBE risk, thus demonstrating that the DCIS Score provides additional information beyond these traditional measures.

25. SUMMARY: DCIS SCORE
Present study validates the DCIS Score as a predictor of an ipsilateral breast event (IBE) and invasive IBE
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

26. SUMMARY: DCIS SCORE
Present study validates the DCIS Score as a predictor of an ipsilateral breast event (IBE) and invasive IBE
DCIS Score quantifies 10-year risk of IBE - Continuous variable or 3 risk groups
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

27. SUMMARY: DCIS SCORE
Present study validates the DCIS Score as a predictor of an ipsilateral breast event (IBE) and invasive IBE
DCIS Score quantifies 10-year risk of IBE - Continuous variable or 3 risk groups
DCIS Score provides independent information on IBE risk beyond clinical and pathologic variables - Including tamoxifen, grade, and negative margin width - Identifies underlying tumor biology
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

28. SUMMARY: DCIS SCORE
Present study validates the DCIS Score as a predictor of an ipsilateral breast event (IBE) and invasive IBE
DCIS Score quantifies 10-year risk of IBE - Continuous variable or 3 risk groups
DCIS Score provides independent information on IBE risk beyond clinical and pathologic variables - Including tamoxifen, grade, and negative margin width - Identifies underlying tumor biology
DCIS Score provides a new clinical tool to guide treatment selection for patients with newly diagnosed DCIS
Solin LJ et al, San Antonio Breast Cancer Symposium 2011; Abstract S4-6.

29. Clinical Applications for DCIS Score™

30. The DCIS Score™ Provides Clinical Insights
Clinicians and patients desire objective evidence regarding risk of local recurrence (DCIS or invasive) to assist in treatment decision-making
In situations where clinicians and patients are uncertain about radiation
In situations where clinical and pathological parameters might indicate treatment and the patient does not wish to receive therapy
In situations where clinicians recommend forgoing therapy and the patient is not convinced

31. DCIS Sample Report: Local Recurrence
Key Points: This is an image of a the first page of the DCIS sample report including graphs depicting the 10-year risk any IBE on the left, and invasive IBE on the right, as a function of the DCIS Score.

32. DCIS Sample Report: Local Recurrence

33. DCIS Sample Report: ER and PR Scores
Key Points: This is an image of a the second page of the DCIS sample report including the ER and PR single gene scores.

34. Future DCIS Research

35. Future Research
Additional research that provides confirmation and more experience in certain groups (e.g., higher risk DCIS and ER negative DCIS)
Identification of predictive genes for radiation sensitivity and/or resistance
Next Generation Sequencing to explore whether new genes might be identified that actually cause progression to invasive disease

36. Additional Information on DCIS
The Oncotype DX® Breast Cancer Assay for DCIS patients will be commercially available starting on December 28, 2011
For additional resources on DCIS visit
If you have further questions, please contact Customer Service at or ONCOTYPE

37. Further Education
In January 2012 you will receive an blast to register for our “On Demand Post-SABCS 2011 Update” summarizing the important developments in breast cancer

38. Closing Remarks
Oncotype DX® reveals the underlying biology that can help guide DCIS treatment decisions
Oncotype DX is the first and only clinically validated commercial genomic assay for patients with DCIS
Oncotype DX predicts the risk of any local recurrence (DCIS or invasive) and the risk of local invasive carcinoma
Oncotype DX allows for personalized treatment based on tumor biology as determined by the DCIS Score™

39. Thank you