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Background: The prognostic abilities of most gene expression signatures in breast cancer are often due to detection of proliferative activity measured from expression of genes regulated as a function of cell cycle progression. Cell Cycle Progression (CCP) score is an important prognostic factor in prostate cancer, and has shown promising results for renal and lung cancer; its role in ductal carcinoma in situ (DCIS) has not been explored. We investigated the prognostic and predictive relevance of CCP Score in DCIS using material from UK/ANZ DCIS trial. Methods: Formalin-fixed paraffin embedded tissues were collected from patients enrolled in the UK/ANZ DCIS trial, a randomised 2X2 factorial design trial investigating role of tamoxifen, radiotherapy (RT) or both as adjuvant treatment in DCIS. mRNA expression of 25 S- and M-phase CCP genes was evaluated by reverse transcription followed by PCR on customized Taqman low-density arrays. CCP score is an un-weighted average of the expression values of CCP genes after normalisation with 14 housekeeping genes. CCP score was analysed as a continuous variable and also as an ordinal variable using tertile-based cut-offs. Exploratory analyses with subgroups defined by HER2 status by immunohistochemistry were performed. Results: CCP scores were evaluable in 521 (134 recurrence events) of 704 available samples (DCIS absent or insufficient RNA in 51, assay failure in 132). Increase in CCP score (median 1.15; IQR 0.71-1.74) was associated with increased risk of ipsilateral breast event (IBE) [Hazard ratio (HR) = 1.28; 95% Confidence Interval (95%CI) 1.08-1.51; p = 0.0049]. CCP score however was not an independent predictor in multivariate analyses [HR = 1.16; 95%CI 0.95-1.42; p = 0.14]. CCP scores were categorised as CCP low (/= 0.87 to < 1.52) and CCP high (>/= 1.52) by tertiles. The benefit of RT in reducing IBE was significant when CCP score was low [HR = 0.35; 95%CI 0.14-0.87; p = 0.024] or intermediate [HR = 0.23; 95%CI 0.09-0.59; p = 0.0023], however, those with high CCP score did not derive significant RT benefit [HR = 0.59; 95%CI 0.31-1.13; p = 0.11]. In exploratory subgroup analyses, HER2 negative DCIS with high CCP score (20.9% of all DCIS cases) did not derive RT benefit and the largest RT benefit was seen for DCIS that expressed HER2 and did not have a high CCP score (23.2% of all DCIS cases). Benefit of RT and 10-year IBE rates by CCP score (categorised) and HER2 status subgroups.SubgroupneventsHR (95%CI)p10-year IBE rates (%) - No RT10-year IBE rates (%) - RTCCP-high & HER2 neg106220.83 (0.35-1.97)0.6722.5 (14.0-35.0)20.0 (10.5-36.0)CCP-high & HER2 pos67210.43 (0.16-1.17)0.09840.6 (27.1-57.6)20.4 (8.9-42.9)CCP-non-High & HER2 neg217300.43 (0.18-0.99)0.04816.2 (10.7-24.0)8.1 (4.0-16.3)CCP- non-High & HER2 pos118330.14 (0.04-0.46)0.001239.5 (29.3-51.6)7.1 (2.3-20.4)CCP-non-High = low or intermediate CCP score Conclusions: CCP score is not independently associated with the risk of IBE but appears to be a predictor of RT benefit. Exploratory analyses suggest that combined with HER2 status, it may help in identifying a large DCIS subgroup where RT is highly indicated and another large subgroup where mastectomy may be merited. Citation Format: Thorat MA, Wagner S, Jones LJ, Levey PM, Bulka K, Hoff R, Sangale Z, Flake II DD, Bundred NJ, Fentiman IS, Forbes JF, Lanchbury JS, Cuzick J. Prognostic and predictive relevance of cell cycle progression (CCP) score in ductal carcinoma in situ: Results from the UK/ANZ DCIS trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-06.

Background: Majority of studies investigating prognostic role of ER and PgR expression in ductal carcinoma in situ (DCIS) have failed to show a significant relationship with recurrence. Branched evolutionary tumour growth and resulting intratumour heterogeneity is now increasingly acknowledged in a variety of cancers. DCIS offers the opportunity to investigate relevance of clonal populations in different ducts that may have evolved differently and may impact outcome differently. We investigated prognostic role of ER and PgR expression in DCIS and impact of clonal variation in ER/PgR expression using material from UK/ANZ DCIS trial. Methods: Formalin-fixed paraffin embedded tissues (FFPETs) were collected from patients enrolled in the UK/ANZ DCIS trial, a randomised 2X2 factorial design trial investigating role of tamoxifen, radiotherapy or both as adjuvant treatment in DCIS. A nested case-control design was used; cases (patients with recurrence) were matched by treatment arm and age to 2 controls each (no recurrence). ER and PgR expression was evaluated on whole sections by immunohistochemistry using 1D5, PGR636 antibodies and EnVisionTM FLEX + detection system (Dako). Assays were scored by Allred method (positive if expression >1%) and clonal method. Clonal method documented presence of ducts with complete lack of ER or PgR expression in otherwise ER or PgR positive (Allred method) DCIS. Analyses categorising such multi-clonal DCIS as ER or PgR positive as per current practice (standard) and categorising these as ER or PgR negative DCIS (clonal method) were performed. Results: Of 540 samples (180 cases, 360 controls), ER and PgR status was evaluable in 504 and 498 patients respectively. ER expression was absent (ER-) in 148 (29.4%), 356 (70.6%) were ER positive (ER+) with 39 (7.7%) of these displaying multi-clonal expression (ERmulti-clonal). PgR expression was absent in 163 (32.7%), 335 (67.3%) were PgR positive with 84 (16.9%) of these displaying multi-clonal expression. ER- DCIS showed increased risk (Table) of in situ ipsilateral breast event (DCIS-IBE) and invasive ipsilateral breast event (I-IBE) (borderline significance). Prognostic discrimination was higher when ER status was determined by clonal method (ΔX2 1.57 for I-IBE and 9.05 for DCIS-IBE). Increases in the risk of I-IBE and DCIS-IBE were similar for ER- and ERmulti-clonal DCIS. TableEndpointComparisonScoring MethodHazard Ratio (95%CI)pI-IBEER- vs. ER+Standard1.75 (0.99-3.09)0.0544 ER- vs. ER+Clonal1.90 (1.10-3.30)0.0224 ER- vs. ER+*Clonal1.89 (1.05-3.41)0.0337 ERmulti-clonal vs. ER+*Clonal1.93 (0.74-5.03)0.1759DCIS-IBEER- vs. ER+Standard2.58 (1.68-3.94) Lack of PgR expression was only associated with increased risk of DCIS-IBE and PgR status did not add to the prognostic information provided by ER status. Conclusions: ER expression is prognostic for recurrence in DCIS. ER+ DCIS with distinct ER- clones has a recurrence risk similar to ER- DCIS. ER scoring should take clonality of expression into account. Citation Format: Thorat MA, Jones LJ, Levey PM, Elia G, Evagora CA, Bundred NJ, Fentiman IS, Forbes JF, Cuzick J. Prognostic role and impact of multi-clonal ER and PgR expression in ductal carcinoma in situ: Results from the UK/ANZ DCIS trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-01.

Background: As compared to invasive breast cancer (IBC), HER2 is much more frequently overexpressed in ductal carcinoma in situ (DCIS). Unlike IBC, the prognostic significance of HER2 overexpression remains to be established in DCIS and large studies to investigate its predictive role are lacking. We investigated the prognostic and predictive relevance of HER2 protein and ERBB2 mRNA expression in DCIS using material from UK/ANZ DCIS trial. Methods: Formalin-fixed paraffin embedded tissues (FFPETs) were collected from patients enrolled in the UK/ANZ DCIS trial, a randomised 2X2 factorial design trial investigating role of tamoxifen, radiotherapy or both as adjuvant treatment in DCIS. ERBB2 mRNA expression was evaluated by reverse transcription followed by PCR on customized Taqman low-density arrays. ERBB2 mRNA expression was analysed as a continuous variable and also as a binary variable using a cut-off to reproduce HER2 expression distribution similar to that observed with immunohistochemistry (IHC). HER2 protein expression was evaluated by IHC using HercepTest™ and scored as per ASCO-CAP 2013 recommendations; HER2 equivocal (IHC2+) were grouped with HER2 negative (IHC 0 or 1+) for main analyses. Additional analyses using binary ERBB2 mRNA expression as a reflex test for HER2 IHC2+ were also performed. Results: HER2 protein expression was evaluable in 713 (181 events) of 755 available samples (DCIS absent or lost during assay in 42). ERBB2 mRNA expression was evaluable in 521 (134 events) of 704 available samples (DCIS absent or insufficient RNA in 51, assay failure in 132). Both results were available in 508 cases (130 events). Increase in ERBB2 mRNA expression (median 0.62; range 0.07-36.76) was associated with increased risk of in situ ipsilateral breast event (DCIS-IBE) [Hazard ratio (HR) = 1.07; 95% Confidence Interval (95%CI) 1.04-1.10; p < 0.0001] but not with increased risk of invasive ipsilateral breast event (I-IBE) [HR = 1.03; 95%CI 0.97-1.10; p = 0.3209]. HER2 positivity by IHC was similarly associated with increased risk of DCIS-IBE [HR = 2.90; 95%CI 1.91-4.40; p < 0.0001] but not with increased risk of I-IBE [HR = 1.40; 95%CI 0. 0.81-2.42; p = 0.2313]. Reclassification of HER2 IHC2+ cases using binary ERBB2 mRNA expression (46 as negative, 16 as positive; 18 expression data unavailable) further improved prognostic discrimination of HER2 IHC [ΔX2 (1d.f.) 5.51; p = 0.0189] for any recurrence. The effect of radiotherapy (RT) for reducing I-IBE was greater in HER2 positive (by ERBB2 mRNA expression) cases [HR = 0.24; 95%CI 0.07-0.83; p = 0.0237] as compared with HER2 negative cases [HR = 0.60; 95%CI 0.23-1.55; p = 0.2925]. Kaplan-Meier estimates of 10-year I-IBE rates with and without RT were 4.5% (2.5%-1.4%) and 15.8% (9.6%-25.3%) in HER2 positive DCIS; rates in HER negative DCIS were 5.2% (2.1%-2.4%) and 7.3% (4.3%-12.2%) respectively. The differential benefit of RT by HER2 status was also seen for reduction in DCIS-IBE. Conclusions: HER2 overexpression is associated with increased risk of DCIS-IBE but not of I-IBE. HER2 status is predictive of radiotherapy response with larger reductions in both I-IBE and DCIS-IBE seen in HER2 positive DCIS. Citation Format: Thorat MA, Wagner S, Jones LJ, Levey PM, Bulka K, Hoff R, Sangale Z, Flake II DD, Bundred NJ, Fentiman IS, Forbes JF, Lanchbury JS, Cuzick J. Prognostic and predictive relevance of HER2 status in ductal carcinoma in situ: Results from the UK/ANZ DCIS trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-02.

Background: Initial results of the UK/ANZ DCIS trial found that adjuvant radiotherapy reduced the recurrence of ipsilateral invasive breast cancer (BC) and ductal carcinoma in situ (DCIS) by 55% and 64% respectively, but the effect of tamoxifen was smaller (Lancet 2003, 362: 95-102). Here, we report an updated analysis of this trial.Methods: 1701 women with completely locally excised DCIS were enrolled in a randomised 2x2 trial of radiotherapy, tamoxifen or both. Elective decision to withhold or provide one of the treatments was also permitted and long-term follow-up was performed by treating hospitals. Seven patients were excluded due to protocol violations leaving 1694 patients available for analysis.Results: After a median follow-up of 12.7 years, a total of 376 breast cancers were diagnosed; 162 invasive (122 ipsilateral; 39 contralateral) and 197 DCIS (173 ipsilateral; 17 contralateral). Radiotherapy continued to reduce the incidence of ipsilateral invasive disease (HR=0.32 (0.19-0.56), P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 34.