Dowsett, Mitch

Abstract Context Prior reports of plasma estradiol concentrations and their reference ranges in postmenopausal women identified marked variation, in part due to assay method and body mass index (BMI). Objective To develop an international reference range for plasma estradiol in postmenopausal women, support accurate assessment of concentrations during systemic and local (eg, vaginal) estradiol therapy, and improve risk prediction for diseases, including osteoporosis, breast, and endometrial cancers. Methodology Estradiol concentrations were measured using primarily liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays in 5 international cohorts of community-dwelling, postmenopausal women of primarily European ancestry aged 38-100 years who were not using exogenous estrogens (n = 7206). Measurements were harmonized to the Centers for Disease Control and Prevention (CDC) reference measurement procedure (RMP) by re-assaying a subset of samples for each cohort and generating recalibration equations. Reference intervals were determined for all women, non-obese women, and specific BMI categories. Results Estradiol concentrations from separate LC-MS/MS assays correlated closely with the CDC RMP, and harmonization minimized inter-assay variability. Estradiol concentrations correlated with BMI but not with chronological age. The reference range (2.5th-97.5th percentile) was 1.1-18.2 pg/mL (4.0-66.8 pmol/L) with median of 4.9 pg/mL (18.0 pmol/L) for all postmenopausal women and 1.1-12.5 pg/mL (4.0-45.9 pmol/L) with median of 4.1 pg/mL (15.1 pmol/L) for non-obese women (BMI < 30). Reference ranges increased progressively with higher BMI categories. Conclusion Cross-calibration of estradiol measurements with the CDC RP enabled the development of harmonized plasma estradiol reference ranges for postmenopausal women who were not using exogenous estrogens, suitable for future studies to establish clinical utility. Increasing obesity in postmenopausal women was associated with increasing estradiol concentration and BMI category-specific reference ranges.

Abstract Background: NET is offered to postmenopausal patients (pts) with clinical stage 2/3 ER+/HER2- BC to promote breast-conserving surgery. Also limited surgical accessibility during the COVID19 pandemic has increased NET utility. Inability to identify ET-resistant disease at diagnosis risks disease progression (PD) and delays more effective treatments. Dowsett et al. recently demonstrated that baseline levels of ER, progesterone receptor (PR), Ki67 (>15% vs ≤15%), and Ki67 (>10% vs ≤10%) 2-4 weeks (wks) after starting NET may improve appropriate patient (pt) selection for NET (PMC7280290). The ER, PR and Ki67-based prediction model divides pts with primary ER+/HER2- BC into 3 groups for appropriateness for NET: (Group 1) NET is likely to be inappropriate (Allred ER <6 or ER 6 and PgR <6), (Group 2) NET may be appropriate and a biopsy for on-treatment Ki67 analysis may be considered after 2-4 wks of NET (2A: ER 7 or 8 and PgR <6 and 2B: ER 6 or 7 and PgR ≥6) given that on-treatment Ki67 >10% has been associated with worse outcome (PMC5455353), or (Group 3) NET is appropriate (ER 8 and PgR ≥6). The ALTERNATE trial (NCT01953588) randomized postmenopausal women with clinical stage II or III, ER+ (Allred score 6-8)/HER2- BC to receive anastrozole (ANA), fulvestrant (FUL), or ANA + FUL for 6 months, unless Ki67 was >10% on wk 4 or 12 biopsy, in which case pts were triaged to receive neoadjuvant chemotherapy (NCT) or surgery. As previously reported, the ET-sensitive disease (mPEPI 0 plus pCR) rates were similar across the treatment arms and overall 22% (286 of 1,299) pts had Ki67 >10% at wk 4 or 12. The ALTERNATE trial therefore provides a large independent data set to evaluate the NET appropriateness model. Results: Among 1,299 eligible pts randomized to receive 6 months of NET, 214 were excluded due to absent HR Allred score (n=41) or absence of pre-treatment and wk 4 Ki67 determinations (n=173). The proportions of the remaining 1,085 pts in Group 1, 2 and 3 were 1% (n=10), 43% (n= 468), and 56% (n=607), respectively. On-study Ki67 >10% prompting conversion from NET to NCT/Surgery occurred in: Group 1 90% (9 of 10), Group 2 30% (141 of 468), and Group 3 17% (104 of 607) (Table 1). Among the 1,075 pts in Groups 2 and 3, 260 (24%) pts had Ki67 ≤15% at baseline (BL), among whom only 14 (5.4%) had Ki67 >10% at wk 4, compared to 231 of the 815 (28.3%) who had BL Ki67 >15% and subsequent Ki67 >10% at wk 4. 2% of pts who remained on NET due to on-treatment Ki67 <10% had PD. Response and PEPI-0 rates by group will be reported. Conclusion: ALTERNATE trial data support a model whereby levels of ER, PR and Ki67 at diagnosis can be used for the identification of postmenopausal pts with primary ER+/HER2- BC who are appropriate for NET. When baseline ER Allred scores are >6 and Ki67 ≤15%, there is a low likelihood of ET-resistant disease. When BL Ki67 is >15%, ET sensitivity is variable, and on-treatment biopsy for Ki67 may assist in triaging regarding NET appropriateness, particularly given the extremely low local PD rates seen in ALTERNATE when on-treatment Ki67 was <10%. Support: U10CA180821, U10CA180882, U24CA196171, UG1CA189856, U10CA180868 (NRG); NCI BIQSFP, BCRF, Genentech, AstraZeneca. https://acknowledgments.alliancefound.org; Clinical Trials.gov Identifier: NCT01953588 Table 1 Baseline levels of ER, PR, and Ki67 in Relation to Wk 4 Ki67 (N=1,085)BaselineWeek 4GroupNERAllred ScorePRAllred ScoreKi67Ki67 ≤10%N (%)Ki67 >10%N (%)1N=26<6≤15%0 (0%)2 (100%)9 (90)N=86<6>15%1 (12.5%)7 (87.5%)2AN=647 or 8<6≤15%61 (95.3%)3 (4.7%)90 (30.1)N=2357 or 8<6>15%148 (63%)87 (37%)2BN=466 or 7≥6≤15%42 (91.3%)4 (8.7%)51 (30.2)N=1236 or 7≥6>15%76 (61.8%)47 (38.2%)3N=1508≥6≤15%143 (95.3%)7 (4.7%)104 (17.1)N=4578≥6>15%360 (78.8%)97 (21.2%) Citation Format: Matthew J Ellis, Vera Suman, A. Marilyn Leitch, Souzan Sanati, Kiran Vij, Gary W Unzeitig, Jeremy Hoog, Mark Watson, Olwen Hahn, Joseph Guenther, Abigail Caudle, Erika Crouch, Horacio Maluf, Mitch Dowsett, Amy Tiersten, Monica Mita, Wajeeha Razaq, Tina J Hieken, Yang Wang, Travis Dockter, Jo Anne Zujewski, Anna Weiss, Clifford Hudis, Eric P Winer, Kelly Hunt, Ann H Partridge, Cynthia X Ma, Lisa A Carey. Validation of a predictive model for potential response to neoadjuvant endocrine therapy (NET) in postmenopausal women with clinical stage II or III estrogen receptor positive (ER+) and HER2 negative (HER2-) breast cancer (BC): An ALTERNATE trial analysis (Alliance A011106) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD2-10.

Abstract Background The immunohistochemical (IHC) 4 + clinical treatment score (C) i.e. IHC4+C assay combines IHC assessment of four routinely measured markers in the clinical setting, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and KI67, with other clinical parameters for aiding decision-making on adjuvant chemotherapy in hormone positive (HR+) breast cancer. It is low cost, widely available and has been shown to provide similar prognostic information to the 21-gene (Oncotype DX) test. Clinical use of IHC4+C is limited by the need for standardized scoring of the IHC markers. Here, we examine the prognostic value of a digital image analysis (DIA) based IHC4+C assay in two study populations and compare this with pathologists' visual assay in a subset of patients. We also evaluated its potential clinical relevance in relation to the Nottingham prognostic index [NPI; i.e. low (<3.4), intermediate (3.4-5.4) and high (>5.4)] and IHC defined breast cancer subtypes (i.e. luminal A-like and B-like). Methods Using DIA data on ER, PR, HER2 and KI67, in addition to clinicopathological characteristics of tumors, we computed DIA based IHC4+C (i.e. DIAIHC4+C) assay for 2,034 women from study populations in Poland (PBCS) and the United Kingdom (SEARCH). A total of 246 deaths occurred over a median follow-up period of 6.7 years. A relevant threshold was determined using quartile analysis and the relationship between dichotomous categories (>75th vs ≤ 75th percentile) of DIAIHC4+C and 10-year breast cancer specific survival (BCSS) was investigated using Kaplan-Meier survival curves and in Cox proportional hazard regression models, overall and stratified by study, NPI and subtype. Results DIAIHC4+C score was associated with worse 10-year BCSS overall [HR (95% CI) >75th vs ≤ 75th percentile = 2.92 (1.77, 4.82); p-value <0.0001] and in both the PBCS [3.48 (1.40, 8.65); p-value = 0.007] and SEARCH [3.87 (2.24, 6.68); p-value <0.0001] study populations. DIAIHC4+C was associated with significantly worse survival outcomes in women with intermediate NPI [1.94 (1.31, 2.88); p-value = 0.001] and luminal A-like subtype of HR+ tumors [5.37 (3.50, 8.26); p-value <0.0001]. DIA and visual IHC4+C results were highly correlated (r = 0.95) and survival associations and model fit parameters were similar for DIA [3.82 (2.08, 7.00); p-value < 0.0001; AIC = 472.3] and visual [4.73 (2.55, 8.76); p-value < 0.0001; AIC = 466.1] IHC4+C scores amongst 444 patients with data from both sources. Conclusion These findings are in support of the prognostic value of DIAIHC4+C assay in HR+ breast cancer and demonstrate its potential clinical relevance as an adjunct to the NPI. Given the advantages of being high-throughput and highly reproducible, DIA based methods could find use in the standardization of the analytical processes for the IHC4+C assay. Citation Format: Mustapha Abubakar, Paul D. Pharoah, Mark E. Sherman, Montserrat Garcia-Closas, Mitch Dowsett. Digital image analysis based IHC4+C assay and prognosis in hormone receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2971.

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) –positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

AbstractAutomated methods are needed to facilitate high‐throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large‐scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37–0.87) and study (kappa range = 0.39–0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p‐value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000–4,500 cells: kappa = 0.78) than those with lower counts (50–500 cells: kappa = 0.41; p‐value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre‐ and post‐analytical quality control procedures are necessary in order to ensure satisfactory performance.

Abstract Background: The Preoperative Endocrine Prognostic Index (PEPI) scores the independent prognostic effects of tumor pathologic staging and expression levels of ER and the “proliferation” marker Ki67 in the surgical sample to predict long term outcomes after completion of neoadjuvant endocrine treatment (Ellis et al JNCI 100:1380, 2008). A limitation of the PEPI is that the prognostic information becomes available only after 4 months of treatment. We therefore evaluated the value of an early assessment of the Ki67 level in a tumor biopsy sample taken two to four weeks after initiating treatment in two neoadjuvant endocrine therapy trials for the purposes of the early identification of non- respondersMethods: A Ki67 cut point of greater than 10% for poor outcome in ER+ breast cancer was derived by comparing the PAM50 intrinsic subtype profile using a qRT-PCR assay with Ki67 data in a 700+ sample data set. A baseline level of 10% or less correlated most closely with a PAM50-based definition of LumA breast cancer and above 10% LumB breast cancer. We subsequently applied the 10% cut point to the baseline and early on-treatment Ki67 data in two trials, POL (Olson et al JACS 208:906, 2009) and IMPACT (Smith et al JCO: 23, 5108, 2005).Results: At baseline the dichotomized Ki67 definition was not significantly predictive for surgical Ki67 level, PEPI score or RFS in this modest size sample set. In contrast, in a result that emphasizes the enhaced prognostic properties of the on-treatment Ki67 approach, the one month POL sample Ki67 values (62 patients) predicted a higher level of Ki67 in the surgical samples at four months after treatment initiation (P=.01), a poorer PEPI score (P=0.01), a smaller number of patients in the PEPI risk point zero group (P=0.08) and worse relapse free survival (P=0.003). The IMPACT data (153 patients) confirmed that a two week Ki67 &gt;10% predicted higher Ki67 in the surgical specimen (P=0.001), a poorer PEPI score (P=0.001), smaller numbers of patients in the PEPI 0 risk point group (P= 0.004) and worse relapse free survival (P=0.008).Ki67 and OutcomePOL 4W Ki67% PEPI 0RFS (events)10%&gt;1/19 (5%)5/21 (23%)10%≤10/36 (28%)1/41 (2.4%)P ValueP=0.08 (Fisher)P=0.003 (log rank)IMPACT 2W Ki67% PEPI 0RFS (events)10%&gt;0/32 (0%)9/35 (26%)10%≤21/101 (21%)13/118 (11%)P ValueP=0.004 (Fisher)P=0.008 (log rank) Conclusions: A tumor Ki67 assessment taken a short time (2 to 4 week window) after the initiation of neoadjuvant AI identifies patients with poor outcome ER+ disease. Amendment 6 of the neoadjuvant endocrine therapy protocol ACOSOG Z1031 will triage patients with an “on treatment” Ki67 value above 10% to chemotherapy in order to assess the pathological response rate to cytotoxic therapy in this important tumor subset.Supported by R01 CA095614, Avon PFP award 3P50 CA68438-07S2, U01 CA114722, ACOSOG U10 CA 76001, Breakthrough Cancer UK and AstraZenica (IMPACT trial). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 78.

Abstract Abstract #12 Background: Meta-analyses (EBCTCG, Lancet 2005; 365:2687) have established in estrogen receptor (ER) positive breast cancer (BC) that 5 yrs of adjuvant tamoxifen (Tam) substantially reduces recurrence and mortality. The aromatase inhibitors (AIs) anastrozole, exemestane and letrozole have been studied in prospective trials in postmenopausal patients (pts) in comparison with Tam either as initial monotherapy (Cohort 1) or after 2 to 3 years of Tam in a switching strategy (Cohort 2); both to a total of 5 years (yrs) of endocrine therapy.&#x2028; Methods: Outcomes data submitted to EBCTCG were used in separate meta-analyses of Cohort 1 (trials: ATAC, BIG 1-98/IBCSG 18-98) and of Cohort 2 (trials: ABCSG 8, ARNO 95, IES/BIG 2-97, ITA). The primary analysis involved pts with tumors reported to be ER positive with endpoints of BC recurrence (all, local only, contralateral BC only, distant), death with and without recurrence, any death. Subgroup analyses included progesterone receptor (PgR) status, age, tumor grade and nodal status. All p-values were 2-sided.&#x2028; Results: Highlights include: Cohort 1: 9,856 pts with 50,000 woman-years of follow-up. At 5 yrs, AI therapy was associated with an absolute 2.7% (standard error [SE] 0.7) decrease in BC recurrence (10.7% vs 13.4%, relative decrease 20% [SE 5], p=0.00004). There appeared to be greater proportional decreases in isolated local recurrence (30% [SE 10], p=0.003) and in contralateral disease (38% [SE 12], p=0.003) than in distant recurrence (12% [SE 6], p= 0.04). AIs yielded an absolute 1.0% (SE 0.5) decrease in BC mortality (5.5% vs 6.5%, relative decrease 7% [SE 7], p=0.28). Cohort 2: 9,015 pts with 33,000 woman-years of follow-up. At 6 yrs from treatment divergence (i.e., 8-9 yrs from diagnosis), AI therapy was associated with an absolute 3.5% (SE 1.1) decrease in BC recurrence (12.6% vs 16.1%, relative decrease 29% [SE 6], p&lt;0.00001). There appeared to be greater proportional reductions in isolated local recurrence (40% [SE 13], p=0.002) and in contralateral disease (35% [SE 16], p=0.03) than in distant recurrence (24% [SE 7], p= 0.001). AIs yielded an absolute 1.6% (SE 0.8) decrease in BC mortality (6.3% vs 8.0%, relative decrease 22% [SE 9], p=0.02). Subset analyses with respect to PgR status, age, tumor grade and nodal status revealed no apparent heterogeneity between the proportional reductions in recurrence and no indication of an increase or decrease in non-breast deaths with AIs in either cohort 1 or 2.&#x2028; Conclusions: In 2 separate meta-analyses considering both the initial monotherapy setting and the switching setting after 2 to 3 yrs of Tam, AIs produced significantly lower BC recurrence rates compared with Tam. Additional follow-up data will provide important information on long-term survival. Meta-analyses can be helpful even in the era of relatively large clinical trials. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 12.

Abstract The majority of candidates for breast cancer prevention have not accepted tamoxifen because of the perception of an unfavorable risk/benefit ratio and the acceptance of raloxifene remains to be determined. One means of improving this ratio is to identify women at very high risk of breast cancer. Family history, age, atypia in a benign biopsy, and reproductive factors are the main parameters currently used to determine risk. The most powerful risk factor, mammographic density, is not presently employed routinely. Other potentially important factors are plasma estrogen and androgen levels, bone density, weight gain, age of menopause, and fracture history, which are also not currently used in a comprehensive risk prediction model because of lack of prospective validation. The Breast Cancer Prevention Collaborative Group (BCPCG) met to critically examine and prioritize risk factors that might be selected for further testing by multivariate analysis using existing clinical material. The BCPCG reached a consensus that quantitative breast density, state of the art plasma estrogen and androgen measurements, history of fracture and height loss, BMI, and waist–hip ratio had sufficient priority for further testing. As a practical approach, these parameters could be added to the existing Tyrer–Cuzick model which encompasses factors included in both the Claus and Gail models. The BCPCG analyzed potentially available clinical material from previous prospective studies and determined that a large case/control study to evaluate these new factors might be feasible at this time.