Suppression Of Ki67 Research Articles

Abstract PO3-19-08: A phase 2 randomized pre-operative,window of opportunity trial investigating the effect of elacestrant with/without triptorelin in premenopausal patients with HR+/HER2- breast cancer – SOLTI-2104-PremiÈRe trial

Abstract Background. Premenopausal women diagnosed with HR+/HER2- breast cancer (BC) often have a different biology and worse prognosis. The SOFT and TEXT studies demonstrated an increase diseasefree survival (DFS) with ovarian function suppression (OFS) plus tamoxifen or exemestane compared to tamoxifen alone. Of note, high-risk clinicopathologic features, adjuvant chemotherapy, or aged ≤35 years correlated with greater OFS benefit. However, adding OFS in this context has intrinsic issues as higher toxicity, which led to treatment discontinuation (20% in SOFT trial), and suboptimal OFS was found in around 25% of patients (pts) with exemestane plus monthly triptorelin (SOFT-EST sub-study). Thus, new effective ET options without OFS are needed for premenopausal patients (pts). Elacestrant is the first oral, non-steroidal, selective estrogen receptor degrader (SERD) to demonstrate improved efficacy to SOC treatments and specifically compared to fulvestrant in postmenopausal pts with HR+/HER2- metastatic BC at the phase III EMERALD trial. In menopausal patients with HR+/HER2- early BC, the window of opportunity SOLTI-1905 ELIPSE trial (NCT04797728), showed that elacestrant was associated with a 27.3% rate of Complete Cell Cycle Arrest (CCCA) and a statistically significant suppression of Ki-67. Among Luminal A tumors, the CCCA rate was 45% and the average decrease in Ki-67 was 64.6%, while no CCCA was reported and the suppression of Ki-67 was less pronounced (31.7%) in the Luminal B population. There is still a need for investigating elacestrant without OFS in the premenopausal scenario. We hypothesize that elacestrant as a single agent or in combination with triptorelin is an effective and safe treatment regimen in premenopausal pts with HR+/HER2-negative early BC capable of achieving an equivalent CCCA rate as a subrogate of effectiveness regardless the use of OFS Study design. PREMIERE is a parallel, non-comparative, two-arm, randomized 1:1, open-label, multicenter, exploratory study in premenopausal women with primary operable HR+/HER2-negative BC. The study aims to evaluate the biological effects of elacestrant with or without triptorelin. Participants must have histologically confirmed HR+ (≥ 10%) and HER2- operable early BC stage I to stage IIB >1 cm with a Ki-67 between 10-35%. The primary objective is to assess the ability of each treatment arm to induce CCCA determined by central assessment of Ki-67 (% Ki-67 ≤ 2.7%). No formal comparison between treatment arms is intended. Secondary objectives include evaluating the biological activity of elacestrant with or without OFS, antiproliferative activity, changes in gene expression including PAM50 subtype changes, and the effect of optimal vs suboptimal suppression on CCCA . Serum E2 and FSH levels will also be evaluated. Safety and tolerability will be assessed based on adverse events and clinical laboratory test results. Pts will undergo screening and randomization, with stratification by PAM50 subtype (Luminal A vs Non-luminal A). Treatment will be elacestrant 400 mg once daily or elacestrant 400 mg once daily plus triptorelin 3.75 mg days +1 and +29 for 30 (+7) days. Surgery or biopsy will be performed after treatment completion, and a post-surgery visit will mark the end of the active follow-up period. Patients will receive SOC treatment after surgery. 48 patients will be recruited in 9 sites within SOLTI Spanish network in 9 months period. This study is financially supported by Menarini-Stemline. Citation Format: Meritxell Bellet- Ezquerra, Cristina Hernando, Pablo Tolosa, Maria Vidal, Yolanda Fernández, Santiago González-Santiago, Pilar Sanchez, Susana De La Cruz, Vanesa Ortega, Xavier Gonzalez-Farré, Milana Bergamino, Alejandra Espinosa, Tomás Pascual. A phase 2 randomized pre-operative,window of opportunity trial investigating the effect of elacestrant with/without triptorelin in premenopausal patients with HR+/HER2- breast cancer – SOLTI-2104-PremiÈRe trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-19-08.

Abstract PD10-06: PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer

Abstract Background: Endocrine therapy (ET) is the mainstay of ER+ BC treatment. However; up to 20% of ER+ BC tumors progress into metastatic disease and develop ET resistance, underscoring the need for combination therapies. Preclinical data suggest that combining phosphatidylinositol 3-kinase (PI3K) inhibitors with ET may overcome resistance. OPPORTUNE, a preoperative phase II window trial, evaluated whether the combination of the PI3K inhibitor pictilisib with anastrozole (PIC+ANA) can increase the antitumor effect of ANA in newly diagnosed operable ER+ BC. Early results showed greater suppression of tumor Ki67 in patients treated with PIC+ANA versus ANA alone. Here, we present gene expression analysis from tumors collected pre- and post-treatment, and their associations with Ki67 outcomes. Methods: Postmenopausal women with newly diagnosed operable ER+/HER2-negative BC were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with ANA 1 mg once per day (n = 47) or the combination of ANA 1 mg with PIC 260 mg once per day (n = 89). The primary end point was inhibition of tumor cell proliferation measured by change in Ki67 protein expression via IHC between tumor samples taken pre- and post-treatment. Samples were analyzed by RNA-sequencing—ER pathway activity, PAM50 intrinsic subtypes, and pathway analyses were assessed by Ki67 outcomes. Elastic net regression analysis and transcription factor activity inference with Dorothea were performed to identify features strongly associated with Ki67 outcomes. Results: 124 patients (ANA, n=43; PIC+ANA, n=81) had paired tumor samples at baseline/week 2 that were evaluable for both Ki67 and RNA-seq. PIC+ANA showed improved suppression of Ki67 compared to ANA alone (-83.78% vs -73.85%, p=0.012) and a greater proportion of patients achieved complete cell cycle arrest (CCCA, as defined by Ki67< 2.7%) in the PIC+ANA arm (45.68% vs 36.59%). ER pathway activity suppression was comparable between treatments. PAM50 classification based on RNA-seq showed that 83.0% of tumors were luminal (Lum) A at baseline and 12.9% were LumB, with the remainder being classified as Normal-like. PIC+ANA showed greater suppression of Ki67 in LumB tumors compared to LumA (-92.29% vs -81.62%). This effect was much greater in the PIC+ANA arm compared to LumB tumors treated with ANA alone (-92.29% vs -37.87%); however, given the low number of LumB tumors in the ANA arm (n=3), we could not determine statistical significance. Bioinformatic analysis of RNA-seq from baseline specimens showed that MYBL2 activity was associated with resistance to ANA (as defined by Ki67≥7.5% at week2). Tumors in the top quartile of MYBL2 activity at baseline showed improved Ki67 outcomes in the PIC+ANA arm compared to ANA alone (-91.71% vs -51.44%), while no differences were observed between treatments for tumors with lower MYBL2 activity (-79.16% vs -76.72%). Single-nucleus (sn)-RNA-seq from untreated ER+ BC tumors showed that MYBL2 was enriched in a single cluster of tumor cells, which also had the highest expression of a subset of actionable targets (including CDK1, CDK2, CDK4, EZH2, and AKT1), as compared to other tumor cells. Expression data from DepMap and drug-sensitivity data from ER+ BC cell lines show a positive trend between high MYBL2 expression and sensitivity to the ER degrader, giredestrant. Conclusions: PIC exhibited greater antiproliferative effects in combination with ANA in ER+/HER2- early BC compared to ANA alone, particularly in LumB and MYBL2-high tumors. Furthermore, the transcriptional profile and in vitro response of tumor cells with high MYBL2 expression suggest potential sensitivity to other combination therapies. Citation Format: Alejandro Martinez Chibly, Alice Shia, Radia Johnson, Michael S. Hwang, Marc Hafner, Ciara Metcalfe, Kalpit Shah, Michal Slyper, Chris Bolen, Sarah E. Pinder, Alastair M. Thompson, Steven Gendreau, Peter Schmid. PD10-06 Clinical outcomes and exploratory gene expression analysis of OPPORTUNE: a phase II window-of-opportunity study to evaluate pictilisib+anastrozole versus anastrozole alone in ER-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-06.

Abstract PD13-02: PD13-02 Exploratory gene expression analysis of coopERA Breast Cancer (BC): a study evaluating neoadjuvant giredestrant versus anastrozole alone and in combination with palbociclib in ER-positive, HER2-negative untreated early BC

Abstract Background: Endocrine therapy remains the mainstay treatment for ER+ BC. CDK4/6 inhibitors induce cell cycle arrest and decrease tumor cell proliferation, as measured by the biomarker Ki67, when used in combination with aromatase inhibitors (AI) such as anastrozole (A). Giredestrant is an oral, well-tolerated, and highly potent selective ER degrader (SERD) that achieves robust ER suppression and has demonstrated antitumor activity in the metastatic setting either as monotherapy or in combination with the CDK4/6 inhibitor palbociclib. The randomized, phase 2 coopERA Breast Cancer study (NCT04436744) evaluated giredestrant in postmenopausal women with untreated ER+/HER2- early BC and met its primary endpoint, demonstrating superior Ki67 suppression with giredestrant vs A after two weeks of single agent treatment. This suppression was maintained at surgery where giredestrant vs A was evaluated in combination with palbociclib. Here, we present gene expression analysis and associations with Ki67 response. Methods: 221 eligible patients with measurable ER+/HER2– untreated early BC and baseline Ki67 ≥ 5% were randomized 1:1 to receive 30 mg oral daily (PO QD) giredestrant or 1 mg PO QD A on Days 1–14 of a neoadjuvant window-of-opportunity phase, followed by four 28-day cycles of PO QD giredestrant or A with 125 mg PO QD palbociclib on Days 1–21 before surgery. FFPE specimens were collected at baseline, week 2 and surgery; and RNA-sequencing (seq) was performed. Gene expression analysis included ER pathway activity, PAM50 intrinsic subtypes, and other pathway analyses, assessed by Ki67 response. Results: 112 and 92 patients had paired tumor samples at baseline/week 2 and baseline/surgery, respectively, that were evaluable for RNA-seq and Ki67. The trend for greater Ki67 protein suppression by giredestrant vs A from baseline to week 2 was maintained in the RNA-seq evaluable subset. Interestingly, the same subset revealed similar suppression of both proliferation gene signatures and ER pathway activity between A and giredestrant. PAM50 subtyping showed that 69% of tumors were luminal (Lum) A and 29% were LumB at baseline. Less than 1% were classified as basal or HER2. Interestingly, giredestrant (G) showed greater suppression of both Ki67 and ER pathway activity vs A in LumB tumors (Ki67: -82% [G] vs -62% [A]; ER activity: -0.83 [G] vs -0.66 [A]) compared to LumA at week 2 (Ki67: -74% [G] vs -71% [A]; ER activity: -0.60 [G] vs -0.70 [A]). Moreover, at week 2, 83% (13/18) of LumB tumors at baseline transitioned into a LumA subtype after giredestrant treatment compared to 46% (5/11) of A-treated tumors. Giredestrant-treated tumors also achieved lower mean ER pathway activity compared to those treated with A at surgery (p=0.023). Gene set enrichment analysis showed downregulation of cell-cycle and ER-related pathways at week 2 and surgery in both treatment arms. A subset of cytokine signaling and immune response pathways were increased at week 2 compared to baseline after treatment with A but not with giredestrant. These pathways were also associated with Ki67 resistance (Ki67 ≥ 7.4%) in A-treated tumors. This was consistent with differential expression analysis of samples collected at week 2, in which cytokine signaling pathways were enriched in A compared to giredestrant. Notably, IL12 signaling was enriched in tumors resistant to A but not giredestrant. Conclusions: Giredestrant has a greater effect on Ki67 protein suppression in ER+/HER2- early BC compared to A, which is more pronounced in LumB tumors. This benefit may involve differential regulation of cytokine and immune responses. These exploratory findings reveal novel mechanisms that may differentiate the activity of SERDs vs AIs, which warrant further validation. Citation Format: Alejandro M. Chibly, Tharu M. Fernando, Ciara Metcalfe, Marc Hafner, Gilbert Owusu-Manu, Sara Hurvitz, Aditya Bardia, Peter A. Fasching, Yeon H. Park, Vanesa Quiroga, Jutta Steinseifer, Pablo Perez-Moreno, Heather M. Moore. PD13-02 Exploratory gene expression analysis of coopERA Breast Cancer (BC): a study evaluating neoadjuvant giredestrant versus anastrozole alone and in combination with palbociclib in ER-positive, HER2-negative untreated early BC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD13-02.

144P Neoadjuvant giredestrant (GDC-9545) + palbociclib (P) vs anastrozole (A) + P in postmenopausal women with oestrogen receptor-positive, HER2-negative, untreated early breast cancer (ER+/HER2– eBC): Biomarker subgroup analysis of the randomised, phase II coopERA BC study

144P Neoadjuvant giredestrant (GDC-9545) + palbociclib (P) vs anastrozole (A) + P in postmenopausal women with oestrogen receptor-positive, HER2-negative, untreated early breast cancer (ER+/HER2– eBC): Biomarker subgroup analysis of the randomised, phase II coopERA BC study

Neoadjuvant giredestrant (GDC-9545) plus palbociclib (P) versus anastrozole (A) plus P in postmenopausal women with estrogen receptor–positive, HER2-negative, untreated early breast cancer (ER+/HER2– eBC): Final analysis of the randomized, open-label, international phase 2 coopERA BC study.

589 Background: Endocrine therapy (ET) is the therapeutic mainstay for ER+ BC. Giredestrant is a highly potent, nonsteroidal, oral, selective ER antagonist and degrader (SERD) which has demonstrated robust ER occupancy, is well tolerated, and has previously shown encouraging antitumor activity as monotherapy and in combination with P in metastatic BC. coopERA BC (NCT04436744) evaluated giredestrant in eBC and met its primary endpoint, highlighting superior Ki67 suppression with single-agent giredestrant vs A at Week 2. Giredestrant was well tolerated. Here, we report the final analysis. Methods: Eligible patients (pts) with measurable ER+/HER2– untreated eBC and baseline Ki67 score ≥5% (202 planned) were randomized 1:1 to receive, on Days 1–14 of a neoadjuvant window-of-opportunity phase, 30 mg oral daily (PO QD) giredestrant or 1 mg PO QD A followed by a 16-week neoadjuvant phase of QD giredestrant or A for four 28-day cycles with 125 mg PO P on Days 1–21. Randomization was stratified by tumor size, baseline Ki67 score, and progesterone receptor status. Endpoints assessed here included Ki67 suppression from baseline to surgery, complete cell cycle arrest (CCCA; Ki67 ≤2.7%) at surgery, objective response rate (ORR), and safety. Results: At final analysis (cutoff: Nov 24, 2021), 112 and 109 pts were randomized to the giredestrant and A arms, respectively (median age: 62 years each; stage I/IIa disease: 60% vs 54%). Consistent with the primary analysis, greater suppression of Ki67 was observed at surgery with giredestrant + P (–81% [95% confidence interval (CI): –86%, –75%]) vs A + P (–74% [95% CI: –80%, –67%]). Similarly, greater CCCA was achieved at surgery with giredestrant + P (20%) vs A + P (14%). ORR was similar between the two arms (giredestrant + P: 50% [95% CI: 40%, 60%]; A + P: 49% [95% CI: 39%, 59%]). ET-related adverse events (AEs) were non-serious and occurred at similar rates between the two arms. Related Grade ≥3 AE rates were also similar at 6% each. Interruption/withdrawal of ET due to AEs was low and similar for both arms. Conclusions: In this final analysis of coopERA BC, the greater suppression of Ki67 with giredestrant vs A observed at Week 2 in the primary analysis was maintained at surgery, and safety data remained consistent with the known safety profile of giredestrant. coopERA BC is the first randomized study to show superior antiproliferative activity of an oral SERD (giredestrant) over an aromatase inhibitor (A) in ER+/HER2– eBC; studies are ongoing to further assess giredestrant’s clinical benefit. Clinical trial information: NCT04436744.

Abstract P1-07-02: Primary results of ONAWA (SOLTI-1802) trial: A window of opportunity trial of onapristone in postmenopausal women with progesterone receptor-positive/HER2-negative early breast cancer (EBC)

Abstract Background PgR expression is a biomarker of ER functionality, cellular progression to malignancy, and response to endocrine therapy (ET) in HR+ BC. Onapristone (ONA), a type 1 antiprogestin, was shown to have a single agent anti-tumor activity in patients with metastatic breast cancer (Robertson et al., 1999; Jonat et al., 2002). However, this once daily immediate-release formulation was associated with liver function test abnormalities in one-third of patients. A new, extended-release formulation (ONA XR) was developed and was evaluated in a BID schedule that reduced peak serum concentrations while sustaining the minimum plasma concentrations previously associated with the higher dose. Safety results of two phase I-II studies confirmed this hypothesis (Cottu et al., 2018; Jayaram et al., 2017; Lewis et al, 2020). Considering BC heterogeneity and that PgR analysis by standard immunohistochemistry (IHC) does not perfectly correlate with PgR target gene expression, the identification of biomarkers allowing the selection of patients with PgR-driven tumors that may benefit from antiprogestins treatment is currently an unmet need. Patients and Methods ONAWA (NCT04142892) is an open-label, single-arm, multicenter window of opportunity clinical trial of ONA XR (50 mg BID for 21 days) for postmenopausal women with EBC amenable to receive a short course of ET before surgery. Ten patients with ER+/PgR+/HER2- and Ki-67 ≥ 15% BC were enrolled. The primary objective is to evaluate the biological activity of ONA by the rate of Complete Cell Cycle Arrest (CCCR) determined by Ki-67 (≤2.7%). Secondary endpoints include safety and correlating biological activity with IHC of tumor expression (ER, PgR, Ser294-PgR, CD24, CD44, ALDH1, Ki-67), estradiol, and progesterone blood levels, and gene expression profile (NanoString nCounter® Breast 360TM panel). Relative Ki-67 suppression was defined as (1 - Ln(Ki-67 Baseline)/Ln(Ki-67 surgery)) Results Assessment of the treatment effects was possible for the 10 patients who successfully completed the protocol and the 10 paired samples (100%) were analyzed. Main patient characteristics were mean age 68 (range 53-81 years), mean tumor size 20.2 mm (range 15-26 mm), stage I (40%) and grade 2 (100%). No patients achieved a CCCR. Tumor Ki-67 expression decreased in six patients was stable in one patient and increased three patients. The mean percentage suppression of Ki-67 was 19.58%. Overall, no statistically significant change was observed in Ki-67 between paired samples (p=0.234). Baseline IHC PgR (%) expression correlated with Ki-67 decrease (r = -0.635). Mean percentage suppression of Ki-67 for tumors with IHC PgR expression ≥90% (N=4) and <90% (N=6) was -25.23.0% and +2.54%, respectively. Six (60%) patients reported AEs at any grade. Most common grade 1 or 2 AEs were post-procedural pain, dry mouth and GGT increased. Grade 3 reversible GGT and AST increase occurred in 1 patient. Conclusion: ONA XR significantly increases suppression of tumor cell proliferation in PgR-high primary breast cancer. The safety profile was consistent with that previously reported. Additional correlative analysis including gene expression will be presented. Acknowledgments: Funding and drug provided by Context Therapeutics Inc., USA. Citation Format: Meritxell Bellet, Serafin Morales, Ariadna Gasol, Kepa Amillano, Nuria Chic, Xavier González-Farré, Patricia Villagrasa, Laia Paré, Claudette Falato, Paolo Nuciforo, Débora Martínez, Juan M Ferrero-Cafiero, Tomás Pascual, Aleix Prat, Carol Lange, Cristina Saura. Primary results of ONAWA (SOLTI-1802) trial: A window of opportunity trial of onapristone in postmenopausal women with progesterone receptor-positive/HER2-negative early breast cancer (EBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-02.

Abstract PS5-01: Biomarkers of resistance to palbociclib in ER+ primary breast cancer in the PALLET trial

Abstract Background: CDK4/6 inhibitors are being used in combination with aromatase inhibitors as therapy for advanced ER+ breast cancer (BC) and are being explored for use in primary BC. Few mechanisms driving resistance to added CDK4/6 inhibitors have been defined. The PALLET phase II randomized neoadjuvant trial of letrozole (LET) ± palbociclib (PALBO) in postmenopausal ER+HER2- primary BC reported that clinical response rate over 14wks was not significantly increased by adding PALBO to LET but suppression of Ki67 was significantly increased (Johnston et al, JCO 2018, 37, 178): after 14wks complete cell cycle arrest (CCCA, Ki67<2.7%) was present in 59% on LET and 90% on LET+PALBO. Here we sought to identify biomarkers of de novo resistance to allow selection of patients most likely to benefit from added PALBO. Methods: 307 patients were randomized to LET (n=103) or LET+PALBO (n=204) for 14 wks. The first 2wks of LET+PALBO patients were randomised to LET, PALBO, or LET+PALBO. Biopsies were taken at baseline, 2wks and 14wks. Biomarker data are presented here for baseline only, other than Ki67 at both baseline and 14wks. IHC analyses were conducted on FFPE biopsies for ER, PgR, RB1, cyclin-E1, and cyclin-D1 (also FISH). RNA-seq was performed on fresh frozen biopsies. Association of each biomarker with CCCA was determined by logistic regression. Differentially expressed genes (DEGs) were identified between patients sensitive (CCCA) (n=94) and resistant (non-CCCA) (n=10) to treatments with or without PALBO at 14wks by DESeq2. Fifty hallmark gene sets were tested for significant enrichment with DEGs and differential gene sets were identified by using Gene Set Enrichment Analysis (GSEA). Results: The association of IHC biomarkers with CCCA is shown in the table. Lower levels of ER, higher levels of cyclin-E1, and amplification of cyclin-D1 were each significantly associated with a greater chance of non-CCCA with LET+PALBO. High cyclin-E1 levels were also associated with reduced chance of CCCA with LET only. Patients with high baseline Ki67 also exhibited higher non-CCCA with LET+PALBO at 14wks (p=0.0002). In the RNAseq data we identified 1973 DEGs between the 14wk CCCA and non-CCCA patients for LET+PALBO. E2F and MYC targets, PI3K/AKT/MTOR signalling and interferon response gene sets were among the hallmark gene sets enriched for genes with higher expression in non-CCCA patients at 14wks for LET+PALBO (FDR<0.05). For LET-only, 311 DEGs were identified and the “Estrogen Response Early” gene set was significantly enriched in genes with higher expression in CCCA samples. At the individual gene level, genes significantly associated with non-CCCA after 14wks LET+PALBO included CCNE1, CDK2 and several E2F-related genes (p<0.05). Their expression was not significantly different between non-CCCA and CCCA patients with LET alone. Conclusion: Biomarkers associated with response/resistance to added PALBO were different from LET only. PALBO resistance was associated with higher baseline expression of cyclin-E1 (both IHC and RNA), CDK2, and genes related to E2F, MYC, interferon and MTOR signalling. These results suggest that multiple identifiable mechanisms of de novo resistance to PALBO are likely to exist in primary ER+ BC. On-going WES analyses will allow the significance of alterations at the DNA level to be presented. Table 1.Continuous measurement in a logistic regression for CCCA at 14 weeks; Oddsratio calculated separately for group A and groups B,C,D and were adjusted forregion (UK vs NA); * amplified vsnon-amplifiedContinuous measurement in a logistic regression for CCCA at 14 weeksBiomarkerLET LET+PALBOOdds ratio95% CIpOdds ratio95% CIpER1.120.36, 3.480.844.471.62, 12.380.004PgR4.381.03, 18.580.053.050.50, 18.560.23RB13.010.24, 38.560.400.420.05, 38.490.83Cyclin-E10.100.01, 0.840.030.020.00, 0.200.001Cyclin-D1 IHC3.090.51, 18.490.222.560.28, 23.330.40CyclinD1 FISH*1.470.43, 4.990.530.280.06, 0.860.03 Citation Format: Eugene F Schuster, Hui Xiao, Elena Lopez-Knowles, Lucy Kilburn, Mothaffar Rimawi, David A Wheeler, Katherine Pogue-Geile, Yuan Lui, Samuel A Jacobs, Chet Cornman, Shannon Puhalla, Maggie Cheang, Judith Bliss, Stephen Johnston, Mitch Dowsett, On behalf of the PALLET Trialists. Biomarkers of resistance to palbociclib in ER+ primary breast cancer in the PALLET trial [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 PS5-01.

In vivo exposure to codeine induces reproductive toxicity: role of HER2 and p53/Bcl-2 signaling pathway

Several studies have implicated codeine use in the aetiopathogenesis of male infertility. The purpose of this study was to investigate the role of HER2, Ki67, oestrogen and p53/Bcl-2 signaling pathways and the possible outcome of codeine cessation on codeine-induced reproductive toxicity. Thirty adult male Wistar rats of comparable ages and weights were randomly allocated into 5 groups. The control animals received distilled water per os (p.o), while animals in the low-dose (LDC) and high dose (HDC) codeine-treated groups received 2 and 5 mg/kg/day of codeine respectively p.o for 6 weeks. The animals in the low-dose codeine recovery (LDC-R) and high-dose codeine recovery (HDC-R) groups received treatment as LDC and HDC respectively followed by another drug-free six weeks, recovery period. Cessation of codeine exposure led to a partial reversal of codeine-induced poor sperm quality, reduced litter size and weight, increased oxidative testicular injury, testicular apoptosis, and testicular DNA damage caused by codeine administration. Codeine-induced gonado-spermotoxicity was associated with a reduction of circulatory testosterone, suppression of testicular HER2, Ki67, and Bcl-2 expression, down-regulation of oestrogen signaling, and upregulation of testicular caspase 3 activities and p53 signaling pathway. Conclusion: Upregulation of oestrogen signaling associated with enhanced testicular HER2 and Ki67 expression during the recovery period is seemingly beneficial in protecting against codeine-related testicular injury and infertility.

Anti-Cancer Potential of Oxialis obtriangulata in Pancreatic Cancer Cell through Regulation of the ERK/Src/STAT3-Mediated Pathway.

As a plant medicine, Oxalidaceae has been used to treat various diseases in Korea. However, there is little data on the anti-cancer efficacy of Oxalidaceae, particularly O. obtriangulata. This study aimed to investigate the anti-cancer effect of O. obtriangulata methanol extract (OOE) and its regulatory actions on pancreatic carcinoma. OOE showed anti-proliferative effects and induced cell death in the colony formation and cell viability assays, respectively. The Fluorescence-activated cell sorting (FACS) data confirmed that OOE significantly induced cell cycle accumulation at the G2/M phase and apoptotic effects. Additionally, OOE inhibited the activated ERK (extracellular-signal-regulated kinase)/Src (Proto-oncogene tyrosine-protein kinase Src)/STAT3 (signal transducers and activators of transcription 3) pathways including nuclear translocation of STAT3. Furthermore, suppression of Ki67, PARP(Poly ADP-ribose polymerase), caspase-3, P27(Cyclin-dependent kinase inhibitor 1B), and c-Myc as well as the STAT3 target genes CDK(cyclin-dependent kinase)1, CDK2, Cyclin B1, VEGF-1(vascular endothelial growth factor-1), MMP-9(Matrix metallopeptidase 9), and Survivin by OOE was observed in BxPC3. We speculate that these molecular actions might support an anti-cancer effect of OOE. In this study, we demonstrated that OOE may be a promising anti-cancer material and may serve as a natural therapy and alternative remedy for pancreatic cancer treatment.

Abstract OT2-02-03: NSABP FB-13: An assessment of the biological and clinical effects of palbociclib with ovarian suppression and letrozole in the neoadjuvant treatment of pts (pts) with premenopausal (preM) estrogen-receptor positive/HER2-negative primary breast cancer

Abstract Background: There have been few studies of neoadjuvant endocrine therapy in premenopausal (preM) women despite low pathologic complete response (pCR) rates using neoadjuvant chemotherapy in ER+/HER2− pts. Suppression of Ki67, particularly complete cell cycle arrest (CCCA) defined as Ki67 <2.7% after 2 wks of neoadjuvant endocrine therapy has been demonstrated to correlate with recurrence-free survival. PALLET, a randomized, neoadjuvant endocrine trial evaluated letrozole (AI) v palbociclib (pal) in combination with AI for 14 wks in post-menopausal women. This study demonstrated a modest improvement in clinical response (54.3% with the combination v 49.5% with AI alone) but showed a significant difference in achievement of CCCA (Ki67<2.7%) with the combination of pal and AI v AI alone (90% v 59%). We seek to evaluate this combination, given over a longer duration in the neoadjuvant setting, in preM women with early-stage breast cancer. Trial design: FB-13 is a phase II, open-label study to examine the biological and clinical effect of neoadjuvant endocrine therapy with AI, pal, and ovarian suppression in preM pts with ER+, HER2-negative early invasive breast cancer. 76 pts will be stratified into one of two approximately equal sized cohorts based on Oncotype DX Breast Recurrence Score (RS) (cohort 1: pts with RS <11 or cohort 2: pts with RS 11 to <26). A baseline tumor biopsy prior to start of therapy (tx) for RS and Ki67 will both be centrally determined. Pts in both cohorts will receive letrozole 2.5 mg by mouth daily, pal 125 mg by mouth daily for 21 days of a 28-day cycle, and goserelin 3.6 mg SQ injection every 28 days. At wk 6, pts will have core-cut biopsies for real-time evaluation of Ki67. Pts from both cohorts who have Ki67 <10% will continue tx for a total of 6 cycles. Pts with a persistent Ki67 ≥10% will permanently discontinue tx and begin neoadjuvant chemotherapy or proceed to surgery at physician discretion. Eligibility: FB-13 is enrolling preM women newly diagnosed with ER+/HER2− early breast cancer who are candidates for neoadjuvant endocrine therapy. Pts must be preM based on standard definitions. Primary tumor size must be >2 cm. RS must be <26. Mandatory pre-treatment and post-surgical samples will be collected and analyzed for RS and Ki67, as well as additional biomarkers to correlate with response. Blood will be collected at baseline, 4 wks, and at 24 wks for estradiol levels to demonstrate ovarian suppression. A 4-wk estradiol level in the postmenopausal range will be required to receive further treatment. Specific aims: The primary aim is to determine complete cell cycle arrest as indicated by a Ki67 <2.7% at 6 wks. Secondary aims are to determine overall response rate (ORR) by clinical exam and ultrasound, the pCR, to generate a Preoperative Endocrine Prognostic Index (PEPI score), to compare the change in Ki67 at baseline, after 6 wks and 24 wks, and to determine the rate of breast-conservation therapy. Statistical methods: We anticipate that the addition of palbociclib to ovarian suppression and AI will result in a measurable drop in Ki67 and a high ORR. The null hypothesis is ORR <40% and alternative hypothesis is ORR >60%. We anticipate that >60% of pts in each cohort will have 6 wk Ki67 <2.7% and clinical response >35%. Present accrual and target accrual: Currently 12 of a planned 76 pts have been enrolled. NSABP Operations contact information: Diana Gosik, Diana.Gosik@nsabp.org NCT03628066 SUPPORT: Genomic Health Inc, Pfizer, NSABP Foundation Citation Format: Shannon Puhalla, Greg Yothers, Amy P Sing, Thomas B Julian, Norman Wolmark, Samuel A Jacobs. NSABP FB-13: An assessment of the biological and clinical effects of palbociclib with ovarian suppression and letrozole in the neoadjuvant treatment of pts (pts) with premenopausal (preM) estrogen-receptor positive/HER2-negative primary breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-02-03.

Abstract ES5-1: Neoadjuvant therapy for hormone receptor-positive breast cancer: challenges and advances

Abstract Neoadjuvant therapy in breast cancer has evolved from a modality able to facilitate surgery into a sophisticated treatment approach, where patterns of response influence the extent of surgery (i.e. breast and axilla) and inform treatment decisions in the adjuvant setting. This is particularly true for patients diagnosed with HER2-positive or triple-negative breast cancer (TNBC). In the subset of estrogen receptor-positive (ER+) disease, the neoadjuvant treatment model has proven to be a successful clinical research tool. Studies investigating pharmacodynamic markers (i.e., Ki67) demonstrated the differences in the relative effectiveness of tamoxifen versus aromatase inhibitor and, more recently, neoadjuvant studies with endocrine therapy plus CDK4/6 inhibitors demonstrated the efficacy of CDK4/6 inhibitors in potentiating the effects of endocrine therapy through a profound suppression of Ki67. However, in clinical practice neoadjuvant endocrine therapy remains underutilized. Routinely, ER+ candidates for neoadjuvant therapy are treated with chemotherapy with minimal attention to disease biology. By contrast, disease biology plays a major role in the adjuvant setting, and the “old notion” of disease burden predicting chemotherapy benefit is fading away. The disconnect between the choices of systemic therapy in the neoadjuvant versus adjuvant setting in ER+ breast cancer has multiple roots: 1) Physicians and patients may expect higher response rates to neoadjuvant chemotherapy; 2) genomic biomarkers to guide chemotherapy treatment decisions were developed using material from surgical specimens; 3) there are limited data with neoadjuvant endocrine therapy in premenopausal women; and 4) the objectives with neoadjuvant endocrine therapy are not always clear. While additional clinical studies are warranted, the available clinical data suggest neoadjuvant endocrine therapy is an appropriate treatment approach for patients with ER+ disease. When selecting patients by ER+ status only, response rates to neoadjuvant endocrine therapy are similar or higher than chemotherapy. Clinical studies have successfully used diagnostic biopsies for genomic profiling, and if anything, the quality of FFPE material should be superior due to pre-analytical variables (e.g., reduced ischemic time). Additionally, neoadjuvant endocrine therapy for ER+ breast cancer and favorable disease biology fulfills the promise of a tailored treatment approach and avoids unnecessary use of cytotoxic therapy. When selecting neoadjuvant endocrine therapy for patients, it is important to understand some key points regarding this treatment approach. The maximum treatment response is not expected before four to six months of therapy, and the interpretation of pathologic response should not be confounded by data from HER2+ and TNBC. Residual disease after neoadjuvant endocrine therapy is not a predictor of survival outcomes. And, lastly, if the patient was not deemed as an ideal candidate for neoadjuvant chemotherapy at baseline, the extent of residual disease should not be used a predictor of chemotherapy benefit. There are, however, caveats and extensive disease at the time of surgery (e.g., four or more positive nodes) might justify a course of adjuvant chemotherapy. In summary, neoadjuvant endocrine therapy should not be a treatment of exception for elderly patients when trying to avoid the toxicities of chemotherapy. Neoadjuvant endocrine therapy is an effective treatment modality with proven clinical utility for postmenopausal patients with ER+ breast cancer. Citation Format: O Metzger. Neoadjuvant therapy for hormone receptor-positive breast cancer: challenges and advances [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES5-1.

MiR-326 inhibits the progression of papillary thyroid carcinoma by targeting MAPK1 and ERBB4.

Papillary thyroid carcinoma (PTC) is the prevalent histotype of thyroid cancer, with increasing incidence worldwide. MicroRNAs (miRNAs) could play an important role in the development and progression of human cancers. Interestingly, miR-326 was validated as one of the downregulated miRNAs in PTC. Therefore, it is necessary to research the function of miR-326 involved in the progression of PTC. In the current study, we detected the downregulation of miR-326 in PTC tissues and cell lines. The miR-326 overexpression or knockdown was conducted in TPC-1 or HTh83 PTC cells. miR-326 mimics decreased the proliferation, clone formation ability and caused G1-phase accumulation. In addition, the reduction of migration and invasion abilities was induced by miR-326 mimics. Western blot analysis showed that the cells with miR-326 mimics exhibited the inhibition of vimentin and N-cadherin, as well as enhancement of E-cadherin. Importantly, miR-326 could directly target mitogen activated protein kinase 1 (MAPK1) and epidermal growth factor receptor 4 (ERBB4). MAPK1 or ERBB4 overexpression rescued the effects of miR-326 on proliferation, migration, and invasion in PTC cells. Notably, miR-326 reduced tumorigenesis in vivo, including the decrease of tumor volume and weight, suppression of Ki-67, N-cadherin, MAPK1 and ERBB4. In all, these results might provide a new therapeutic target for the diagnosis of PTC.

Amelioration of estrogen-induced endometrial hyperplasia in female rats by hemin via heme-oxygenase-1 expression, suppression of iNOS, p38 MAPK, and Ki67.

Although heme oxygenase-1 (HO-1) is part of an endogenous defense system implicated in the homeostatic response, its role in cell proliferation and tumor progression is still controversial. Endometrial hyperplasia (EH) is associated with high risk of endometrial cancer (EC). Therefore, we aimed to evaluate the effect of hemin, a HO-1 inducer, against EH. Thirty-two female rats (60-70 days old) were divided into 4 groups treated for 1 week: vehicle control group, hemin group (25 mg/kg; i.p. 3 times/week), estradiol valerate (EV) group (2 mg/kg per day, p.o.), and hemin plus EV group. Sera were obtained for reduced glutathione level. Uterine malondialdehyde, superoxide dismutase, total nitrite/nitrate, and interleukin-1β levels were estimated. HO-1 and p38 mitogen-activated protein kinase expressions were obtained in uterine tissue. Uterine histological and immunohistochemical assessment of iNOS and Ki67 were also done. Results demonstrated that upregulation of HO-1 expression in hemin plus EV rats led to amelioration of EH which was confirmed with histological examination. This was associated with significant decrease in oxidative stress parameters, p38 mitogen-activated protein kinase expression, and interleukin-1β level. Also, uterine iNOS and Ki67 expressions were markedly suppressed. In conclusion, upregulation of HO-1 expression via hemin has ameliorative effect against EH through its antioxidant, anti-inflammatory, and antiproliferative actions.

Abstract P6-20-04: Activation of AR inhibits growth of endocrine-resistant breast cancer

Abstract Introduction Resistance to endocrine therapy is a major clinical problem in estrogen receptor positive (ER+) breast cancer. The androgen receptor (AR) is expressed in ˜90% of all ER+ breast cancers and high expression of AR is associated with a better patient outcome in this subtype. In agreement, AR activation in breast cancer cell line models reduces proliferation of cells via antagonism of ER signaling. However, uncertainty surrounding the role of AR in endocrine resistance is reflected in current clinical trials in which both AR agonists and antagonists are being investigated. In this study, we sought to investigate the optimal approach in targeting AR in endocrine-resistant breast cancer. Methods We evaluated the consequences of AR activation, using AR cognate ligand 5α-dihydrotestosterone (DHT) and selective AR modulator enobosarm, and AR antagonism using enzalutamide on in vitro and in vivo models of endocrine-resistance. The efficacy of these AR modulators were assessed in vitro using tamoxifen-resistant (TamR) and long-term estrogen derived (LTED) models of MCF7 cells, and in vivo using ESR1 mutant E2-dependent (HCI-005) and ESR1 wild-type E2-independent (Gar15-13) endocrine-resistant PDX models Results Treatment with DHT and enobosarm inhibited the growth of MCF7 TamR and LTED cells but enzalutamide had no effect. AR activation was associated with loss of ER in MCF7 TamR cells and loss of ER-regulated PR expression in MCF7 LTED which suggests that this growth suppression was mediated through the antagonism of ER signaling. Notably, an additive anti-proliferative effect was observed with the combination of enobosarm and CDK4/6 inhibitor palbocilib in the MCF7 TamR cells. A similar pattern was observed in vivo with DHT strongly inhibiting the proliferation of both PDX models. Enobosarm similarly suppressed the proliferation of HCI-005, and to a lesser extent in Gar15-13. The benefit of enobosarm in Gar15-13 was significant given that this model is fulvestrant-resistant. Antagonizing AR with enzalutamide had no effect on growth of Gar15-13 model, similar to our in vitro data. AR agonists reduced expression levels of ER and PR in HCI-005, and transcriptomic analysis of AR agonist-treated Gar15-13 identified significant negative enrichment of genes related to proliferation and estrogen response. These observations indicate that the growth-suppressive effects of AR activation in vivo were mediated through inhibiting ER signaling. We identified an AR gene signature, through RNA-seq analysis of DHT-treated Gar15-13 PDX, which is strongly associated with good outcome in the METABRIC dataset, supporting the hypothesis that an active canonical AR signaling is tumor suppressive in both endocrine-sensitive and -resistant disease contexts. Lastly, we present in vivo data demonstrating enhanced suppression of Ki-67 with the combination of enobosarm and palbociclib in the Gar15-13 PDX. Conclusion We have demonstrated that activating AR is an effective therapeutic approach in endocrine-resistant breast cancer, and the combination of an AR agonist with a CDK4/6 inhibitor warrants further investigation in this breast cancer subtype. Citation Format: Chia KM, Milioli H, Portman N, Laven-Law G, Yong A, Swarbrick A, Caldon L, Tilley W, Hickey T, Lim E. Activation of AR inhibits growth of endocrine-resistant breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-04.

Long noncoding RNA APTR contributes to osteosarcoma progression through repression of miR-132-3p and upregulation of yes-associated protein 1.

A growing amount of evidence has shown that long noncoding RNAs (lncRNAs) play crucial roles in osteosarcoma (OS). However, little knowledge is available about the functional roles and molecular mechanisms of lncRNA Alu-mediated p21 transcriptional regulator (APTR) in OS. Herein, APTR expression was demonstrated to be significantly upregulated in OS tumor tissues and four OS cell lines (including MG63, 143B, Saos-2, and HOS) compared with the adjacent tissues and human osteoblast cell line hFOB1.19, respectively. We confirmed miR-132-3p to be a target for APTR, and its expression was demonstrated to be inhibited by APTR. In functional terms, knockdown of APTR and overexpression of miR-132-3p both, remarkably repressed human OS cell proliferation, invasion and migration, and induced apoptosis. Also, Yes-associated protein 1 (YAP1) was determined as an inhibitory target of miR-132-3p. Moreover, our findings demonstrated that the repression of YAP1 protein expression and the suppression of Ki-67, MMP9, and Bcl2 expression induced by APTR knockdown required increased miR-132-3p. Thus, APTR contributed to OS progression through repression of miR-132-3p and upregulation of YAP1 expression. Therefore, we have uncovered a novel regulatory mechanism by which the APTR/miR-132-3p/YAP1 axis can regulate OS progression.

A Pre-Clinical Large Animal Model of Sustained Liver Injury and Regeneration Stimulus

A feasible large animal model to evaluate regenerative medicine techniques is vital for developing clinical applications. One such appropriate model could be to use retrorsine (RS) together with partial hepatectomy (PH). Here, we have developed the first porcine model using RS and PH. RS or saline control was administered intraperitoneally to Göttingen miniature pigs twice, two weeks apart. Four weeks after the second dose, animals underwent PH. Initially, we tested different doses of RS and resection of different amounts of liver, and selected 50 mg/kg RS with 60% hepatectomy as our model for further testing. Treated animals were sacrificed 3, 10, 17 or 28 days after PH. Blood samples and resected liver were collected. Serum and liver RS content was determined by Liquid Chromatograph-tandem Mass Spectrometer. Blood analyses demonstrated liver dysfunction after PH. Liver regeneration was significantly inhibited 10 and 17 days after PH in RS-treated animals, to the extent of 20%. Histological examination indicated hepatic injury and regenerative responses after PH. Immunohistochemical staining demonstrated accumulation of Cyclin D1 and suppression of Ki-67 and PCNA in RS-treated animals. We report the development of the first large animal model of sustained liver injury with suppression of hepatic regeneration.

Inhibition of basal-like breast cancer growth by FTY720 in combination with epidermal growth factor receptor kinase blockade

BackgroundNew molecular targets are needed for women with triple-negative breast cancer (TNBC). This pre-clinical study investigated the combination of the EGFR inhibitor gefitinib with the sphingosine kinase (SphK) inhibitor FTY720 (Fingolimod), aiming to block tumorigenic signaling downstream of IGFBP-3, which is abundantly expressed in basal-like TNBC.MethodsIn studies of breast cancer cell growth in culture, proliferation was monitored by IncuCyte live-cell imaging, and protein abundance was determined by western blotting. In vivo studies of mammary tumor growth used two models: orthotopic xenograft tumors derived from three basal-like TNBC cell lines, grown in immune-deficient mice, and syngeneic murine 4T1 tumors grown in immune-competent mice. Protein abundance in tumor tissue was assessed by immunohistochemistry.ResultsQuantitated by live-cell imaging, the inhibitor combination showed synergistic cytostatic activity in basal-like cell lines across several TNBC molecular subtypes, the synergy being decreased by IGFBP-3 downregulation. Suppression of the tumorigenic mediator CD44 by gefitinib was potentiated by FTY720, consistent with CD44 involvement in the targeted pathway. In MDA-MB-468 and HCC1806 orthotopic TNBC xenograft tumors in nude mice, the drug combination inhibited tumor growth and prolonged mouse survival, although this effect was not significant for the gefitinib-resistant cell line HCC70. Combination treatment of murine 4T1 TNBC tumors in syngeneic BALB/c mice was more effective in immune-competent than immune-deficient (nude) mice, and a relative loss of tumor CD3 (T-cell) immunoreactivity caused by FTY720 treatment alone was alleviated by the drug combination, suggesting that, even at an FTY720 dose causing relative lymphopenia, the combination is still effective in an immune-competent setting. Immunohistochemistry of xenograft tumors showed significant enhancement of caspase-3 cleavage and suppression of Ki67 and phospho-EGFR by the drug combination, but SphK1 downregulation occurred only in MDA-MB-468 tumors, so is unlikely to be integral to treatment efficacy.ConclusionsOur data indicate that targeting IGFBP-3-dependent signaling pathways through gefitinib-FTY720 co-therapy may be effective in many basal-like breast cancers, and suggest tissue IGFBP-3 and CD44 measurement as potential biomarkers of treatment efficacy.

Abstract S4-06: Biological effects of abemaciclib in a phase 2 neoadjuvant study for postmenopausal patients with HR+, HER2- breast cancer

Abstract Background: Abemaciclib is a potent oral CDK4 and 6 inhibitor, which demonstrated evidence of clinical activity and an acceptable safety profile on a continuous dosing schedule either as a single agent (MONARCH 1; NCT02102490) or in combination with endocrine therapies in heavily pre-treated women with HR+ metastatic breast cancer (BC).1,2 Methods: neoMONARCH (NCT02441946) is a randomized, multicenter, open-label phase 2 neoadjuvant study comparing the biological effects of abemaciclib plus anastrozole vs abemaciclib monotherapy vs anastrozole monotherapy in women with early-stage HR+, HER2- BC. Patients (pts) were stratified by progesterone receptor status and tumor size and randomized 1:1:1 ([abemaciclib 150mg orally [PO] every 12 hours [Q12H] plus anastrozole 1 mg PO daily [QD]], [abemaciclib 150mg PO Q12H], and [anastrozole 1mg PO QD]). Each regimen was given for 2 weeks; followed by all pts receiving abemaciclib 150mg PO Q12H plus anastrozole 1mg QD for the subsequent 14 weeks. All pts received prophylactic loperamide during the first 28 days of abemaciclib therapy, then at the discretion of the investigator. Eligible pts included postmenopausal women with HR+, HER2- clinical Stage I breast tumor ≥1 cm in diameter, Stage II, Stage IIIA, or IIIB BC. The primary objective: compare biological activity by assessing the percent change from baseline value in Ki67 protein expression after 2 weeks of therapy with the three initial regimens. Clinical activity and safety of the subsequent 14 weeks of therapy of abemaciclib plus anastrozole are evaluated as secondary objectives at surgery. Exploratory objectives included assessment of changes in cell cycle mRNAs by Modaplex (QIAGEN) analyses and PIK3CA and ESR1 mutational analysis in core biopsy samples. Statistical methods: The design provides 80% power to detect superiority of the combination vs anastrozole monotherapy, and 80% power to detect superiority of abemaciclib monotherapy vs anastrozole monotherapy, at a 1-sided alpha level of 0.1. Secondary and exploratory objectives will be presented as descriptive data. Results: Enrollment of 223 pts began August 2015 and was completed August 2016. At a 9 month interim analysis, abemaciclib, given either as monotherapy or in combination with anastrozole showed significantly (p<0.001, n=64) greater suppression of Ki67 after 14 days of dosing than anastrozole alone. The safety profile of the combination differed from that previously reported for abemaciclib 200mg BID monotherapy in the MONARCH 1 study1 and the Phase Ib2 with reduced incidence of diarrhea and hematologic events. Change in proliferation gene mRNAs after 2 weeks of treatment (n=38), in both tumor and blood, appeared to correlate with change in Ki67 expression, with a greater reduction in the abemaciclib-containing arms. Updated data will be presented including safety data for all 223 pts, molecular data on approximately 150 pts who are evaluable for change in Ki67 and mRNA expression at 2 weeks and approximately 100 pts evaluable for clinical efficacy, final Ki67 and RNA expression at surgery.

Abstract P2-05-01: Clinico-pathological relationships with Ki67 in POETIC (CRUK/07/015) – Critical lessons for assessing Ki67 for prognosis and as a pharmacodynamic marker

Abstract Background Higher levels of the proliferation marker Ki67 at breast cancer (BC) diagnosis are increasingly recognised to indicate poorer prognosis. Change in ΔKi67 in response to endocrine treatment reflects response. The precise relationship between Ki67 and other clinico-path factors and how associations are affected by short exposure to aromatase inhibitor (AI) has been unclear. Methods POETIC was a UK-wide, phase III, randomised trial which tested perioperative use of AI (anastrozole (A), letrozole (L)) in postmenopausal women with early BC (Dowsett JNCI Monogr 2011). Ki67 was measured in a single central lab at diagnosis (B=baseline) and 2 weeks later at surgery (S) allowing in vivo assessment of AI sensitivity. POETIC recruited 4483 women from 130 UK centres. Paired biopsies were available for 96%. Relationship between Ki67 and clinico-path factors is described by summary statistics (median) and independent associations explored in multivariable linear regression models (MVM). Analyses of Ki67 at S and ΔKi67 (reduction) were adjusted for B Ki67 and surgical sample type. Results Absolute Ki67 (B) was associated with each classic prognostic factor. Factors affecting ΔKi67 in control pts were Ki67 (B), grade (B) and surgical sample type (core cut 4.1%; excision 17.7% p<0.001). After 2 weeks of AI, variation in extent of ΔKi67 was dominated by PgR, HER2 and grade (B). Centres using L more often supplied excision samples (p<0.001). In spite of this both surgical sample type and choice of AI (both p<0.001) independently influenced ΔKi67 (core cut: A 65.1% L 73.8% excision A 75.2% L 81.3%). Pattern of change in grade between B and S differed for AI and control. Baseline Ki67 %Ki67 change (%) with 2 weeks AIVariableNMedianln(Ki67) P value (MVM)NMedianln (Ki67(S)/Ki67 (B)) P Value (MVM)All patients391315.2 252877.6 PgR (local) 0.01 <0.001Positive221914.6 141180.0 Negative47720.4 32661.1 Not known121715.1 79177.0 HER2 (local) <0.001 <0.001Negative345914.3 224079.2 Positive40226.8 26552.9 Unknown5213.9 2385.5 Tumour grade (B) <0.001 <0.001G15489.1 34378.6 G2227514.5 149479.6 G370330.1 44762.1 Unknown38714.0 24475.4 Tumour size (S) 0.002 0.49<2cm150312.9 98379.1 2-5cm222117.5 142576.4 >5cm17416.9 11474.1 Histological type (B) <0.001 0.58Ductal313816.3 203077.5 Lobular54911.9 35379.0 Other/Not known22613.1 14574.0 Nodes involved 0.03 0.530235714.1 153577.6 1-3110815.8 70878.2 4+43320.3 27876.1 Vascular invasion <0.001 0.03Yes112119.9 71175.9 No260313.6 169578.2 Unknown18914.6 12275.8 Summary POETIC provides the largest multi-centre series of women in whom centrally assessed Ki67 has been correlated with classic clinico-path factors and impact of short term AI exposure explored. Choice of AI and surgical sample type are both dictated by participating site and their inter-relationship requires further review. Whether the greater suppression of Ki67 following L, a drug associated with better E2 suppression and aromatisation than A has clinical consequences is beyond the scope of this work. Relationships found (e.g. sample type, grade) are critical for interpretation of studies using Ki67 for prognosis and ΔKi67 as a pharmacodynamic response marker. Citation Format: Bliss JM, Morden J, Evans A, Holcombe C, Horgan K, Mallon E, Raghavan V, Skene A, Dodson A, Hills M, Detre S, Zabaglo L, Graf M, Banerji J, Gillman A, Robertson J, Dowsett M, Smith I, On Behalf of the POETIC Trialists. Clinico-pathological relationships with Ki67 in POETIC (CRUK/07/015) – Critical lessons for assessing Ki67 for prognosis and as a pharmacodynamic marker [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 P2-05-01.

Calpain mediated pulmonary vascular remodeling in hypoxia induced pulmonary hypertension

To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms. Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR. RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression. Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.