Search

Contains supplementary tables and methods

Purpose:Addition of alpelisib to fulvestrant significantly extended progression-free survival in PIK3CA-mutant, hormone receptor–positive (HR+) advanced/metastatic breast cancer in the phase III SOLAR-1 study. The combination of alpelisib and letrozole also had promising activity in phase I studies of HR+ advanced/metastatic breast cancer. NEO-ORB aimed to determine whether addition of alpelisib to letrozole could increase response rates in the neoadjuvant setting.Patients and Methods: Postmenopausal women with HR+, human epidermal growth factor receptor 2-negative, T1c-T3 breast cancer were assigned to the PIK3CA-wild-type or PIK3CA-mutant cohort according to their tumor PIK3CA status, and randomized (1:1) to 2.5 mg/day letrozole with 300 mg/day alpelisib or placebo for 24 weeks. Primary endpoints were objective response rate (ORR) and pathologic complete response (pCR) rate for both PIK3CA cohorts.Results:In total, 257 patients were assigned to letrozole plus alpelisib (131 patients) or placebo (126 patients). Grade ≥3 adverse events (≥5% of patients) in the alpelisib arm were hyperglycemia (27%), rash (12%), and maculo-papular rash (8%). The primary objective was not met; ORR in the alpelisib versus placebo arm was 43% versus 45% and 63% versus 61% in the PIK3CA-mutant and wild-type cohorts, respectively. pCR rates were low in all groups. Decreases in Ki-67 were similar across treatment arms and cohorts. In PIK3CA-mutant tumors, alpelisib plus letrozole treatment induced a greater decrease in phosphorylated AKT versus placebo plus letrozole.Conclusions:In contrast to initial results in advanced/metastatic disease, addition of alpelisib to 24-week neoadjuvant letrozole treatment did not improve response in patients with HR+ early breast cancer.

Introduction: The PI3K pathway is often hyperactivated in cancer as a result of an altered PI3K isoform and/or loss of phosphatase and tensin homolog function. Approximately 40% of patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2−) advanced breast cancer (ABC) have tumors with mutations in PIK3CA, which encodes the α-isoform of PI3K, p110α. These mutations are known to cause hyperactivation of the PI3K pathway, which contributes to cell proliferation, drug resistance, and poor prognoses. Alpelisib (ALP) is an α-selective PI3K inhibitor that, in combination with fulvestrant (FUL), prolonged median progression-free survival (mPFS) in pts with HR+, HER2−, PIK3CA-mutant ABC following progression on/after prior aromatase inhibitor in the phase 3 SOLAR-1 trial. In SOLAR-1, prospective PIK3CA mutation testing was performed on tumor tissue using PCR-based assays. Through retrospective analysis, efficacy of ALP was demonstrated in subgroups of pts with PIK3CA alteration(s) in tumor tissue and mutation(s) in ctDNA, detected by next-generation sequencing (NGS) and PCR, respectively. In this exploratory biomarker analysis, we assessed clinical outcomes of pts with PIK3CA alteration(s), detected in ctDNA by NGS.Methods: SOLAR-1 is a phase 3, randomized, double-blind, placebo-controlled study of ALP 300 mg vs placebo taken daily with FUL 500 mg every 28 days + Cycle 1 Day 15 in men and postmenopausal women with HR+, HER2- ABC. Retrospectively, the full exonic region of the PIK3CA gene was sequenced using the Foundation Medicine 324-gene ctDNA panel in plasma ctDNA collected at baseline. mPFS was assessed using Kaplan-Meier methodology per investigator assessment.Results: Of 572 pts in SOLAR-1, 381 pts (66.6%) across both PIK3CA-mutant and nonmutant cohorts had valid plasma ctDNA data. Of these pts, 193 (50.7%) had a PIK3CA alteration; 168 (87%) had PCR-detectable mutations and 147 (76%) had a single alteration. A total of 70 (36%) and 102 (53%) pts had alterations in exons 9 and 20, respectively. ALP plus FUL prolonged mPFS in pts with PIK3CA alterations detected in plasma ctDNA by NGS (n=101; Table). Clinical benefit was also observed in pts with PCR-detectable mutations (n=88), single mutations (n=83), and pts with mutations in exon 9 (n=34) and exon 20 (n=54). Pt numbers were low, and wide 95% CIs were observed in groups with alterations not detectable by PCR (n=13) and in pts with multiple alterations. Some limitations of this retrospective plasma analysis include that this is a subgroup (66.6%) of the SOLAR-1 pt population and that the subgroup of pts with non-altered PIK3CA included pts with a PIK3CA mutation in their tumor tissue. Conclusions: ALP plus FUL demonstrated clinical benefit in pts with PIK3CA mutations detected in plasma ctDNA by NGS, in pts with single alterations, and in pts with alterations in exons 9 and 20. Results were consistent across pt groups, except in those with alterations not detectable by PCR. In conclusion, these data demonstrate a consistent clinical benefit of ALP plus FUL in various groups of pts with PIK3CA alterations detected in ctDNA by NGS. Clinical Outcomes of Patients With PIK3CA Alterations Detected in Plasma ctDNA by NGS in SOLAR-1Alpelisib + fulvestrantPlacebo + fulvestrantHR (95% CI)Events/N (%)mPFS, mo (95% CI)Events/N(%)mPFS, mo (95% CI)PIK3CA altered vs non-alteredAltered58/101(57.4)11.04(7.72-16.16)73/92(79.3)3.65(2.86-6.80)0.47(0.33-0.67)Non-altered40/87(46.0)10.87(5.59-16.76)60/101(59.4)5.45(3.75-9.00)0.60(0.40-0.91)PIK3CA: alteration detectable by PCR vs alteration not detectable by PCRDetectable52/88(59.1)12.48(7.36-18.37)66/80(82.5)3.58(2.37-5.65)0.44(0.30-0.64)Not detectable6/13(46.2)8.48(2.69-NA)7/12(58.3)7.39(1.87-12.98)1.12(0.35-3.56)PIK3CA: number of alterationsSingle45/83(54.2)12.88(7.36-18.50)50/64(78.1)3.58(1.87-6.11)0.43(0.28-0.65)Multiple13/18(72.2)9.00(3.68-18.37)23/28(82.1)4.63(3.48-9.63)0.55(0.25-1.20)PIK3CA alterations in exon 9 or exon 20Exon 918/34(52.9)15.21(7.03-NA)29/36(80.6)3.66(2.86-7.36)0.31(0.16-0.61)Exon 2034/54(63.0)10.91(5.72-18.37)40/48(83.3)3.52(1.87-6.11)0.51(0.31-0.82)CI, confidence interval; ctDNA, circulating tumor DNA; HR, hazard ratio; mPFS, median progression-free survival; mo, months; NA, not available; NGS, next-generation sequencing. Citation Format: Eva M. Ciruelos, Sibylle Loibl, Ingrid A. Mayer, Mario Campone, Hope S. Rugo, Monica Arnedos, Hiroji Iwata, Pier Franco Conte, Fabrice André, Albert Reising, Chong Ma, Michelle Miller, Naveen Babbar, Dejan Juric. Clinical outcomes of alpelisib plus fulvestrant in hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer with PIK3CA alterations detected in plasma ctDNA by next-generation sequencing: Biomarker analysis from the SOLAR-1 study [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 PD2-06.

Background: The prognostic and predictive value of the 4 main intrinsic subtypes of breast cancer (ie, luminal A [LumA], luminal B [LumB], human epidermal growth factor receptor 2 enriched [HER2E], and basal-like) in hormone receptor-positive, HER2− advanced breast cancer (ABC) treated with endocrine therapy (ET) and ribociclib (RIB) is currently unknown. The MONALEESA-2, -3, and -7 trials all showed a significant benefit in progression-free survival (PFS) with RIB over placebo (PBO; Hortobagyi et al. Ann Oncol. 2018; Slamon et al. J Clin Oncol. 2018; Tripathy et al. Lancet Oncol. 2018). Here, we correlate ABC intrinsic subtypes with the PFS benefit of RIB in the MONALEESA trials. Methods: Patient samples from the MONALEESA-2, -3, and -7 trials underwent PAM50-based subtyping (blinded from clinical data), and the correlation between intrinsic subtype and PFS was analyzed. Gene expression profiling of formalin-fixed, paraffin-embedded tumor samples was performed using a customized NanoString nCounter GX 800-gene panel. The prognostic and/or predictive relationship of PAM50-based subtypes with PFS and the risk of tumor progression by subtype were evaluated using univariate and multivariable Cox proportional hazards models. Multivariable models were adjusted for known clinical prognostic factors, including age, prior chemotherapy, prior ET, ECOG performance status, visceral disease (presence of liver/lung metastases), bone-only metastases, histological grade, number of metastatic sites, and de novo metastatic disease. Results: A total of 1160 tumor samples from both the RIB (n = 672) and PBO (n = 488) treatment arms of the MONALEESA trials were profiled. Subtype distribution was generally consistent across treatment arms (Table). The associations between intrinsic subtypes and PFS were statistically significant in both treatment arms (P < .0001). Compared with patients with LumA subtype, which is the subtype that is the most prevalent and has the best prognostic outcome, patients with LumB, HER2E, and basal-like subtypes had a 1.41, 2.30, and 3.97 times higher risk of tumor progression, respectively, after adjusting for other clinical-pathologic variables and treatment arm. In terms of treatment benefit, all subtypes except for basal-like showed a significant PFS benefit with RIB treatment (Table). Patients with HER2E (hazard ratio [HR], 0.389; P < .0001), LumB (HR, 0.521; P = .0001), LumA (HR, 0.633; P = .0007), and normal-like (HR, 0.467; P = .0005) subtypes all derived benefit from RIB treatment, with HER2E demonstrating the greatest benefit. Patients with the basal-like subtype (n = 30) did not derive benefit from RIB (HR, 1.15; P = .767), although these results should be interpreted with caution due to the small sample size (RIB: 2%; PBO: 3%). Conclusions: This is the largest analysis evaluating the correlation of intrinsic ABC subtype with efficacy outcomes in patients treated with CDK4/6 inhibitors. Patients with HER2E, LumA, LumB, and normal-like subtypes all exhibited a consistent PFS benefit with RIB treatment, while patients with basal-like ABC (RIB: 2%; PBO: 3%) did not. The HER2E subtype (RIB: 14%; PBO: 11%) exhibited the greatest relative reduction in risk of progression or death (61%) with RIB plus ET. Table.SubtypeTreatment ArmDistribution, n (%)Median PFS, months, (95% CI)HRP ValueLuminal ARIB320 (48)29.60 (23.03-NA)0.63.0007PBO222 (45)19.48 (15.61-24.80)Luminal BRIB154 (23)22.21 (18.79-NA)0.52< .0001PBO124 (25)12.85 (10.98414.82)HER2-enrichedRIB95 (14)16.39 (12.71-24.6)0.39< .0001PBO52 (11)5.52 (3.12-9.17)BasalRIB16 (2)3.71 (1.91-13)1.15.77PBO14 (3)3.58 (1.87-NA)NormalRIB87 (13)22.34 (16.56-NA)0.47.0005PBO76 (16)11.10 (7.39-16.56)NA, not achieved. Citation Format: Aleix Prat, Anwesha Chaudhury, Nadia Solovieff, Laia Paré, Debora Martinez, Nuria Chic, Olga Martínez, Fara Brasó-Maristany, Karen Rodriguez-Lorenc, Tetiana Taran, Naveen Babbar, Faye Su. Correlative biomarker analysis of intrinsic subtypes and efficacy across the MONALEESA Phase III studies [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 GS1-04.

Introduction: Hyperactivation of the phosphoinositide-3-kinase (PI3K) pathway is a frequent event in human cancers and can be a result of phosphatase and tensin homolog (PTEN) loss, activation of receptor tyrosine kinases, or alterations in PI3K isoforms. About 40% of hormone receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2-) breast cancers harbor alterations in the PIK3CA gene, which encodes the alpha subunit of PI3K (p110α). Mutations in PIK3CA can cause PI3K pathway hyperactivation, which may contribute to endocrine resistance. Alpelisib (ALP) is an α-selective PI3K inhibitor that, in combination with fulvestrant (FUL), demonstrated improved median progression-free survival (PFS) vs placebo (PBO) + FUL (11.0 vs 5.7 mo, respectively; hazard ratio [HR] 0.65; 95% confidence interval [CI], 0.50-0.85; P Methods: SOLAR-1 is a phase 3, randomized, double-blind study of ALP 300 mg (or PBO) once daily + FUL 500 mg every 28 days + Cycle 1 Day 15 in men and postmenopausal women with HR+, HER2- ABC. Retrospectively, PIK3CA alterations (mutations and amplifications) were assessed by NGS and PTEN levels were assessed by immunohistochemistry. PTEN loss was defined based on an H-score Results: Of 572 pts in SOLAR-1, 341 (60%) had a PIK3CA mutation in tumor tissue by PCR, valid NGS results were available for 404 (71%), and PTEN levels were available for 401 (70%). Of the pts with available PTEN results, 34 (8%) had PTEN loss. PFS results and HRs, by PIK3CA alteration status as detected by NGS and PTEN status, are shown in the Table. In pts whose tumors had any PIK3CA alteration (n=239), which included a small group of pts with alterations not detectable by the PCR-based test used for screening in SOLAR-1 (n=31), mPFS was improved in the ALP + FUL vs PBO + FUL arms. In the small group of pts (n=19) whose tumors had PTEN loss but no PIK3CA alteration, ALP showed an improved mPFS and favorable HR vs the placebo arm. In pts whose tumors had multiple alterations (n=44), HR and mPFS benefits of ALP were observed, albeit with wide 95% CIs. Conclusions: These data demonstrate the efficacy of ALP in pts whose tumors harbor PIK3CA alterations as detected by NGS, including alterations that were not detectable by the PCR-based test used in SOLAR-1. Additional analyses suggest that ALP is also effective in pts with single and multiple PIK3CA mutations and in those whose tumors do not harbor a PIK3CA alteration but do have PTEN loss; however, low patient numbers in the PTEN loss subgroups may limit conclusions. Alpelisib + FulvestrantPlacebo + FulvestrantHR (95% CI)Events/N (%)mPFS, mo (95% CI)Events/N(%)mPFS, mo (95% CI)PIK3CA:altered vs non-altered (by NGS)Altered68/121 (56.2)11.01 (8.05 - 15.21)85/118 (72.0)5.52 (3.55 - 7.36)0.59 (0.43 - 0.82)Non-altered36/82 (43.9)7.29 (5.22 - 9.17)39/83 (47.0)7.16 (5.03 - 11.01)0.99 (0.62 - 1.57)PIK3CA:single vs multiple alterations (by NGS)Single60/100 (60.0)11.01 (7.49 - 14.55)66/94 (70.2)4.63 (3.38 - 7.39)0.59 (0.41 - 0.84)Multiple8/20 (40.0)9.36 (6.31 - NA)19/24 (79.2)7.29 (2.07 - 12.85)0.56 (0.23 - 1.33)PIK3CA: alteration detectable by PCR vs alteration not detectable in SOLAR-1 (by NGS)Detectable64/105 (61.0)11.01 (7.49 - 15.21)79/103 (76.7)3.81 (3.52 - 7.29)0.56 (0.40 - 0.79)Not detectable4/16* (25.0)8.48 (5.55 - NA)6/15 (40.0)12.98 (3.38 - NA)0.75(0.21 - 2.73)PIK3CA alterations (by NGS) and/or PTEN loss (by IHC)PIK3CA non-altered, PTEN non-loss31/72 (43.1)7.39 (5.16 - 9.26)32/71 (45.1)7.16 (5.06 - 11.01)1.03 (0.62 - 1.70)PIK3CA altered, PTEN non-loss63/113 (55.8)11.01 (8.31 - 15.21)78/111 (70.3)5.52 (3.61 - 7.39)0.63 (0.45 - 0.88)PIK3CA non-altered, PTEN loss5/9 (55.6)6.23 (2.79 - NA)7/10 (70.0)1.86 (1.64 - NA)0.50 (0.13 - 1.89)PIK3CA altered, PTEN loss5/8 (62.5)7.72 (2.69 - NA)7/7 (100.0)3.55 (1.05 - 7.23)0.24 (0.06 - 0.97)CI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry; mPFS, median progression-free survival; NA, not available; NGS, next-generation sequencing; PTEN, phosphatase and tensin homolog.*These 16 alterations not detectable by PCR include 15 mutations and 1 PIK3CA amplification. Citation Format: Dejan Juric, Fabrice Andre, Christian F. Singer, Joohyuk Sohn, Mario Campone, Sibylle Loibl, Pierfranco Conte, Hiroji Iwata, Eva Ciruelos, Ingrid A. Mayer, Albert Reising, Chong Ma, Michelle Miller, Naveen Babbar, Hope S. Rugo. Clinical outcomes of alpelisib in hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer by next-generation sequencing-detected PIK3CA alteration status and phosphatase and tensin homolog loss: Biomarker analysis from the SOLAR-1 study [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 P4-10-04.

Background: In the Phase III MONALEESA-3 study (NCT02422615),the cyclin-dependent kinase (CDK) 4/6 inhibitor ribociclib(RIB) + fulvestrant significantly prolonged progression-free survival (PFS) vs placebo (PBO) + fulvestrant in postmenopausal patients (pts) with hormone receptor–positive, human epidermal growth factor receptor 2–negative (HR+/HER2−) advanced breast cancer (ABC; median, 20.5 vs 12.8 mo; hazard ratio 0.593 [95% CI, 0.480-0.732]; P< .001; Slamon D et al. J Clin Oncol. 2018). Previous MONALEESA-3 biomarker analyses, including those based on immunohistochemistry and next-generation sequencing of baseline circulating tumor DNA (ctDNA), showed consistent PFS benefit with ribociclib (Neven P et al. ESMO 2018; Neven P et al. SABCS 2018). We present analyses of gene expression in baseline tumor samples from the MONALEESA-3 trial and potential correlations with PFS. Methods: Postmenopausal pts with ≤1 prior line of ET for advanced disease were enrolled and randomized 2:1to receive either RIB or PBO plus fulvestrant. Gene expression data were generated for 531 patients using tumor samples collected before treatment using the NanoString 800-gene nCounter® GX Customized Panel. Correlations between PFS and mRNA expression levels were assessed for various genes, including those relevant to breast cancer, the CDK4/6 pathway, the mitogen-activated protein kinase (MAPK) pathway, receptor tyrosine kinases (RTKs), estrogen receptor (ER) signaling and response to estrogen, and proliferation; specific genes are listed in the Table. Pts were classified into low and high mRNA expression subgroups, with median values of gene expression levels or pathway scores as the cutoff. The Kaplan-Meier method was used to calculate median PFS with 95% CIs in each treatment arm by biomarker subgroup. Cox proportional hazards models were used to estimate hazard ratios and 95% CIs. Results: PFS hazard ratios for all biomarker subgroups favored RIB + fulvestrant vs PBO + fulvestrant (Table). There appeared to be a trend toward greater PFS benefit with RIB in patients with lower expression of BCL2 or genes involved in ER signaling. Similar RIB PFS benefit was observed regardless of the expression level of the CCNE1 gene, RTK genes, genes involved in tumor cell proliferation, CDK4/6 pathway genes, or MAPK pathway genes. Conclusions: The PFS benefit observed with RIB vs PBO across gene expression subgroups in this analysis was generally consistent with that seen in the primary analysis of MONALEESA-3. Table. Progression-Free Survival by Gene Expression SubgroupGene/PathwayLow ExpressionHigh ExpressionPBORIBPBORIBCCNE1an9017682183Median PFS, mo14.5220.0412.7816.62HR (95% CI)0.62 (0.44-0.87)0.66 (0.46-0.94)BCL2an7918793172Median PFS, mo9.4317.9418.4620.04HR (95% CI)0.47 (0.33-0.66)0.8 (0.55-1.14)Proliferationbn9017682183Median PFS, mo12.6819.0912.9120.04HR (95% CI)0.58 (0.41-0.81)0.72 (0.5-1.03)ER signalingcn8018692173Median PFS, mo9.4619.2914.8219.12HR (95% CI)0.53 (0.37-0.75)0.78 (0.55-1.11)Estrogen responsivedn8518187178Median PFS, mo9.116.3619.3823.46HR (95% CI)0.53 (0.38-0.74)0.76 (0.52-1.11)RTK genesen8717985180Median PFS, mo12.29NA16.4916.62HR (95% CI)0.55 (0.38-0.79)0.73 (0.51-1.03)CDK4/6 pathwayfn9017682183Median PFS, mo16.5622.0810.8416.49HR (95% CI)0.63 (0.43-0.9)0.67 (0.48-0.94)MAPK pathwaygn9117581184Median PFS, mo12.2922.0812.9117.94HR (95% CI)0.66 (0.47-0.94)0.65 (0.45-0.92)aBreast cancer–related gene; b MKI67, TYMS, MYC, TOP2A; c GATA3, FOXA1, AR, ESR1; d CCND1, PGR, NAT1, FOXA1, RARA, BCL2, BAG1, GATA3, MYC, STC2, XBP1; e AREG, EGFR, ERBB2, ERBB3, ERBB4, FGFR1, FGFR2, FGFR3, FGFR4, FYN, IGF1, KDR, KIT, PDGFA, PDGFRA, PDGFRB, TYRO3; f CCNA2, CCND1, CCND2, CCND3, CCNE1, CDK2, CDK4, CDK6, E2F1, E2F3, TFDP1, CCNB1, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CDKN3, RB1, RBBP8, RBL1, RBL2, TP53; g BRAF, HRAS, KRAS, NRAS, RAF1, MAP3K8, NF1. Citation Format: Stephen Chia, Faye Su, Patrick Neven, Seock-Ah Im, Katarina Petrakova, Giulia Val Bianchi, Wei He, Karen Rodriguez-Lorenc, Tetiana Taran, Naveen Babbar, Dennis Slamon. Gene expression analysis and association with treatment response in postmenopausal patients with hormone receptor-positive, HER2-negative advanced breast cancer in the MONALEESA-3 study [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 PD2-08.

Background: Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that is a major negative regulator of the Phosphatidylinositol 3-kinase (PI3K) pathway. Loss of PTEN protein expression has been mechanistically linked to tumor progression. Blocking the PI3K pathway might inhibit the growth and proliferation of cells that have deletions in PTEN. Thus characterization of PTEN expression in patient tumor samples may assist prediction of potential response to PI3K inhibitor therapies. Methods: We developed and validated an immunohistochemical assay on Ventana BenchMark XT to detect PTEN in formalin fixed and paraffin-embedded (FFPE) tissue by utilizing a rabbit monoclonal antibody (clone 138G6) from Cell Signaling Technologies that recognizes the carboxy-terminal domain of PTEN. PTEN immunohistochemical staining was performed on 1577 breast tumor specimens to determine PTEN protein loss. A subset of these cases (n=663) was also assessed for PTEN mutation. Results: Cellular localization of PTEN expression was observed in both the cytoplasm and the nucleus. Both cellular compartments were scored and used in the staining intensity determination. We observed that PTEN staining is sensitive to variation in tissue handling, fixation and antigen retrieval. PTEN staining was affected significantly by antigen retrieval method. Optimal staining conditions were determined to be 1:60 antibody dilution using Citrate pH 6.0 as antigen retrieval buffer. In a cohort of 1577 hormone receptor positive HER2-negative locally advanced or metastatic breast cancer patients, loss of PTEN protein was observed in 8.6% (135/1577) of patients. There was a positive correlation between PTEN mutation rate (17 out of 66 PTEN IHC positive cases vs 14 out of 587 negative cases) and loss of PTEN protein expression. PTEN loss was observed to correlate with better clinical response to PI3K inhibitor. Conclusions: Pre-analytical handling of samples is important for PTEN IHC staining. PTEN mutations and insertions/deletions contribute to PTEN protein loss. This study validates a simple method to interrogate PTEN status in clinical specimens and supports the utility of this test in selecting patients who are likely to respond to PI3K inhibitor treatment. Citation Format: Hua Gong, Emmanuel Pacia, Sharmila Manjeshwar, Beiru Chen, Xun Li, Bashar Dabbas, Jelveh Lameh, Naveen Babbar, Shabnam Tangri. Development and characterization of PTEN IHC assay for testing breast cancer patients specimens [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 P1-09-08.

PURPOSE The prognostic and predictive value of intrinsic subtypes in hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer treated with endocrine therapy and ribociclib (RIB) is currently unknown. We evaluated the association of intrinsic subtypes with progression-free survival (PFS) in the MONALEESA trials. METHODS A retrospective and exploratory PAM50-based analysis of tumor samples from the phase III MONALEESA-2, MONALEESA-3, and MONALEESA-7 trials was undertaken. The prognostic relationship of PAM50-based subtypes with PFS and risk of disease progression by subtype and treatment were evaluated using a multivariable Cox proportional hazards model, adjusting for age, prior chemotherapy, performance status, visceral disease, bone-only metastases, histological grade, number of metastatic sites, prior endocrine therapy, and de novo metastatic disease. RESULTS Overall, 1,153 tumors from the RIB (n = 667) and placebo (n = 486) cohorts were robustly profiled. Subtype distribution was luminal A (LumA), 46.8%; luminal B (LumB), 24.0%; normal-like, 14.1%; HER2-enriched (HER2E), 12.6%; and basal-like, 2.4% and was generally consistent across treatment arms and trials. The associations between subtypes and PFS were statistically significant in both arms ( P < .001). The risks of disease progression for LumB, HER2E, and basal-like subtypes were 1.44, 2.32, and 3.87 times higher compared with those for LumA, respectively. All subtypes except basal-like demonstrated significant PFS benefit with RIB. HER2E (hazard ratio [HR], 0.40; P < .0001), LumB (HR, 0.52; P < .0001), LumA (HR, 0.63; P = .0007), and normal-like (HR, 0.47; P = .0005) subtypes derived benefit from RIB. Patients with basal-like subtype (n = 28) did not derive benefit from RIB (HR, 1.14; P = .78). CONCLUSION In this retrospective exploratory analysis of hormone receptor–positive and human epidermal growth factor receptor 2–negative advanced breast cancer, each intrinsic subtype exhibited a consistent PFS benefit with RIB, except for basal-like.

PURPOSE This analysis evaluated the genomic landscape of premenopausal patients with hormone receptor–positive and human epidermal growth factor receptor 2–negative advanced breast cancer and the association of genetic alterations with response to ribociclib in the phase III MONALEESA-7 trial. METHODS Premenopausal patients were randomly assigned 1:1 to receive endocrine therapy plus ribociclib or placebo. Plasma collected at baseline was sequenced using targeted next-generation sequencing for approximately 600 relevant cancer genes. The association of circulating tumor DNA alterations with progression-free survival (PFS) was evaluated to identify biomarkers of response and resistance to ribociclib. RESULTS Baseline circulating tumor DNA was sequenced in 565 patients; 477 evaluable patients had evidence of ≥ 1 alteration. The most frequent alterations included PIK3CA (29%), TP53 (19%), CCND1 (10%), MYC (8%), GATA3 (8%), receptor tyrosine kinases (18%), and the Chr8p11.23 locus (13%). A treatment benefit of ribociclib was seen with wild-type (hazard ratio [HR] 0.44 [95% CI, 0.32 to 0.61]) and altered (HR 0.56 [95% CI, 0.36 to 0.89]) PIK3CA. Overall, patients with altered CCND1 had shorter PFS regardless of treatment, suggesting CCND1 as a potential prognostic biomarker. Benefit with ribociclib was seen in patients with altered (HR 0.22 [95% CI, 0.08 to 0.55]) or wild-type (HR 0.50 [95% CI, 0.38 to 0.67]) CCND1, but greater benefit was observed with altered, suggesting predictive potential of CCND1. Alterations in TP53, MYC, Chr8p11.23 locus, and receptor tyrosine kinases were associated with worse PFS, but ribociclib benefit was independent of alteration status. CONCLUSION In this study—to our knowledge, the first large study of premenopausal patients with hormone receptor–positive and human epidermal growth factor receptor 2–negative advanced breast cancer—multiple genomic alterations were associated with poor outcome. A PFS benefit of ribociclib was observed regardless of gene alteration status, although in this exploratory analysis, a magnitude of benefits varied by alteration.

BackgroundCombined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2− breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance.MethodsIn this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance.ResultsWe show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of bothPIK3CAmutant and wild-type ER+/HER2− breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2− breast cancer models.ConclusionsThese data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2− breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

International audience; An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

International audience; Metastasis is the main cause of death for patients with breast cancer. Many studies have characterized the genomic landscape of breast cancer during its early stages. However, there is evidence that genomic alterations are acquired during the evolution of cancers from their early to late stages, and that the genomic landscape of early cancers is not representative of that of lethal cancers1-7. Here we investigated the landscape of somatic alterations in 617 metastatic breast cancers. Nine driver genes (TP53, ESR1, GATA3, KMT2C, NCOR1, AKT1, NF1, RIC8A and RB1) were more frequently mutated in metastatic breast cancers that expressed hormone receptors (oestrogen and/or progesterone receptors; HR+) but did not have high levels of HER2 (HER2-; n = 381), when compared to early breast cancers from The Cancer Genome Atlas. In addition, 18 amplicons were more frequently observed in HR+/HER2- metastatic breast cancers. These cancers showed an increase in mutational signatures S2, S3, S10, S13 and S17. Among the gene alterations that were enriched in HR+/HER2- metastatic breast cancers, mutations in TP53, RB1 and NF1, together with S10, S13 and S17, were associated with poor outcome. Metastatic triple-negative breast cancers showed an increase in the frequency of somatic biallelic loss-of-function mutations in genes related to homologous recombination DNA repair, compared to early triple-negative breast cancers (7% versus 2%). Finally, metastatic breast cancers showed an increase in mutational burden and clonal diversity compared to early breast cancers. Thus, the genomic landscape of metastatic breast cancer is enriched in clinically relevant genomic alterations and is more complex than that of early breast cancer. The identification of genomic alterations associated with poor outcome will allow earlier and better selection of patients who require the use of treatments that are still in clinical trials. The genetic complexity observed in advanced breast cancer suggests that such treatments should be introduced as early as possible in the disease course.

1009 Background: Biomarker analyses have been presented separately for each Phase III ML trial, which tested efficacy and safety of ribociclib (RIB) with different endocrine therapy (ET) combination partners as first- or second-line treatment for hormone receptor–positive, HER2-negative (HR+/HER2−) ABC. Here, using the largest pooled biomarker dataset of a CDK4/6 inhibitor in ABC to date, we identify potential biomarkers of response or resistance to RIB across ML trials. Methods: Baseline ctDNA from 1503 patients (pts) enrolled in ML-2, 3, and 7 was assessed using next-generation sequencing with a targeted panel of 557 genes. Genes with an alteration frequency ≥2% and in ≥15 pts per treatment arm were included (83 genes). Genetic alteration was defined as presence of a mutation, short insertion/deletion, or copy number alteration. Cox proportional hazard model of progression-free survival (PFS) was fit with gene-by-treatment interaction. Genes with interaction P< 0.10 and genes of interest were investigated. Results: Pts with alterations in FRS2 and PRKCA (treatment interaction P< 0.05) as well as MDM2, ERBB2, AKT1, and BRCA1/2 ( P> 0.05 but considered actionable) had a trend for increased PFS benefit of RIB vs PBO (Table). Pts with alterations in CHD4, BCL11B, ATM, or CDKN2A/2B/2C derived little to no added PFS benefit with RIB vs PBO ( P interaction < 0.10; hazard ratio [HR] > 0.80). Data on genes implicated in the literature as potential mechanisms of resistance to ET and/or CDK4/6 inhibition ( ESR1, PTEN, FAT1, RB1, and NF1) will be presented. Conclusions: Results of this pooled analysis of the ML-2, 3, and 7 trials, the largest biomarker analysis of any CDK4/6 inhibitor in ABC, revealed several potential biomarkers of response ( FRS2, MDM2, PRKCA, ERBB2, AKT1, and BRCA1/2) or resistance ( CHD4, BCL11B, ATM, or CDKN2A/2B/2C) to RIB. Clinical trial information: NCT01958021; NCT02422615; NCT02278120 . [Table: see text]

Background: The sensitivity of plasma-based oncological assays is influenced by the amount of tumor DNA shed into the circulation (ctDNA fraction). Higher ctDNA fraction has been shown to correlate with later-stage cancers and poorer overall survival (Bettegowda C, et al. Sci Transl Med. 2014; Dawson SJ, et al. N Engl J Med. 2014). Other factors affecting ctDNA fraction are poorly understood. We present results from three large Phase III breast cancer studies (MONALEESA-2, -3, and -7) to evaluate correlations between ctDNA fraction and clinical characteristics and impact on progression-free survival (PFS) and best overall tumor response. Methods: Baseline plasma samples were collected from patients (pts) in the MONALEESA-2, -3, and -7 studies, which are Phase III registration trials testing ribociclib, a cyclin-dependent kinase 4/6 inhibitor, in hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. For each trial, cell-free DNA was extracted from plasma and sequenced using a validated next-generation sequencing assay targeting approximately 550 genes. ctDNA fraction was estimated by analyzing short-read sequences using PureCN (Riester M, et al. Source Code Biol Med. 2016) and tested for correlations with various tumor characteristics (size at baseline, Ki67 expression, number and type of metastatic sites, hormone receptor status) and pt characteristics (type of prior therapy, line of therapy, ECOG performance status, age, body mass index, race, histological grade, menopausal status). We also evaluated the prognostic value of ctDNA fraction on PFS and best overall response. To evaluate the prognostic effect on PFS, ctDNA fraction was binned into 3 categories (not detectable; low, 0.5%-10%; high, > 10%). Results: Higher ctDNA fractions were observed in pts with more metastatic sites (P < 0.0001), larger tumor diameter (P < 0.0001), higher Ki67 expression (P = 0.0001), higher lactate dehydrogenase levels (P < 0.0001), higher-grade tumors (P = 0.0004), worse ECOG performance status (P < 0.0001), progesterone receptor–negative tumors (P = 0.01), presence of liver metastases (P < 0.0001), and shorter disease-free intervals (P < 0.0001). ctDNA fractions were also higher in younger pts (continuous variable; P < 0.0001) and pts with lower BMI (P = 0.0003), prior chemotherapy (P = 0.002), and prior endocrine therapy (P = 0.01). Lower ctDNA fractions were observed in pts with better overall response (P < 0.0001), with median ctDNA fractions lowest in pts with complete responses followed by partial response, stable disease, and progressive disease. Pts with low (0.5%-10%) ctDNA fractions had longer PFS than pts with high (> 10%) ctDNA fractions (HR [95% CI], 0.63 [0.48-0.82], 0.64 [0.51-0.80], and 0.67 [0.52-0.86] in the MONALEESA-2, -3, and -7 trials, respectively). A more pronounced effect on PFS was observed in pts with undetectable levels of ctDNA vs pts with higher ctDNA fractions (HR [95% CI], 0.35 [0.23-0.52], 0.34 [0.21-0.55], and 0.31 [0.21-0.47] in the MONALEESA-2, -3, and -7 trials, respectively). Conclusions: Generally, higher ctDNA fraction was associated with more aggressive tumor characteristics and prior exposure to therapy. Lower ctDNA fraction was associated with longer PFS and better overall response. Together these data suggest that ctDNA fraction is a robust prognostic factor. Citation Format: Nadia Solovieff, Faye Su, Rebecca Leary, Alejandro Balbin, Arunava Chakravartty, Karen Rodriguez Lorenc, Tetiana Taran, Naveen Babbar. Association of tumor DNA in circulation with clinical characteristics and treatment response in HR+/HER2− advanced breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A031. doi:10.1158/1535-7163.TARG-19-A031

Introduction: Approximately 40% of patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2-) advanced breast cancer (ABC) exhibit mutations in the PIK3CA gene, leading to phosphatidylinositol-3-kinase (PI3K) pathway hyperactivation. Use of the oral α-specific PI3K inhibitor alpelisib (ALP) + fulvestrant (FUL) in SOLAR-1 significantly improved progression-free survival (HR 0.65; P Methods: SOLAR-1 (N=572) is a randomized, double-blind, phase 3 study of ALP 300 mg once daily + FUL 500 mg every 28 days + Cycle 1 Day 15, in men and postmenopausal women with HR+, HER2- ABC whose disease progressed on/after an aromatase inhibitor. Tumor tissue was analyzed by PCR-based Novartis clinical trial assay or QIAGEN therascreen® PIK3CA RGQ PCR to determine PIK3CA status and assign pts to cohorts. Pts in the mut cohort had mutations in hotspots in exons 7, 9, and 20 (Table), reflecting mutations common in pts with HR+ BC and sensitive to ALP in vitro. Results: Using archival tumor tissue (92%) and fresh biopsies (8%) from randomized pts, 341 had a detectable PIK3CA mutation per primary (n=262) or metastatic (n=74) tumor or unknown (n=5); 231 had no detectable PIK3CA mutation per primary (n=174) or metastatic (n=51) tumor or unknown (n=6). Mutations in exon 20, H1047, were most prevalent (n=193; 57%); E545 (n=106; 31%), E542K (n=60; 18%), C420R (n=6; 2%), and Q546 (n=5; 1%) were also observed. Conclusions: Analysis of these data is the first step in investigating PIK3CA alterations in SOLAR-1. Further results beyond PIK3CA mutation hotspots from next-generation sequencing will be shared at the congress. NCT02437318. Table.PIK3CA MutationsExon 7C420RExon 9E542K; E545A; E545D; E545G; E545K; E545Xa; Q546E; Q546R; Q546XaExon 20H1047L; H1047R; H1047Xa; H1047YaThe Novartis clinical trial assay does not differentiate all mutations and reports E545X for E545A/D/G/K mutations, Q546X for Q546E/K/R mutations, and H1047X for H1047L/R/Y mutations. Citation Format: Hope S. Rugo, Ingrid Mayer, Pierfranco Conte, Sibylle Loibl, Mario Campone, Dejan Juric, Fabrice Andre, Marilyn Fritzemeier, Wei He, Naveen Babbar, Eva Ciruelos. Prevalence of PIK3CAmutations in patients with hormone receptor-positive, human epidermal growth factor-2-negative advanced breast cancer from the SOLAR-1 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT142.

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6. Citation Format: Neil A. O'Brien, Martina SJ McDermott, Dylan F. Conklin, Alex Gaither, Tong Luo, Raul Ayala, Suruchi Salgar, Emmanuelle DiTomaso, Naveen Babbar, Faye Su, Sara A. Hurvitz, Ronald Linnartz, Kristine Rose, Samit Hirawat, Dennis J. Slamon. Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3825.

Background: The genetic landscape of premenopausal hormone receptor-positive (HR+) ABC is not well understood. The Phase III MONALEESA-7 study (NCT02278120), the first trial of endocrine therapy ± a cyclin-dependent kinase 4/6 inhibitor for premenopausal patients (pts) with HR+/human epidermal growth factor receptor 2-negative (HER2-) ABC, demonstrated that the addition of ribociclib (RIB) to a nonsteroidal aromatase inhibitor (NSAI) or tamoxifen (TAM) + goserelin (GOS) significantly extended progression-free survival (PFS; Tripathy D, et al. Lancet Oncol. 2018). We conducted a comprehensive ctDNA genomic analysis from MONALEESA-7. Methods: Premenopausal pts with HR+/HER2- ABC were randomized 1:1 to RIB or placebo (PBO) + NSAI (letrozole [LET] or anastrozole) or TAM + GOS. Plasma samples for ctDNA analysis were collected at baseline and end of treatment. ctDNA was analyzed using next-generation sequencing (targeted panel of 550 genes). Results: Among the 489 pts with ctDNA analyzed at baseline, the most common alterations were in PIK3CA (28%), TP53 (19%), CCND1 (11%), MYC (8%), and GATA3 (8%). Poorer prognosis in both treatment groups was most evident in patients with TP53 and MYC alterations. A PFS treatment effect in favor of RIB was noted in all subsets, independent of biomarker status (Table). However, based on HR, a trend for more pronounced benefit with RIB + NSAI/TAM + GOS was observed in pts with altered CCND1, GATA3, and genes involved in receptor tyrosine kinase signaling. Conclusions: RIB + NSAI/TAM + GOS provided PFS benefit irrespective of baseline biomarker alteration status and represents recommended first-line therapy for pts with premenopausal HR+/HER2- ABC. The genetic landscape of premenopausal ABC might modulate the magnitude of therapeutic benefit; these novel findings require confirmation in additional biomarker studies. RIB + NSAI/TAM + GOSPBO + NSAI/TAM + GOSEvents, n/NPFS, median monthsEvents, n/NPFS, median, monthsHRa (95% CI)PIK3CAWT68/18024.6798/17012.190.45 (0.33-0.62)Alt38/6914.7546/7012.850.57(0.36-0.9)TP53WT78/20324.67109/19412.980.48(0.36-0.65)Alt28/469.2335/467.160.47(0.27-0.82)CCND1WT91/22122.11126/21712.880.52(0.39-0.68)Alt15/2811.2718/235.520.21(0.08-0.54)MYCWT90/22924.67125/22112.880.49(0.37-0.65)Alt16/207.3419/197.160.57(0.25-1.31)GATA3WT96/22622.11131/22212.850.52(0.39-0.68)Alt10/23NA13/185.520.18(0.05-0.62)Receptor tyrosine kinasesbWT76/19827.53114/20614.520.5(0.37-0.67)Alt30/5114.5530/345.650.26(0.14-0.47)8p11.23cWT84/21523.03124/21412.780.47(0.36-0.63)Alt22/3412.5220/269.130.51(0.26-1)8p11.23, chromosome 8, short arm, region 11.23; alt, alteration; CCND1, cyclin D1; CI, confidence interval; GATA3, GATA binding protein 3; GOS, goserelin; NA, not applicable; NSAI, nonsteroidal aromatase inhibitor; MYC, MYC proto-oncogene, bHLH transcription factor; PBO, placebo; PFS, progression-free survival; PIK3CA, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α; RIB, ribociclib; TAM, tamoxifen; TP53, tumor protein p53; WT, wild type.a HR for PFS of RIB vs PBO. b Receptor tyrosine kinase genes include EGFR, ERBB2, ERBB3, ERBB4, FGFR1, IGF1, IGF1R, KDR, KIT, PDGFRA, PDGFRB, and VEGFA. c Includes FGFR1, WHSC1L1, and ZNF703. Citation Format: Aditya Bardia, Faye Su, Nadia Solovieff, Seock-Ah Im, Joohyuk Sohn, Keun Seok Lee, Saul Campos-Gomez, Kyung Hae Jung, Rafael Villanueva Vazquez, Yen-Shen Lu, Fabio Franke, Sara Hurvitz, Nadia Harbeck, Louis Chow, Karen Rodriguez Lorenc, Tetiana Taran, Naveen Babbar, Debu Tripathy. Genetic landscape of premenopausal HR+/HER2- advanced breast cancer (ABC) based on comprehensive circulating tumor DNA analysis and association with clinical outcomes in the Phase III MONALEESA-7 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT141.

1018 Background: The Phase III MONALEESA-7 study (NCT02278120) is the first dedicated trial of endocrine therapy (ET) ± a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor in premenopausal patients (pts) with hormone receptor–positive (HR+)/human epidermal growth factor receptor 2–negative (HER2−) ABC. The study demonstrated that the addition of ribociclib (RIB) to a nonsteroidal aromatase inhibitor (NSAI) or tamoxifen (TAM) + goserelin (GOS) significantly extended progression-free survival (PFS; hazard ratio [HR] 0.55; Tripathy D, et al. Lancet Oncol. 2018). Here we present a gene expression analysis of baseline tumor mRNA from MONALEESA-7. Methods: Premenopausal pts with HR+/HER2− ABC were treated with RIB or placebo (PBO) + GOS with either an NSAI (letrozole or anastrozole) or TAM. Baseline archival tumor samples from 360 of 672 intent-to-treat (ITT) pts were evaluated for gene expression (RIB n = 185; PBO n = 175) using a customized NanoString nCounter® GX 800-gene panel containing relevant breast cancer, CDK, and proliferation pathway–related genes. Pt subgroups were classified as having low or high mRNA expression using median expression as the cutoff. Results: PFS benefit in the biomarker-assessed group was similar to that in the ITT population. A trend toward a more pronounced PFS benefit with RIB was observed in pts with high vs low expression of CCND1 (HR 0.38 vs 0.67, respectively), IGF1R (HR 0.33 vs 0.77), and ERBB3 (HR 0.33 vs 0.76). The PFS benefit seen with RIB also trended to be greater in pts with low vs high expression of CCNE1 (HR 0.38 vs 0.65, respectively) and MYC (HR 0.37 vs 0.69). The PFS benefit with RIB was similar in pts with high vs low expression of FGFR1 (HR 0.45 vs 0.61, respectively), ESR1 (HR 0.57 vs 0.57), and tumor proliferation genes, such as MKI67 (HR 0.50 vs 0.51). Conclusions: This is the first gene expression analysis of a large set of premenopausal pts with ABC. The benefit with RIB was generally consistent across gene expression subgroups, although the magnitude varied in certain subsets. This analysis suggests that there may be unique resistance mechanisms to ET ± CDK4/6 inhibitors in premenopausal pts with ABC. Clinical trial information: NCT02278120.

Inflammation has been implicated in the development of many human epithelial cancers, including those of the stomach, lung, colon, and prostate. Tumor necrosis factor-α (TNF-α) is a potent pleiotropic, proinflammatory cytokine produced by many cells in response to injury and inflammation. Here, we show that TNF-α exposure results in increased production of reactive oxygen species (ROS), with a concomitant increase in the production of 8-oxo-deoxyguanosine, a marker for oxidative DNA damage, in human lung bronchial epithelial cells. The source of the ROS in TNF-α–treated cells was determined by both pharmacologic and small interfering RNA (siRNA) strategies to be spermine oxidase (SMO/PAOh1). SMO/PAOh1 oxidizes spermine into spermidine, 3-aminopropanal, and H2O2. Inhibition of TNF-α–induced SMO/PAOh1 activity with MDL 72,527 or with a targeted siRNA prevented ROS production and oxidative DNA damage. Further, similar induction in SMO/PAOh1 is observed with treatment of another inflammatory cytokine, interleukin-6. The data are consistent with a model that directly links inflammation and DNA damage through the production of H2O2 by SMO/PAOh1. Further, these results suggest a common mechanism by which inflammation from multiple sources can lead to the mutagenic changes necessary for the development and progression of epithelial cancers. (Cancer Res 2006; 66(23): 11125-30)

Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5' SSAT promoter-flanking region led to the identification of two NF-kappaB (nuclear factor kappaB) response elements. Electrophoretic mobility-shift assays showed binding of NF-kappaB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-kappaB signalling and increased binding at these NF-kappaB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirin-induced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.

An activated Ki-ras was expressed in the human colon adenocarcinoma cell line Caco-2 to study the effects of Ki-ras oncogene on polyamine metabolism during gastrointestinal tumorigenesis. Multiple clones selected for expression of the mutant Ki-ras transgene displayed a suppression of transcription of a key catabolic enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Gene expression analysis, with cDNA microarrays, showed that Ki-ras transfected clones had decreased levels of expression, compared to mock transfected cells, of peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation. The activated Ki-ras suppressed SSAT expression by a mechanism involving the PPARgamma response element (PPRE) located at +48 bp relative to the transcription start site of the SSAT gene. Transient expression of the PPARgamma protein in Ki-ras expressing Caco-2 clones, or treatment with the PPARgamma ligand ciglitazone, led to an increase in the SSAT promoter activity. A MEK1/2 inhibitor PD98059 induced transcription of both PPARgamma and SSAT genes in the activated Ki-ras clones, suggesting that the mitogen-activated protein kinases (MAPKs) were involved in the regulation of SSAT expression by PPARgamma. We concluded that mutated Ki-ras suppressed SSAT via a transcriptional mechanism involving the PPARgamma signaling pathway.

Introduction Colorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance. Materials and Methods A multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days. Results Using one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%. Conclusion These results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance.

Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia, which are risk factors for cancer development in humans. Targeting polyamine metabolism (by use of polyamine synthesis inhibitors or polyamine catabolism activators) and inflammation (by use of nonsteroidal anti-inflammatory drugs) has been studied for many cancers, including colon, prostate, and skin. Genetic epidemiology results indicate that a genetic variant associated with the expression of a polyamine biosynthetic gene is associated with risk of colon and prostate cancers. A clinical trial of difluoromethylornithine (DFMO), a selective inhibitor of polyamine synthesis, showed that the 1 year treatment duration reduced prostate volume and serum prostate-specific antigen doubling time in men with a family history of prostate cancer. A second, clinical trial of DFMO in combination with sulindac, a NSAID in patients with prior colon polyps found that the 3-year treatment was associated with a 70% reduction of all, and over a 90% reduction of advanced and/or multiple metachronous colon adenomas. In this chapter, we discuss that similar combination prevention strategies of targeting polyamines and inflammation can be effective in reducing risk factors associated with the development of human cancers.

Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

As more data emerge, the significance of polyamine catabolism in polyamine homeostasis, drug response, and disease etiology is expanding. Importantly, the regulation and function of the polyamine catabolic pathway has emerged as a rational target for drug intervention in both chemotherapeutic and chemopreventive strategies. Mammalian intracellular polyamine catabolism had long been thought to be a two-step process primarily regulated by a rate-limiting acetyltransferase, spermidine/spermine N1-acetyltransferase (SSAT), followed by the activity of a constitutively expressed acetylpolyamine oxidase (PAO). However, as recent reports have clearly demonstrated, mammalian polyamine catabolism also includes the activity of a previously unrecognized spermine oxidase (SMO/PAOh1). The production of reactive oxygen species (ROS) and other toxic products by these various polyamine catabolic enzymes can result in both useful and potentially dangerous consequences. This chapter will examine some of the most recent findings related to polyamine catabolism and will address the cloning and characterization of mammalian polyamine oxidases, including the newly discovered SMO/PAOh1. Additionally, further characterization of the highly regulated SSAT, as facilitated by many recent advances with transgenic models, will be discussed with respect to the potential role that it and the oxidases play in determining response to various drugs and stimuli. Although the polyamine catabolic pathway is well described and being studied in multiple organisms, this work will focus primarily on results directly related to mammalian systems, with special emphasis given to the relationship between polyamine catabolism and human disease. Specifically, data indicating that the induction of polyamine catabolism by specific antitumor polyamine analogs plays a direct role in determining drug response will be discussed. Also to be examined is the recent recognition that the oxidation of polyamines contributes to disease processes, and the potential targeting of polyamine catabolism as a strategy for chemoprevention.

Sulindac, a non-steroidal anti-inflammatory prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Sulindac sulfide, but not sulindac sulfone, inhibits cyclooxygenase (COX) enzyme activities, yet both derivatives have growth inhibitory effects on colon cancer cells. Microarray analysis was used to detect COX-independent effects of sulindac on gene expression in human colorectal cells. Spermidine/sperm-ine N1-acetyltransferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was induced by clinically relevant sulindac sulfone concentrations. Northern blots confirmed increased SSAT RNA levels in these colon cancer cells. Deletion analysis and mutational studies were done to map the sulindac sulfone-dependent response sequences in the SSAT 5'-flanking sequences. This led us to the identification of two peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the SSAT gene. PPRE-2, at +48 bases relative to the transcription start site, is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPAR gamma in the Caco-2 cells as shown by transfection and gel shift experiments. PPRE-1, at-323 bases relative to the start site, is not required for the induction of SSAT by sulindac sulfone but can be bound by both PPAR delta and PPAR gamma. Sulindac sulfone reduced cellular polyamine contents in the absence but not in the presence of verapamil, an inhibitor of the export of monoacetyl diamines, inhibited cell proliferation and induced apoptosis. The induced apoptosis could be partially rescued by exogenous putrescine. These data suggest that apoptosis induced by sulindac sulfone is mediated, in part, by the COX-independent, PPAR-dependent transcriptional activation of SSAT, leading to reduced tissue polyamine contents in human colon cancer cells.

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene ( APC ) and show defects in APC -dependent signaling. APC influences the expression of several genes, including the c- myc oncogene and its antagonist Mad1 . Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c- myc and a modifier of APC -dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc -binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.