Goetz, Matthew P.

Abstract Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in HR+ MBC, but progression of disease ultimately occurs. Apart from HR+ status, there are no clinically available biomarkers that enable oncologists to determine prognosis and predict response to CDK4/6i. An emerging biomarker is serum thymidine kinase 1 (TK1), a secreted marker of proliferation that is prognostic in pts with HR+ HER2- MBC. High levels of TKa are associated with inferior PFS, whereas pts with low TKa levels pretreatment, or TKa levels that decrease on ET and a CDK4/6i, have superior PFS. Notably, TKa levels rebound ≥ 5 days off Pb, with resumption of cell cycling. PROMISE (NCT0281902) is a prospective study that enrolled women with HR+ MBC starting Pb + letrozole (L) in the 1st line [FL] or Pb + fulvestrant in the 2nd line [SL] setting. The trial includes a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify novel genomic variants and pathways associated with an early decline in TKa (measured after 2 months or end of cycle [C]2) and PFS. Here, we report the association between i) pre-treatment TKa (pre-TKa) levels and PFS (i.e. from registration to the 1st disease event) and ii) TKa levels at the end of C2 (C2-TKa) and PFS-2 (i.e. from the start of C3 to the 1st disease event).Methods: TKa testing was performed using the DiviTum assay (Biovica). TKa+ disease was defined as ≥ 200 Du/L and TKa- disease as below limit of detection to 200 Du/L. Log-rank test and univariate Cox modeling were used to assess the association between pre-TKa levels and PFS and between end of C2-TKa levels and PFS-2. The database was locked on June 28, 2021. Results: Of 68 pts enrolled, 4 were ineligible and pre-TKa data was unavailable for 4. Of the remaining 60 pts (45 FL, 15 SL), the percentage of pts with pre-TKa+ disease was 33.3% in FL (15/45, 95% CI: 20.0-49.0%), and 46.7% (7/15, 95% CI: 21.4-71.9%) in the SL. The median follow-up time for pts on study was 24 months (range: 2-42 months). There were 22 disease events (13 in FL, 9 in SL). In the FL setting, PFS was significantly shorter for preTKa+ pts compared to preTKa- pts (HR: 4.15, 95% CI:1.35-12.74; p=0.007), but not for SL pts (HR: 1.11, 95% CI: 0.30-4.18, p=0.875). End of C2 TKa data was obtained for pts while on Pb (n=5), or after stopping Pb as follows: 1-4 days (n=9), 5-8 days (n=28) and 9-36 days (n=11). PFS-2 was not associated with C2-TKa in the FL (p=0.834) or SL (p=0.454) settings. An analysis of TKa levels by metastatic site will be presented at the meeting.Conclusions: A secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the treatment of HR+ MBC is associated with PFS in pts receiving 1st line Pb + L, but not in those receiving 2nd line Pb + fulvestrant. While the end of C2 TKa levels were not associated with PFS, the interpretability of these data are limited, given treatment delays (0-36 days) prior to starting C3 that may result in TKa rebound. Future studies evaluating the predictive nature of TKa and Pb response should focus on earlier timepoints while on drug. Citation Format: Ciara C O'Sullivan, Jun He, Jason Sinnwell, Vera J Suman, Krishna R Kalari, Peter T Vedell, Ann M Moyer, Xiaojia Tang, Kevin J Thompson, Abe Eyman Casey, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Brendan McMenomy, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Mattias Bergqvist, Adrian Nordmark, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Serum thymidine kinase 1 activity (TKa) levels and progression-free survival (PFS) in patients (pts) with hormone receptor positive (HR+) HER2-negative metastatic breast cancer (MBC) on palbociclib (Pb) and endocrine therapy (ET) [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 P5-13-22.

Abstract Background: Hormone receptor-positive (HR+) breast cancer (BC) accounts for ~60-70% of all BC cases. CDK4/6i combined with endocrine therapy (ET) is the standard of care for first- and second-line therapy of patients with HR+ metastatic breast cancer (MBC) based on substantial improvements in progression free survival (PFS) and overall survival compared to ET alone. Unfortunately, most patients (pts) develop resistance to CDK4/6i. The mechanisms underlying CDK4/6i resistance have not been fully elucidated. Robust in vivo CDK4/6i resistant animal models from pts with de novo and acquired CDK 4/6i resistance are lacking. Patient-derived xenografts (PDXs) are important preclinical tools in oncology to advance our understanding of cancer biology and to evaluate drug response phenotypes. Unfortunately, the establishment of sustainable PDXs from pts with HR+ BC is much more difficult than from HR-negative BC. In order to obtain clinically relevant models that can be utilized to better understand mechanisms of primary and acquired resistance to ET and CDK4/6i, we report the development of PDXs from a prospective study in which percutaneous biopsies were obtained from women receiving palbociclib for the treatment of HR+ advanced or MBC (PROMISE; NCT03281902). Methods: PROMISE (Prospective Study to Evaluate the Role of Tumor Sequencing in Women Receiving Palbociclib for Advanced Hormone Receptor (HR)-Positive Breast Cancer) is a prospective trial which enrolled pts with HR+, HER2-negative MBC who planned to start treatment with palbociclib and letrozole (1st line [FL]) or palbociclib and fulvestrant (2nd line [SL]). For pts enrolled at Mayo Clinic Rochester (MCR), serial tumor biopsies were obtained prior to the start of treatment and at disease progression to establish PDXs. Because baseline take rate was low, we amended the protocol to attempt PDX generation after the 2nd cycle (C2) of therapy. Tissues were received within an hour of excision and were orthotopically implanted in the mammary fat pad of 6~8-week-old nonobese diabetic (NOD)/SCID/IL-2g-receptor null (NSG) mice. Tumor growth was defined as tumor volume ≥ 50 mm3. Tumor take rate was defined as percent of patients with development of at least one stably transplantable (passed at least for two generations) xenograft that was pathologically confirmed as BC. Results: Of the 50 pts enrolled at MCR, 37 were FL pts (2 ineligible) and 13 were SL pts (1 ineligible). Fresh tumor samples obtained pre-treatment from 43 eligible pts (32 FL), after C2 from 17 eligible pts (13 FL), and at disease progression from 10 eligible pts (5 FL) were implanted. Tumor growth was seen in at least one mouse from a pre-treatment specimen in 1 (3.1%) FL pt (confirmed BC) and 4 (36.4%) SL pts (2 confirmed BC); from end of C2 specimens in 2 (15.4%) FL pts and 2 (50%) SL pts (1 confirmed BC); and at progression in 1 (20%) FL pt and 2 (40%) SL pts (1 confirmed BC). The take rate from pre-treatment samples among FL pts was 1/32 (3.1%; 95% CI: 0.08-16.2%). Take rates in other groups are pending histological review. Discussion: In pts receiving palbociclib for HR+ MBC, the take rate using a pre-treatment percutaneous biopsy was low (3.1%) in the FL setting. However, higher growth rates were seen in the SL setting. The observation of higher growth rates at the end of C2 and at progression may relate to the activation of G1/S that occurs with withdrawal of palbociclib, and should be further studied. PDX remains a valuable tool for the evaluation of tumor biology and the development of new therapeutics, especially for those models established from pts with palbociclib and ET resistance. Proteogenomic and sequencing studies are ongoing to fully characterize these PDXs. Citation Format: Jia Yu, Vera J. Suman, Jun He, Jason P. Sinnwell, Ann M. Moyer, Bo Qin, Yayun Gu, Huan Zhang, Jodi M. Carter, Krishna R. Kalari, Brendan P. McMenomy, Minetta C. Liu, Tufia C. Haddad, Kathryn J. Ruddy, Fergus J. Couch, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard Weinshilboum, Ciara O'Sullivan, Matthew P. Goetz, Liewei Wang. Patient-derived xenografts (PDXs) generated from hormone receptor-positive breast cancer (BC) before and after cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) treatment: Initial findings from the PROMISE study [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 P5-01-07.

Abstract Background: Quality of life (QOL) in breast cancer patients (pts) can be greatly impacted by initial treatment and ongoing therapy, particularly if side effects and symptoms are not well tolerated. The PALLAS trial investigated whether the addition of 2 years of palbociclib (palbo) to adjuvant endocrine therapy (ET) improved invasive disease-free survival (iDFS) over adjuvant ET alone. We report on the main patient-reported outcome (PRO) quality of life (QOL) and symptom severity results of this trial by treatment arm. Methods: PALLAS is an ongoing multicenter, open label phase 3 trial that enrolled hormone receptor positive, HER2-negative, stage II-III breast cancer patients at 406 cancer centers across 21 countries. Patients were randomly assigned (1:1) to either 2 years of palbo (125mg/day, 3 weeks on 1 week off) plus ongoing provider or patient-choice adjuvant ET (palbo+ET) versus ongoing ET alone. The primary study endpoint was iDFS. Treatment with palbo in all pts stopped at the time of the second interim analysis (5/2020) due to futility; all pts then moved to follow up. The PRO analyses were triggered for completion after awareness of the pre-specified 469 iDFS cases in November 2020. Eight PRO endpoints were measured serially (i.e., day 1 of each monthly cycle for the first 3 months, then every 3 months for the first 2 years, and once at year 3). The endpoints were the EORTC QLQ-C30 global health status/QOL score, the Brief Fatigue Inventory score, the modified Brief Pain Inventory severity and pain interference scores, the EORTC QLQ-BR23 alopecia score, and the Breast Cancer Prevention Trial hot flash symptoms, vaginal problems, and musculoskeletal pain scores. Linear mixed models compared the average difference between arms across time points during the initial 2-year treatment period adjusting for cycle 1 day 1 (C1D1) PRO scores, demographic and clinical variables. The average differences between arms (palbo+ET vs ET alone) and the two-sided (1-[0.05/8]) x 100% confidence intervals, adjusted for multiple comparisons, were calculated. Analysis of covariance compared the average between-arm differences by endpoint at 3 years. Results: The PRO intention to collect population included 4688 (81%) of the overall trial pts, and was clinically and demographically representative of the remaining 1073 pts. Each analysis population, with measures at C1D1 and at least 1 post-C1D1 assessment for each PRO endpoint, comprised ≥ 89% of the 4688 pts and the proportions were similar between arms. After adjustment for baseline covariates, on average, no clinically important differences between arms were observed for any of the eight endpoints over the 2 year treatment period (Table 1). All effect sizes were below the pre-specified 0.2 threshold. These PRO results were similar at the 3 year time point. Conclusions: No clinically significant differences in either patient-reported QOL or symptom severity were found, on average, between participants in the two PALLAS treatment arms while either taking palbo+ET or ET alone, or after study-wide termination of palbo. In general, the addition of palbociclib in the adjuvant breast cancer setting did not contribute to increased symptom burden within this survivorship population. Further analyses will examine the relationship between PROs and treatment discontinuation by arm and study time point. Support: AFT, Pfizer, https://acknowledgments.alliancefound.org Table 1.Results of the Patient-Reported QOL and Symptom Severity Analyses Between the Two Treatment Arms During the First 2 Years of PALLASPRO Endpoint *Palbo + ET Adjusted average score (95% CI)ET alone Adjusted average score (95% CI)Average Difference + (Palbo + ET vs ET alone)[99.38% CI] Clinically ++ Important DifferenceEORTC QLQ-C30 Global Health Status/QOL71.7 (71.2, 72.2)74.0 (73.5, 74.5)-2.3 (-3.3, -1.4)**NoBrief Fatigue Inventory Score2.3 (2.2, 2.3)2.1 (2.0, 2.1)0.2 (0.1, 0.3)NoModified Brief Pain Inventory - Severity Score2.3 (2.2, 2.3)2.4 (2.4, 2.5)-0.2 (-0.3, -0.1)NoModified Brief Pain Inventory - Interference Score1.7 (1.6, 1.7)1.7 (1.6, 1.7)0.0 (-0.1, 0.1)NoEORTC QLQ-BR23 Alopecia1.4 (1.4, 1.4)1.3 (1.3, 1.3)0.1 (0.1, 0.1)NoBreast Cancer Prevention Trial - Hot Flash Symptoms1.2 (1.2, 1.3)1.2 (1.2, 1.3)0.0 (-0.1, 0.1)NoBreast Cancer Prevention Trial - Vaginal Problems0.8 (0.8, 0.8)0.8 (0.7, 0.8)0.0 (0.0, 0.1)NoBreast Cancer Prevention Trial - Musculoskeletal Pain1.2 (1.2, 1.2)1.3 (1.3, 1.3)-0.1 (-0.2, 0.0)No* For the EORTC QLQ-C30 Global Health Status/QOL subscale, higher scores indicate better QOL. For all other PRO endpoints, higher scores indicate worse symptom levels. ** The lower bound of the one-sided CI (adjusted for multiple comparisons) was -3.3. Because the lower limit is greater than the pre-specified non-inferiority margin of -3.44, non-inferiority of palbo+ET relative to ET-alone was concluded. The non-inferiority margin corresponds to a 0.2 SD in the EORTC QLQ-C30 global health/QOL score. +The average difference was adjusted for the following baseline covariates: Cycle 1 day 1 score, region (if applicable), age category, first adjuvant ET, race, ethnicity, N-stage, T-stage, histological grade, PgR, prior chemotherapy, ECOG Performance Status. ++Based on each instrument’s published clinically relevant cut-offs, if available. After calculating Cohen’s d treatment effect sizes, (i.e. by dividing the average difference by the ET-alone arm standard deviation from cycle 1 day 1), all effect sizes were below the pre-specified 0.2 threshold, and would not be considered clinically important. Citation Format: Michelle Joy Naughton, David Zahrieh, Michael Gnant, Nicholas Zdenkowski, Julie Lemieux, Jun J Mao, Vesna Bjelic-Radisic, Eileen Shinn, Marija Balic, Christoph Thomssen, Jane Neisel, Manuel Ruiz-Echarri, Sibylle Loibl, Claudine Isaacs, David Cameron, Fernando Manuel Henao Carrasco, Matthew Goetz, Viktor Wette, Gustavo Werutsky, Hope Rugo, Marcus Vetter, Ling-Ming Tseng, Kathy Miller, Florian Fitzal, Juan Miguel Gil Gil, Haeseong Park, Barbro Linderholm, Emilio Bajetta, Zoneddy Dayao, Aleix Prat, Karin Ehrhardt, Otto Metzger, Amal Arahmani, Ernest Law, Ann Partridge, Lisa Carey, Alex Zoroufy, Amylou Dueck, Dominik Hlauschek, Angela DeMichele, Erica Mayer. Quality of life and symptom severity in the PALLAS randomized trial of palbociclib with adjuvant endocrine therapy in early breast cancer (AFT-05) [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 P4-10-01.

Abstract Background: Cyclin-dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor-positive (HR+) metastatic breast cancer (MBC), but endocrine resistance is a major challenge. PROMISE [NCT0281902; n=63] is a multicenter study that enrolled women with HR+ HER2- MBC commencing palbociclib (Pb) with letrozole (1st line [1L]) or fulvestrant (2nd line [2L]), and was designed to perform a comprehensive “omic” assessment of prospectively collected biospecimens (pre-treatment (M1), at 2 months (M2), and at disease progression). The goal is to identify novel genomic variants and pathways associated with resistance to CDK4/6i and ET and PFS outcomes. Here we report the association between the proteomic, metabolomics, and lipidomics data generated from pre-Pb and 2-month serum samples and PFS. Methods: Untargeted mass spectrometry data was generated from Metabolon, assaying 1308 metabolites and 831 lipids. Additionally, 1436 proteins were assayed on the Olink platform. Cox proportional hazard models were used to evaluate the univariate hazard ratio (HR) for all features with respect to PFS. The analyses were performed on samples from 45 patients (N=33 1Lwith 9 progression events and 12 2L with 8 progression events), obtained from M1 and M2 timepoints on Pb + ET. Enrichment analysis p-values are calculated using Fisher’s exact test. Results: Proteomics: In the M1 timepoint, 93 and 43 proteins were associated with PFS in the 1L and 2L settings, respectively; inflammation genes were enriched among the 1L setting (p= 0.034); 33 proteins presented HRs ranging between 0.026 and 0.56. The FABP9 protein (HR of 1.98, 95% CI 1.02-3.83) was associated with worse PFS. Conversely, inflammation genes were not observed to be enriched in 2L. In the M2 timepoint, we observed 60 and 21 proteins significantly associated with PFS, but no biological function was enriched in 1L and 2L. Metabolites: In the M1 timepoint, metabolism of the sulfur-containing amino acids (methionine, cysteine, SAM and taurine) were enriched in the 1L setting (p= 0.035, HR range 0.15-0.33); and the branched-chain amino acids (leucine, isoleucine, and valine) were significantly associated with PFS in the 2L setting (p= 0.028, HR range 0.013-0.33). At the M2 timepoint, the amino acids were no longer enriched, but fatty acid metabolism was significantly enriched for both 1L and 2L (p= 0.048 and 0.067, respectively). Pathways involving lipids, amino acids, and xenobiotics were enriched in metabolites related to PFS (p <0.05) for both treatment lines at M1 and M2. Lipidomics: In the M1 timepoint, 10 and 19 lipids were associated with PFS for 1L and 2L, respectively. The most notable lipid associated with worse PFS in the 1L was an 18 carbon phosphatidylinositol, PI(18:1/18:2), (HR 7.34 (CI 1.27-42.50); 8 triglycerides were associated with improved PFS (HR range 0.39 and 0.55). In 2L, the 19 lipids associated with PFS included 12 phosphatidylcholines (enrichment p = 5.6X10-8). In the M2 timepoint, 15 and 8 lipids were significantly associated with PFS for 1L and 2L. An enrichment of phosphatidylinositols was observed in 1L (p= 1.2X10-5); none were observed in the 2L.Future Directions: Networks are being constructed using the proximity scores of the proteins, lipids, and metabolites associated with PFS in M1 and M2 for 1L and 2L. Network similarities and analyses will be conducted.Conclusion: Distinct multi-omic changes identified in serum samples obtained from PROMISE participants M1 and M2 on Pb correlate with disease progression in both 1L and 2L settings. Additionally, validation studies will determine the significance of these findings. Citation Format: Krishna R. Kalari, Kevin J. Thompson, Jason Sinnwell, Xiaojia Tang, Vera J. Suman, Jun He, Seul Kee Byeon, Akhilesh Pandey, Abe Eyman Casey, Peter T. Vedell, Ann M. Moyer, Alvaro Moreno-Aspitia, Donald W. Northfelt, Minetta C. Liu, Tufia C. Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J. Ruddy, Karthik V. Giridhar, Roberto A. Leon-Ferre, Richard M. Weinshilboum, Liewei Wang, Ciara C. O’ Sullivan, Matthew P. Goetz. Multiomics data reveal novel biomarkers for CDK4/6 resistance [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 P4-01-03.

Abstract Introduction. The standard treatment for non-metastatic triple-negative breast cancer (TNBC) is neoadjuvant chemotherapy (NAC) and nearly 50% exhibit pathological complete response (pCR). However, patients with residual disease after NAC are at increased risk for recurrence and death. Prior studies examining the transcriptome of TNBC pre/post-NAC have examined a limited number of genes (<500) in heterogeneous subgroups of TNBC (e.g. LAR and non-LAR). We explored the transcriptome of androgen-receptor (AR) negative (non-LAR) TNBC subtype both pre/post NAC to identify pathways associated with NAC response. Methods. Tumors obtained pre/post NAC from TNBC patients enrolled in the Breast Cancer Genome Guided therapy study (BEAUTY) underwent RNA sequencing and reverse-phase protein array (RPPA). EdgeR was applied for differentially expressed (DE) analysis and regression methods for RPPA. Digital deconvolution method (CIBERSORTx) and TNBC single-cell data were used to obtain cell types. Pathway analysis was carried out using 2972 gene sets and gene set variation analysis (GSVA). Functional enrichment analysis was conducted with significant genes. Results. Of the 44 TNBC patients, 32 patients were excluded from the analysis cohort due to: LAR tumor (6 pts.), non-LAR tumor with pCR (23 pts.), and cell type issues with RNA-seq data (3 pt.). Paired RNA-Seq data were available for 12 TNBC patients (4 with progression <2 years [EP]) and 8 who were progression-free > 4 years [NP]) and paired RPPA data were available for 9 of these 12 patients. Differentially expressed genes, proteins and cell types between EP and NP in post-NAC. We identified 489 genes differentially expressed (DE) between EP and NP (logFC=|2|, FDR < 0.05). Analysis of cytobands from these 489 genes showed an enrichment of genes on chromosome 6p22.1-2 and 17q25.3 regions (enrichment ratio >5; p-value <10E-4). Critical genes identified in the AR- network (p-value < 10E-3) were IL1RN, SLAMF9, KRT81, BHLHE22, B3GALT5, PCP4, TREM1, AQP9, NRTN, and COL2A1.In addition, preliminary results from RPPA data of post-NAC tumors showed astrocytic phosphoprotein (PEA-15), involved in apoptosis, proliferation, glucose metabolism, as well as cell proliferation and Y box binding (YB1) proteins (involved in metastases), were more DE in EP than NP (p < 0.05). CIBERSORTx was applied to estimate the proportions of different cell types in post-NAC tumors. Cancer-associated fibroblasts iCAFs were low and myCAFs are high in EP vs NP. It is known that the cross-talk between CAFs and tumor cells may induce tumor resistance to chemotherapy. Differentially expressed pathways in post and pre-NAC EP tumors. Using genome-wide expression data from the paired 12 tumors and the GSVA method, we obtained individual pathway scores for 2972 pathways. One hundred ninety pathways were downregulated and 61 pathways were upregulated (p-value <= 0.05) in the post-NAC residual disease of EP relative to NP. We further examined these 190 pathways in the paired EPs and found 71% of those pathways were upregulated in the pre-NAC. These 190 downregulated pathways were enriched with FOXO, TGF-beta, PI3k, FGFR1, insulin and others. The 61 upregulated pathways in post-NAC EP tumors were enriched with mismatch repair, purine, tubulin, telomere, polymerase and gap-junction related pathways; 77% of those 61 pathways were downregulated in pre-NAC. Conclusions. Using a comprehensive “omics” approach, we have identified novel cancer and drug response pathways associated with recurrence in AR-TNBC disease. Further work to evaluate these as markers of outcome and potential drug targets is warranted. Citation Format: Krishna R Kalari, Vera J Suman, Xiaojia Tang, Jason P Sinnwell, Kevin J Thompson, Peter T Vedell, Jodi M Carter, Sarah A McLaughlin, Alvaro Moreno Aspitia, Donald W Northfelt, Richard J Gray, Richard Weinshilboum, Liewei Wang, Judy C Boughey, Matthew Goetz. Multi-omics data shows downregulation of mismatch repair, purin and tublin pathways in AR-negative triple-negative chemotherapy-resistant tumors [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 P4-01-05.

Abstract Background:. Poor patient outcomes in triple negative breast cancer (TNBC) largely stem from a lack of understanding of therapeutic vulnerabilities and an insufficient armamentarium of effective drugs. The standard of care for most early stage TNBC patients continues to be (neo)adjuvant chemotherapy and radiation. Identifying additional therapeutic options for this subset of patients represents a major unmet need. We and others demonstrated that estrogen receptor beta (ERβ) is expressed in about 20% of TN tumors. Prior research has shown that ligand-mediated activation of ERβ decreases proliferation, invasion, and migration in TNBC cell lines. In vivo, ERβ suppresses the growth of cell line xenograft models and prevents the development of metastatic lesions in a ligand dependent manner. Mechanistically, we found that ERβ repurposes EZH2 to suppress oncogenic NFκB signaling and that pharmacologic inhibition of EZH2 diminishes ERβ function and its anti-cancer effects. This research has led to an ongoing clinical trial (NCT03941730) assessing the efficacy of estradiol in ERβ+ TNBC patients with chemorefractory disease. As with all therapies, patients will exhibit de novo and acquired resistance and therefore we sought to understand the mechanisms of resistance to ERβ targeted therapies. Methods:. Using multiple models of ERβ positive TNBC, we developed ERβ resistant cell lines through chronic exposure to estradiol and the ERβ specific agonist LY500307 over a period of 8 months. We employed RNA sequencing to characterize the transcriptomic changes which occurred following ERβ resistance. We profiled XIST expression across multiple publicly available datasets, including TCGA, Metabric, BEAUTY, and GTEx. XIST expression was also modulated using CRISPR/Cas9 to assess subsequent effects on TNBC cell biology, ERβ function, and response to ERβ targeted therapies. Results:. Our resistant cell line models of ERβ positive TNBC maintained ERβ expression, but were no longer growth inhibited by ERβ agonists. RNAseq revealed substantial differences comparing the transcriptome of resistant versus sensitive cell lines, of which the most increased transcript in the resistant setting was the lncRNA XIST. XIST is best known for its role in X-chromosome inactivation through recruitment and association with the PRC2 complex, and thus EZH2. However, little is known about its functions in breast cancer. We therefore assessed the expression levels of XIST in breast tumors and cell lines. XIST expression was highly variable in breast cancer, was found in a proportion of all breast cancer sub-types and did not correlate with ERβ status. However, XIST expression was up-regulated in ERβ expressing cell lines following long term estrogen treatment. No effects on XIST expression were identified in multiple ERα positive models following estrogen, SERM/SERD treatment, or estrogen deprivation. Furthermore, upregulation of XIST was not associated with resistance to Paclitaxel or Doxorubicin, suggesting XIST is not a part of a broad resistance phenotype. Strikingly, CRISPR mediated knockout of XIST in ERβ resistant cells completely re-sensitized cells to ERβ targeted therapies suggesting that XIST expression is a critical component of ERβ resistance. Conclusions:. ERβ represents a relevant therapeutic target that is being tested in clinical trials. Using multiple in vitro models, we provide evidence that XIST expression is sufficient to induce resistance to. ERβ targeted therapies in TNBC and may therefore represent a relevant biomarker for patient stratification. Further strategies to suppress XIST expression may elicit anti-cancer effects on their own and may resensitize a sub-set of ERβ resistant tumors to ERβ agonists. Citation Format: Michael J Emch, Kirsten GM Aspros, Elizabeth S Bruinsma, Krishna R Kalari, Calley J Jones, Brandon W Simone, Matthew P Goetz, John R Hawse. The lncRNA XIST mediates sensitivity to ERβ targeted therapies in triple negative breast cancer [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 P4-02-09.

Abstract Background: FGFR dysregulation is observed in multiple cancers and targeting FGFR is an emerging therapeutic strategy with FDA approved treatments in bladder and cholangiocarcinoma. Here we examined the prevalence of FGFR mutations, fusions, and high-level amplifications in breast cancer, stratified by receptor subtype and local/metastatic status, in both Foundation Medicine (FM) and institutional Mayo Clinic (MC) cohorts. Methods: For the FM cohort, comprehensive genomic profiling (CGP) examining at least 324 genes for all classes of alterations, including FGFR1-4 was carried out for 32,048 breast cancers during the course of routine clinical care in a Clinical Laboratory Improvement Amendments (CLIA)-certified lab (Foundation Medicine Inc., Cambridge, MA, USA). Tumor mutational burden (TMB) was determined on up to 1.1 Mb, microsatellite instability high (MSI-High) was determined on up to 114 loci and predicted ancestry from >10,000 SNPs. Estrogen receptor (ER) and HER2 status were available for a subset of FM samples. Additionally, 131 patients with metastatic breast cancer from a subset of patients at three Mayo Clinic sites (MC cohort) with clinical characteristics and cancer-panel DNA sequencing data from a CLIA-certified lab (Tempus, Chicago, IL) were included. Results: In the FM cohort, the prevalence of FGFR1-4 high-level amplification (CN≥10) was 10.1%, while mutations (1.5%) and fusions (0.72%) were rare. Most amplifications occurred in FGFR1 (9.2%); most fusions and mutations occurred in FGFR2 (0.46%, 0.77%). FGFR alteration prevalence was highest in ER+/HER2- subtype (14.4%) and lowest in HER2+ disease (7.7%). FGFR alterations were more common in IDC (11.7%) than ILC (7.7%), p<3E-08. FGFR alterations were more prevalent in the metastatic setting relative to breast-biopsied disease (13.6% v 10.1%; OR = 1.4; p=2E-17), especially in the HER2+ (OR =1.9, p=0.004) and ER-/HER2- (OR = 1.9, p = 0.05) disease; no enrichment was seen in the ER+/HER2- metastases (OR =1.0, p = 1). FGFR amplifications were observed at a higher prevalence in patients with predicted East Asian ancestry, relative to patients with European ancestry (12.1% v 10.0%; p = 0.03). Overall, the most common activating mutations in FGFR were FGFR2 N549K (n=85), FGFR1 N546K (n=78), FGFR4 V510M (n=28), FGFR2 K659E (n=28), FGFR4 V510L (n=20), and FGFR2 Y375C (n=15). The most common recurrent fusions were FGFR3:TACC3 (n=36), FGFR2:TACC2 (n=17), FGFR1:TACC1 (n=9), FGFR1:BAG4 (n=6), and FGFR2:ATE1 (n=5). In patients with FGFR amplifications, the most frequently co-occurring alterations were ZNF703 (78.4%), TP53 (51.5%), CCND1 (36.1%), FGF3/4/19 (32.9 - 34.4%), PIK3CA (30.7%), MYC (29.6%), ESR1 (17.2%), EMSY (16.3%), and PTEN (10.6%). Significant co-occurrence was observed for a number of genes including FGF3/4/19, CDK4, and CDK8 (all OR>2, p<1E-07); mutual exclusivity was observed with PIK3R1, BRCA1, and BRCA2 (all OR <0.5, p<4E-13), among other genes. In the 131 metastatic tumors from MC, the prevalence of FGFR1-4 high-level amplifications was 19.8% [FGFR1 (12.4%), FGFR2 (7.4%), and FGFR3 (0.8%)]. The prevalence of high-level FGFR amplifications did not differ by clinical subtypes: HR-/HER2- (7/31), HR+/HER2- (15/79), and HER2+ (2/11), p=0.68. Conclusions: High-level FGFR amplifications are observed in >11% of breast cancers, especially the ER+/HER2- subtype, while mutations/fusions are rare. These data support the ongoing studies evaluating targeted therapies for FGFR amplified ER + breast cancer. Correlations with clinical information (MC cohort) and associations with actionable alterations are ongoing and may inform potential combination strategies. Citation Format: Karthik V Giridhar, Ethan S Sokol, Peter T Vedell, Jason P Sinnwell, Aakash Desai, Tufia C Haddad, Ciara C O’Sullivan, Roberto A Leon-Ferre, Siddhartha Yadav, Kostandinos Sideras, Brenda Ernst, Minetta C Liu, Abe Eyman Casey, Xiaojia Tang, Zoe Fleischmann, Karthikeyan Murugesan, Krishna R Kalari, Matthew P Goetz. The frequency and somatic mutation landscape of Fibroblast growth factor receptor (FGFR) alterations in breast cancer [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 P3-08-02.

Abstract Background: The importance of the antitumor immune response in TNBC is well established. TNBC with higher TILs are less likely to recur and more responsive to systemic therapy. Likewise, PD-L1+ TNBC are more likely to benefit from chemoimmunotherapy. However, TNBC is highly heterogeneous. Of the TNBC molecular subtypes, LAR TNBC is less sensitive to systemic therapy, has lower TILs and lower rates of PD-L1 positivity. The role of other immune related proteins in LAR TNBC is not well established. Here, we evaluated differentially expressed (DE) immune related proteins in the stromal and intratumoral compartments of LAR vs non-LAR TNBC tumors. Methods: We used the Nanostring GeoMX DSP platform to quantitate 58 proteins within spatially distinct intraepithelial, cytokeratin (CK)-positive tumor segments and adjacent CK-negative/nuclei-positive stromal segments in 248 TNBC tumors included in a tissue microarray generated from a cohort of pts with centrally confirmed TNBC who underwent breast surgery without prior neoadjuvant therapy. A subset (n=111) underwent bulk tumor RNA sequencing and were classified as LAR or non-LAR TNBC. DE proteins were identified using a negative binomial generalized linear model (SNR>2, p<0.05). A targeted set of DE proteins was dichotomized at the 80th percentile. Results: Of 111 TNBC tumors, 17 (15%) were LAR and 94 (85%) non-LAR. Compared to non-LAR TNBC, pts with LAR TNBC were older (age ≥50: 82% vs 52%, p<0.01), with tumors that were more often of apocrine histology (35% vs 0%, p <0.01), grade 1-2 (24% vs 1%, p<0.01), and had lower Ki67 (Ki67 ≤15: 24% vs 11%, p=0.06). Most tumors were T1-2 (94% vs 93%, p=0.82) and N0 (53% vs 62%, p=0.09), respectively. As expected, expression of most immune-related proteins was higher in the stromal vs the intratumoral compartment for both LAR and non-LAR TNBC. When focusing on the stromal compartment, expression of multiple immune related proteins was significantly lower in LAR compared to non-LAR TNBC, including the pan-leukocyte marker CD45 (log-2 fold change [log2FC]: 0.552, p=0.05), the macrophage marker CD14 (log2FC: 0.834, p=0.06), CD44 (lof2FC: 0.637, p=0.07), and the immune checkpoint proteins IDO1 (log2FC: 0.914, p=0.04), VISTA (log2FC: 0.471, p=0.07), ICOS (log2FC: 0.444, p=0.08), and STING (log2FC: 0.544, p=0.09). Proteins with expression levels too low for comparisons included PD-L1, LAG3, FOXP3 and BCL-2. When focusing on the intratumoral compartment, expression of most immune-related proteins was very low in both LAR and non-LAR TNBC. Like in the stromal compartment, CD45 expression was lower in LAR TNBC (log2FC: 0.78, p=0.02). Expression of the immune checkpoint B7-H3 was lower in LAR TNBC (log2FC: 0.737, p=0.02), while expression of the T cell marker CD127 was higher (log2FC: -0.528, p=0.34). With regards to relevant non-immune markers, expression of Ki67 was lower in LAR TNBC (log2FC: 0.5498, p=0.05), consistent with the clinical assay. Conclusion: In this ultra high-plex spatial analysis, we provide first insights into the differential expression at the protein level of several targetable immune checkpoint molecules in LAR vs non-LAR TNBC. The lower expression of several immune related proteins in LAR TNBC is consistent with the hypothesis that LAR TNBC exhibits a “cold” immune microenvironment compared to other TNBC subtypes, potentially rendering itself less susceptible to immunotherapy-based strategies. These data support the need to consider TNBC molecular subtypes in future evaluations of immune-based therapeutic approaches. Funding: This work was supported by NIH grant P50CA116201 to RLF, JMC, KRK, FJC, DZ, JNI, and MPG; BCRF grant 19-161 to EAT and NCATS grant CTSA KL2 TR002379 to RLF. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH Citation Format: Roberto A Leon-Ferre, Jodi M. Carter, David M. Zahrieh, David W. Hillman, Saranya Chumsri, Yaohua Ma, Jennifer M. Kachergus, Xue Wang, Judy C. Boughey, Minetta C. Liu, James N. Ingle, Krishna R. Kalari, Jose C. Villasboas Bisneto, Fergus J. Couch, E. Aubrey Thompson, Matthew P. Goetz. Digital spatial profiling of immune-related proteins in luminal androgen receptor (LAR) vs non-LAR triple-negative breast cancer (TNBC) [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 P1-04-01.

Abstract Background: Although major advances have been made in the treatment of HER2+ metastatic breast cancer (MBC), the goal of care remains largely palliative, therefore better treatments are needed. Given encouraging preclinical and clinical data, the combination of cyclin dependent 4/6 kinase inhibitors and HER2-directed therapy is further being evaluated in this trial. Trial Design: In this phase I/II multicenter trial, we will determine the maximum tolerated dose (MTD) of abemaciclib (Abem) combined with T-DM1. Three Abem dose levels will be examined, 50 mg, 100 mg and 150 mg. The phase 2 portion of this trial will examine whether PFS is increased with addition of Abem to T-DM1 in two pt cohorts - those with ER+ HER2+ MBC and those with ER-HER2+ MBC. For phase 2, a pre-registration biopsy is required to confirm ER and HER2-status and to determine levels of tumor infiltrating lymphocytes; vimentin (an epithelial-mesenchymal transition marker); and CD8 and FOXP3 expression. Blood samples will be collected pretreatment , at 6 weeks, and at progression for all pts. Eligibility Criteria: Phase I&II: All pts must have HER2+ MBC per ASCO-CAP guidelines and prior treatment with a taxane, trastuzumab and pertuzumab. For the phase Iportion, pts can have measurable or non-measurable disease with no restriction on the number of prior lines of therapy. In the phase II portion, pts must have measurable disease with ≤1-2 prior lines of chemotherapy alone, ≤1 HER2-directed therapy alone, and/or chemotherapy with HER2-directed therapies. There is no limit on prior endocrine therapy. Specific Aims: The primary objective of the phase II trial is to assess whether addition of Abem to T-DM1 increases PFS in one or both patient cohorts. Secondary objectives include an assessment of toxicity, objective response rates and overall survival. Correlative studies will assess the association between baseline TIL levels, vimentin expression, and CD8/FOXP3 expression with PFS. Changes in peripheral blood mononuclear cells, CTCs, ctDNA and serum thymidine kinase 1 during the course of treatment will be examined to determine if there is a link with PFS outcomes overall and separately for each cohort. Pharmacogenomic studies will determine if pts with the FCGR3A-158 polymorphism derive less benefit from T-DM1 and have inferior PFS outcomes compared with pts who do not have this polymorphism. Statistical Methods:Phase I: Standard 3+3 design, with dose limiting toxicities as per protocol. Phase II: For each pt cohort, a stratified randomization scheme will be used to assign pts to treatment with liver mets as a stratification factor. For each pt cohort, a stratified log rank test will be used to assess whether PFS is increased with the addition of Abem to T-DM1. A non-binding futility analysis will be applied in each cohort after 58 events in the ER+ HER2+ MBC study cohort and 48 events in the ER- HER2+ MBC study cohort. Present Accrual: 0; target accrual: minimal 120 pts., maximal 140 pts CohortOne-sidedalphaPowerAccrual Period (accrual rate)Follow-up after close of enrollmentPFS with T-DM1PFS with abema and T-DM1Number of eligible patientsER+/HER2+0.100.912 months (5-6 pts per month)12 months12 weeks24 weeks64 (32 per arm)ER-/HER2+0.100.8512 months (3-4 pts per month)12 months6 weeks12 weeks50 (25 pts per arm) Citation Format: Ciara C O Sullivan, Jun He, Vera J Suman, Krishna R Kalari, Roberto A Leon-Ferre, Jose C Villasboas-Bisneto, Pratima Chalasani, Demet Gokalp Yasar, Daniel M Anderson, Philip J Stella, Anthony J Jaslowski, Susan H Tannenbaum, Angela Saverimuthu, Donald Northfelt, Alvaro Moreno-Aspitia, Jodi M Carter, Minetta C Liu, Liewei Wang, Zhenkun Lou, Matthew P Goetz. A phase I/II trial of abemaciclib and T-DM1 in women and men with HER2-positive advanced or metastatic breast cancer that has progressed on treatment with a taxane, trastuzumab and pertuzumab (THP) (ACCRU-BR-1801) [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 OT2-19-05.

Abstract Background: More than two thirds of patients with advanced breast cancer (ABC) have estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2−) tumors. In most countries, current standard-of-care first-line treatments include an aromatase inhibitor or fulvestrant, a selective ER degrader, combined with cyclin-dependent kinase 4/6 inhibitors. Concurrent use of luteinizing hormone-releasing hormone agonists is recommended for men and premenopausal women with ABC. Nevertheless, almost all ABCs eventually become resistant to endocrine therapy and the disease is incurable in these cases. New therapies are needed to combat endocrine therapy resistance, maintain patient health-related quality-of-life, and delay the need for chemotherapy. AZD9833 (camizestrant) is a highly potent, next-generation selective ER degrader and pure ER antagonist that has demonstrated anticancer properties across a range of preclinical models, including those with ER-activating mutations (Scott et al, AACR 2020). A Phase I dose-escalation and expansion study (SERENA-1) has demonstrated that AZD9833 is well tolerated and has a promising antitumor activity when administered alone or in combination with the cyclin-dependent kinase 4/6 inhibitor palbociclib (Baird et al, SABCS 2020). SERENA-4 (NCT04711252) is a randomized, multicenter, double-blind, Phase III trial to evaluate the safety and efficacy of AZD9833 in combination with palbociclib for patients with ER+/HER2− ABC who have not received systemic treatment in the advanced disease setting. Methods: SERENA-4 will enroll 1342 patients with de novo or recurrent ER+/HER2− ABC who have not previously received systemic treatment for their locoregionally recurrent or metastatic disease. Patients with recurrent disease must have received adjuvant aromatase inhibitor or tamoxifen therapy for at least 24 months without relapse. Patients will be randomized 1:1 to receive oral treatment with either (a) AZD9833 75 mg once daily, palbociclib 125 mg once daily for 21 days followed by 7 days off treatment and a placebo for anastrozole 1 mg once daily or (b) anastrozole 1 mg once daily, palbociclib (same as active arm) and a placebo for AZD9833 75 mg once daily. Men and premenopausal women will also receive a luteinizing hormone-releasing hormone agonist. The primary endpoint is progression-free survival (PFS; up to 5 years). Secondary endpoints include overall survival (up to 8 years), second PFS, time to chemotherapy, objective response rate, and changes in health-related quality-of-life measures. Enrollment began in January 2021. As of 02 July 2021, the number of open sites is 57 across 15 countries. Acknowledgments: We thank Julia Mawer, PhD, of Oxford PharmaGenesis, Oxford, UK, for providing medical writing support funded by AstraZeneca Funding: The SERENA-4 trial is funded and overseen by AstraZeneca. © 2021 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2021 ASCO Annual Meeting. All rights reserved. Citation Format: Fabrice André, Seock-Ah Im, Patrick Neven, Richard D Baird, Johannes Ettl, Matthew P Goetz, Erika Hamilton, Hiroji Iwata, Zefei Jiang, Anil Abraham Joy, Vincent Haddad, Andrew Walding, Manuel Selvi Miralles, Cynthia Huang Bartlett, Antonio Llombart-Cussac. SERENA-4: A Phase III comparison of AZD9833 (camizestrant) plus palbociclib, versus anastrozole plus palbociclib, for patients with ER-positive/HER2-negative advanced breast cancer who have not previously received systemic treatment for advanced disease [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 OT2-11-06.