Negative Metastatic Breast Cancer Patients Research Articles

(CDK) 4/6 inhibitors: An effective treatment for homologous recombination deficient (HRD) luminal breast cancers (BC)?

e13100 Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) still represents the best 1st-line choice for hormone receptor (HR) positive(+)/HER2 negative(-) metastatic breast cancer (MBC) patients (pts). Approximately 8% of pts with HR+ BC harbors germline BRCA mutation (g BRCAmt). The interaction between Cyclin/CDK pathway, endocrine receptor (ER) and Breast Cancer gene 1/2 (BRCA1/2) is complex and evidence reported so far, await confirmation in the context of future randomized clinical trials. So far, limited data exist about treatment effectiveness of CDK4/6i in homologous recombination deficiency (HRD) Luminal BC. A current frequent question in clinical practice is which additional adjuvant therapy to prioritize for luminal BRCA tumors (tu), considering the approval of both CDK4/6i andpoly ADP ribose polymerase (PARP) inhibitors in this scenario. Considering potential impacts of genetic instability in tu biology, such as potential loss of Rb protein related to Loss of Heterozygosity (LOH) of BRCA2 as a primary resistance mechanism in CDK4/6i, we sought to analyze the clinical results of that drug in tu with genetic instability. Methods: We retrospectively analyzed real-world outcomes of CDK4/6i performance in HR+/HER2- MBC treated in a single institution, with focus in clinical outcomes of HRD BC. germline HR genes status and somatic phosphatidylinositol 3-kinase (PIK3CA) status were also analyzed. Results: A total of 384 consecutive MBC pts treated with CDK4/6i were included in this study. The most used CDK4/6i was palbociclib (40,6%), followed by ribociclib (29,8%) and abemaciclib (29,2%). Considering the whole cohort PFSm 30months (95%IC 26-34) and OSm were 56 months (95%IC 52-60) No differences in PFS or OS was observed among the 3 CDK4/6i. Median age of the cohort was 60 years old. 38.5% were premenopause Pts and 59.9% were postmenopause. A total of 149 (39%) pts performed germline screening. Between 19 pts with gHR mt, 12 were BRCA1/2 mt (just one BRCA1). The other were: 2 PALB2, 2 CHEK2, 1 ATM, 1 RAD51D, 1 RAD51C mt. So far, no differences in PFS ou OS was observed in the presence of BRCA 1/2 or other gHR mt. Nevertheless, PIK3CA mt was tested in 142 pts, 51 were positives. That mt was a predictor of worse OS (55m; 95%IC 45-65) x 63m (95%IC 56-69), p 0,019. Conclusions: The results of this real-world study suggest that treatment outcomes with CDK4/6i may be worse in pts with HRD tu due to potential differences in tu biology. Future exploration in larger samples of patients who have had biomarker testing is ongoing. Patients with Pik3CA mt treated with CDK4/6i presented worse clinical outcomes.

Prognostic and Predictive Significance of HER2-low Expression in Metastatic Hormone Receptor Positive Breast Cancer Patients Receiving CDK4-6 Inhibitor Therapy

Objective: This study aimed to analyze the predictive and prognostic value of HER2-low expression in hormone receptor (HR) positive human epidermal growth factor receptor-2 (HER2) negative metastatic breast cancer patients receiving cyclin-dependent kinase-4/6 inhibitor (CDK4/6i) therapy. Methods: This retrospective study included patients who received CDK4/6i plus endocrine therapy (ET). The pathological and clinical characteristics and survival times of the patients were compared and analyzed. Results: Our study included 122 patients. There were HER2-zero 88(72%) and HER2-low 34 (28%) patients. The median progression free survival (mPFS) of all patients who received CDK4/6i+ET was 21 (95% confidence interval (CI),18.5–23.5) months, while mPFS was not reached in the HER2-zero group, and mPFS in the HER2-low group was 12 (95%CI, 6.8–17.1) months (p=0.001). The mPFS was shorter in patients with primary endocrine resistance (6 vs. 21 months, p=0.001). There was a change in the HER2-low status of 26(45%) patients with recurrence compared to the first biopsy. In the HER2-zero and HER2-low groups, 22(25%) and 24(71%) patients, respectively, progressed with CDK4/6i+ET (p=0.001). Estrogen receptor (ER) levels less than and greater than 50% resulted different mPFS (6 and 21 months, respectively) (p=0.025). Median PFS differed based on CDK4/6i+ET combination, treatment line, and best treatment response (all p=0.001). In multivariate analysis, HER2 status(p=0.018), chemotherapy status(p=0.006), best response status with CDK4/6i (p=0.001) for PFS, and best response status with CDK4/6i therapy (p=0.007) for OS were significant. Conclusions: In patients with HR+HER- metastatic breast cancer receiving CDK4/6i therapy, the duration of mPFS was lower in the HER2-low group than that in the HER2-zero group. HER2-low expression is a predictive biomarker of response to CDK4/6 inhibitor therapy.

Clinicopathological Evaluation of Patients with Hormone Receptor–Positive HER2-Negative Metastatic Breast Cancer Progressing on Endocrine Treatment: A Real-World Retrospective Study from a Regional Cancer Center

Metastatic breast cancer (MBC) is an incurable disease with the primary aim of treatment being the improvement of the patient's quality of life and the delay of disease progression. A substantial proportion of patients with hormone receptor (HR)-positive MBC eventually experience progression despite endocrine treatment. As endocrine resistance remains a significant challenge, we aim to comprehend the intricate relationship between clinicopathological characteristics and the utility of various parameters as predictive markers for hormonal treatment response. This study, conducted at a single center, is ambispective in nature and includes hormone receptor (HR)-positive, human epidermal growth factor 2–negative MBC patients who progressed while on endocrine treatment, selected through purposeful sampling. Nominal data were analyzed in terms of frequency distribution, and continuous variables were represented as median/mean ± standard deviation. Spearman's correlation test and chi-square test were employed to examine variable dependencies. Data comparisons were performed using the independent t-test, one-way analysis of variance, or Mann–Whitney's test. The majority of our study participants (n = 44, 64.70%) presented with de novo metastasis, while the remainder (n = 24, 35.29%) were patients who progressed from early-stage breast cancer to metastasis. The overall mean age of our study population at presentation was 47 ± 11 years. Patients with upfront stage 4 tumors presented at an older age, exhibited grade 2 tumors, had a higher frequency of bone-only metastasis, and experienced longer progression-free survival (PFS) compared to patients who progressed from the early stage to metastasis. Multiple visceral involvements had a significant negative impact on PFS in contrast to cases with single visceral or bone-only involvement. No significant associations with PFS were observed for the Ki-67 index, first-line chemotherapy, or endocrine therapy. The extent of metastasis to various organs emerged as the most influential factor in determining PFS. Consequently, we propose the necessity for larger prospective studies aimed at identifying superior or additional biomarkers.

Efficacy of anlotinib in Chinese patients with metastatic breast cancer: A retrospective observational study.

Anlotinib, a multitarget tyrosine kinase inhibitor, can inhibit tumour angiogenesis proliferation, metastasis, promote vascular normalization, increase T cell and NK cell activity and infiltration, remodel tumour microenvironment and synergistic immune enhancement. Our study aimes to evaluate the efficacy of anlotinib in the treatment of advanced metastatic breast cancer (MBC) after multiple lines of therapy. Patients included were treated with anlotinib for advanced MBC in the Affiliated Cancer Hospital of Zhengzhou University from 1 January 2019 to 30 June 2023. The objective remission rate, disease-free progression survival and adverse reactions were analysed. We compared and analysed the efficacy of anlotinib in the treatment of advanced metastatic breast cancer, which showed that ORR was 23.6% and DCR was 69.1%. The DCR of monotherapy was 66.7% and that of combination therapy was 69.6% in MBC patients. The combination therapy, combined with chemotherapy had the best effect (79.3%), combined with immunotherapy came second. In addition, the DCR (88.9%) was higher in MBC patients having received prior antiangiogenic therapy. According to the Kaplan-Meier (K-M) survival estimate analysis, the mPFS was 4.17 months (95% CI, 1.758-6.582 months) in Her-2 positive MBC patients, and 7.83 months (95% CI, 2.416-9.104) in Her-2 negative MBC patients. The mPFS was 5.76 months (95% CI, 3.231-8.298 m) in HR positive MBC patients, 7.83 months (95% CI, 3.182-12.478 m) in TNBC patients. Fatigue (20.0%), hypertension (21.8%) and liver dysfunction (18.2%) were common adverse reactions, followed by bone marrow suppression (16.4%), anorexia (14.5%), hypothyroidism (14.5%) and diarrhoea (14.5%). Altogether, Anlotinib monotherapy or combination therapy provides a viable third (or above)-line therapeutic strategy in patients with metastatic breast cancer. The adverse reactions of anlotinib are well tolerated and controllable.

Late onset toxicities associated with the use of CDK 4/6 inhibitors in hormone receptor positive (HR+), human epidermal growth factor receptor-2 negative (HER2-) metastatic breast cancer patients: a multidisciplinary, pan-EU position paper regarding their optimal management. The GIOCONDA project

The personalization of therapies in breast cancer has favoured the introduction of new molecular-targeted therapies into clinical practice. Among them, cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have acquired increasing importance, with the approval in recent years of palbociclib, ribociclib, and abemaciclib in combination with endocrine therapy. Currently, no guidelines are available to monitor and manage potential long-term toxicities associated with the use of these drugs. A multidisciplinary panel of European oncologists, was supported by a pharmacologist, a hematologist, a hepatologist and a pulmonologist to discuss the management of long-term toxicities, based on the literature review and their clinical experience. The panel provided detailed roadmaps to manage long-term toxicities associated with the use of CDK4/6 inhibitors in clinical practice. Knowing the frequency and characteristics of the toxicity profile associated with each CDK4/6 inhibitor is important in the decision-making process to match the right drug to the right patient.

Tumor-agnostic ctDNA levels by mFAST-SeqS in first-line HR-positive, HER2 negative metastatic breast cancer patients as a biomarker for survival

This prospective cohort study reports aneuploidy score by mFast-SeqS as a strong prognostic marker in MBC patients. mFAST-SeqS is an affordable and easily implementable method for the assessment of total ctDNA levels and, as such, provides an alternative prognostic tool. One mixed cohort (cohort A, n = 45) starting any type of treatment in any line of therapy and one larger cohort (cohort B, n = 129) consisting of patients starting aromatase inhibitors (AI) as first-line therapy were used. mFAST-SeqS was performed using plasma of blood in which CTCs (CellSearch) were enumerated. The resulting aneuploidy score was correlated with categorized CTC count and associated with outcome. The aneuploidy score was significantly correlated with CTC count, but discordance was observed in 31.6% when applying cut-offs of 5. In both cohorts, aneuploidy score was a significant prognostic marker for both PFS and OS. In the Cox regression models, the HR for aneuploidy score for PFS was 2.52 (95% CI: 1.56–4.07), and the HR for OS was 2.37 (95% CI: 1.36–4.14). Results presented here warrant further investigations into the clinical utility of this marker in MBC patients.

Abstract P4-01-13: A retrospective study of anlotinib in patients with HER-2 negative metastatic breast cancer after prior two or more lines treatment

Abstract Background: Anlotinib is a novel small molecule antiangiogenic drug that can inhibit multiple tyrosine kinase receptor activities. It has demonstrated antitumor activity in various cancers. This retrospective study explored the efficacy and safety of anlotinib combined chemotherapy in HER-2 negative metastatic breast cancer patients who have disease progressed after prior two or more lines of therapy. Methods: This retrospective study enrolled twenty-two HER-2 negative metastatic breast cancer patients including TNBC and HR+ subtypes. These patients pretreated with at least two lines of chemotherapy and/or endocrine drugs before receiving anlotinib. These patients received anlotinib combination with investigator-selected chemotherapy agents, including docetaxel, vinorelbine, gemcitabine, etoposide, alibrine and so on. Anlotinib was administered 12mg d1-d14 for a 21-day cycle. The primary end point of this study was progression free survival (PFS). Secondary end points included objective response rate (ORR), disease control rate (DCR), overall survival (OS), and toxicity. The treatment continued until disease progression or intolerability or the patient withdraw informed consent. Results: The enrolled breast cancer patients received anlotinib with a median age of 52.5 (rang 30 to 73). The patients with multiple metastases were 59.1% (13/22). All patients were evaluated and the ORR was 50% (95% Cl 0.282-0.718), DCR 95.5% (95% Cl 0.772-0.999) and the mPFS was 8 months (95% Cl 5.15-10.85). We also analyzed the efficacy of anlotinib combination with chemotherapy in different subtypes. For HR+HER2- metastatic breast cancer patients, the ORR was 77.8% (95% Cl 0.4-0.972), DCR 100% (95% Cl 0.664-1.0) and the mPFS was 8.5 months (95% Cl 7.217-9.783). And for TNBC patients, the ORR was 30.8% (95% Cl 0.091-0.614), DCR 92.3% (95% Cl 0.640-0.998) and the mPFS was 6 months (95%Cl 4.405-7.595). The most common adverse events were grade 1/2 including fatigue, hand-foot syndrome and thrombocytopenia et.al., and no serious adverse events occurred. Conclusion: Anlotinib is safe and effective in patients with HER-2 negative metastatic breast cancer after prior two or more lines treatments, and it is well tolerated. Citation Format: Jing Sun, Yijun Tang, Xiaohui Liu, Jiangli Li, Jin Xia, Liang xu, Junlan Guo, Jing Wang, Shengnan Guo, Zhaojie Sheng, Yanfang Zhang, Yuan Yuan. A retrospective study of anlotinib in patients with HER-2 negative metastatic breast cancer after prior two or more lines treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-13.

Abstract P3-05-45: Circulating tumor cells, immunohistochemical subtypes, and genes mutation as prognostic markers in HER2 negative metastatic breast cancer patients candidates to chemotherapy

Abstract Background: In the era of targeted therapies, chemotherapy (CT) is still a valuable treatment option for patients (pts) with HER2 negative metastatic breast cancer (MBC). The identification of predictive and prognostic markers might improve treatment response and survival. Here we investigated the role of tumor subtypes, circulating tumor cells (CTCs), and mutations in genes or pathways of interest, in predicting response to CT and prognosis of HER2 negative MBC pts within AARES trial. Methods: AARES is an open label multicentric randomised phase 2 trial comparing a DNA-damaging (arm A: cisplatin 25 mg/m2/day, day 1-3 + cyclophosphamide 600 mg/m2 day 1) versus (vs) a non-DNA-damaging (arm B: capecitabine 1000 mg/m2 bid/day, day 1-14 + vinorelbine 60 mg/m2/day, day 1,8) CT regimen in pts with HER2 negative MBC. Archival tumor tissue samples and blood samples were collected at baseline. The Cell SearchTM system was used for CTCs isolation and enumeration and a cut-off of 5 CTC/7,5 ml was used. Tumor subtypes were based on locally assessed hormone receptors and HER2 status. The luminal-like subtype (Lum) was defined by ER or PR positivity, while the triple negative subtype (TN) by ER, PR, and HER2 negativity. Tissue samples were used for DNA extraction and next generation sequencing analysis using a target enrichment panel of 170 genes (TruSight 170, Illumina). Primary endpoint was objective response rate (ORR) assessed for the two treatment arms. Progression free survival (PFS) and overall survival (OS) were evaluated as secondary endpoints, and estimated with the Kaplan-Meier method. ORR, PFS and OS were correlated with CTC counts, tumor subtypes, and mutational status of genes/pathway of interest (PIK3CA, TP53, BRCA1, BRCA2, PI3K/AKT pathway (PI3K/AKT)). Results: AARES enrolled 102 pts from 2011 to 2016 across 9 Italian centers. Of these, 77 pts were evaluable for ORR. Median follow up was 32 months. Overall, median age was 57. The majority of pts had Lum tumors (73%), while 27% had TN. 86% of pts had visceral metastases, and 52% had 3 or more metastatic sites. 48% of pts received the study treatment as 1st line, 32% as 2nd line, and 19% as 3rd line. Out of the 77 pts with ORR data, 41 (53%) were CTC- and 36 (47%) CTC+. A larger proportion of pts with Lum tumors were CTC+ (54% n=30), while TN tumors were mainly CTC- (71% n=15). 49 pts had adequate tumor tissue for sequencing analysis. TP53 mutations (mut) were found in 33% of pts, BRCA1/2 mut in 14%, PIK3CA mut in 35%, and PI3K/AKT mut in 47%. ORR was 24% and 37% in arm A vs arm B, respectively (p= 0.2), and no difference in ORR was observed by tumor subtypes (Lum vs TN), and CTCs count (+ vs -). PFS and OS were assessable on the whole population (n=102) and did not significantly differ by treatment arms. Median OS (mOS) was higher in Lum pts compared to TN (24 months (mo) vs 15.4 mo, p=0.048) while median PFS (mPFS) did not differ according to tumor subtypes (p=0.28). Overall, CTCs count was not significantly associated with mPFS or OS however in pts with TN tumors and CTC-, mPFS and mOS were significantly longer (mPFS: 4.44 mo vs 2.56 mo in CTC- and CTC+ subgroups, respectively, p=0.00087 and mOS: 22.07 mo vs 3.57 mo in CTC- and CTC+ subgroups, respectively, p< 0.0001). On the other hand, no differences were observed in Lum pts categorized according to CTC status (p=0.83 for mPFS, p=0.54 for mOS). Finally, pts with PI3K/AKT mut had a significantly worse PFS compared to wild type in the overall population (p=0.0091) as well as in Lum (p=0.034) and TN pts (p=0.0052). Conclusions: CTCs and tumor subtypes were not predictive of response to CT regimens in HER2 negative MBC pts. A number of CTC >5 in TN MBC, and mutations in the PI3K/AKT pathway, identified a subgroup of pts with worse prognosis, potentially candidates for alternative treatments. Further studies are needed to confirm these results in a larger population. Citation Format: Emanuela Risi, Marta Pestrin, Chiara Biagioni, Dario Romagnoli, Ilenia Migliaccio, Francesca Galardi, Francesca De Luca, Matteo Benelli, Erica Moretti, Giuseppina Sanna, Luca Livraghi, Silvia Cappadona, Roberta Di Marsico, Domenico Amoroso, Angelo Martignetti, Angela S. Ribecco, Elena Rota Caremoli, Luigi Coltelli, Fabio Puglisi, Luca Malorni, Laura Biganzoli. Circulating tumor cells, immunohistochemical subtypes, and genes mutation as prognostic markers in HER2 negative metastatic breast cancer patients candidates to chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-45.

Abstract P3-05-10: Disease features, genomic profiles and outcomes of younger vs. older Chinese hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients

Abstract Background: Breast cancer in younger women has been characterized as a more aggressive disease with relatively poor outcomes compared to breast cancer in older women. This pattern has been attributed to a variety of clinicopathologic characteristics including the enrichment of aggressive HER2+ and TNBC subtypes in younger patients. However, similar to older breast cancer patients, the majority of young breast cancer patients are HR+/HER2-. Here we have evaluated the clinicopathologic characteristics and genomic profiles of real-world HR+/HER2- MBC patients to examine the determinants of outcome for younger vs. older patients in this subtype. Patients and Methods: This study included 65 Chinese patients presenting with HR+/HER2- MBC at the Peking University Cancer Hospital. Blood samples were collected upon metastatic disease diagnosis before treatment and profiled with the targeted 152-gene PredicineCARETM sequencing panel. Fourteen patients were < 40 years, 19 were 40-50 years, and 32 were > 50 years at the time of primary cancer diagnosis. Kaplan-Meier survival analysis was performed to analyze outcomes including overall survival (OS), disease free survival (DFS) and progression free survival (PFS) in association with clinicopathologic and genomic variables. OS was also evaluated in association with age in separate multivariate models using Cox proportional hazards regression. Results: Significant variation across age groups was observed for several clinicopathological features, including molecular subtype classification (p = 0.045), de novo Stage IV disease (p = 0.011), type of adjuvant endocrine therapy (p = 5.56E-06), resistance to adjuvant endocrine therapy (p = 0.015) and receipt of adjuvant chemotherapy (p = 0.042). Luminal B status was most frequent in patients < 40 years (92.31%), whereas de novo Stage IV disease was more prevalent in patients > 50 years (37.5%). Aromatase inhibitors (AIs) were administered as adjuvant endocrine therapy (ET) to 73.68% of women > 50 years vs. 16.67% and 0% of women aged 40-50 years and < 40 years, respectively. Resistance to adjuvant ET was observed in 23.08% of patients < 40 years but was not observed in the other age groups. Adjuvant chemotherapy was received by a higher proportion of patients in the 40-50 year age group (94.44%) compared to patients < 40 years (69.23%) and patients > 50 years (60%). No differences in somatic gene alteration frequencies were observed across age groups, menopausal status, germline mutation status or tumor grade. However, a higher frequency of FGFR1 alterations was observed in patients classified as Luminal B vs. A (p = 0.048). Comparison of profiles across women who received adjuvant ET revealed a higher prevalence of FGFR1 (p = 0.012), ATM (p = 0.047) and CCND2 (p = 0.04) alterations in patients treated with AIs vs. SERMS. In addition, a higher frequency of APC alterations was observed in patients with high (≥ 20%) vs. low (< 20%) Ki67 index (p = 0.035). At the univariate level OS was significantly associated with age (p = 0.039). OS was shortest in patients > 50 years, intermediate in patients < 40 years, and longest in patients 40-50 years. Across all patients, shorter OS was also associated with de novo Stage IV disease (p = 0.0001), Luminal B subtype (p = 0.008), adjuvant ET with AIs vs SERMS (p = 0.028) and the presence of an FGFR1 (p = 0.028) or CCND2 (p = 0.033) alteration. In multivariate analyses, OS remained significantly longer for patients aged 40-50 years after adjustment for de novo Stage IV disease, Luminal B subtype and FGFR1 status (all p < 0.05). Conclusions: In this group of real-world HR+/HER2- MBC breast cancer patients, younger age was not associated with worse outcomes. While current guidelines recommend treatment decisions based on tumor biology rather than age, young HR+ breast cancer patients are more likely to receive chemotherapy. Our findings support the development of biomarker-driven treatment strategies for these patients. Citation Format: Jinhao Wang, Yaxin Liu, Bin Shao, Hang Dong, Tiantian Zheng, Pan Du, Shidong Jia, Bonnie King, Jing Wang, Xiaoran Liu, Huiping Li. Disease features, genomic profiles and outcomes of younger vs. older Chinese hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-10.

Abstract P5-02-50: Do thymidine kinase 1 (TK1) plasma concentration and activity play a role in therapy management of metastatic breast cancer patients treated with CDK4/6 inhibitors?

Abstract Introduction: CDK4/6 is a checkpoint kinase, regulating the transition of the S into the G2 phase of the cell cycle. As a cell transverses through the cell cycle, thymidine kinase 1 (TK1) is expressed and represents a direct marker of proliferative activity.which might indicate therapy efficiency of CDK4/6 inhibitors (CDK4/6i), the first choice of therapy in case no sign of visceral crisis is detected in hormone receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients. We here determined whether the activity and/or concentration of TK1 in plasma samples of MBC patients treated with CDK4/6i harbours predictive, monitoring or prognostic value. Methods: Blood of 90 HR+/HER2-MBC patients drawn at baseline of CDK4/6i (Ribociclib/Palbociclib) plus endocrine treatment and 18 HR+/HER2-MBC patients drawn before initiation of endocrine monotherapy (control), as well as available matched blood samples of these patients after six months under TX (n=90), 12/24 months before progression (n=72/24) and at the time of progression (n=52) are available. TK1 concentration in plasma samples was measured by competitive ELISA (ABIN809094, Shanghai BlueGene Biotech Co., LTD, China) and TK1 activity of matched plasma samples by competitive, two-step chemiluminescence immunoassay (REF 310960, Diasorin, Stillwater, US). Statistical analysis was conducted via log-rank test and univariate or multivariate Cox regression. Results: Currently, baseline samples were evaluated for TK1 concentration (n=106) and activity (n=90). The mean value for TK1 activity was 12.69 U/L and the median value was 8.37 U/L in the entire cohort. Values ranged from 0.89 to 79.50 U/L and the standard deviation was determined to be 12.64 U/L, which is similar to the mean value. In contrast to the control group, high TK1 activity (Cut-off: mean or determined by ROC analysis and maximal Youden’s Index for PFS < six months) was significantly correlated with decreased progression free survival (PFS) in the CDK4/6i cohort (p-value < 0.05 in log rank test and univariate Cox regression). In addition, using multivariate Cox regression analysis including clinical parameters (e.g. therapy line, prior chemo- or endocrine therapies, type of CDK4/6i accompanying endocrine therapy, number of sites of metastases, visceral metastases, menopausal status), TK1 activity was found to be an independent marker for PFS.High TK1 activity (Cut-off: mean or determined by ROC analysis and maximal Youden’s Index for PFS < six months or already deceased patients) at baseline was significantly correlated with shorter time from baseline to death in the CDK4/6i cohort (p-value < 0.05 in log rank test and univariate Cox regression) but not in the control cohort. For TK1 concentration analysis, the mean value was 21.76 ng/ml and the median value was 18.57 ng/ml in the entire cohort. Values ranged from 7.35 ng/ml to 46.66 ng/ml and the standard deviation was determined to be 11.16 ng/ml, which is half of the mean value. In contrast to TK1 activity measurements, no significant association of TK1 concentration with PFS or overall survival (OS) was detected in the CDK4/6i cohort. Currently, all remaining samples during the course of treatment are evaluated for TK1 activity and concentration and final statistical analysis of the complete data set will be available at the meeting. Conclusion: Our preliminary results indicate that TK1 activity, in contrast to TK1 concentration, determined before starting CDK4/6 inhibtion could be a suitable predictive and prognostic biomarker for estimating response to treatment. Our final analysis will clarify, whether TK1 could also serve as a monitoring marker during treatment. Citation Format: Stefanos Moukas, Merle Dohn, Rainer Kimmig, Mitra Tewes, Hans-Christian Kolberg, Sabine Kasimir-Bauer, Corinna Keup. Do thymidine kinase 1 (TK1) plasma concentration and activity play a role in therapy management of metastatic breast cancer patients treated with CDK4/6 inhibitors? [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-50.

Abstract P4-01-41: Clinical outcome and toxicity profile of Cyclin-dependent kinase 4/6 inhibitors in combination with hormonal treatment in management of metastatic breast cancer patients: A Middle-East Real World Experience

Abstract Background: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors in combination with hormonal treatment is the standard of care in the management of hormone receptor positive, human epidermal growth factor receptor-2 (Her2) negative metastatic breast cancer (MBC) patients. Data coming from the Middle east region is very rare and our patients’ population were poorly represented in the CDK4/6 inhibitors landmark clinical trials, This retrospective study is to evaluate our real-world experience with this treatment combination in our patients’ population. Patients and methods: A retrospective study was conducted in a middle eastern tertiary institution, where the clinical data of treatment outcome and toxicity were collected from the electronic medical records of 164 patients with hormone receptor positive, Her2 negative MBC who were managed with CDK4/6 inhibitors in combination with hormonal treatment, at and beyond first line of treatment. Progression free survival (PFS) was the study primary objective, Objective response rate (OR), Overall survival (OS), and toxicity profile were the secondary objectives. Results: From January 2017 to April 2021, 164 patients (5 men and 159 women) were managed with CDK4/6 inhibitors in combination with hormonal treatment for MBC, median age was 52 years (IQR: 45-59), 63% of patients were postmenopausal, 60% had luminal type B tumors, 22% had Her2 expression of 2+ by IHC, 55% had de Novo metastases, 67% had visceral metastases, 61% defined to have endocrine resistant disease (13% primary and 48% secondary resistance), CDK inhibitors (46% Palbociclib, 54% Ribociclib) were given as 1st, 2nd and beyond 2nd line in (48%, 28% and 24% respectively), at a median duration of follow up of 25.3 months (IQR: 13.8-33.9 months), the median PFS for the overall group was 14.2 months (CI 09.8-18.7), it was highest for 1st line treatment 19 months vs 15 and 5 months for the 2nd and beyond 2nd line treatment respectively. The median overall survival was 51.6 months (CI 35.8-67.5) for the whole group, 50.3 and 51.6 months for 1st and 2nd line vs 33.1 months for beyond 2nd line treatment. 5% of patients had achieved complete response, 37% partial response, and 24% had stable disease, with a clinical benefit rate of 74%. A body mass index (BMI) < 30, KI67 < 20%, luminal type A, de Novo metastases, Ribociclib as a CDKi, Aromatase inhibitor (AI) as the hormonal partner, and introducing the treatment combination in early treatment lines (1st and 2nd lines) were all associated with significantly higher PFS in univariate analysis. BMI and AI were the only factors associated with significantly higher PFS in multivariate analysis. Efficacy was consistent regardless of the menopausal status, type of endocrine resistance, and site of metastases. The most common toxicities were neutropenia, fatigue, vomiting, diarrhea, QTC prolongation and hepatic toxicity, the most common G3&4 toxicity were Neutropenia 56%, hepatic toxicity 5.4% and QTC prolongation in 4.3%. Treatment was permanently stopped because of toxicity in 12 patients (7.3%). Conclusion: Our real-life data came consistent with the pivotal clinical trials’ results, encouraging and supporting the use of CDK 4/6 inhibitors in combination with hormonal treatment in our patient population with metastatic breast cancer, especially in the early treatment lines, deferring the start of more toxic therapies to a later stage. Citation Format: Ahmed Mostafa Gad, Adhar AlSayed, Aisha AlShibani, Taher Twegieri, Kausar Suleman, Dahish Ajarim. Clinical outcome and toxicity profile of Cyclin-dependent kinase 4/6 inhibitors in combination with hormonal treatment in management of metastatic breast cancer patients: A Middle-East Real World Experience. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-41.

Abstract OT3-11-01: TK IMPACT: Treatment Monitoring of Hormone Receptor Positive (HR+), HER2 Negative (HER2-) Metastatic Breast Cancer (MBC) Patients Receiving CDK 4/6 Inhibitors (CDK4/6i) with DiviTum® Thymidine Kinase 1 Activity

Abstract Background: CDK 4/6i have altered the therapeutic landscape of HR+, HER2- MBC, improving progression free and overall survival (PFS and OS) compared to endocrine therapy (ET) alone. Despite durable responses to CDK 4/6i in a large majority of patients, treatment response monitoring in this population has historically included numerous serial blood-based and imaging studies at frequent time points. There is a growing global interest in utilizing novel non-invasive biomarker-driven disease monitoring assessments to improve patient outcomes and reduce health care costs. Thymidine kinase 1 (TK1), a key cell-cycle regulated enzyme important for nucleotide metabolism during DNA synthesis, is regulated by the E2F pathway, downstream of CDK 4/6. Studies have shown that DiviTum® TK1 activity (TKa) may serve as both a prognostic and predictive biomarker of CDK 4/6i treatment response (McCartney et al, Clin Canc Res, 2020; Malorni et al, Eur J Cancer, 2022; Bagegni et al, Breast Cancer Res, 2017). Early TKa suppression within 2 weeks (wk) post CDK 4/6i therapy initiation is associated with improved PFS, suggesting a subgroup of patients who may be able to de-escalate imaging frequency. Elevated TKa at baseline and post CDK 4/6i may identify patients with CDK 4/6i-resistant disease and disease progression (PD) requiring early therapy modification. TK IMPACT is a prospective, single-arm trial designed to assess the impact of incorporation of DiviTum® TKa on a physician’s decision regarding subsequent timing of routine disease monitoring modalities in patients with advanced HR+, HER2- MBC receiving ET plus CDK 4/6i (NCT04968964). Methods: Blood sample collections will be analyzed using DiviTum® TKa at baseline (bl), wk 2, 4, 6, 8, and Q 4 wks thereafter beginning at wk 8 during the first 24-wk time period of study enrollment (+/- 3 days); followed by Q 12 wks thereafter, until PD or 36 months, whichever occurs first. Optional repeat TKa within 2-4 wks (+/-3 days) is permitted in case of rising TKa. Research blood (bl, wk 2, 12, 24, 48, and PD) and optional archival tumor tissue collection at diagnosis and PD will be obtained for correlatives. The investigator will record intended imaging modalities and timing prior to receipt of TKa, followed by documentation of any changes in imaging testing interval after receipt of TKa. Key eligibility criteria include postmenopausal women age ≥18 years with HR+, HER2- MBC, to initiate (Cohort 1) or are currently receiving (≤24 months, Cohort 2) any FDA approved first line ET plus CDK 4/6i with a life expectancy > 6 months. The primary endpoint is any physician-reported intended change in imaging testing interval post TKa by study cohort, within the first 48-wk period of study participation. Key secondary endpoints are concordance rate between TKa values and progression status at first on-study imaging and longitudinal TKa dynamics. Key exploratory endpoints include plasma and tumor tissue-based biomarkers of CDK 4/6i response and resistance. A total of 40 patients will be enrolled (n=20/Cohort). The expected change rate is 20% with a 95% Wilson confidence interval of 0.105~0.248 across all patients and if within each cohort, with a 95% Wilson confidence interval of 0.081~0.416 for N=20. N=40 allows the lower limit of the 95% CI > 10% and that of the N=20 in Cohort 1 to be ~10%, indicating some clinically meaningful influence of TKa progression on patient management. The study is open to accrual and has presently enrolled 5 patients. Citation Format: Nusayba A. Bagegni, Isabella Grigsby, Leslie Nehring, Jingqin Luo, Jennifer Powers Carson, David W. Gibson, Meghan Horvath, Katherine K. Clifton, Foluso O. Ademuyiwa, Rama Suresh, Ashley Frith, Andrew A. Davis, Lindsay L. Peterson, Ron Bose, Amy Williams, Mattias Bergqvist, Cynthia Ma. TK IMPACT: Treatment Monitoring of Hormone Receptor Positive (HR+), HER2 Negative (HER2-) Metastatic Breast Cancer (MBC) Patients Receiving CDK 4/6 Inhibitors (CDK4/6i) with DiviTum® Thymidine Kinase 1 Activity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-11-01.

Abstract OT2-01-01: Trial in Progress: Clinical Utility of Fluoroestradiol F18 PET/CT in Metastatic Breast Cancer Patients with ER-Positive and HER2-Negative Primary Lesions after Progression on First Line Hormonal Therapy

Abstract BACKGROUND: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors combined with endocrine therapy are the recommended first-line standard-of-care (SOC) treatment for estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) patients. After progression on first-line treatment, current clinical practice guidelines (NCCN, ESMO) recommend to sequence endocrine therapy (in absence of visceral crisis) until there is no clinical benefit after up to 3 regimens, and subsequently initiate chemotherapy regimens. However, clinical benefit for endocrine monotherapy after progression on first-line standard-of-care is limited, with median progression-free survival (PFS) of 2-5 months. Multiple factors can contribute to endocrine therapy resistance, including changes in ER expression, (epi)genetic mutations, and/or clonal selection under therapeutic pressure. ER heterogeneity can occur over time (temporal) as well as between lesions in a patient (spatial). As confirmed by tissue biopsy, greater than 20% of MBC patients have discordance in ER expression between their primary tumor and a metastatic lesion. Measuring total ER heterogeneity in a patient – the variation in ER expression at whole body level across all MBC lesions – is not clinically feasible by tissue biopsy alone. Fluoroestradiol F18 (Cerianna), also called 18F-FES, is a radioactive diagnostic agent that can be used with PET/CT for in vivo detection of ER-positive lesions in MBC with high accuracy. Results from 18F-FES PET/CT studies have shown that ER expression across MBC lesions at whole body level is heterogeneous in approximately half of ER-positive, HER2-negative MBC patients. In this study, we aim to evaluate the clinical utility of 18F-FES PET/CT to guide second-line treatment decision in ER-positive, HER2-negative MBC patients with progressive disease on first-line SOC hormonal therapy. METHODS: Fluoroestradiol F18 (Cerianna) is being evaluated in a phase 4, open-label, prospective cohort study enrolling 206 patients at 20-30 centers in the United States. Patients diagnosed with ER-positive, HER2-negative MBC who have progressive disease on first-line aromatase inhibitor therapy with or without CDK4/6 inhibitor will be included in the study; patients with isolated hepatic metastases will be excluded. All patients will undergo an 18F-FES PET/CT scan. The treating physician will complete a standardized questionnaire to indicate the second-line therapeutic management plan before the scheduled 18F-FES PET/CT. After interpretation of 18F-FES PET/CT by local radiologist or nuclear medicine physician, imaging results will be provided to the treating physician who will then fill out a similar questionnaire to specify the final therapeutic decision. The proportion of patients with a change in therapeutic management plan based on incorporation of 18F-FES PET/CT results will be the primary endpoint. During the study follow-up period of 18 months, data on SOC imaging, treatments/procedures received, and clinical outcomes will be collected. Secondary endpoints include visual and quantitative heterogeneity assessment of tumor 18F-FES uptake, and PFS rates at 6 months and 18 months after 18F-FES PET/CT, which will be assessed between patients with and without a change in therapeutic management plan. The trial is planned to begin enrollment in Q3 2022. Clinical trial information: NCT05068726 Citation Format: Stephanie van de Ven, Feng Luo, Nicholas DiGregorio, Adolfo Fuentes-Alburo, Francois Tranquart. Trial in Progress: Clinical Utility of Fluoroestradiol F18 PET/CT in Metastatic Breast Cancer Patients with ER-Positive and HER2-Negative Primary Lesions after Progression on First Line Hormonal Therapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-01-01.

Palbociclib plus endocrine therapy in hormone receptor-positive and HER2 negative metastatic breast cancer: a multicenter real-world study in the northwest of China

BackgroundReal-world data of Palbociclib are insufficient in China. This study aimed to investigate the treatment pattern and real-world outcomes in hormone receptor positive and human epidermal growth factor 2 receptor negative (HR+/HER2-) metastatic breast cancer (MBC) patients treated with Palbociclib in the northwest of China.MethodsHR+/HER2- MBC patients who received Palbociclib in 8 centers from July 2017 to September 2019 were retrospectively included in this study. Real-world objective response rate (ORR), progression-free survival (PFS) and safety profiles were analyzed. The survival curves were plotted by the Kaplan-Meier method to analyze PFS, which was verified by the log-rank test.ResultsIn total, 211 women were eligible for the analysis. A total of 85 patients (40.3%), 78 (37.0%), and 48 (22.7%) received Palbociclib in the first-, second-, third- or later-line setting, respectively. 46 patients achieved partial response and 145 patients experienced stable disease, with an ORR of 21.8% and a disease control rate of 90.5%. Following a median follow-up period of 14.2 months, the median PFS was 12.2 months (95% confidence interval, 10.1-14.3 m), and the median overall survival was not reached. Early Palbociclib initiation, sensitivity or acquired resistance to endocrine therapy, estrogen receptor and progesterone receptor double positivity, less than 3 metastatic sites, without visceral metastasis, bone metastasis only, without prior chemotherapy or endocrine therapy were associated with a prolonged PFS in MBC (All P < 0.05). The most common grade 3 or 4 adverse events (AE) was neutropenia (36.5%), and the most common nonhematologic AE was fatigue (10.9%). No patient experienced AE leading to treatment discontinuation.ConclusionPalbociclib plus endocrine therapy exhibited favorable effectiveness and manageable toxicities in the real-world setting, supporting their use in Chinese patients with HR+/HER2 − MBC.

PIK3CA mutation status, progression and survival in advanced HR + /HER2- breast cancer: a meta-analysis of published clinical trials

BackgroundApproximately 40% of hormone receptor positive/human epidermal receptor 2 negative (HR + /HER2-) metastatic breast cancer (mBC) patients harbor phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. However, associations between PIK3CA mutation status and clinical outcomes among patients with HR + /HER2- mBC have been heterogeneous across clinical trials. This meta-analysis was conducted to survey recently available trial data to assess the prognostic effects of PIK3CA among patients with HR + /HER2- mBC. MethodsRandomized clinical trials reporting progression-free survival (PFS) or overall survival (OS) stratified by PIK3CA status in HR + /HER2- mBC were identified via systematic literature review. Trial arms receiving phosphatidylinositol 3-kinase (PI3K)-targeted therapies were excluded. Meta-regression analysis was used to estimate the association between PIK3CA status and PFS and OS among included studies.ResultsThe analyzed data included 3,219 patients from 33 study arms across 11 trials (PIK3CA mutated: 1,386, wild type: 1,833). PIK3CA mutation was associated with shorter median PFS (difference [95% CI] (months): -1.8 [-3.4, -0.1], I2 = 35%) and shorter median OS (-8.4 [-13.4, -3.5], I2 = 58%, N = 1,545). Findings were similar for PFS rates at 6 months (odds ratio [95% CI]: 0.74 [0.59, 0.94], I2 = 42%, N = 3,160) and 12 months (0.76 [0.59, 0.99], I2 = 42%, N = 2,468) and directionally consistent but not statistically significant at 18 months (N = 1,726).ConclusionsPooling evidence across multiple studies, PIK3CA mutation was associated with shorter PFS and OS. These findings suggest a negative prognostic value of PIK3CA mutations in patients with HR + /HER2- mBC.

64Cu-SAR-Bombesin PET-CT Imaging in the Staging of Estrogen/Progesterone Receptor Positive, HER2 Negative Metastatic Breast Cancer Patients: Safety, Dosimetry and Feasibility in a Phase I Trial.

Breast cancers are most frequently oestrogen receptor (ER) and progesterone receptor (PR) positive and [18F]Fluorodeoxyglucose PET-CT (FDG) has lower sensitivity for these subtypes. The gastrin-releasing peptide receptor (GRPR) is overexpressed in ER+/PR+ breast cancers. This study assessed the safety and potential of [64Cu]Cu-Sarcophagine (SAR)-Bombesin PET/CT (BBN) in re-staging metastatic ER+/PR+/human epidermal growth-factor-2-negative (HER2-) breast cancer. Seven patients with metastatic ER+/PR+/HER2- breast cancer undergoing staging underwent [64Cu]Cu-SAR-BBN PET-CT. Bloods, vital signs and electrocardiogram, blood tracer-clearance and dosimetry were undertaken. GRPR status was assessed in available metastatic biopsy samples. Staging with conventional imaging ([18F]FDG, bone scan and diagnostic CT) was within 3 weeks of [64Cu]Cu-SAR-BBN PET/CT. PET scans were assessed visually and quantitatively. Seven patients underwent imaging. One of the seven had de-novo metastatic breast cancer and six of the seven recurrent metastatic disease. Two of the seven had lobular subtype. No adverse events were reported. All seven patients were positive on conventional imaging (six of seven on FDG). [64Cu]Cu-SAR-BBN imaging was positive in five of the seven. Both [64Cu]Cu-SAR-BBN-negative patients had disease identified on [18F]FDG. One patient was [64Cu]Cu-SAR-BBN positive/[18F]FDG negative. Four of seven patients were [64Cu]Cu-SAR-BBN positive/[18F]FDG positive. In these four, mean SUVmax was higher for [64Cu]Cu-SAR-BBN than [18F]FDG (SUVmax 15 vs. 12). In the classical lobular subtype (two of seven), [64Cu]Cu-SAR-BBN was more avid compared to [18F]FDG (SUVmax 20 vs. 11, and 20 vs. <3). Dosimetry calculations estimated whole-body effective dose for 200 MBq of [64Cu]Cu-SAR-BBN to be 1.9 mSv. [64Cu]Cu-SAR-BBN PET/CT appears safe and may have diagnostic value in metastatic ER+/PR+/HER2- breast cancer, particularly the lobular subtype. Further evaluation is warranted.

Defining resistance mechanisms to CDK4/6 inhibition in hormone receptor-positive HER2-negative metastatic breast cancer (MBC) through a machine learning approach applied to circulating tumor DNA (ctDNA).

3055 Background: Although cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are a primary treatment for hormone receptor-positive/HER2 negative MBC, data regarding resistance mechanisms are still an unmet need. The aim of the study was to highlight new resistance pathways using machine learning (ML) to detect multiparametric patterns in complex datasets. Methods: The study retrospectively analyzed a cohort of 610 hormone receptor positive HER2 negative MBC patients (pts) at Northwestern University, Massachusetts General Hospital and Washington University in St. Louis between 2015-2020 with baseline ctDNA testing by Guardant360. Pathways were defined based on previous work (Sanchez-Vega F et al, Cell. 2018) (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Only pathogenic variants according to OncoKB were included in the models. Associations among single nucleotide (SNVs) and copy number (CNVs) variations, pathway classification and previous exposure to CDK4/6i were explored through logistic regression and Gradient boosted machines (GBMs) ML algorithm. Results: at baseline, 322 pts (52.8%) were previously treated with CDK4/6i. The most detected pathway alterations were SNVs in PI3K (37.1%), P53 (31.8%), ER (29.2%) and RTK (22.3%). After stepwise logistic regression, RB1, NF1 and ESR1 SNVs were associated with previous exposure to CDK4/6i (respectively OR: 3.55 P = 0.017; OR: 3.06 P = 0.026 and OR: 1.82 P &lt; 0.001), while SNVs in the ER pathway were associated with CDK4/6i (1.56 P &lt; 0.001). Two GBMs models were designed based on gene variants (training AUC: 0.695, cross validation AUC: 0.631) and oncogenic pathways (training AUC: 0.713, cross validation AUC: 0.619). The highest relative importance (RI) was observed for ESR1 SNVs (RI: 35.35), TP53 SNVs (RI: 11.33), NF1 SNVs (RI: 3.45), SMAD4 SNVs (RI: 3.39) and RB1 SNVs (RI: 3.33). Alterations at a pathway level with the highest RI were ER SNVs (RI: 33.50), P53 SNVs (RI: 14.98), PI3K SNVs (RI: 14.40), RTK SNVs (RI: 10.55), RTK CNVs (RI: 10.26), cell cycle CNVs (RI: 6.99), cell cycle SNVs (RI: 6.77) and RAS SNVs (RI: 6.54). Of the previously highlighted pathway alterations, a significant impact on PFS after ctDNA collection was observed among de novo pts treated with CDK4/6i (165 pts) for ER SNVs (P &lt; 0.0001), RTK SNVs (P = 0.0011), RTK CNVs (P = 0.0006), Cell cycle CNVs (P = 0.0010) and Cell cycle SNVs (P = 0.0143). No impact was observed on PFS for pts who had not received a CDK4/6i-based regimen. Conclusions: The combination of ctDNA-based datasets and machine learning algorithms defined novel resistance pathways for patients treated with CDK4/6i. Although preliminary, these results suggest that alterations of the ER, RTK and Cell cycle pathways might be crucial to optimize treatment strategy and drug development.

Serial circulating tumor DNA monitoring of CDK4/6 inhibitors response in metastatic breast cancer.

Cyclin‐dependent kinase 4/6 inhibitors (CDK4/6i) significantly improve progression‐free survival and have become the standard therapy for estrogen receptor‐positive/human epidermal growth factor receptor 2‐negative metastatic breast cancer patients. Treatment surveillance by radiological imaging has some limitations in detection and repeated biopsy genomic profiling is not clinically feasible. Serial circulating tumor DNA (ctDNA) analysis may provide insights into treatment response. Here we performed serial ctDNA analysis (n = 178) on 33 patients. Serial ctDNA analysis identified disease progression with sensitivity of 75% and specificity of 92%. In eight of 12 patients (61%) responding to CDK4/6i who eventually developed progressive disease, serial sampling every 3 or 6 months captured the initial rise of ctDNA with an average lead time of 3 months. In three of eight patients that did not respond to CDK4/6i (progressive disease at first radiological assessment, 3 months), biweekly sequencing within the first cycle of CDK4/6i treatment (1 month) detected sustained ctDNA levels (≥0.2% variant allele frequency), with lead time of 2 months. Serial ctDNA analysis tracked RECIST response, including clinically challenging scenarios (bone metastases or small‐sized target lesions), as well as detecting acquired genetic alterations linked to CDK4/6i resistance in the G1 to S transition phase. Circulating tumor DNA analysis was more sensitive than carcinoembryonic antigen or cancer antigen 15‐3 serum tumor markers at monitoring tumor response to CDK4/6i treatment. Our findings indicated the possible clinical utility of serial ctDNA analysis for earlier progressive disease detection and real‐time monitoring of CDK4/6i response.

Abstract P2-07-01: Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i)

Abstract Background: CDK4/6i combined with ET improves survival for pts with HR+, HER2- MBC. However, biomarkers to predict efficacy and resistance are needed. We hypothesized that a comprehensive next-generation sequencing (NGS)-based liquid biopsy assessment of ctDNA mutation and copy number analysis may identify novel prognostic and predictive biomarkers. Methods: We collected serial blood for ctDNA testing from the 51 pts with HR+, HER2- MBC enrolled in the Alt Dose Palbo trial (NCT03007979), a single-arm phase II study of palbociclib plus letrozole or fulvestrant at 5 days on/2 days off weekly schedule as the first- or second-line ET. The median follow up was 38.2 months at data cutoff for this analysis. Plasma collected at baseline (BL) from all 51 pts, at progression (PD) from all 20 pts who have progressed, and at additional interim timepoints from 2 pts to explore longitutinal changes, with paired germline DNA, were subjected to the PredicineWES+ assay and low-pass whole-genome sequencing (lpWGS). The PredicineWES+ assay provides deep sequencing of 600 genes in the PredicineATLAS panel combined with whole-exome sequencing (WES) of all exonic regions of 20,000 genes enabling genome-wide detection of somatic single nucleotide variation (SNV), indels, copy number variation (CNV) and determination of bTMB. LpWGS offers an unbiased and high-throughput assessment of CNVs and tumor fraction in cell free DNA to derive bCNB. Statistical associations of bTMB, bCNB, and individual alterations with clinical benefit (CB), defined as no PD at 24 weeks by RECIST 1.1, and progression-free survival (PFS) were examined by Fisher’s exact test, Kaplan-Meier analysis, and Cox model. P values were adjusted to control false discovery rate (FDR). Results: The BL median bTMB was 1.6 mutations per megabase pair [IQR 0.6-3.5]. Pts with CB (N=41, 80%) had significantly lower bTMB scores (median [IQR] 1.2 [0.6-2.6] vs. 8.3 [2.4-21.4], (P&amp;lt;0.01) and significantly lower bCNB (P=0.03). bTMB and bCNB were highly correlated (Spearman correlation ρ =0.77, P=6.2E-11). bTMB above the predefined cutoff of 16 identified pts with significantly shorter PFS (4.7 vs 18.2 months, HR 3.73, CI 1.24-11.27, P=0.01). In contrast, BL clinical features including ET sensitivity, bone only vs. visceral, or de novo vs. recurrent MBC, were not associated with CB. BL alterations (SNV or CNV) in 91 genes, occurring in at least 3 pts each, were significantly associated with worse PFS (adjusted P&amp;lt;0.05), 61 of which were outside of the 600-gene panel. The 91 genes included both novel and previously reported alterations implicated in CDK4/6i and ET resistance. Examples include AR, ATM, AURKA, BRCA2, CCND1, DDR2, ESR1, FAT1, FGFR4, FOXP1, MYC, RB1, and RUNX1T1, some of which were also enriched at PD. Additional novel variants were enriched at PD, including ABCC12, ABCA13, SHANK1, and TET1. Serial analysis at BL, cycle 1 day 15 (C1D15), C2D1, the last Q3-month staging scan without PD, and at imaging detection of PD from 2 pts revealed a decline in bCNB at C1D15, then plateau, followed by an increase in bCNB that preceded imaging detection of PD. Conclusion: The comprehensive ctDNA profiling approach with PredicineWES+ and lpWGS identified high BL bTMB and bCNB as poor prognostic biomarkers in pts on CDK4/6i and ET. Our study also discovered novel candidate genes involved in cell cycle and DNA damage repair pathways in association with poor outcome. Results from this study highlight the genomic heterogeneity of HR+, HER2- MBC and provide important insights in the understanding of CDK4/6i and ET resistance mechanisms to guide therapeutic development in pts with resistant disease. Citation Format: Andrew Davis, Jingqin Luo, Tiantian Zheng, Lu Tan, Amy Wang, Rama Suresh, Foluso Ademuyiwa, Caron Rigden, Timothy Rearden, Katherine Clifton, Katherine Weilbaecher, Ashley Frith, Pavan K Tandra, Tracy Summa, Brittney Haas, Shana Thomas, Leonel Hernandez-Aya, Lindsay Peterson, Shujun Luo, Bonnie L King, Shidong Jia, Jianjun Yu, Pan Du, Jairam Krishnamurthy, Cynthia X Ma, C Dai. Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i) [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 P2-07-01.

Abstract P2-01-17: Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot study

Abstract Background and aim Minimally invasive liquid biopsies, usually referring to the analysis of tumor-derived material such as circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), have already proven their individual prognostic value in metastatic breast cancer. Whereas CTCs can be quantified by enumeration, levels of ctDNA are often expressed as the variant allele frequency of the dominant mutation, determined by rather costly targeted next generation sequencing. The modified fast aneuploidy screening test-sequencing system (mFAST-SeqS) represents a fast and cost-effective assay to estimate the fraction of ctDNA by measuring chromosomal aneuploidy instead of mutations. Next to the low costs (EUR 50, USD 60) additional advantages of the mFAST-SeqS method include the independency of mutational status of the tumor and the comprehensive overview of the tumor genome. Our aim was to investigate the potential concordance and complementarity between CTC-count and the mFAST-SeqS-based genome-wide aneuploidy score (GWA score). Material and methods Consecutive blood samples obtained from ER positive, Her2 negative metastatic breast cancer patients, starting a new line of therapy, (n=23) were enumerated for CTCs using the CellSearch-system (Menarini Silicon Biosystems, Bologna, Italy) and screened for the percentage of ctDNA using mFAST-SeqS. This method provides a GWA-score by amplification of long interspaced nuclear elements (LINE-1 elements) throughout the genome and calculating the overall deviation from the average profile in normal controls. Spearman correlation between CTC-count and GWA-score was calculated. For survival analysis, CTC-count was divided into high (CTChigh) and low (CTClow) using 5 CTCs/7.5 ml blood as the cut-off as before; similarly GWA-score was divided in high (GWAhigh)and low (GWAlow) based on the previously described cut-off of 5. Kaplan-Meier analysis was performed to assess overall survival (OS) in subgroups. Results Mean patient age was 58 (range 42-78). Median follow up was 15 months (range 0-37). Median number of previous treatment lines of patients was 2 (range 0-11). Median CTC-count was 22 (range 0-4438) and median GWA-score was 3.94 (range 0.87-114.31). The Spearman correlation coefficient between CTC count and GWA-score was 0.44 (p=0.02). 3 out of 20 patients were alive at end of follow-up. OS was significantly different in the CTChigh group versus the CTClow group (p=0.01, log-rank test, median OS 31 and 7 months, respectively), as well as in the GWAhigh versus the GWAlow group (p =0.01, log-rank test, median OS 27 and 7 months). There was concordance in 12 out of 23 samples (52.2%). 4 samples had both scores low and 8 both high, 9 samples were CTChigh/GWAlow and 2 were CTClow/GWAhigh. OS was significantly different in these groups (p &amp;lt; 0.01, log-rank test), with a median OS of 6 months in the group with both scores high, a median OS of 18 months for the cases with a discordant score and median OS not reached in the group with both scores low.Conclusions Our pilot study confirmed that both CTC-count and mFAST-SeqS GWA-score are prognostic and indicate that a combination of these measurements may yield complementary prognostic value. It should be noted however that the sample size was limited thus far. Our current preliminary results will be extended to 98 cases, results of which will be presented at the meeting. Citation Format: Noraly Verschoor, Elisabeth M Jongbloed, Jaco Kraan, Vanja de Weerd, Ngoc M Van, Agnes Jager, Stefan Sleijfer, Saskia M Wilting, John WM Martens. Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot 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 P2-01-17.